View
3
Download
0
Category
Preview:
Citation preview
An empirical framework for combining
surgery with immune therapy
Matthew David Brown MBBS (Hons)
This thesis is presented to The University of Western Australia
for the degree of Doctor of Philosophy
School of Surgery & Pathology
2007
- 2 -
To my parents, who have always inspired me. To my friends and
mentors, who spurred me on when things seemed hopeless. And to
my darling Sarah, who shares the journey with me.
- 3 -
Table of Contents
DECLARATION .................................................................................... - 9 -
PROFILE .............................................................................................. - 10 -
ABSTRACT .......................................................................................... - 15 -
INDEX OF FIGURES ......................................................................... - 18 -
1. INTRODUCTION ......................................................................... - 21 -
1.1. RATIONALE FOR SURGERY/IMMUNE THERAPY ......................................... - 25 -
1.1.1. Effects of surgery on inflammation........................................................ - 25 -
1.1.2. Effects of surgery on innate immunity ................................................... - 25 -
1.1.3. Effects of surgery on adaptive immunity ............................................... - 26 -
1.1.4. Surgery and overall immune function .................................................... - 26 -
1.1.5. Surgery and immunity: conflicting paradigms ....................................... - 27 -
1.1.6. Surgery improves anti-tumour immunity ............................................... - 27 -
1.1.7. Other benefits of surgery/immune therapy ............................................ - 28 -
1.1.8. Against surgery/immune therapy ........................................................... - 29 -
1.2. IMMUNE EFFECTS OF SENTINEL NODE BIOPSY ......................................... - 29 -
1.2.1. The evolution of lymph node surgery .................................................... - 29 -
1.2.2. Sentinel lymph nodes ............................................................................. - 30 -
1.2.3. Immune function of sentinel lymph nodes ............................................. - 30 -
1.2.4. Immune consequences of sentinel node surgery .................................... - 31 -
1.2.5. Nodal invasion and tumour proximity ................................................... - 32 -
1.2.6. Lymphadenectomy and immune therapy ............................................... - 33 -
1.3. SURGERY AND TUMOUR-SPECIFIC IMMUNITY .......................................... - 33 -
1.3.1. Surgery and antigen presentation ........................................................... - 34 -
1.3.2. Surgery and immune suppression networks ........................................... - 38 -
1.3.2.1. Alleviate tumour-derived suppressive factors............................................... - 38 -
1.3.2.2. Reduce effector requirements ....................................................................... - 38 -
1.3.2.3. Reduce MSC ................................................................................................. - 39 -
1.3.2.4. Provide “antigen holiday” ............................................................................. - 39 -
- 4 -
1.3.2.5. Change memory phenotype .......................................................................... - 41 -
1.3.2.6. Release effectors ........................................................................................... - 42 -
1.3.2.7. Improve overall cell mediated immunity ...................................................... - 42 -
1.4. AIMS AND HYPOTHESES ............................................................................. - 42 -
2. METHODS .................................................................................... - 45 -
2.1. CELL LINES AND CULTURE TECHNIQUES ................................................... - 45 -
2.1.1. Cell harvest............................................................................................. - 45 -
2.1.2. Mycoplasma screening ........................................................................... - 45 -
2.1.3. AB1 ........................................................................................................ - 46 -
2.1.4. AB1HA .................................................................................................. - 46 -
2.1.5. Renca ...................................................................................................... - 47 -
2.1.6. RencaHA ................................................................................................ - 47 -
2.2. MURINE SPECIES ........................................................................................ - 48 -
2.2.1. BALB/c and BALB/c nu-/-
mice ............................................................. - 48 -
2.2.2. CL4 TCR transgenic mice ...................................................................... - 49 -
2.2.3. HNT TCR transgenic mice .................................................................... - 49 -
2.3. IN VIVO PROCEDURES ................................................................................ - 49 -
2.3.1. Anaesthesia ............................................................................................ - 49 -
2.3.2. Analgaesia .............................................................................................. - 50 -
2.3.3. Subcutaneous inoculation....................................................................... - 50 -
2.3.4. Axillary inoculation ............................................................................... - 51 -
2.3.5. Intranodal inoculation ............................................................................ - 51 -
2.3.6. Intravenous inoculation .......................................................................... - 51 -
2.3.7. Intracardiac inoculation .......................................................................... - 52 -
2.3.8. Intrarenal inoculation ............................................................................. - 52 -
2.3.9. Resection studies .................................................................................... - 53 -
2.3.10. Lymphadenectomy ................................................................................. - 53 -
2.3.11. Monitoring ............................................................................................. - 54 -
2.3.12. Tumour size assessments ....................................................................... - 54 -
2.3.13. Survival analysis .................................................................................... - 54 -
2.3.14. Adoptive cell transfers ........................................................................... - 55 -
2.4. IN VIVO CFSE PROLIFERATION ASSAY ...................................................... - 55 -
- 5 -
2.4.1. Preparation of CL4 cells for adoptive transfer ....................................... - 55 -
2.4.2. Adoptive transfer for antigen presentation ............................................. - 56 -
2.4.3. Analysis for in vivo antigen presentation............................................... - 56 -
2.4.4. Statistical analysis for antigen presentation ........................................... - 57 -
2.5. IN VIVO CTL ASSAY .................................................................................... - 57 -
2.5.1. Pulsed and target reference peaks .......................................................... - 57 -
2.5.2. Adoptive transfer for in vivo CTL lysis assay ....................................... - 58 -
2.5.3. Analysis for in vivo CTL lysis ............................................................... - 58 -
2.5.4. Statistical analysis for in vivo CTL Assay ............................................. - 59 -
2.6. DC PHENOTYPING ....................................................................................... - 59 -
2.6.1. Isolation of DCs from lymph nodes ....................................................... - 59 -
2.6.2. Staining of DCs ...................................................................................... - 60 -
2.6.3. DC flow cytometry ................................................................................. - 60 -
2.6.4. Analysis of DC phenotypes .................................................................... - 60 -
2.7. TREG ASSAYS ................................................................................................ - 60 -
2.7.1. Cell surface and intracellular staining for Treg ....................................... - 61 -
2.7.2. Flow cytometry for Treg .......................................................................... - 61 -
2.7.3. Statistical analysis of Treg ....................................................................... - 62 -
2.8. MSC STUDIES ............................................................................................. - 62 -
2.9. HA-SPECIFIC CD8+: DETECTION/PHENOTYPE .......................................... - 63 -
2.9.1. Pentamer calibration............................................................................... - 63 -
2.9.2. Assessment of tumour-specific memory cells ....................................... - 63 -
2.10. THERAPIES .................................................................................................. - 65 -
2.10.1. Toll Like Receptor (TLR) ligand therapy .............................................. - 65 -
2.10.2. poly I:C .................................................................................................. - 65 -
2.10.3. CpG-ODN 1668 ..................................................................................... - 65 -
2.10.4. 3M019TM
................................................................................................ - 66 -
2.10.5. Activating anti-CD40 antibody therapy ................................................. - 66 -
2.11. IN VIVO DEPLETION STUDIES ...................................................................... - 66 -
2.11.1. CD4+/CD8
+T cell depletions .................................................................. - 67 -
2.11.2. Treg depletion .......................................................................................... - 67 -
- 6 -
2.12. IDENTIFICATION OF SENTINEL NODES ........................................................ - 68 -
2.12.1. Methylene blue ....................................................................................... - 68 -
2.13. DC TRACKING ............................................................................................. - 68 -
2.14. HISTOLOGY ................................................................................................. - 69 -
2.14.1. H&E staining .......................................................................................... - 69 -
2.14.2. Resection specimens .............................................................................. - 70 -
2.14.3. Kidneys .................................................................................................. - 70 -
2.14.4. Lymph nodes .......................................................................................... - 70 -
2.14.5. Lungs ...................................................................................................... - 70 -
2.15. CULTURE OF NECROPSY SPECIMENS .......................................................... - 71 -
2.15.1. Lung ....................................................................................................... - 71 -
2.15.2. Lymph nodes .......................................................................................... - 71 -
2.16. HA-SPECIFIC REAL TIME PCR ................................................................... - 71 -
2.16.1. Extraction of DNA ................................................................................. - 72 -
2.16.2. PCR of DNA templates .......................................................................... - 72 -
3. SURGERY AND CROSS PRESENTATION ............................ - 74 -
3.1. INTRODUCTION ........................................................................................... - 74 -
3.2. RESULTS ...................................................................................................... - 75 -
3.2.1. AB1HA in wild type and immunodeficient mice .................................. - 75 -
3.2.2. HA-specific presentation during AB1HA growth.................................. - 76 -
3.2.3. Specificity of CL4 proliferation ............................................................. - 76 -
3.2.4. HA presentation after surgery ................................................................ - 77 -
3.2.5. Completeness of resection...................................................................... - 80 -
3.2.6. HA presentation from recurrent AB1HA ............................................... - 84 -
3.2.7. Antigen presentation to CD4+ T cells post-op ....................................... - 84 -
3.2.8. Post-operative DC phenotype ................................................................ - 86 -
3.2.9. Cross presentation and in vivo CTL function ........................................ - 86 -
3.2.10. Recurrent tumour and systemic CTL responses..................................... - 91 -
3.3. DISCUSSION ................................................................................................. - 91 -
3.4. SUMMARY ................................................................................................... - 94 -
- 7 -
4. SINECOMITANT IMMUNITY .................................................. - 97 -
4.1. INTRODUCTION ........................................................................................... - 97 -
4.2. RESULTS ...................................................................................................... - 98 -
4.2.1. Concomitant immunity in the AB1HA model ....................................... - 98 -
4.2.2. Sinecomitant immunity in the AB1HA model ....................................... - 98 -
4.2.3. Sinecomitant immunity in the wounded flank ....................................... - 99 -
4.2.4. Sinecomitant immunity and re-challenge dose .................................... - 100 -
4.2.5. Surgical trauma and sinecomitant immunity........................................ - 101 -
4.2.6. HA in sinecomitant immunity to AB1HA ........................................... - 101 -
4.2.7. T cell dependence of sinecomitant immunity ...................................... - 103 -
4.2.8. Persistent tumour and sinecomitant immunity ..................................... - 104 -
4.2.9. Persistent antigen and sinecomitant immunity ..................................... - 107 -
4.2.10. Distribution of HA specific effectors post-op ...................................... - 108 -
4.2.11. Suppression and sinecomitant immunity ............................................. - 110 -
4.2.12. Sinecomitant immunity and immune therapy ...................................... - 120 -
4.3. DISCUSSION ............................................................................................... - 122 -
4.4. SUMMARY ................................................................................................. - 132 -
5. TUMOUR IMMUNITY & SENTINEL NODES ..................... - 135 -
5.1. INTRODUCTION ......................................................................................... - 135 -
5.2. RESULTS .................................................................................................... - 136 -
5.2.1. Identification of sentinel nodes ............................................................ - 136 -
5.2.2. Dendritic tracking and the sentinel nodes ............................................ - 137 -
5.2.3. Tumour proximity and node function .................................................. - 138 -
5.2.4. Tumour invasion and nodal function ................................................... - 141 -
5.2.5. Surgical dissection of the sentinel nodes ............................................. - 141 -
5.2.6. Antigen ablation and sentinel node excision ........................................ - 141 -
5.2.7. Tumour antigen presentation after node removal ................................ - 146 -
5.2.8. Sentinel node removal and re-challenge .............................................. - 147 -
5.2.9. Sentinel sampling and staged lymphadenectomy ................................ - 149 -
5.3. DISCUSSION ............................................................................................... - 149 -
- 8 -
5.4. SUMMARY ................................................................................................. - 161 -
6. RENCAHA ................................................................................... - 163 -
6.1. INTRODUCTION ......................................................................................... - 163 -
6.2. RESULTS .................................................................................................... - 164 -
6.2.1. Initial experience with RencaHA ......................................................... - 164 -
6.2.2. Subcutaneous RencaHAM ................................................................... - 165 -
6.2.3. Intravenous RencaHAM....................................................................... - 167 -
6.2.4. Orthotopic (intra-renal) RencaHAM .................................................... - 169 -
6.2.5. Lymph node metastases from RencaHAM .......................................... - 170 -
6.3. DISCUSSION ............................................................................................... - 176 -
6.4. SUMMARY ................................................................................................. - 180 -
7. THESIS SUMMARY .................................................................. - 183 -
7.1. PRINCIPAL FINDINGS ................................................................................ - 184 -
7.1.1. Effects of surgery on antigen presentation ........................................... - 184 -
7.1.2. Surgery & tumour-specific CTLs......................................................... - 184 -
7.1.3. Sentinel lymph nodes & anti-tumour immunity................................... - 185 -
7.1.4. Properties of sinecomitant immunity ................................................... - 185 -
7.2. CONCLUSIONS ........................................................................................... - 186 -
7.3. FUTURE DIRECTIONS ................................................................................ - 188 -
APPENDIX A: REFERENCES ........................................................ - 190 -
APPENDIX B: ABBREVIATIONS ................................................. - 216 -
- 9 -
Declarat ion
The experiments in this thesis constitute work carried out by the candidate unless
otherwise stated. The thesis is less than 100,000 words in length (inclusive of tables,
figures, bibliography and appendices) and complies with the stipulations set out for the
degree of Doctor of Philosophy of The University of Western Australia.
Dr Andrew Currie, Dr Robert van der Most, and Dr Kathy Heel have aided with the
calibration of flow cytometry instruments when four colour flow cytometry was
performed. Immune therapy protocols for experimentation in mice were provided by
Professor Bruce Robinson and Dr Delia Nelson. Finally, Irma Larma (the Urological
Research Centre Research Assistant) worked under the supervision of the candidate for
the final twelve months of experimentation. She prepared histological sections and
performed the RT-PCR experiments in their entirety.
The following organisations are gratefully acknowledged for their financial support:
Sporting Chance Cancer Foundation, Royal Australasian College of Surgeons, Abbott
Australasia, Australasian Urological Foundation, Sir Charles Gairdner Hospital Clinical
Staff Association and The University of Western Australia (Athelstan & Amy Saw
Fellowship). These organisations did not influence the research direction or
experimental design of this thesis.
- 10 -
Prof i le
Grants, Scholarships & Awards
Villus Marshall Prize, USANZ Annual Scientific Meeting, 2007
Finalist (Villus Marshall Prize), USANZ Annual Scientific Meeting, 2006
Finalist (ASI Young Scientist of 2006), Australasian Society of Immunology, 2006
Best Registrar Scientific Paper, Royal Australasian College of Surgeons (WA), 2006
SCGH Young Investigator Award, Sir Charles Gairdner Hospital, 2006
Inaugural SCGH Clinical Staff Association PhD Scholarship, SCGH, 2005
Best Surgical Oncology Paper, Royal Australasian College of Surgeons ASC, 2005
AUF/Abbott Registrar Scholarship, Australasian Urological Foundation, 2005
Raelene Boyle Scholarship, Royal Australasian College of Surgeons, 2004
Athelstan and Amy Saw Fellowship, University of Western Australia, 2004
Best Registrar Research Paper, Urological Society of Australasia State Meeting, 2004
- 11 -
Associated Papers and Published Abstracts
Brown M, Vivian J, Currie A, Robinson B, Hall J. Post operative tumour antigen
presentation. ANZ Journal of Surgery 2005;75(Suppl):A106-A107
Broomfield S, Currie A, van der Most R, Brown M, van Bruggen I, Robinson BWS,
Lake RA. Partial, but not complete, tumour debulking promotes protective anti-tumour
immunity when combined with chemotherapy and adjuvant immunotherapy. Cancer
Research 2005;65:7580-7584
Brown MD, Vivian JB, Currie AJ, Robinson BWS. Surgery re-sets the anti-tumour
immune response. Tissue Antigens 2005;66(5):367
Brown MD, Vivian JB, Currie AJ, Robinson BWS. Post-operative tumour antigen
presentation in murine models of malignancy. BJU International 97(Supp 1):9
- 12 -
Conference Presentations
Are Tumour Vaccines Logical? Urological Society of Australasia State Meeting (WA),
Bunker Bay Western Australia, November 5-7 2005
Postoperative Tumour Neo-Antigen Presentation in Murine Models of Primary and
Metastatic Malignancy, Australasian Society of Immunology National Convention,
Adelaide South Australia, December 12-16 2005
Postoperative Tumour Neo Antigen Presentation, Keystone Cancer Symposium,
Keystone Colorado USA, March 19-24 2005
Postoperative Tumour Antigen Presentation, Royal Australasian College of Surgeons
Annual Scientific Congress, Perth Western Australia, May 9-14 2005
Postoperative Tumour Antigen Presentation, Australasian Medical Science Research
Symposium (WA), Perth Western Australia, June 7 2005
Surgery Re-Sets the Anti-Tumour Immune Response, Australasian Society of
Immunology National Convention, Melbourne Victoria, December 4-7 2005
Post-operative tumour antigen presentation in murine models of malignancy. Urological
Society of Australasia Annual Scientific Meeting, Brisbane Queensland, March 26-30
2006
Intra-tumoural anti-CD40 agonist regresses local recurrence and metastasis after
surgery. Australasian Medical Science Research Symposium (WA), Perth Western
Australia June 9 2006
Sculpting the immune system with lymph node surgery. Royal Australasian College of
Surgeons Annual Registrar’s Day (WA), Perth Western Australia, July 22 2006
Intra-tumoural anti-CD40 agonist regresses local recurrence and metastasis after
surgery. Royal Australasian College of Surgeons Annual Registrar’s Day (WA), Perth
Western Australia, July 22 2006
- 13 -
Intra-tumoural anti-CD40 agonist regresses local recurrence and metastasis after
surgery. Royal Australasian College of Surgeons State Meeting (WA). Bunker Bay
Western Australia, August 5–6 2006
Sculpting anti-tumour immunity with sentinel lymph node surgery. Australasian Society
of Immunology Annual Conference, Auckland New Zealand, December 2–7 2006
The immune benefit of cancer surgery: lessons from sinecomitant immunity.
Australasian Society of Immunology Annual Conference (Young Scientist Session),
Auckland New Zealand, December 2–7 2006
Locally delivered agonistic anti-CD40 antibody in murine models of post-operative
recurrence and metastasis. Urological Society of Australia and New Zealand Annual
Scientific Meeting, Adelaide South Australia, February 18-22 2007
Immune implications of sentinel lymphadenectomy. Urological Society of Australia and
New Zealand State Meeting (WA), Mandurah Western Australia, October 26-28th
2007
- 14 -
Papers in Preparation
Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. Immune implications of
sentinel lymph node surgery
Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. The anti-tumour immune
benefit of surgery: conflicting paradigms and potential mechanisms
Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. Post-operative tumour
antigen presentation
Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. The immune benefit of
cancer surgery: lessons from sinecomitant immunity
Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. Empirical evidence for
combined surgery and immune therapy approaches in solid malignancy
Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ. Anti-tumour immunity from
the surgeon’s perspective
Brown MD, Vivian JB, Robinson BWS, Hall JC, Currie AJ, Nelson D. Locally
delivered agonistic anti-CD40 antibody in murine models of post-operative recurrence
and metastasis
- 15 -
Abstract
Only 50% of patients diagnosed with cancer can expect to survive more than five
years,1 and overall cure rates have remained static for some thirty years.
2,3 Within the
last decade, there has been resurgence in interest for the use of immune therapy to
improve oncologocial outcomes. While immune treatments have been disappointing as
monotherapies (responses are usually <20%),4-9
there is emerging evidence that immune
therapy can be effective when combined with surgery.10-19
Although there is clear
evidence that surgery impacts on general aspects of immunity, little is known about how
surgery affects key parameters of tumour-specific immunity. Until such insights are
obtained, the optimum strategy for combining surgery and immune therapy is likely to
remain unclear.
In this thesis, a haemagglutinin (HA) transfected murine mesothelioma tumour
(AB1HA)20
was employed to study the effects of surgery (primary resection and/or
sentinel node biopsy) on tumour-specific immunity. Using this tumour model, the HA-
specific immune response was tracked in vivo, to “spy” upon endogenous tumour-
specific immunity. Particular parameters of focus were: tumour antigen cross
presentation, tumour-specific cytotoxic T lymphocytes (CTL), sentinel nodes, and
resistance to re-challenge.
Antigen presentation was found to be highly efficient since both micro-metastases and
small primary tumours were associated with robust antigen presentation. Antigen
presentation was directly related to tumour size, but always confined to the sentinel
lymph nodes. Surgery, in the forms of primary resection and/or sentinel node biopsy,
had a profound effect on antigen presentation. Primary tumour resection produced a
gradual decline in antigen presentation, until it was no longer detectable at two weeks
after surgery. As was the case pre-operatively, tumour antigen presentation was
confined to the sentinel nodes for all post-operative time points. When sentinel node
biopsy was combined with primary resection, antigen presentation could be
immediately and completely ablated. If tumour remained in situ when sentinel nodes
were removed, antigen presentation shifted to more distant (or systemic) sites. It was
not possible to predict the nodes which presented tumour antigen after sentinel node
biopsy, but a lag phase of three to five days preceded the shift.
- 16 -
This thesis highlights the complex role of tumour antigen in the adaptive immune
response. While surgery could reduce tumour antigen, this reduction correlated with an
improvement in CTL function and tumour resistance. Not only did CTL function
improve, but unlike pre-operatively, it was detectable systemically after surgery. It was
postulated that tumour antigen presentation had a suppressive effect on tumour-specific
immunity, and may tether tumour-specific CD8+ to the sentinel node. By extension,
surgery could “slip the antigen tether”, releasing CD8+ from the sentinel nodes and
improving systemic CD8+-mediated tumour resistance.
The contribution of sentinel nodes was similarly complex. As previously described by
others,21,22
there seemed to be a topography of immunological function across sentinel
nodes. The sentinel nodes closest to a tumour exhibited poor tumour-specific CD8+
proliferation and tumour target lysis in vivo, but the nodes of intermediate distance
functioned well. Moreover, while sentinel nodes were a reservoir for tumour antigen,
and while a decline in antigen presentation correlated with enhanced tumour resistance
after surgery, sentinel biopsy was detrimental to tumour immunity. That apparent
paradox could be related to the disproportionate representation of tumour-specific CTL
within the sentinel nodes. Thus while sentinel node biopsy could reduce tumour antigen,
it may also remove the pool of CD8+ that would otherwise egress from the sentinel
nodes and acquire more effective cytotoxic characteristics.
To integrate the concepts of this thesis, the principle that surgery enhanced tumour-
specific immunity should be highlighted. This phenomenon, previously described as
“operation immunity”23,24
or “sinecomitant immunity”25
, provides a rationale for
combining surgery with immune therapy. Importantly, sinecomitant immunity was
strongest away from the surgical site and beyond the early post-operative phase. It
depended on three, logically linked factors: a decline in antigen presentation after
surgery, the presence of sentinel lymph nodes, and CTL. By uncovering these
requirements for sinecomitant immunity, and by identifying the effects of surgery on
tumour specific immunity more generally, this thesis provides an empirical framework
by which surgery and immune therapy may be combined.
Specifically, it is now hypothesised that the early and intermediate post-operative phase
may present a “window of opportunity” for immune therapy. During that period,
patients may be optimally responsive to immune treatments: tumour associated
- 17 -
suppressive factors may decline,26,27
and tumour antigen presentation is falling. The
importance of antigen decline during this phase suggests that tumour vaccines may
initially be inappropriate. In the early post-operative phase, it may rather be suitable to
aid the effector arm of the immune system, e.g. by activating anti-CD40 antibody (as
demonstrated in this thesis), CD4+ regulatory T cell (Treg)-targeting therapy (e.g.
cyclophosphamide), or adoptive transfer therapy. At delayed time points after surgery,
immune suppression and tumour antigen load may be re-constituted by recurrent
disease. In that setting, strategies to further debulk tumours (e.g. chemotherapy,
radiotherapy and surgery) and/or more complex immune interventions (e.g.
lymphodepletion + adoptive transfer + tumour vaccination) may be required.28
- 18 -
Index of Figures
Figure 1.1. William Coley – the father of immunotherapy. ....................................... - 23 -
Figure 1.2. Simplified view of anti-tumour immunity ............................................... - 37 -
Figure 3.1. Growth kinetics of subcutaneous AB1HA in wild type and nude mice .. - 77 -
Figure 3.2. Location of HA-specific presentation in AB1HA tumour bearing mice . - 78 -
Figure 3.3. CL4 proliferation during tumour growth ................................................. - 79 -
Figure 3.4. Specificity of CL4 proliferation. ............................................................. - 80 -
Figure 3.5. Surgery for AB1HA tumours .................................................................. - 81 -
Figure 3.6. CL4 proliferation before and after surgery .............................................. - 82 -
Figure 3.7. Completeness of resection. ...................................................................... - 83 -
Figure 3.8. CL4 proliferation with locally recurrent or “metastatic” AB1HA. ........ - 85 -
Figure 3.9. HA-specific CD4+ proliferation before and after surgery. ...................... - 87 -
Figure 3.10. Flow cytometry for DC cell phenotyping. ............................................. - 88 -
Figure 3.11. DC phenotype before and after surgery. ................................................ - 89 -
Figure 3.12. Post-operative in vivo CTL ................................................................... - 90 -
Figure 3.13. Primed in vivo CTL after surgery .......................................................... - 92 -
Figure 4.1. Concomitant immunity in the AB1HA model. ........................................ - 99 -
Figure 4.2. Sinecomitant immunity in the AB1HA model. ..................................... - 100 -
Figure 4.3. Sinecomitant immunity in the surgical site. .......................................... - 102 -
Figure 4.4. Sinecomitant immunity: significance of re-challenge dosage. .............. - 103 -
Figure 4.5. Effect of surgical wounding on sinecomitant immunity........................ - 105 -
Figure 4.6. HA specific immunity does not dominate the sinecomitant response ... - 106 -
Figure 4.7. Sinecomitant immunity in BALB/c nu-/-
............................................... - 106 -
Figure 4.8. The effect of T cell depletion on sinecomitant immunity ...................... - 107 -
Figure 4.9. Incomplete surgery did not protect against new tumour challenges...... - 109 -
Figure 4.10. Tumour antigen persistence partially ablated sinecomitant immunity - 110 -
Figure 4.11. Distribution of HA-specific CD8+ T cells. .......................................... - 112 -
Figure 4.12. CD127 and CD44 analysis of CD8+Pentamer
+ cells ........................... - 113 -
Figure 4.13. Expression of CD44+ in Pentamer+ and Pentamer
- CD8
+ ................... - 114 -
Figure 4.14. Expression of CD127 in CD8+CD44
+ populations .............................. - 115 -
Figure 4.15. Representative flow cytometry for Treg quantification. ....................... - 116 -
Figure 4.16. Treg frequency pre- and post-operatively ............................................. - 117 -
Figure 4.17. Treg remained despite PC61 mAb. ....................................................... - 118 -
Figure 4.18. Correlation of Treg depletion with tumour emergence ......................... - 119 -
- 19 -
Figure 4.19. Effect of Treg depletion on local recurrence. ........................................ - 120 -
Figure 4.20. MSC in tumour bearing and post-operative mice. ............................... - 121 -
Figure 4.21. Response to immune therapy after surgery. ........................................ - 123 -
Figure 4.22. Response to immune therapy in the healthy flank and surgical site. ... - 124 -
Figure 5.1. Transit of methylene blue dye into the sentinel nodes. ......................... - 137 -
Figure 5.2. Traffic of DC to the sentinel nodes........................................................ - 139 -
Figure 5.3. Cross presentation and in vivo CTL function after surgery. .................. - 142 -
Figure 5.4. Tumour proximity and nodal function. .................................................. - 143 -
Figure 5.5. Viability and assay cell penetrance in the axillary and inguinal nodes. - 144 -
Figure 5.6. Nodal invasion: effects on antigen presentation and in vivo CTL. ........ - 145 -
Figure 5.7. Primary resection with sentinel node excision. ..................................... - 146 -
Figure 5.8. Antigen presentation after resection and sentinel node biopsy. ............ - 147 -
Figure 5.9. Cross presentation from local recurrence, after sentinel node removal. - 148 -
Figure 5.10. Effect of sentinel node removal on survival from re-challenge .......... - 150 -
Figure 5.11. Re-challenge after sentinel node sampling or delayed biopsy............. - 151 -
Figure 5.12. The post-operative CD8+ effector egress postulate. ........................... - 157 -
Figure 5.13. Predicted implications of sentinel biopsy: scenario 1.......................... - 158 -
Figure 5.14. Predicted implications of sentinel biopsy: scenario 2.......................... - 159 -
Figure 5.15. Predicted implications of sentinel biopsy: scenario 3.......................... - 160 -
Figure 6.1. Derivation of RencaHA sub-clone......................................................... - 166 -
Figure 6.2. RencaWT and RencaHAM in wild type and congenic BALB/c nu-/-
.... - 167 -
Figure 6.3. Presentation of HA from subcutaneous RencaHAM. ............................ - 168 -
Figure 6.4 Pulmonary morphology and histology post intravenous RencaHAM .... - 171 -
Figure 6.5. Antigen presentation from i.v. RencaHAM. .......................................... - 172 -
Figure 6.6. Antigen presentation from intra-cardiac RencaHAM............................ - 173 -
Figure 6.7. Gross morphology and histology of orthotopic RencaHAM. ................ - 174 -
Figure 6.8. HA presentation from orthotopic RencaHAM. ..................................... - 175 -
Figure 6.9. Pulmonary micrometastases from orthotopic RencaHAM. ................... - 176 -
Figure 6.10. Evidence of nodal invasion from RencaHAM..................................... - 177 -
Figure 7.1. The window of opportunity for post-operative immune therapy. ......... - 187 -
- 20 -
Chapter 1
- 21 -
1. Introduction
Cancer surgery involves the removal of primary tumour mass, with or without a
regional lymph node procedure. Surgery is much maligned as an immune suppressive
treatment,29-33
but this seems at odds with several key phenomena. Firstly, the presence
of occult tumour cells and/or metastases in patients with clinically localised breast
cancer,34
colon cancer,35
melanoma,36
and prostate cancer37,38
has been well described.
In such instances, at least a proportion of patients may never present with recurrence
after primary resection, or they may enjoy a considerable period of remission - a
phenomenon known as “cancer dormancy”.3,39-41
This would seem impossible if surgery
was detrimental to tumour resistance.
Secondly, the spontaneous regression of metastases after primary resection has been
well described in melanoma and renal cell carcinoma.42
It has also been documented in
many other malignancies including: oesophageal cancer,43
gastric cancer,44
and
mesothelioma.45
If surgery was immune suppressive, such a phenomenon would be
unlikely.
Thirdly, long term survival is achievable if patients are able to undergo surgical
resection of metastases (metastectomy) in virtually every solid malignancy.3 While such
patients would have occult residual disease by definition, many enjoy prolonged
remission and some 15 – 20% are cured.3 If surgery was detrimental to the control of
micrometastases, this would seem inconsistent with the benefit of metastectomy.
Finally, there is increasing evidence that surgery boosts tumour-specific immunity by
numerous mechanisms, including: a reduction in myeloid-derived suppressor cell
(MSC) levels,46,47
a shift in CD4+ memory phenotype,
48 and a decline in the effector
requirements for tumour eradication.26
Given this immune benefit of cancer surgery,
combined surgery/immune therapy strategies for malignancy are advocated in this
thesis.
The concept of combining surgery with immune therapy dates back to the 19th
century.
In the 1800s, Verneuil recognised that post-operative infection delayed the onset of
recurrence and/or improved cure rates from resection.49,50
Typically, Verneuil would
leave cancer resections open or loosely approximated, intentionally facilitating post-
- 22 -
operative suppuration.49
As it is now recognised, bacterial infections elute molecular
danger signals (e.g. cytosine phosphorothioate guanine oligodeoxynucleotides, CpG-
ODN) which upregulate tumour immunity by numerous mechanisms (including the
activation of antigen presenting cells (APC).51
Indeed, in the era of Verneuil, the
practical application of infection (with or without surgical resection) became a popular
strategy for the amelioration and/or eradication of cancer.49,52
With the popularisation of aseptic technique, the use of bacterial infections to eradicate
cancer and/or improve results from cancer surgery declined.49
It was then in the late 19th
century, that the New York surgeon William Coley noted regression of sarcoma in a
patient who developed Streptococcal infection.49
This led Coley to embark on a
systematic study into the use of bacterial products with or without surgery, for the
treatment of malignancy.
Coley (see Figure 1.1), who is now credited to be the father of immunotherapy,49
developed a number of bacterial vaccines, including heat-killed Streptococcus pyogenes
and Serratia marcescens (Coley‟s Toxin), which was applied intra-tumourally and/or
topically.49
However, after Coley‟s death in 1936 and following the popularisation of
antibiotic use in surgery,49
there was a decline in efforts to combine surgery with
immune activating agents.
The later parts of the 20th
century and the early 21st century have seen a resurgence in
interest for the use of immune treatments in combination with surgery.3 There is
persisting research in the use of non-specific immune activating agents (like bacterial
toxins, as used by Coley),53
but many modern approaches employ a tumour-specific
approach (active specific immunotherapy).49
Indeed, the contemporary literature reports
a vast array of treatments that have been combined with surgery, in attempts to improve
the static cancer cure rates of the last 30 years.49
Most solid malignancies have been tackled with combined surgery/immune therapy,
including: melanoma54
and carcinomas of the breast,55
colon,56
prostate,57
lung,58
and
kidney.9,17
There are innumerable permutations in the type of immune treatment used,
the timing of immune therapy, mode of therapy (systemic,59
topical,60
or intra-
tumoural),61
extent of resection, accuracy/extent of staging,3 duration of immune
intervention, and whether the immunotherapy was combined with conventional
- 23 -
strategies. Some trials restricted therapy to cytokines,59
others trialled non-specific
immune activating treatments,62
some tried tumour vaccines as monotherapies,54
and
others used a combination of these approaches.63
Treatments were sometimes
commenced pre-operatively,64
sometimes started in the early post-operative phase,56
and
occasionally treatments were extended out to six months or more post-operatively.65
Figure 1.1. William Coley – the father of immunotherapy.
Dr William Coley practiced surgery at the New York Memorial (Sloan Kettering) Hospital between 1890
and 1936. He was the first to systematically study the use of immune treatments (in his case, heat-killed
bacteria) with or without surgery, for the treatment of solid malignancy.49,52
- 24 -
In this thesis, the immune benefit of cancer surgery is highlighted, and the essential
elements of this phenomenon are identified. This work further provides an empirical
framework by which surgery and immune therapy can be combined, rectifying
numerous deficits in our knowledge about the interaction of surgery with tumour-
specific immunity. Hopefully, the findings of this thesis will prove useful for the future
design of combined surgery/immune therapy strategies for malignancy, accelerating
developments in the field.
Subsequent discussion in this introduction will comprise three parts. In the first part, the
controversies about whether surgery boosts tumour immunity will be critiqued, because
improved tumour immunity would be the premise for combining surgery with immune
therapy. The dogma that surgery is immune suppressive will then reconciled with the
recent and historical evidence that resection benefits anti-tumour immunity. The
concept of sinecomitant immunity will also be highlighted, because the immunological
components of post-operative tumour immunity will be investigated in this work.
(Chapter 4).
In the second component of this Introduction, the evolution and anti-tumour immune
impact of sentinel node biopsy will be examined. Sentinel node biopsy is an
increasingly utilised technique of surgical prognostication, yet as will be explained,
little is known about the effects of sentinel biopsy on anti-tumour immunity. The
immune impact of sentinel node biopsy will be studied in Chapter 5 of this work.
The final aspect of Chapter 1 will provide a brief sketch of anti-tumour immunity,
highlighting the nexus of interaction between APC, CTL and helper T cells (TH). The
effects of surgery upon the tumour antigen presentation nexus have not previously been
studied in vivo, and examining this will be a major objective of this thesis.
The potential role of surgery in the disruption of tumour associated suppressive
networks will also be elucidated, because this phenomenon accounts for the anti-tumour
immune benefit of cancer surgery. Certain interactions between resection and tumour
associated suppression will subsequently be investigated in this thesis, including: the
role of MSCs, dendritic cell (DC) phenotypes, Treg, and antigen itself.
- 25 -
1 .1 . Rat ionale for Surgery / Immune Therapy
If surgery is to be combined with immune therapy, it is important to understand the
effects of operation on anti-tumour immune function. There is an existing body of
research that addresses the profound effects surgery can have on general immune
function. However, there is limited information about how surgery affects tumour-
specific immunity. In this section, current dogma about the effects of surgery on
immune function is dissected, and then reconciled with the central argument of this
thesis: that surgery improves tumour immunity.
1 . 1 . 1 . Effects of surgery on inf lammation
Surgery represents controlled trauma, producing inflammation proportionate to the
degree of the trauma.29,68
When trauma increases in magnitude, the inflammatory signal
shifts from immune stimulation to feedback loops of immune suppression.29,30
Thus the
effect of surgery on immune function may relate to numerous variables, including: type
of anaesthesia,69
presence/absence of blood transfusion,70-72
size of incision, amount of
tissue disruption/dissection, vascularity of the field, operation time, temperature
changes, and surgical approach (open or laparoscopic).73
To induce inflammation, surgery produces a flurry of endocrine, neural and cytokine
signals. These signals include local elution of PgE2,74
disseminated catecholamine
release,75
increase in adrenal corticosteroid production,74,76
and changes in cytokine
levels.74
Such cytokines are released from monocytes and macrophages at the wound
site, especially TNF, Il-1, and Il-6.68
Cytokine release occurs within hours of surgery,
and levels remain elevated for up to 3 days. An increase in hepatic acute phase reactant
production also occurs 77
(e.g. CRP, fibrinogen, haptoglobin) as well as elevated
systemic inflammatory markers (fever, elevated white cell count, and tachycardia).68,77
1 . 1 . 2 . Effects of surgery on innate immunity
Of the acute phase reactants released after surgery, CRP is perhaps the most studied. It
rises within hours of the surgery, peaks at 72 hours post-op, and remains elevated for 2
weeks.30,78
CRP may enhance neutrophil function and phagocytosis,78,79
but overall,
surgery is thought to depress neutrophil function.73
The cause of depressed neutrophil
function is unknown73
but a reduction in chemotaxis and phagocytosis has been
observed.80
- 26 -
Prostaglandin release from surgical wounds may also modulate natural killer cell (NK)
counts and function after surgery.81
One study suggests a 2 day nadir in natural killer
cell counts and cytotoxicity after hysterectomy,73
but a post-cholecystectomy82
study
paper suggested that decreased natural killer function could persist for up to 30 days
post-op. Therefore, the extent of impaired NK immunity after surgery remains
unknown.
1 . 1 . 3 . Effects of surgery on adapt ive immunity
Like innate immunity, surgery may have profound effects on adaptive immunity.
Several studies have assessed delayed type hypersensitivity (DTH) skin reaction after
surgery, demonstrating a depression in DTH, that was proportional to the extent of the
surgery (laparotomy versus laparoscopy or mini-laparotomy).83
The effect on DTH was
assessed early in each case: immediately,83
two days post-op,83
or three days post-op84
.
The time to recovery remains unclear, but is thought to be brief.85
The mechanism of depressed cell mediated immunity is undefined. Surgery and/or
trauma may be associated with a brief shift in the TH/Treg ratio (less than 7 days post-
op),85
with reduced production of Il-2,86
downregulation of MHC Class II expression on
macrophages,87,88
reduced induction of immature DC from peripheral blood
monocytes,89
and a reduction in the efficiency of antigen presentation.90
In addition to impairing antigen presentation and overall cell mediated immunity,
surgery may induce a preponderance to humoral immunity (a “TH1 to TH2 shift”).91
This
shift is mediated by a downregulation of TH1 cytokines (Il-2, Il-12, IFN, TNF, Il-
1)92
along with increased production of TH2 cytokines (Il-10, Il-1rA, sTNFr, sIL-
2r).93,94
Once again, the duration of the TH1 to TH2 shift is unknown.
1 . 1 . 4 . Surgery and overal l immune funct ion
The existing weight of literature suggest that surgery impairs general immune function.
By disrupting tissue, surgery may evoke a monocyte-derived cytokine profile that
enhances TH2-based inflammation, hinders antigen presentation, reduces cell mediated
immunity, and promotes a humoral response. However, this impairment is thought to be
brief, and reversible.85
- 27 -
Prevailing dogma also suggests that surgery impairs the anti-tumour immune response.
Many publications document accelerated tumour growth after surgery. For instance,
surgery accelerates pulmonary metastases from tail vein injection,31,95
produces faster
growth of spontaneous pulmonary metastases,32,33
enhances the growth of
intraperitoneal tumours,96
increases the number of hepatic metastases after portal vein
injection,97
and accelerates flank tumour growth.98
1 . 1 . 5 . Surgery and immunity: confl ic t ing paradigms
If surgery impairs anti-tumour immunity, the duration of that impairment is unknown.
Existing papers hint that surgical impairment of tumour immunity may be a short term
phenomenon (less than two weeks in rodents),96
and one group‟s data even suggests that
general immunity recovers to baseline at 24 hours post-operatively.99
Moreover, if surgery harms general aspects of immune function, modern techniques and
contemporary drug treatments might greatly attenuate that harm. In animal models,
blocking the immune mediators of surgery (e.g. non-steroidal anti-inflammatories100
and
corticosteroid inhibitors101
) can reduce surgical immune suppression. In addition,
changing to regional anaesthesia102
and minimally invasive techniques 73,103,104
could
further minimise the immunologic harm of surgery.
Finally, and more fundamentally, in almost every paper reported to describe accelerated
tumour growth with surgery, the surgery of interest was a sham procedure. As discussed
in 1.3.2, tumours normally suppress the immune response. Since cancer surgery reduces
tumour burden, it may improve the immune response rather than impair it. To assess the
effect of surgery on anti-tumour immune function more accurately, the surgery must
involve tumour resection – not irrelevant trauma. When this approach is taken, the
weight of literature shifts to suggest surgery improves anti-tumour immunity.
1 . 1 . 6 . Surgery improves ant i - tumour immunity
The anti-tumour immune benefit of cancer surgery was discovered nearly one hundred
years ago.23,24
Uhlenhuth and his co-authors reported that surgically treated rats could
resist a second challenge of the same tumour, and they dubbed this phenomenon
“operation immunity”. They noted that resection had to be complete, or else immunity
did not develop.24
- 28 -
Some 60 years later, Fisher et al proposed the term “sinecomitant immunity” to describe
the anti-tumour reaction against a tumour re-challenge after surgery.25
Sinecomitant
immunity was distinguished from “concomitant immunity”,25,105
where rejection of a
second tumour challenge is sometimes seen in animals with progressive primary
tumour. As such, the immune benefit of surgery can be understood as the excess of
sinecomitant immunity (immunity against re-challenge after primary tumour removed)
over concomitant immunity (immunity against re-challenge with primary tumour left in
situ).
Preceding research suggests the extent of sinecomitant immunity depends on the
antigenicity of the tumour, the size of the primary tumour, the time of re-challenge, and
the strength of the re-challenge.24,106,107
In previous publications, resistance to re-
challenge was best seen in immunogenic tumours (i.e. tumour models where protection
occurs after irradiated vaccination),108
relatively small tumours (<10mm in diameter),109
and when the re-challenge was delayed at least seven days after the surgery.110,111
Sinecomitant immunity has previously been thought to be weak, since inoculums >105
cells could overcome resistance in some models.108
1 . 1 . 7 . Other benef i t s of surgery/ immune therapy
Not only might surgery improve tumour immunity,112,113
but it may offer a number of
other benefits. For instance, de-bulking could improve a patient‟s overall function and
reduce symptoms.112,113
It can also eliminate the primary as a source of pain, para-
neoplastic syndromes, and haemorrhage.112
Primary resection also prevents further
metastases from that site,112
and reduces the number of tumour cells. Reduced tumour
burden may enable lesser doses of systemic therapy and better response.112
Moreover,
cancers are metabolically active and are associated with a degree of cachexia. By
removing metabolically active tumour and reducing the levels of cachexia-inducing
cytokines (e.g. Il-1)114
surgery could alleviate the catabolic state. Speculatively, this
might improve the availability of conditionally essential amino acids (e.g. glutamine)115
and improve immune function. Finally, tumour resection provides tissue. Not only does
this provide a definitive diagnosis and assist with prognostication, but it also facilitates
research (e.g. the preparation of tumour vaccines, tumour infiltrating lymphocyte
therapy etc.)112
- 29 -
1 . 1 . 8 . Against surgery/ immune therapy
There are numerous arguments against combining surgery with immune therapy, and
these should be acknowledged. Surgery exposes the patient to the risk of operative
mortality, and to the potential for considerable morbidity. The latter morbidity may
even preclude subsequent immune therapy.14,112,113
Most worryingly, many tumours
promote angiogenesis, but some tumours may release anti-angiogenic and non-specific
metastasis-suppressing factors107
(e.g. angiostatin, endostatin).116,117
Thus, in some
cancers, metastases may grow more rapidly after primary resection, because of
alleviated angio-suppression.107,116,117
Finally, surgical manipulation of tumours may
promote seeding and new tumours,34,118,119
, abrogating the benefit of immune therapy.
Indeed, surgical wounds are a rich environment for tumour seeding: hypoxia, fibroblast
activation, and paracrine factors promote tumour growth and/or suppress immunity.120
1 .2 . Immune Effec ts o f Sent ine l Node Biopsy
In the first section of this introductory Chapter, it was argued that surgery improves
tumour immunity rather than detracts from it. However, discussion has focussed on the
effects of tumour resection only. Oncological procedures may also entail a lymph node
procedure, and more recently, a sentinel node biopsy. Thus to understand the effects of
surgery on tumour immunity, it is also important to consider lymphadenectomy.
Lymph nodes are anatomically and functionally elegant collections of immune tissue,
strategically located on the efferent lymphatics of regional tissues. Lymph nodes are
frequently frequent sites for metastasis and for this reason, cancer surgery has
traditionally involved lymphadenectomy.22
The practice of lymphadenectomy has
undergone considerable evolution in recent decades, but the immune implications of
lymph node removal remain unknown.
1 . 2 . 1 . The evolut ion of lymph node surgery
In the early 1700s, Valsalva proposed that cancer was a local lesion which spreads via
the lymphatics to the regional nodes.121,122
This led to the practice of wide local
resection and regional node clearance, which has prevailed until the last few decades.122
However, tumours may spread by the haematogenous route, or they may bypass
regional nodes via lymphatico-lymphatic and lyphatico-venous shunts. Thus cancers
may avoid lymph glands and disseminate systemically, even at an early stage.123
From
- 30 -
the modern view-point, cancer is considered a highly complex, heterogeneous, and
systemic disease.122
Loco-regional lymph nodes are no longer thought as “barriers to
metastasis”, but simply organs that are frequently affected by cancer spread.122
Thus
node dissection is beneficial only to provide staging information about the cancer, or to
improve survival for particular cancers (e.g. testicular cancer,124
, melanoma,125
and
penile cancer)126,127
where nodes can be the solitary sites of metastasis. Given that
lymphadenectomy is primarily for prognostication, an assessment of node status without
extensive dissection is preferred. To this end, sentinel node biopsy has been
popularised.
1 . 2 . 2 . Sent inel lymph nod es
The sentinel node concept was first proposed by Cabanas in 1977.128
Sentinel nodes are
those lymph glands in direct lymphatic communication with the tumour site.129
The
value of sentinel node biopsy is that its tumour status (involved or not involved),
predicts the status of the entire regional node group.129
By sentinel biopsy, it is possible
to provide prognostication without exposing the patient to the risks and morbidity of
extended lymphadenectomy.
Notably, the sentinel nodes include not only the first lymph node seen on dynamic
lymphoscintigraphy, but all nodes in direct communication with the tumour site. Thus
identification of the sentinel node is best accomplished by visualising the lymphatic
channels130
(e.g. using isosulphan blue dye), together with radioactive isotope (to assist
in finding the nodes).131-133
Sentinel lymph node biopsy has been validated for a number
of malignancies, most rigorously in breast carcinoma,134
melanoma,135
and squamous
cell carcinoma. 132,136
1 . 2 . 3 . Immune funct ion of sent inel lymph nodes
By definition, the sentinel lymph node would encounter a tumour first. Therefore,
sentinel nodes are probably the site of initial tumour antigen presentation.137
Indeed, the
sentinel node has been shown to produce the greatest volume of IFN, GM-CSF and Il-
2.137,138
However, as the sentinel nodes are the earliest affected by the tumour, they
might also be most susceptible to immune suppression.21
Consistent with this, T cells of the sentinel lymph nodes have the highest levels of
TCR downregulation (evidence of T cell suppression) in nodes of patients with breast
- 31 -
cancer.139
Sentinel nodes also produce more Il-10 than non-sentinel nodes in
melanoma140,141
which may induce immune suppressive DCs142,143
, and reduce T cell
activation.22
Additionally, relative to mature DC, the DC of the sentinel nodes are less
dense,141,144
have reduced dendrite length,141,144
low co-stimulatory molecule
expression, 27
and decreased MHC Class II expression.145
A greater proportion of those
DCs also express indoleamine-2,3-dioxygenase (IDO),146
which reduces T cell
responses.147
1 . 2 . 4 . Immune consequences of sent inel node surgery
With numerous advances in tumour immunology, the immune impact of node removal
can be examined with new insight. Firstly, the tumour draining lymph nodes are the
principal site of cell associated tumour antigen presentation20,148,149
(although not soluble
antigens)150
. If tumour draining nodes are actually sentinel nodes (Chapter 5), then
sentinel biopsy might dilute antigen below immunogenic thresholds (through passage
into the vasculature),151
completely ablate antigen presentation,152
or force antigen
presentation to secondary lymph nodes.153
The validity nor sequelae of these hypotheses
have never been tested in vivo.
If tumour antigen can be eradicated by sentinel node biopsy, the impact of that
phenomenon is unknown. In one approach for colorectal cancer, dubbed “immune
corrective surgery”, (ICSTM
, Biocrystal, Columbus Ohio USA), investigators attempt to
resect all lymph nodes where tumour antigen is found.154
The technique uses radio-
isotype labelled anti-tumour antigen antibody (anti-TAG-72) to localise sites of antigen
presentation. Those nodes are then removed at the time of surgery. The authors report
an improvement in survival with this technique in a phase 1 study,154
but a larger study
has yet to be published.
Secondly, T lymphocytes localise in the sentinel lymph nodes of primary
tumours,48,155,156
possibly because of tumour-derived chemokine signals, inflammatory
signals, or persisting tumour antigen. Evidence for effector localisation to the draining
nodes includes not only modern data (adoptive transfer studies, tetramer staining etc.)155
but also the simple fact that draining lymph nodes (rather than naïve nodes and non-
draining nodes) convey protection against that tumour to syngeneic, naïve mice.153
Therefore, if sentinel node biopsy is performed, a patient may be depleted of tumour-
specific effectors. The result may be increased susceptibility to local recurrence and
- 32 -
metastases. Alternatively, the local lymph nodes might only be a component of a
generally activated lymphoreticular immune network.157
Removing the sentinel nodes
might therefore be harmless, as remaining components of the network provide tumour
surveillance. Which of these theories are correct, remains unknown.
Sentinel node biopsy may also impair immunological memory, particularly the
generation of memory CD4 cells. While established memory cells are maintained in the
absence of lymphoid tissue, CD4+ cells require secondary lymphoid organs for memory
differentiation.158
Only a handful of studies from the 1960s and 1970s have examined the role of lymph
node dissection on tumour growth using syngeneic models. Sentinel node biopsy had
not been established at that stage and results were highly variable. Several publications
suggested that lymph node dissection had an insignificant effect on the growth of
primary tumours, local re-challenges, distant re-challenges, or metastases.152,153,157,159
Others reported that node dissection accelerated the growth of nearby re-challenges, but
not distant tumours.160
Further publications suggested that node dissection slowed the
onset of concomitant immunity161
and impaired the response to re-challenge (local and
distant).161-163
In humans, the picture is even more confusing. In some tumours, node
dissection may reduce local recurrence164
and/or metastases.164,165
In other tumours (e.g.
breast) variations of node dissection made no difference to recurrence and/or survival
from metastasis.166-169
Inconsistencies between the findings may relate to whether
lymph node metastases were present or not, whether primary resection was complete or
not, the extent and timing of node dissection, species and subspecies variation,
immunogenicity of the tumours,170
size of the primary, sites of re-challenge, and the
strength of the re-challenge inoculum.161
1 . 2 . 5 . Nodal invas ion and tumour proximity
The location and tumour status of sentinel nodes may affect their contribution to tumour
resistance, and by extension, the effects of sentinel node removal. Numerous studies
demonstrate that tumour proximity affects node function. Those lymph nodes closest to
a tumour have less mitogenic reactivity for allo-reactive CTL,171
reduced CD4+:CD8
+
ratios,172,173
higher concanavalin-A induced suppressor cells,174
and less tumour
reactivity.175
- 33 -
Based on immune reactions from melanoma and breast cancer node specimens, Cochran
and colleagues proposed a topography of anti-tumour immunity across the lymph node
basin.21,22
Specifically, those nodes closest to a tumour may be exposed to the greatest
antigen stimulation, but also the strongest tumour-derived suppression. Those nodes
furthest away react poorly also, because they are too distant to be engaged by the
tumour. Thus the intermediate zone lymph nodes function best – encountering tumour
antigen, but relatively spared from tumour-derived suppression.21,22
The effect of node
dissection may therefore depend on the design of the resection: removal of closest nodes
may alleviate suppression, but removal of more distant nodes might impair immunity.
Secondly, tumour invasion may impact on the contribution of nodes to tumour
immunity. By invading the nodes, tumours may tip the profile of antigen processing
away from cross presentation and towards direct presentation. Since tumour cells have
poor MHC class I expression and low co-stimulatory molecule expression,176
the result
may be reduced priming and/or anergy. Supporting this notion, previous research
demonstrates that nodal metastasis are associated with the accumulation of suppressor
cells174
,T lymphocyte anergy, 177-179
, and the ablation of concomitant immunity.180
However, tumour-specific parameters of in vivo CTL function and antigen presentation
by tumour-invaded nodes have not been tested previously.
1 . 2 . 6 . Lymphadenectomy and immune therapy
As the immune impact of sentinel node dissection has not previously been elucidated, it
is unsurprising that little is known about how sentinel dissection affects response to
immune therapy. There are only two publications, neither of them looking at sentinel
node biopsy. Specifically, Harada and colleagues found a reduced response to Il-12
therapy after node dissection in the B16 melanoma model.181
Then, in a small clinical
trial of recurrent head and neck cancer, a reduced response to local Il-2 was observed in
patients after bilateral lymph node dissection.182
1 .3 . Surgery and Tumour-Speci f i c Immuni ty
The anti-tumour immune response provides a “last line of defence” against malignancy,
swinging into action once the normal intracellular and genetic safety mechanisms have
failed to combat carcinogenesis.39
Innate immunity may contribute to tumour
immunity,183,184
particularly through inflammation.185
However, this thesis emphasises
the effects of surgery on adaptive immunity, because tumour-specific immunity is the
- 34 -
basis for most successful immune strategies to date.186-188
Understanding adaptive
tumour immunity requires an appreciation of the antigen presentation nexus, which
consists of APC, TH, and CTL. However, no previous study describes the effects of
surgery upon the levels of antigen presentation in vivo, and in this thesis, a primary aim
is to rectify that deficit. Potential interactions of surgery with the antigen presentation
nexus are now explained, followed by a critique of the potential mechanisms by which
surgery boosts tumour immunity.
1 . 3 . 1 . Surgery and ant igen presentat ion
The antigen presentation nexus involves the processing and recognition of tumour
antigen. Those tumour antigens may be unique to an individual tumour (a new antigen,
or “neo-antigen”), tumour-specific shared antigens (e.g. MAGE), antigens common to a
particular type of tissue (e.g. PSA), tissue differentiation antigens (e.g. CEA), or
ubiquitous antigens (e.g. HER-2/neu).66
The effects of surgery upon tissue
differentiation and normal tissue antigens has been well described for various tumours
(e.g. PSA after prostatectomy,189
post-operative CEA in colon cancer190
). However,
such antigens are not foreign to the host, and are therefore subject to tolerance. Rather it
is the foreign (neo-antigens) that are most crucial to tumour rejection, and therefore
potentially the most important to consider. Unfortunately, the kinetics of post-operative
tumour neo-antigens have not previously been studied in vivo, and until this deficit can
be rectified, our understanding of post-operative tumour immunity remains deficient.
Neo-antigens may be directly presented to CD8+ T cells by the endogenous or “classical
MHC Class I” pathway.191
As with healthy cells, tumour cellular proteins are
continuously turned over. Tumour proteins marked for degradation are tagged with
ubiquitin molecules, taken up by proteosomes, and degraded into oligopeptide residues.
Those tumour oligopetides are hydrolysed by cytosolic peptidases into peptides. Most
of those peptides are recycled by the cell for protein synthesis and energy, but a
proportion are transported into the endoplasmic reticulum (ER) via the “transporter
associated antigen processing protein” (TAP). Once there, the peptides are trimmed by
ER aminopeptidase into 8 or 9 amino acid peptides, associated with MHC Class I, and
transported to the cell surface.192-197
Once on the cell surface, the tumour peptides are
presented in the context of MHC Class I. The tumour peptide is then recognised by
CD8+ cells, and that cell is targeted for destruction.
- 35 -
For direct presentation to predominate in priming, intra-lymphatic and intra-nodal
tumour must occur.198-200
However, antigen presentation can be robustly detected in the
absence of intra-lymphatic tumour and nodal metastases, suggesting a cross priming
predominance.198
Additionally, direct presentation from tumour cells is probably a weak
phenomenon, due to downregulation of co-stimulatory molecules201
and low MHC class
I expression.202
Indeed, there are many examples where direct presentation is shown to
have a minor role in tumour surveillance. For instance, chimeric mice lacking functional
APCs are unable to generate CD8+ T cell responses against tumours in numerous
models. In contrast, when APCs are provided to those mice (facilitating cross
presentation), antigen presentation is evident. 203-205
Thus while direct presentation may contribute in the process of immune surveillance,
another mechanism of antigen presentation seems to be important. This form of neo-
antigen presentation, “cross presentation”, involves the uptake and processing of
exogenous (class I) tumour antigens, using the machinery normally reserved for the
endogenous (class II) pathway. Cross presentation occurs predominantly by
professional APC (DC, macrophages),206
although B cells, endothelial cells, and
neutrophils may also cross-present.191,203,207-212
The downstream consequence of cross presentation is the delivery of tumour antigen
epitopes on class I, to CD8+ T cell receptors (TCRs). For the naïve CD8
+, the outcome
of cross presentation may be tolerance (“cross-tolerance”) or priming (“cross-
priming”).213
Which of the two occurs depends on the activation status of the APC.
That, in turn, depends on the presence of immune stimulatory (danger) signals. Those
danger signals may be from cellular products (e.g. heat shock proteins), from CD4 cells
(e.g. CD40 ligand), or microbial derivatives (e.g. CpG).191,214-216
To unify these concepts, and to understand how surgery might affect antigen
presentation, a simplified model is proposed (Figure 1.2). The first step of the process is
infiltration of the tumour by APCs, of which the DC is probably the most important.217-
220 Those APCs phagocytose tumour antigen from dead or dying tumour cells, or by
“nibbling” tumour cells.217-220
Laden with tumour antigen, APCs traffic to the draining
lymph nodes, 221
where they present to both CD4+ and CD8
+ T cells. Providing
appropriate co-stimulation is present, CD8+ cells will become activated, proliferate, and
differentiate into CTL effectors.214,215
Those CTLs then traffic back to the tumour,
- 36 -
exiting the tumour vasculature and accessing the neoplastic parenchyma. The CD8+
cells then effect tumour cytolysis by numerous mechanisms (e.g. FasL). 222,223
Helper T
cells may augment the process at numerous steps (Figure 1.2), e.g. activation of the
APC,224-229
assisting CD8+ differentiation,
230,231 and/or exerting direct anti-tumour
activity.232-234
Surgery may impact on numerous facets of this process. Firstly, by definition, cancer
resection will reduce tumour mass. While antigen presentation has been related to
certain threshold values of antigen in previous work,235
the correlation between antigen
presentation and tumour mass has not previously been studied in vivo. Also the
relationship between surgery and tumour antigen presentation in vivo has only been
mentioned in one conference abstract (no formal publications as yet).236
This shortfall in
knowledge presents a conundrum to planning post-operative immune therapy. For
instance, 50% of patients suffer progressive renal cancer after resection of apparently
localised tumour.237
In this situation, immune therapy may be useful to aid tumour
clearance. However, it is unknown whether sufficient tumour antigen remains after
macroscopic tumour clearance (i.e. an immune boosting adjuvant is most appropriate),
or whether antigen signal is a limiting factor after surgery (i.e. a vaccine is required).
Secondly, from the model presented in Figure 1.2, antigen presentation occurs within
so-called “tumour draining” lymph nodes. However, it has yet to be established whether
tumour draining lymph nodes are regional nodes (i.e. all adjacent lymph nodes to the
tumour), sentinel nodes, or other nodes entirely. Understanding this relationship
becomes increasingly important, as cancer surgery may involve removal of some (or all)
of these nodes. In addition, if the nexus of priming occurs in the sentinel lymph nodes,
then sentinel node biopsy could have profound implications for tumour immunity. To
date, no-one has examined the effects of sentinel node biopsy on tumour antigen
presentation or even tumour resistance in vivo. Finally, the interaction of CTL with the
tumour itself may be affected by surgery. If tumour associated CTL are predominantly
located in sentinel nodes, then removing sentinel nodes could have dire effects on
tumour resistance and anti-tumour immune memory. These issues are tackled in
Chapter 5.
- 37 -
Figure 1.2. Simplified view of anti-tumour immunity
Modified from reference 238. Adaptive anti-tumour immunity is thought to revolve around the nexus of
antigen presentation and CTL priming. CD4+
“help” the process at multiple steps. A: DCs acquire tumour
antigen by phagocytosis, and traffic to the draining lymph nodes. B: DCs present tumour epitopes to both
CD4+ and CD8
+ lymphocytes. C: CD8
+ cells become activated and differentiate to effector CTL. D:
CD8+ cells traffic back to the tumour and egress the tumour vasculature. E: CTL infiltrate the tumour and
effect cytolysis.
In summary, the nexus of interaction between APC, CTL, and DC has previously been
localised to the tumour draining lymph node. As surgery removes tumour bulk and/or
sentinel lymph nodes, this will likely impact on antigen presentation and tumour
immunity. By uncovering the effects of surgery on key elements of anti-tumour
immunity in vivo, particularly antigen presentation and in vivo CTL function, this thesis
provides an empirical framework for combining surgery with immune therapy. This
- 38 -
work may also assist in understanding the mechanisms by which surgery boosts tumour
resistance, further validating the principle of combining immune therapy with surgery.
Current insights into the mechanisms by which surgery boosts tumour immunity are
now discussed.
1 . 3 . 2 . Surgery and immune suppress ion networks
The anti-tumour immune benefit of surgery may arise from the disruption of “tumour
associated immunosuppressive networks”.27
While cancers may engage the host
immune system by the mechanisms discussed in 1.3, from the regrettable frequency of
cancer it is clear that tumours have the means to frustrate tumour resistance, including:
ignorance,179,206,239-243
selection of non-immunogenic clones,39,201,202,244,245
production of
immune suppressive agents (e.g. PgE2, soluble phosphatidylserine, VEGF),27
induction
of humoral TH (TH2 predominance),246-248
MSC27,246,249-256
tumour associated
macrophages (TAM),27,146,254,257
IDO+ plasmacytoid DC,
258 Treg,
259-266 expression of Fas
ligand,222,267-270
exclusion of effector cells/altered tumour vasculature,27,271-274
blocking
of effector function (e.g. release of soluble phosphatidyl serine etc.),27,39,275-279
and the
induction of immune anergy.176
Surgery may impact on numerous elements of these
“escape” mechanisms.
1 . 3 . 2 . 1 . A l l e v i a t e t u m o u r - d e r i v e d s u p p r e s s i v e f a c t o r s
Tumours are known to produce a number of factors that suppress the immune system,
including soluble phosphatidylserine, Il-10, Il-4, VEGF, and TGF.27
The accumulation
of those factors is thought to parallel tumour burden27
and by extension, reducing
tumour burden should decrease the production of those factors.3,26,27
Whether this
occurs is yet to be determined, but theoretically, a reduction in tumour derived soluble
factors should produce downstream disruption of the tumour‟s immune suppressive
network27
(MSC, IDO+ plasmacytoid DC, Treg and TAM).
1 . 3 . 2 . 2 . R e d u c e e f f e c t o r r e q u i r e m e n t s
A certain critical ratio of lymphocytes to tumour cells may be required to control
tumour growth. In theory, reducing tumour cell burden tips the ratio favourably,
increasing the probability that host immunity can control tumour.26
Furthermore, fewer
tumour cells should mean fewer mutations,26
and therefore less probability that immune
evasion characteristics will develop. This has not been tested in vivo.
- 39 -
1 . 3 . 2 . 3 . R e d u c e M S C
MSC were formerly known as “early myeloid cells”249,250
or “inhibitory
macrophages”.250,251
These are a heterogeneous population of immature, bone-marrow
derived cells that characteristically are CD11b+
and Gr-1+.246
While additional markers
for MSC have been described (CD31+
251 and Il-4R
+
252), they have yet to be
definitively typed.
MSC are thought to be recruited by the production of VEGF, GM-CSF, Il-3, and Il-6.246
They progressively accumulate in the blood and spleen, in parallel with tumour
burden.253
MSC may also localise in the tumour and in secondary lymphoid organs in
response to chemokine signals, forming an “immunosuppressive network”.27
MSC
mediate immune suppression by several mechanisms. First, MSC may produce T cell
anergy by the uptake of soluble tumour antigens and the presentation of those antigens
in a tolerogenic manner.254
However, MSC may also produce CD8+ inhibition by
antigen independent mechanisms246
that require cell to cell contact.280,281
Such mechanisms include IDO27
and arginase I.27,255
Those enzymes deplete tryptophan
and arginine in the tumour microenvironment respectively, impairing CD8+
proliferation and maintenance of the CD3 chain.256
Moreover, MSC produce
peroxynitrites (which induce CD8+ apoptosis).
246 In addition to hampering the anti-
tumour immune response, MSC can support tumour growth through the release of
tumour nutritive polyamines.282,283
Surgery reduces MSC counts in mice and humans with cancer. This occurs somewhere
between 10 days46
and 4 weeks47,284,285
after surgery. In a chemically induced BALB/c
sarcoma model, reduction in MSCs was paralleled by an improvement in cell mediated
immunity and resistance to tumour.285
However, a reduction of MSC alone is probably
insufficient to convey effective anti-tumour immunity after surgery.281
1 . 3 . 2 . 4 . P r o v i d e “ a n t i g e n h o l i d a y ”
Tumours characteristically induce anergy of the tumour-specific CD8+ repertoire.
176
One mechanism for that anergy is by the failure to provide “second signal”.176
Tumour
cell death may occur in an immunologically bland manner,238
such that “danger signals”
are not delivered and APC are not activated. Co-stimulation is probably similarly absent
with direct presentation by the tumour itself,201
adding to the problem.
- 40 -
A second postulated mechanism of tumour-induced anergy is the release of a
continuous stream of antigen. Continuous antigen exposure has been shown to induce a
state of immune hyporesponsiveness in viral models. 286-291
Indeed, maintenance of that
hyporesponsiveness requires persistence of the antigen, and T cells may recover when
removed from that chronic antigen.288,291-293
Numerous associations of antigen
associated anergy have been identified, including: TCR downregulation,286,291,294-296
TCR desensitization,293
and increased expression of negative regulatory molecules
(CD5291
and PD-1297
).
In recent years, based on findings in virology and by the detection of up-regulated PD-
L1 (B7-H1) on numerous tumours, there has been increased interest in the role of PD-1.
With acute infectious disease, the immune system is exposed to a burst of foreign
antigen. This is followed by an immune response, a wane of infection, and a reduction
in antigen levels. In this scenario, CD8+ cells are activated, produce cytokines, and
ultimately form memory.297
The immune response to chronic viral infection is different.
With chronic antigen exposure, cells are tolerised and gradually acquire an “exhausted”
phenotype.298
The exhausted phenotype features an up-regulation of the inhibitory
receptors PD-1 and CTLA4, downregulation of co-stimulatory proteins, defects in
accessory and cytokine signals, and disrupted proximal T cell receptor signalling.298
Exhausted cells function less well as effectors, do not form useful memory cells, and are
unable to control the viral infection.297
Of the two inhibitory receptors, PD-1 signalling seems critical to the exhaustion
process.299
PD-1 acts in peripheral tissues, binding two receptors: PD-L1 (B7-H1) and
PD-L2 (B7-DC).299
PD-L1 is thought to be inhibitory and PD-L2 can be inhibitory or
activating.297,299
The importance of PD-1/PD-L1 signalling is highlighted by the
recovery of exhausted cells to full effector function using a blocking monoclonal
antibody. After PD-1/PD-L1 blockade, despite chronic viral infection, there is increased
CD8+ proliferation, increased viral control, and improved cytokine production.
300
In short, tumours may induce anergy/tolerance by providing a continuous source of
antigen and the absence of a danger context. Mechanisms that reverse this phenomenon
(e.g. Il-2 and “antigen rest”) may be beneficial in overcoming the exhaustion of
continuous tumour antigen exposure.300,301
Surgery might affect antigen rest by reducing
- 41 -
the tumour burden. The degree and duration of antigen rest for the recovery of immune
responsiveness is unclear, and it may differ for memory302
and effector292
CTL.
Memory CTL may recover if antigen is absent for two days,302
whereas effector CTL
may require between 26 days and 2 months of an “antigen-free” environment.292
It is unresolved whether surgery can in fact produce antigen rest. At least in theory, if
some tumour cells are left behind, they may be below the threshold required for antigen
presentation.235
Thus an antigen-free environment may be possible after surgery, even in
the setting of residual disease. This has never been confirmed or refuted in vivo.
1 . 3 . 2 . 5 . C h a n g e m e m o r y p h e n o t y p e
The majority of antigen specific T cells that participate in an initial antigen response
will undergo apoptosis,303
leaving a small subset of memory T cells. Memory cells have
a great avidity for antigen,304
are capable of responding quickly to a recurring threat,305
have faster cell divisions,305
and more rapidly acquire effector function.306
Memory
subsets remain an area of intense research and debate.307
However, the classification
into central and effector memory subtypes seems well accepted. Central memory (CM)
cells have lymph node homing molecules expressed on their cell surface (CCR7 and
CD62L) and are found in the lymph nodes, blood, and spleen. Effector memory (EM)
cells do not have these molecules, and are found in the peripheral tissue.308
The significance of this distinction is somewhat contentious. Whether central and
effector memory develop independently308
or inter-convert is unresolved.309,310
However, central memory tends to predominate in relatively low antigen
environments.48,298,299,308
In some models at least, central memory cells control
infections more effectively, proliferate more rapidly, and generate more delayed type
hypersensitivity.299,311
In the case of tumours, antigen titres are (possibly) high and
persistent. This may mould the memory response towards an effector phenotype.
Effector memory cells may be inferior for tumour control because they tend to
proliferate more slowly, and home to regional lymph nodes more avidly.299,311
It has
already been demonstrated that surgery enables the predominance of central memory
phenotype in the CD4 compartment.48
However, this study is based on a transgenic
mouse strain that had a high frequency of tumour-specific CD4+ cells. Additionally, the
effect of surgery on post-operative CD8+ memory phenotypes has never been
investigated.
- 42 -
1 . 3 . 2 . 6 . R e l e a s e e f f e c t o r s
In response to tolerogenic priming,176,230
persistent antigen,312
inflammation,313
and/or
chemokine signals,230
tumours might trap effector cells in the draining lymph nodes.
Surgical resection might remove that trap for activated lymphocytes,112,113
enabling
dissemination of T cells,48
and enhanced systemic immunity. Systemic release of CTL
has previously been seen with CD40 therapy155,156
and CD4+ adoptive transfer
therapy.314
However, it is unknown whether this occurs after surgery.
1 . 3 . 2 . 7 . I m p r o v e o v e r a l l c e l l m e d i a t e d i m m u n i t y
Theoretically, the combination of surgical-associated improvements in immune function
should translate into enhanced tumour-directed cell mediated immunity. Direct evidence
for this remains limited, but Salvadori and colleagues demonstrated improved
allogeneic tumour rejection after primary resection when metastases were present.285
This encouraging finding suggests that surgery could be beneficial to tumour-specific
immunity, even when disease is extensive.
1 .4 . Aims and Hypotheses
The notion that surgery boosts tumour immunity remains the rationale for combining
surgery with immune therapy. The mechanisms by which surgery improves tumour
resistance remain largely hypothetical, and relate to the disruption of tumour associated
suppression networks. The impact of sentinel node biopsy on the tumour-specific
immune response is also unknown, and there has been no previous in vivo study of this
issue. These shortcomings in current knowledge will be rectified in the four
experimental Chapters of this thesis. Collectively, these studies describe the effects of
surgery (primary resection and sentinel node biopsy) upon key elements of tumour-
specific immunity, providing an empirical framework for the combination of surgery
and immune therapy in the treatment of solid malignancy.
Chapter 3 aims to determine the effects of surgery on tumour antigen presentation, and
the implications of those antigen changes on CTL function, phenotype, and distribution.
It is hypothesised that surgery reduces cross presentation and that reduced post-
operative tumour antigen presentation could improve CTL function and tumour
immunity.
- 43 -
In Chapter 4, the effects of surgery on key mediators of the immune suppressive
network (MSC, plasmacytoid DC, Treg) and overall anti-tumour immunity are explored.
Furthermore, the possible relationships between tumour antigen presentation and overall
tumour resistance is examined. Building on the findings of Chapter 3, it is hypothesised
that tumour antigen presentation and tumour resistance are inversely proportional. It is
also postulated that tumour resistance is inversely related to post-operative MSC, Treg,
and plasmacytoid DC levels.
Chapter 5 examines the immune significance of sentinel node excision. Building on
Chapters 3 and 4, it is hypothesised that sentinel node biopsy will shift antigen
processing to the next most proximal nodes, and that this will be detrimental to tumour
resistance overall. As a secondary objective, the hierarchy of antigen presentation and
CTL function of the sentinel nodes, based on tumour proximity, are explored.
Chapter 6 discusses an orthotopic murine kidney cancer model, providing insights into
tumour-specific immunity that are not possible with AB1HA. The model is especially
useful for future research because kidney cancer is a promising target for combined
surgery/immunotherapy, as facilitated by this thesis. The orthotopic model should allow
analysis of combined surgery/immunotherapy strategies in kidney cancer, and the direct
testing of principles outlined in Chapters 3 – 5.
The main findings of this thesis are summarised in Chapter 7, which is the final chapter.
Conclusions are also stated, and some future directions for research are identified. The
methods used in these experiments are detailed in the next chapter, and then the results
are presented. Secondary aims and hypotheses of each experiment are detailed in the
relevant chapters.
- 44 -
Chapter 2
- 45 -
2. Methods
In this section, the methods used in Chapters 3 – 6 are detailed. This research
emphasises the tracking of tumour specific immunity in vivo. HA tumour transfectants
and the corresponding, HA-specific transgenic mice are used extensively. Most of the
methods have been previously published, and these are referenced accordingly.
2 .1 . Cel l l ines and cul ture techniques
Cell lines were cultured in standard culture medium (denoted “R10”), comprised of
RPMI 1640 (Gibco/BRL, Grand Island, New York USA) supplemented with 20 mM N-
2-hydro-2ethylpiperazine-N-2ethane (HEPES, Gibco/BRL), 0.05 mM 2-
mercaptoethanol, 100 µ/mL (Sigma-Aldrich, St Louis, Minnesota USA),
benzylpenicillin 60 mg/L (CSL, Melbourne, Victoria, Australia), 48 µg/mL gentamicin
(Sigma-Aldrich) and 5-10% foetal calf serum(FCS, CSL Melbourne, Victoria
Australia). The cell culture media were prepared sterile, and adjusted to 300 mOsm.
Where HA transfectants were used (RencaHA and AB1HA), culture medium was
supplemented with the neomycin analogue Geneticin (Invitrogen, Mount Waverley,
Victoria Australia) at the concentration of 400 g/mL. All cell lines were maintained at
37°C and 5% CO2 in monitored incubators.
2 . 1 . 1 . Cel l harves t
Adherent cells were detached by brief exposure to gamma irradiated trypsin (JRH,
Lenexa, Kansas USA), and washed twice with sterile phosphate buffered saline
(PBS, Invitrogen). Cell counts and viability were assessed by trypan blue exclusion
(Sigma-Aldrich). Viabilities of 95% or greater were deemed acceptable for
inoculation. Unless otherwise stated, tumour inoculums consisted of 1x106 cells,
suspended in 100L of 0.9% sterile normal saline (Astra Zeneca, North Ryde, New
South Wales Australia).
2 . 1 . 2 . Mycoplasma screening
All cell lines were screened for mycoplasma on a three monthly basis. Once cell lines
were growing in antibiotic-free medium for 72 hours, they were trypsinised, washed
twice in normal saline (Astra Zeneca) and re-suspended at a concentration of 1x106 cells
- 46 -
per 100L sterile saline (Astra Zeneca). Samples were tested by Pathcentre (SCGH
Hospital, Nedlands, Western Australia) for the presence of mycoplasma DNA by PCR.
If two successive PCR results for mycoplasma were negative and the cell culture
remained healthy, experimentation proceeded until the next screening.
2 . 1 . 3 . AB1
The AB1 mouse mesothelioma model has been described previously.315
Briefly; IUCC
reference samples of Wittenoom Gorge crocidolite (asbestos) were injected
intraperitoneally (i.p.). After a latency period of 7-25 months, 35% of BALB/c subject
mice developed ascites. At that stage, disease affected mice were culled, exudates
harvested, and cell lines maintained in culture. The AB1 mesothelioma tumour line
shared biological characteristics of human mesothelioma: variable and long latency,
asbestos as a causative agent, ultra-structural microvilli, and low immunogenicity.
Similar to human mesothelioma, AB1 was class I positive and had low expression of
MHC class II.315
After subcutaneous inoculation of 1x106 AB1 cells into BALB/c mice, solid and
vascularised tumours were reliably produced. When the tumour was inoculated
intraperitoneal, a plaque lesion forms at the site of inoculation, together with exudates
consistent with malignant ascites.
2 . 1 . 4 . AB1HA
AB1HA was produced as previously described.148
In short, the Mount Sinai PR8
haemagglutinin (HA) gene was sub-cloned into the expression vector phBApr-1-neo,
and transfected into AB1 cell line using DOTAP (a cationic lipid). Stable transfectants
were selected using geneticin (neomycin analogue) in serial dilutions. HA expression
was verified using biotinolyated HA-specific monoclonal antibody (H18), originally
donated by Dr Walter Gerhard (Wistar Institute, Philadelphia, Pennsylvania USA).
Three clones of HA expression profile were isolated: ABHAHI
, AB1HAMEDIUM
, and
AB1HALO
. The sub-clone AB1HAMEDIUM
was used throughout this thesis, and was
denoted as “AB1HA”.
- 47 -
2 . 1 . 5 . Renca
The Renca cell line was provided by Dr Mark Smyth and Mr Jeremy Swann (Peter
MacCallum Cancer Centre, Melbourne, Victoria Australia). Renca arose spontaneously
as a renal cortical adenocarcinoma in a BALB/c mouse, and was subsequently passaged
subcutaneously and in vitro. The tumour formed solid, grey and vascular tumours, often
undergoing central necrosis and/or haemorrhage.316
Renca has been successfully transplanted intra-renally, subcutaneously, intravenously,
and intraperitoneal. However, the behaviour of Renca is affected strongly by its route of
transplantation. When Renca is implanted subcutaneously, it does not metastasise to the
lungs. However, when placed orthotopically (i.e. into the kidney), Renca grows rapidly
and metastasises aggressively.317
Renca is similar to human renal cancer in its metastatic targets, histological appearance,
immunogenicity (displays sinecomitant immunity), and tendency to produce
polycythaemia.316
When implanted intra-coelomically, Renca has a tendency to invade
adjacent structures and metastasise to the lymph nodes, lung, liver, spleen,
mediastinum, bladder, and the serosa of the gastrointestinal organs.316
Like AB1, Renca
expresses MHC Class I and not MHC Class II.198
It also does not express CD80 (co-
stimulatory molecule).198
2 . 1 . 6 . RencaHA
RencaHA was kindly donated by Dr Eduardo Sotomayer and Dr Fengdong Cheng (H.
Lee Moffitt Cancer Centre & Research Institute, Tampa, Florida USA) and was
originally developed by Dr Linda Sherman and colleagues (The Scripps Research
Institute, La Jolla California USA).318
Briefly, wild type Renca was transfected with
haemagglutinin (HA) of PR8 influenza H1N1 (A/PR/8/34) using calcium phosphate
mediated plasmid transfection. HA positive cells were selected by culture in 400 g/mL
G418 (a neomycin analogue), and tested for HA expression using H18 mAb.198
Like
AB1, RencaHA expresses MHC Class I and not MHC Class II. It also does not express
CD80 (co-stimulatory molecule).198,318
Due to possible contamination of the RencaHA
cell line during transport, it was passaged in vivo by subcutaneous administration into 5
BALB/c mice. In each instance, the tumour grew, but only one had retained HA
positivity on H18 staining and flow cytometry. Previously, RencaHA had been grown
unreliably subcutaneously, and was associated with loss of HA expression.318
- 48 -
The stably expressing HA cell line was then cultured in vitro, using R10 culture
medium supplemented with 400 g/mL G418. After one passage, repeat H18 staining
and flow cytometry were performed, revealing a broad HA expression profile. After
further passage and expansion, the cell line was sorted thrice on HA expression levels,
with the assistance of Dr Kathy Heel (BIAF, University of Western Australia, Nedlands
Australia) and the FACS Vantage Cell Sorter (Becton Dickinson, Mountain View,
California USA).
The resultant clones of RencaHA (RencaHALO
, RencaHAMEDIUM
, and RencaHAHI
) were
passaged in vitro and frozen at low passage numbers of 3 – 7. The RencaHAMEDIUM
cell
line was chosen for experimentation because of its favourable in vitro and in vivo
growth characteristics. Herein, RencaHAMEDIUM
was denoted as RencaHAM.
RencaHAM, was successfully transplanted by intracardiac, tail vein, intraperitoneal and
intra-renal administration. The relative growth properties of RencaHAM are detailed in
Chapter 6, Sections 6.2.2 - 6.2.4.
2 .2 . Murine Species
2 . 2 . 1 . BALB/c and BALB/c nu- / -
mice
Female BALB/c and congenic BALB/c nu-/-
mice were obtained from the Animal
Resource Centre (Perth, Western Australia). BALB/c mice are a common in-bred
laboratory mouse strain, with MHC Class I and Class II denotations of 1-Ad and H-
2Kd respectively. BALB/c nu
-/- have both BALB/c and nude characteristics. As
BALB/c nu-/-
lack a thymus, they are unable to produce T cells.
All animals were maintained under standard clean conditions at the University of
Western Australia Animal Care Unit in Sir Charles Gairdiner Hospital.
All protocols were approved by the University of Western Australia Animal Ethics
Committee, and were compliant with National Health and Medical Research
Committee (NHMRC) and Office of Gene Technology Regulation (OGTR)
regulations.
- 49 -
2 . 2 . 2 . CL4 TCR transgenic mice
The CL4 TCR transgenic murine line has been previously described.319
CL4 mice are
class I restricted, recognising the dominant H-2d restricted HA epitope
(IYSTVASSL). Breeding pairs were obtained from Dr Linda Sherman (The Scripps
Research Institute, La Jolla, California) and backcrossed for at least five generations
onto the BALB/c genetic background at the University SPF animal facility. Purity of
CL4 transgenic progeny was typed prior to experimental use, using mAbs anti-CD8-PE
(53-6.7) and anti-Vb8.1-FITC (MR5-2) (Pharminogen, San Diego, California).
2 . 2 . 3 . HNT TCR transgenic mice
The derivation of the HNT TCR transgenic murine line has similarly been described
previously.320
HNT lines are class II restricted, recognising the HNTNGVTAACSHE
epitope of HA in the context of H-2Kd. Breeding pairs were obtained from Dr D Lo
(The Scripps Research Institute, La Jolla, California) and backcrossed for at least five
generations. HNT mice were typed using the mAbs anti-CD4-PE (RM4-5) and anti-
Vb8-FITC (F23.1) (Pharminogen, San Diego, California)
2 .3 . In Vivo Procedures
2 . 3 . 1 . Anaesthes ia
For prolonged surgery (nephrectomy and lymph node dissection), chloral hydrate
(Orion Laboratories, Balcatta, Western Australia) was used as primary agent. The syrup
preparation was used, diluted in sterile normal saline to a concentration of 3.5%. Each
mouse was weighed and administered 0.1mL/10g intra-peritoneally. Over a period of
five to ten minutes, general anaesthesia would occur. This was supplemented with
inhalational methoxyflurane (Medical Developments International Limited, Springvale,
Victoria Australia)
Methoxyflurane (Penthrane, Medical Developments International Limited) was used as
primary agent for anaesthesia for all other surgical procedures. Briefly, animals were
placed in a glass jar, with paper tissues as a base. The tissue base was impregnated with
approximately 0.5mL of methoxyflurane, and the jar sealed. Animals were monitored
- 50 -
for the onset of anaesthesia, evidenced by depressed respirations and the absence of
movement.
No procedure was commenced unless the animals were deeply anaesthetized. The depth
of anaesthesia was deemed adequate if the following applied: no righting or startle
reflex, no response to pinprick, and slow respiratory rate. Animals were carefully
monitored during anaesthesia for evidence of lightening. In that instance, supplementary
methoxyflurane (Penthrane, Medical Developments) was administered via nose cone.
During anaesthetic maintenance, the operator monitored the reflexes and respiratory rate
regularly. The nose cone was shifted on and off to maintain a steady depth of
anaesthesia.
Once anaesthesia was complete, animals were shifted to a recovery area. Mice were
placed on their sides, on top of a heat pad. The head, neck and torso were maintained in-
line. A moist dab was placed on each exposed eye. The animal was watched for
adequate respiratory effort and occasionally stimulated (by rubbing) if under-breathing.
Once mobilizing and capable of shifting posture, mice were returned to the cage.
Animals were further monitored for an hour, then twice daily for the first three days.
2 . 3 . 2 . Analgaes ia
Intraperitoneal 0.05 mg/kg buprenorphine (Reckitt & Coleman, Chiswick, UK)
analgaesia was administered intraperitoneal (i.p.). Analgaesia was routinely
administered to all animals undergoing laparotomy or lymph node dissection, just prior
to the completion of anaesthesia. All other animals were monitored in the early
postoperative phase (days 0 – 3). During that period, analgaesia was administered at a
dosage of 0.05 mg/kg four times daily on a discretionary basis. Few animals required
such analgaesia.
2 . 3 . 3 . Subcutaneous inoculat ion
Subcutaneous injections were undertaken to administer immune therapies or transplant
tumour cells. Prior to injection, the flank region was shaved and prepared with 70%
ethanol. The most common site of inoculation was the caudal flank region,
approximately in the mid axillary line. A 29 gauge needle (0.5mL, BD UltraFineTM
,
Becton Dickinson Pty Ltd, Scoresby, Victoria Australia) was placed into the subcutis of
- 51 -
this area and a bleb of inoculum was gradually raised. Animals were monitored in the
post-procedural period for distress and bleeding.
2 . 3 . 4 . Axil lary inoculat ion
To study the effects of tumour proximity on node function, a near-axillary injection site
was chosen. Animals were restrained supine, and an area just ventral and caudal to the
axilla was chosen. The skin was depilated by clippers, and prepared with 70% of
alcohol. As with flank inoculums, a 29 gauge needle (0.5mL, BD UltraFineTM
, Becton
Dickinson Pty Ltd) was placed at 30 degrees to the skin, and an injection bleb raised in
the subcutis.
2 . 3 . 5 . Intranodal inoculat ion
To determine the effect of nodal invasion on antigen presentation and in vivo CTL
function, tumour was transplanted into axillary nodes. Animals were subject to general
anaesthesia, and the axillary region was shaved. After preparation with 70% alcohol, an
oblique incision of 5mm was centred over the axilla. Superficial fascia was dissected
clear to demonstrate the free border of pectoralis major. That border was grasped with
haemostat artery forceps, and retracted superiorly and towards the midline. By blunt
dissection and with the aid of 3x prism operating loupes, the axillary node was exposed.
The axillary node was bi-lobulate or somewhat pear shaped in appearance, and
invariably plastered onto the axillary vein. By careful traction on the capsule, a second
operator injected the node with tumour cells using a 29 gauge needle (0.5mL, BD
UltraFineTM
, Becton Dickinson Pty Ltd). The concentration of inoculum was 1x106 cells
per 50 L of sterile saline (Astra Zeneca). Invariably the node swelled with inoculum,
and some cells leaked into the fossa. The wound was closed using four cutaneous 5-0
vicryl (polyglactin 910, Ethicon Australia) interrupted sutures.
2 . 3 . 6 . Intravenous inoculat ion
Intravenous inoculation of immune therapies and tumour cells was performed using the
lateral tail vein. Animals were heated under a heat lamp, and restrained using a standard
restraint cylinder. The lateral tail vein was then identified and injected under vision with
a 29 gauge needle (0.5mL, BD UltraFineTM
, Becton Dickinson Pty Ltd). Flashback into
the bevel of the syringe, the absence of resistance to plunger depression, and a lack of
tail swelling were indicators of successful injection.
- 52 -
2 . 3 . 7 . Intracardiac inocu lat ion
Intracardiac injections were occasionally required when intravenous inoculation was not
possible and/or to study the RencaHAM system. Animals were subject to general
anaesthesia, and secured in a supine position. The midpoint of the sternum was located
by digital palpation. A 29 gauge needle (0.5mL, BD UltraFineTM
, Becton Dickinson Pty
Ltd) was then slowly inserted perpendicular to the skin, in the mid-clavicular line, at the
level of the sternal midpoint. Steady aspiration was continued as the needle was
advanced into the mediastinum. Ready flashback of dark red blood indicated entry of
the needle into the right ventricle, at which time the required volume was slowly
injected. The needle was then gradually withdrawn from the animal, and the animal was
monitored closely for complications. Animals were euthanased if respiratory distress or
cardiovascular compromise occurred.
2 . 3 . 8 . Intrarenal inoculat ion
To study the behaviour of intra-renal RencaHAM, orthotopic (i.e. into the kidney)
tumour implantation was required. Animals were subject to general anaesthesia using
methoxyflurane. The right flanks of the animals were shaved and prepared with 70% of
alcohol. Animals were taped into the right lateral position using masking tape. A
transverse loin incision was fashioned, 3 – 4 mm caudal to the lowermost limit of the
thoracic cage and centred over the mid-axillary line. Incision of the skin and abdominal
musculature exposed the erector spinae muscles posteriorly, and peritoneum.
Peritoneum was incised, and the ovaries swept clear of the operative field.
Where necessary, the right lobe of the liver was gently supported off the lateral aspect to
the kidney. Invariably with this technique, the anterior and lateral surfaces of the kidney
were seen well. Under direct vision, a 29 gauge needle (0.5mL, BD UltraFineTM
, Becton
Dickinson Pty Ltd) was advanced deep to the renal capsule. Cell suspensions were
gradually injected: 1x106 Renca or RencaHA cells in 50 L of sterile saline (Astra
Zeneca). The kidneys swelled slightly from the injection, and sometimes took on a
slightly mottled appearance. The capsule of the kidney dissected off the parenchyma
from the injection, particularly adjacent to the puncture. Most inoculum seemed to
collect under the capsule.
- 53 -
2 . 3 . 9 . Resect ion s tudies
Subcutaneous tumours were excised on day 16 after tumour cell inoculation. At that
time point, tumours were solid and vascularised, with a diameter of 5.57 0.30 mm.
Incisions were elliptical, longitudinal, and centred over the lesion. Incisions were
approximately 15mm in length, with lateral margins of 3 – 4mm. Once the incisions
were fashioned, the rostral tip of the cutaneous flaps were elevated and teased clear of
deep fascia. With steady outwards and caudal tension, skin and adherent tumours were
removed en bloc. Tumour pedicles were dissected clear of the adjacent deep fascia, and
ligated with 5-0 vicryl. Inguinal nodes were identified and preserved, unless
lymphadenectomy was to be included in the procedure. Once the tumour and cutaneous
flap were removed, adjacent skin was undermined and approximated with 5-0 vicryl
(Polyglactin 910, Ethicon) sutures.
2 . 3 . 10 . Lymphadenectomy
Lymphadenectomy was undertaken for axillary and/or inguinal nodes. For the axillary
node, the axillary area was shaved and prepared with 70% ethanol. An oblique incision
of 5mm in diameter was centred over the axilla, and superficial fascia was dissected
clear of the free border of pectoralis major. That border was then retracted, and blunt
dissection into the axillary fossa was undertaken to expose the axillary node and vein.
Gentle traction on the axillary node capsule, together with delicate dissection enabled
delivery of the axillary node off the vessels. The skin was closed with four 5-0 vicryl
(Polyglactin 910, Ethicon) interrupted sutures.
The inguinal node was removed with or without tumour. When the tumour was absent
or left behind, a longitudinal inguinal incision was performed. The incision was 10mm
in length, located in the anterior axillary line, and just rostral to the hindquarter. The
skin edges were elevated and retracted to expose fascia and the lateral thoracic vessels.
The inguinal node was located at the confluence of the inferior epigastric and lateral
thoracic vessels. Blunt dissection was used to carefully remove the node, without
damaging the closely applied vessels. Skin was closed with 5-0 vicryl (Polyglactin 910)
suture. Where tumour was present, the skin and adherent tumour were elevated as
previously described. The inguinal node was identified and the pedicle was ligated and
incised, ensuring the inguinal node was included in the resected pedicle.
- 54 -
2 . 3 . 11 . Monitor ing
Animals with tumour in situ were monitored every three days for weight loss, poor
grooming, huddled behaviour, elevated or depressed respirations, and reduced activity.
These signs were triggers for euthanasia.
Animals were also monitored post-operatively, initially twice daily and then once daily.
All checks were done by the author, who having performed the procedure, was familiar
with potential problems. For instance, the potential complications of lymph node
dissection were swelling of the foreleg (on the affected side), ischaemia of the foreleg,
and reduced mobilization of the nearby joints. The more general aspects of animal well-
being were also checked post-operatively: behaviour/activity (especially huddling), free
mobilization of all limbs and head, feeding, interactions with the other members of the
colony, grooming, and the presence of subcutaneous fat. In addition, tumours were
closely examined for size and evidence of necrosis, bleeding, inflammation and/or
infection.
2 . 3 . 12 . Tumour s ize assessm ents
Tumour sizes were assessed at a minimum of three daily intervals, via standard
callipers. Measurements were taken in millimetres, using two perpendicular directions
that were denoted length (l) and width (w). Tumour product was calculated by the
multiplication of (l) by (w), and tumour diameter represented the square root of that
value.
Tumour diameters of each cohort were expressed as mean SEM. Differences in tumour
diameter between any one intervention group and its matched control arm were tested
using the student‟s t test. Where relevant, tumour growth was compared between
cohorts by two way analysis of variance (ANOVA), removing cured animals from
analysis and considering the interactions of time and treatment. Differences were
considered significant for all statistical tests when P values were < 0.05.
2 . 3 . 13 . Survival analys is
Euthanasia was considered “mortality” and was necessary when tumour diameters
reached 10mm, and/or animals showed any indicators of distress (2.3.11). Survival was
compared between groups using the Log Ranks test, and differences were considered
significant when P < 0.05. Survival benefits for treatments were expressed as hazard
- 55 -
ratios (H.R.) relative to untreated or placebo control groups, and 95% confidence
intervals for H.R. were provided. When follow-up was short (dual-tumour experiments:
see Chapter 4) tumour-free survival was used as proxy for overall survival. Tumour-free
survival was similarly compared using the Log Ranks test, with the same conditions for
statistical significance.
2 . 3 . 14 . Adopt ive cel l t ransfers
CL4 cells were prepared for adoptive transfer as per “CFSE Proliferation Assay” and
HA peptide pulsed targets were formulated as per the “In Vivo CTL Assay” (Sections
2.4 and 2.5). Cells were suspended at a concentration of 2x107 per 100 L of sterile
normal saline. Intravenous access was via the lateral tail vein (or failing that,
intracardiac injection) by 29 gauge needle (0.5mL, BD UltraFineTM
, Becton Dickinson
Pty Ltd)
2 .4 . In v ivo CFSE prol i ferat ion assay
The CFSE proliferation assay was based on the principle that adoptively transferred
tumour-specific CD8+ cells proliferate on antigen encounter. The technique for in vivo
antigen presentation analysis has been previously described,321
but is accounted below.
2 . 4 . 1 . Preparat ion of CL4 cel l s for adopt ive t ransfer
One CL4 transgenic mouse was sacrificed for every three subject BALB/c mice
assayed. Extensive lymph node harvest was undertaken for each CL4, including the
axillary, brachial, inguinal, cervical, facial, jugular, para-aortic, iliac, mediastinal,
celiac, and renal nodes.
CL4 nodes were collected into chilled PBS (Invitrogen) on ice, and mechanically
digested using sterile glass slides. Resultant single cell suspensions were filtered via
40m nylon cell strainer (Falcon Cell Strainer, Becton Dickinson and Company,
Franklin Lakes, New Jersey USA), and centrifuged.
Pellets were subject to red cell lysis by incubating and agitating with in vivo red cell
lysis buffer (5% NH4Cl in PBS, pH 7.2; BDH, Victoria Australia). A further two
washes with PBS followed, and then cells were suspended in RPMI/HEPE
- 56 -
(Gibco/BRL) at a concentration of 2x107 per mL.
322 10L of 5mM 5,6-Carboxy-
fluoroscein-succinimidyl-ester (CFSE; Molecular Probes, Eugene, Oregon, USA) was
added for each 20mL of suspension. CFSE was incubated with the cells for a total of 10
minutes at room temperature. Solutions were then centrifuged through FCS (CSL)
underlay.
A further two spins of RPMI/HEPE (Gibco/BRL) with FCS (CSL) underlay were
performed, and the solution were washed twice with PBS (Invitrogen). The pellet was
then re-suspended in normal saline (Astra Zeneca) and adjusted to a concentration of
2x108 per mL. The adequacy of CFSE staining was checked before adoptive transfer by
flow cytometry (FACScan; Becton Dickinson, Franklin Lakes, New Jersey USA).
2 . 4 . 2 . Adopt ive t ransfer for ant igen presentat ion
2x107 CFSE stained CL4 cells (suspended in 100 L of sterile saline) were adoptively
transferred into each subject mouse and each relevant control. Cells were administered
by tail vein (2.3.6) and/or direct intracardiac (2.3.7) injection.
2 . 4 . 3 . Analys is for in v ivo ant igen presentat ion
Total lymphadenectomy and splenectomy were performed on each subject mouse, three
days after CL4 transfer. Those nodes were similarly collected into chilled PBS
(Invitrogen), mechanical digested, filtered, washed, and red cell lysed. Samples were
suspended in PBS/5% FCS (Invitrogen & CSL) and incubated with 1:200 anti-CD8a-
PE-Cy5 (BioLegend, San Diego, California USA), or rat IgG2aK-PECy5 (BioLegend)
isotype control. All samples were processed using FACscan (Becton Dickinson), Cell
Quest V3.1 (Becton Dickinson) and FlowJo V7.1.3 (Tree Star Inc, Ashland, Oregon
USA). Samples were gated to the lymphocyte population, and the forward scatter and
side scatter distribution. A minimum of 2,500 CFSE positive events were collected in
any one sample. Thresholds were set by CD8+ single stain and isotype controls, and
lymphocyte populations were sub-gated for CD8 positivity. CD8+ cells were examined
for CFSE intensity using a histogram distribution. Lymphocyte proliferation, as a
readout of in vivo antigen presentation, was quantified using the technique described by
Lyons and Parish.321
Subject samples were compared to normal BALB/c, sham operated
BALB/c, and unstained BALB/c controls.
- 57 -
Antigen presentation was quantified as the proportion of CFSE positive cells of lesser
fluorescence than the brightest (parental) population. Antigen presentation in subject
animals was compared to naïve and sham surgery controls. A background proliferation
rate was considered normal, possibly attributable to the division of transfused B cells.321
2 . 4 . 4 . Stat is t ical analys is for ant igen presentat ion
The proportion of lymphocytes proliferating was treated as a continuous variable,
representative of cross presentation levels. Different mouse groups were considered
categorical variables. Explanatory variables included: presence/absence of tumour,
tumour size, post-operative time point, presence/absence of lymph node dissection, and
tumour location. Groups were compared using matched pairs student t tests, using
Prizm 3 statistical software (Graphpad Software, San Diego, California USA).
Differences were considered significant when the two tailed P value was < 0.05.
2 .5 . In v ivo CTL assay
Analysis of in vivo CTL activity was performed similarly to that previously
described,323
and is outlined below.
2 . 5 . 1 . Pulsed and target reference peaks
Full lymphadenectomy and splenectomy were performed on one BALB/c for every
three mice studied. Tissues were collected into chilled PBS (Invitrogen), mechanically
digested, filtered, and red cell lysed as described (Section 2.4.1.) Single cell suspensions
were made up to 4mL in volume and divided into two equal volumes. After
centrifugation, each pellet was re-suspended in R10. CL4 peptide from HA (Chiron
Technologies, Clayton, Victoria Australia) was added to one fraction (pulsed peptide
fraction) at a concentration of 1 g/mL. The other fraction was left unpulsed. Both
fractions were incubated at 37°C for 90 minutes, and periodically agitated. The two
suspensions were then topped up with R10 and centrifuged. Pellets were re-suspended
in RPMI/HEPE (Gibco/BRL) to a final concentration of 2x107 cells per mL.
The two fractions were labelled as either 2.5 M CFSE (pulsed peak) or 0.50 M CFSE
(non-pulsed peak). Following CFSE incubation, solutions were centrifuged thrice over a
FCS (CSL) underlay, and washed twice with PBS (Invitrogen). Each pellet was re-
- 58 -
suspended in sterile saline (Astra Zeneca) and cell counts were performed to ensure
equal numbers. The two fractions were combined in equal portions, and flow cytometry
was performed (FACSscan, Becton Dickinson) to ensure adequate peak separation and
equivalence. A final suspension of 2x108 cells per mL was formulated using sterile
normal saline (Astra Zeneca).
2 . 5 . 2 . Adopt ive t ransfer for in v ivo CTL lys is assay
2x107 cells were then administered to each subject animal by lateral tail vein (2.3.6)
and/or intra-cardiac (2.3.7) injection. Several naïve BALB/c mice were injected with
labelled targets each time the experiment was performed, to provide controls for non-
specific target killing (see also, 2.5.3).
2 . 5 . 3 . Analys is for in v ivo CTL lys is
After a period of 16 hours, total lymphadenectomy and splenectomy was performed on
each recipient mouse. Nodes and spleens were prepared into individual single cell
suspensions, by collection into chilled PBS (Invitrogen). Mechanical digestion,
filtration, washing, and red cell lysis followed, as per 2.5.1. Samples were suspended in
PBS/5% FCS (Invitrogen and CSL), at an approximate concentration of 1x107 per mL.
Flow cytometry was performed using FACScan (Becton Dickinson), Cell Quest V3.1
(Becton Dickinson), and FlowJo V7.1.3 (Tree Star Inc). The percentage of tumour
targets lysed was calculated as follows:
Calculated % Lysis = (Calculated #Lysed)/(Calculated #Pulsed) x 100
Calculated lysed and pulsed values were obtained via correcting assay values for
background killing. Specifically, the ratio of pulsed to non-pulsed cells was calculated
for normal animals. This ratio was then used to calculate the theoretical number of
pulsed cells that were present, prior to CTL lysis, in any one subject animal tissue:
Calculated # Pulsed = (Observed #Pulsed) x (Ratio from Normal Animal)
The number of cells lysed could then be determined by subtracting the number of
pulsed cells at flow cytometry from the calculated number of cells originally present:
Calculated # Lysed = (Calculated #Pulsed) - (Observed #Pulsed)
- 59 -
Those values were then substituted into “Calculated % Lysis” equation above, to
determine in vivo CTL killing for each animal and tissue. Since values were corrected
against normal animals in each assay, it was possible to compare cytotoxicity
percentages between experiments across time.
2 . 5 . 4 . Stat is t ical analys is for in v ivo CTL Assay
The primary outcome for statistical analysis was the percentage cytotoxicity of HA
pulsed cells in each tissue studied (axillary, inguinal, non-draining nodes, spleen). As
subject animals were matched with sham surgery controls, explanatory variables were
primary tumour, cancer surgery, and the presence/absence of nodal invasion.
Cytotoxicity was analysed as a continuous variable, with different groups of mice
examined as categorical variables. Statistical analysis was performed using the Prism 3
statistical programme (GraphPad Software) and the matched pairs student‟s t test.
Differences were considered significant when the two-tailed P value was < 0.05.
2 .6 . DC phenotyping
DCs were extracted from lymph nodes as published previously,324,325
and detailed
below.
2 . 6 . 1 . Isola t ion of DCs from lymph nodes
Lymph nodes were collected into cold RBMI (Invitrogen) with 2% FCS (CSL), “R2”,
and then diced in minimal volume using sterile scalpels. Segments were re-suspended in
2mL of R2 with 1 mg/mL Collagenase (Worthington Biochemical Corporation,
Lakewood, New Jersey USA) and 5 g/mL DNase (DNase I, grade 2, Roche Applied
Science, Basel, Switzerland) and digested for 20 minutes at room temperature, on a
shaking roller. Next, 430 L of 0.1M EDTA (Sigma-Aldrich) was added to each
sample, and further homogenised by repeated pipetting through glass transfer pipettes.
Undigested fragments were removed by passage through 40 m nylon cell strainer
(Falcon Cell Strainer, Becton Dickinson and Company), and suspensions were topped
up with 4.5mL of R10 with 20mM HEPES (Gibco/BRL). Samples were underlaid with
1:9 0.1M EDTA (Sigma Aldrich) and FCS (CSL) and centrifuged. A single wash with
FACS/EDTA solution preceded antibody staining.
- 60 -
2 . 6 . 2 . Staining of DCs
Samples were incubated in 50 L of FACS/EDTA with 1:200 CD45RA-FITC
(0.2mg/mL BD PharMingen, San Diego, California USA), 1:50 CD11c-PE
(eBioscience, San Diego, California USA), and 1:200 CD8PECy5 (BioLegend).
Additional samples were unstained, incubated with single stain, or stained with the
appropriate isotype controls: rat IgG2bK-FITC (BD PharMingen), Armenian hamster
IgG1K-PE (eBioscience), and IgG2aK-PECy5 (BioLegend).
2 . 6 . 3 . DC flow cytom etry
Samples were processed for four colour flow cytometry with the assistance of Dr Kathy
Heel (BIAF, University of Western Australia, Nedlands Australia) using the FACS
Vantage (Becton Dickinson) flow cytometer.
Flow cytometry voltages and compensation were set with single stain samples, and
specificity was verified using unstained and isotype controls. Analyses were performed
with FlowJo V7.1.3 (Tree Star Inc). CD11c positive cells were sub-gated from the
lymphocyte population on forward scatter and side scatter. CD11c positive cells were
plotted for CD45-RA positivity (x-axis) against CD8a positivity (y-axis). As has been
published previously,326,327
plasmacytoid, CD8a positive, and double negative DCs were
then assessed.
2 . 6 . 4 . Analys is of DC phenotypes
DC samples were pooled from five animals for each experimental group. Naïve animals
were compared to tumour bearing and post-operative subjects. In the absence of
individual data points, it was impossible to make statistically reliable comments on the
significance of the trends.
2 .7 . T r e g assays
Treg frequency (as a proportion of lymph node CD4+ cells) was assessed in naïve,
tumour bearing, and post-operative animals. Treg depletion was also verified in depletion
experiments. Peripheral blood and lymph node samples were examined as single cell
suspensions, using the currently recognised Treg cell surface labels: CD4, CD25, and
Foxp3.328,329
- 61 -
2 . 7 . 1 . Cel l sur face and intracel lular s taining for T r e g
Lymph nodes were dissected from subject animals (via biopsy or at the time of
euthanasia) and collected into chilled PBS (Invitrogen). Nodes were mechanically
digested using frosted glass slides, and prepared into single cell suspension. Cells were
filtered with 40 m nylon filter (Falcon Cell Strainer) and centrifuged. Pellets were
washed twice with PBS (Invitrogen) and suspended into 200 L of PBS (Invitrogen),
with 10% FCS (CSL). Cell surface stains of CD4-FITC (0.5mg/mL, PharMingen) and
CD25-PE (0.2mg/mL, eBioscience) were incubated with the samples at concentrations
of 1:500 and 1:200 respectively. Specificity of staining was validated, using the isotype
controls of mouse IgG1–FITC (PharMingen) and rat IgG1–PE (eBioscience).
After half an hour of incubation at 4°C, samples were washed once with PBS/10% FCS
(CSL), followed by one wash with PBS (Invitrogen). Following surface staining, cells
were stained for intracellular Foxp3, according to manufacturer‟s instructions
(BioLegend). Wash buffer was comprised of PBS (Invitrogen) with 2% FCS (CSL), 1%
BSA (Sigma-Aldrich) and 0.01% sodium azide (Sigma). Samples were incubated with
200 L of Fix/Perm Buffer (BioLegend) and then centrifuged for five minutes. Samples
were then washed with Wash buffer, followed by Perm Buffer (BioLegend). Samples
were then incubated with Perm Buffer (BioLegend) for 15 minutes at room temperature.
After incubation with Perm Buffer (BioLegend), samples were centrifuged and stained
with 1:50 Foxp3-Alexa Fluor® 488 (BioLegend) for 30 minutes. Foxp3 labelled
samples were washed twice with Wash buffer, and then fixed with 4%
paraformaldehdye (BDH Chemicals, Kilsyth, Victoria Australia) for ten minutes.
2 . 7 . 2 . Flow cytometry for T r e g
Flow cytometry was performed on the FACSVantage (Becton Dickinson) flow
cytometer, with calibrations performed using single stain and isotype controls with the
assistance of Dr Robert van der Most. CD4, CD25 and Foxp3 were considered markers
for Treg cells, as generally accepted.328,329
Using FlowJo V7.1.3 (Tree Star Inc), Treg
were identified by triple positivity for these markers, along with appropriate cell size
and granularity. In some instances, combinations of CD4+ and Fox P3
+, Foxp3
+ alone,
and/or CD4+ and CD25
+ were used to identify Treg.
- 62 -
2 . 7 . 3 . Stat is t ical analys is o f T r e g
Treg relative frequencies (proportions) were calculated by dividing the number of
CD25+CD4
+Foxp3
+ cells, by the number of CD4
+ cells in each sample. Mouse groups
were treated as categorical variables, and Treg proportions were treated as continuous
variables. Explanatory variables included the presence/absence of tumour, post-
operative time point, and the presence/absence of Treg depletion. Differences between
groups were examined using the matched pairs t test in Prism 3 (GraphPad Software).
Differences were considered statistically reliable if they were significant at the 5% level
or better.
2 .8 . MSC Studies
Splenic MSC were identified in subject animals using flow cytometry and the
characteristic markers of CD11b and Gr-1. Spleens of normal animals, subject animals,
and controls, were collected into chilled PBS (Invitrogen), mechanically digested (using
glass slides), filtered with 40 m nylon cell strainer (Becton Dickinson and Company),
red cell lysed with 5% NH4Cl (BD) and washed twice with PBS (Invitrogen). A tenth of
each sample was re-suspended in 250 L of PBS (Invitrogen), with 5% FCS (CSL).
Each sample was incubated for 30 minutes with 1:200 Gr-1/Ly-6G/Ly-6C-PECy5
(BioLegend, San Diego, California USA) and 1:200 CD11b-APC (BioLegend),
centrifuged and washed in PBS/Turbo (Invitrogen/CSL). Flow cytometry was
performed using the BD FACSCalibur Flow Cytometer (Becton Dickinson), Cell Quest
V3.1 (Becton Dickinson) and FlowJo V7.1.3 (Tree Star Inc). Flow cytometer voltages,
threshold fluorescence values and compensation levels were set with reference to
unstained, single stain CD11b-APC (BioLegend) and single stain Gr-1- PECy5
(BioLegend) samples. Specificity of staining was verified by irrelevant isotype control
mAb; specifically rat IgG2bK-PECy5 (BioLegend) for Gr-1, and rat IgG2bK-APC
(BioLegend) for CD11b.
In accordance with previously published work, the Gr-1+CD11b
+ population was a
heterogeneous cell group.246
As it is unknown which cell type of the Gr-1+CD11b
+
population is responsible for immune suppression,246
the group was quantified as a
whole. Quantification was performed by dividing the number of Gr-1+CD11b
+ cells by
the number of viable, non red cell splenocytes collected over the same period, in that
- 63 -
sample.281
Percentage splenic MSC were expressed as mean values SEM where
relevant. Percentages of MSC were considered continuous variables and compared
between post-operative, normal, and tumour bearing animals. Explanatory variables
were the presence/absence of tumour, tumour size, and post-operative time point.
Statistical analysis was performed using Prism 3 (GraphPad) and the matched pairs
student t test. Differences were considered significant at the singled tailed 5% level.
2 .9 . HA-Speci f i c CD8+: de tec t ion/phenotype
Tumour-specific CD8+ cells were identified by fluorescently labelled IYSTVASSL-
MHC I pentameric complexes (Pro5® MHC Pentamer, ProImmune, Oxford United
Kingdom) in conjunction with CD8 staining in the HA tumour transfectant system
(AB1HA). Tumour-specific CD8+ cells were further characterised by the memory
marker CD44330
and the CD127 homeostatic proliferation receptor.298
Homeostatic
proliferation is canonical to a true memory cell,298
and the combination of HA
Pentamer, CD8, CD44 and CD127 enables a precise delineation of true, tumour-specific
memory cells.
2 . 9 . 1 . Pentam er cal ibrat ion
CD44, CD8, CD127, and IYSTVASSL Pro5® MHC Pentamer staining was calibrated,
prior to examining for tumour-specific CD8+ cells. 1x10
6 tumour-specific CD8
+ cell
samples (obtained from preparations of CL4 lymph nodes), were used to test varying
dilutions of pentamer (4:100, 8:100, 12:100, 16:100, 1:5, and 1:4). Of these, a 1:5
pentamer concentration (maximum of 1x106 tumour-specific cells) was selected, as it
was associated with minimum background binding and near maximal detection of
IYSTVASSL-specific CD8+ cells.
2 . 9 . 2 . Assessm ent of tumour -speci f i c memory cel l s
Samples of axillary, inguinal and non-draining (e.g. mediastinal, mesenteric) lymph
nodes and spleens were harvested from naïve, tumour bearing, post-operative, and post-
sham surgery BALB/c mice of various time points. Lymph nodes were harvested into
chilled PBS (Invitrogen), mechanically digested (using frosted glass slides), filtered
with 40mcm nylon cell strainer (Becton Dickinson and Company), red cell lysed with
5% NH4Cl (BD) and washed with wash buffer. The wash buffer was comprised of
- 64 -
0.1% sodium azide (Sigma-Aldrich), 0.1% BSA (Sigma-Aldrich) and 2% FCS (CSL) in
PBS (Invitrogen).
Residual fluid on the pellet (approximately 50L) was used as suspension volume for
pentamer staining. To remove protein aggregates, the pentamer reagent was micro-
centrifuged at 14,000g, and 4°C for 3 minutes prior to staining. Pentamer was added to
the concentrated cell suspensions at a dilution of 1:5, agitated, and incubated at room
temperature (22 degrees) for 10 minutes. Solutions were shielded from light during
incubation. After pentamer incubation, cells were washed and re-suspended in 100 L
of wash buffer for antibody staining. Samples were incubated with 1:200 CD8a-PECy5
(BioLegend), 1:200 CD44-FITC (BioLegend) and 1:50 CD127-APC (eBioscience) for
20 minutes, shielded from light.
Unstained, single stain samples, and isotype controls were used to verify specificity of
the staining procedure: rat IgG2b-FITC (BioLegend), rat IgG2b-PECy5
(BioLegend), and rat IgG2a-APC (eBioscience). Samples were processed by four
colour flow cytometry (BD FACSCalibur, Becton Dickinson) with the assistance of Dr
Andrew Currie (Tumour Immunology Group, Sir Charles Gairdiner Hospital).
Samples were analysed using FlowJo V7.1.3 (Tree Star Inc). Tumour-specific CD8+
cells were expressed as a proportion of lymph node CD8+ cells, to a maximum of two
decimal places. CD44+ and/or CD127
+ were also calculated proportionately to a
denominator of Pentamer+, CD8
+ lymphocytes. CD44
+ and CD127
+ percentages were
expressed to a maximum of two decimal places, and denoted as means SEM, where
pertinent.
As outcomes were proportionate and all samples were processed concurrently,
comparisons could be made between subject groups. Tumour-specific CD8+
proportions, CD44+ proportions, and CD127
+ proportions were all considered
continuous variables. Different mouse groups were considered categorical variables.
Proportions were compared between subject groups and controls, using the paired
student t test and Prism 3 (GraphPad). Differences were considered significant at the
5% level.
- 65 -
2 .10 . Therapies
2 . 10 .1 . Tol l Like Receptor (TLR) l igand therapy
TLR therapy doses were in accordance with previously published values, and were
optimised in the Tumour Immunology Group at Sir Charles Gairdner Hospital
laboratory by Dr Andrew Currie and Mr Steven Broomfield.
2 . 10 .2 . poly I :C
The TLR3 ligand, polyriboinosinic acid-polyribocytidylic acid (poly I:C, InvivoGen,
San Diego, California USA) was administered in the scar of postoperative animals, or
into experimental metastases after surgery. The maximum dosage was 10 g in 100 L
of saline (saline), every day, for up to six treatments. This was consistent with the range
of dosages seen in the literature, including a clinical trial of intramuscular poly I:C for
malignant glioma in humans.331
When tumours were being treated, therapy was
delivered intra-tumourally once the tumour was visible and palpable. If tumours were
not present, doses were skipped until such time as they reappeared. The maximum
frequency of treatments was daily, and the maximum number of treatments was six.
Saline and inactive nucleic acid, CpG 1720 (nCpG, Tib-Molbiol, Hamburg, Germany)
were administered at comparable frequency and dosage, to matched control animals.
Comparisons of survival were made between poly I:C treated animals and controls
using the log rank test.
2 . 10 .3 . CpG-ODN 1668
The TLR9 ligand, cytosine phosphorothioate guanine oligodeoxynucleotide 1668 (CpG-
ODN 1668, Tib-Molbiol, Hamburg, Germany) was administered intra-tumourally to
experimental post-operative metastases, or intravenously at the time of surgery. When
CpG was administered intravenously, a single injection of 10 g was provided at the
time of surgery. This dosage (0.5mg/kg) approximates the upper range of published
intravenous treatments in humans, but is low in comparison to other published dosages
in mice.332
Intra-tumoural CpG was given at a dosage of 10 g in 100 L of saline,
comparable to previously published regimens for mice.53
When tumours were absent,
doses were skipped. The maximum frequency of dosage was daily, and each mouse
received up to six treatments.
- 66 -
As with poly I:C experiments, saline or inactive nucleic acid nCpG (Tib-Molbiol) was
administered at comparable frequency and dosage to matched control animals.
Comparisons of survival were made between CpG treated animals and controls using
the log rank test.
2 . 10 .4 . 3M019T M
3M019 (3M Pharmaceuticals, St Paul, Minneapolis USA) is a recently developed
imidazoquinoline. The intra-tumoural mode of administration in this research (rather
than topical) differs significantly to all previously published literature on TLR7
analogues. In collaboration with Mr Steven Broomfield (TIG) and 3M Pharmaceuticals,
3M019 was injected into post-operative experimental metastases. A dosage of 50 g
was used, suspended in 100 L of sterile saline (Astra Zeneca). When tumours were
absent, doses were skipped. The maximum frequency of treatment was every second
day, and each mouse was treated up to six times. As with the other treatment
experiments, comparisons were made between 3M019 treated animals and controls
using the log rank test.
2 . 10 .5 . Act ivat ing ant i -CD40 ant ibody therapy
Murine agonistic anti-CD40 monoclonal antibody (FGK45 mAb, Monoclonal Antibody
Facility, Perth Western Australia) was administered intra-tumourally or peri-tumourally
at a dosage of 40 g in 100 L of sterile saline, every 2nd
day, for a maximum of six
treatments. FGK45 was also given intravenously at the dosage of 100 g every third
day, once tumours had emerged. This was consistent with previously published doses
for systemic FGK45 (e.g. 100 g q2dx311,333
and 100 g q2dx5334
). Survival amongst
FGK45 treated animals and controls were compared using the log rank test.
2 .11 . In v ivo deple t ion s tudies
CD4+, CD8
+, and Treg depletions were performed in vivo. Previously described mAb
reagents were used for all depletions. Complete depletion for each period of interest was
verified on peripheral blood (CD4+, CD8
+, Treg) and lymph node biopsy (Treg).
- 67 -
2 . 11 .1 . CD4+/CD8
+T cel l deplet ions
CD8+ T cells were depleted using YTS169 mAb (Monoclonal Antibody Facility, Perth
Western Australia). CD4+ cells were depleted with GK1.5 mAb (Monoclonal Antibody
Facility). For both CD4+ and CD8
+ depletions, 200 g of mAb was administered by
intraperitoneal injection, 24 hours before the depletion period. The same dose was also
given on the first day of the depletion period. Depletion was maintained by
intraperitoneal mAb, at a dosage of 150 g every second day. Completeness of CD4+ or
CD8+ depletion was assessed by flow cytometry on peripheral blood. Briefly, glass
capillary tubes were used to collect blood from the retro-orbital sinus. Each blood
sample was diluted with PBS (Invitrogen)/10% FCS (CSL), up to a volume of 1mL.
Diluted samples were placed atop 1mL of Ficoll® (Sigma-Aldrich) and centrifuged at
4°C , 2000rpm, for 20 minutes. After centrifugation, a buffy coat (lymphocytes)
appeared on the Ficoll® surface, which was harvested and transferred to FACS tubes
(Round Bottom 12mm x 75mm with cell strainer cap, BD FalconTM
, BD Biosciences).
2mL of PBS/Turbo was then added to each FACS tube, and the mix was centrifuged at
4 degrees, for 5 minutes.
Pellets were re-suspended in 100 L of PBS (Invitrogen)/10%FCS (CSL) with 1/500
CD4-FITC (PharMingen), CD8-PeCy5 (BioLegend), or isotype controls: rat IgG2a-
FITC (PharMingen) and rat IgG2bK-PECy5 (BioLegend). Antibodies were incubated at
4 degrees Celsius for 30 minutes, shielded from light. After incubation, samples were
washed with PBS/Turbo, and flow cytometry was performed. Calibration of the flow
cytometer (FACScan, Becton Dickinson) was performed with unstained and single stain
samples, and the specificity of staining was verified using the isotype controls.
Depletion was considered successful when >90% of CD4+ cells were removed and/or
>95% of CD8+ cells were absent from treated samples
2 . 11 .2 . T r e g deplet ion
PC61 Anti-CD25 mAb was obtained from the Monoclonal Antibody Facility (Perth,
Western Australia) and used to deplete Treg in vivo, as per standard protocols.335
Briefly,
PC61 was administered intra-peritoneally at a dosage of 500 g, 24 hours prior to the
period of required Treg depletion. Successful Treg depletion was confirmed on the first
day of the period of interest by retro-orbital bleeds and processing for
CD4+CD25
+Foxp3
+ flow cytometry, as described earlier. The period of Treg depletion
was approximately 10-14 days and the gradual re-emergence of Treg at 7-10 days was
- 68 -
verified by lymph node biopsy and flow cytometry. While CD25+ cells were absent on
flow cytometry after anti-CD25 mAb therapy, some preservation of the Foxp3+CD4
+
population was found, consistent with recently published data.336
2 .12 . Ident i f icat ion of sent ine l nodes
Vital dyes are a recognised method of visualising lymphatics and identifying sentinel
lymph nodes.129,130
Methylene blue was used to identify the lymphatics and sentinel
nodes for the BALB/c flank.
2 . 12 .1 . Methylene b lue
10mg/mL methylene blue (Sigma) was formulated into sterile PBS and 100 L was
injected into the subcutaneous flank of anaesthetised BALB/c mice. Animals were
systemically dissected and photographed at 5 minutes, 15 minutes, 30 minutes, 1 hour
and 24 hours after inoculation.
2 .13 . DC tracking
Skin DC migration was tracked in vivo using CFSE, GM-CSF and the standard
cutaneous (Langerhans) DC markers of CD11c and DEC205.337
Animals were
preconditioned at the typical tumour injection site using 10 g of recombinant murine
GM-CSF (ProSpec-Tany Technogene Ltd, Rehovot, Israel). 24 hours after treatment
with GM-CSF, 100 L of 10 M CFSE (Molecular Probes) was injected into the same
site.
A further 24 hours after CFSE inoculation, regional lymph nodes were harvested and
processed for DC isolation as per 2.6.1. Samples were then incubated with 1:50 CD11c-
PE (eBioscience), 1:200 CD11b-APC (0.2mg/mL, BioLegend), and 1:125 DEC-205-
Biotin (CedarLane Labs, Hornby, Ontario Canada). Additional samples were incubated
as single stained, unstained controls, or IgG1-PE (eBioscience) isotype control and rat
IgG2b -APC (BioLegend) isotype controls.
Samples were washed twice with PBS, and fixed with 4% paraformaldehyde (Sigma-
Aldrich) prior to flow cytometry. Flow cytometry was performed using the FACS
- 69 -
Vantage (Becton Dickinson) instrument, with associated CellQuest V3.1 software
(Becton Dickinson). The mononuclear population was selected from the forward scatter
and side scatter plot, and sub-gated for CD11c positivity (x-axis) against DEC-205 (y-
axis). CFSEHI
CD11cHI
DEC-205HI
CD11bHI
cells were quantified as a proportion of total
CD11c positive cells. Different node groups within the animal were compared and
correlated with findings on methylene blue injection.
2 .14 . Histology
Histological sections were undertaken to assess the completeness of resection, the
presence of lymph node invasion, and to document the morphology of RencaHAM in
vivo. Slides were prepared for histology by standard haematoxylin and eosin (H&E)
staining, as below.
2 . 14 .1 . H&E staining
Specimens were fixed in OTC (Sakura Finetek, Tokyo Japan) and frozen at -80C.
Frozen specimens were cut at 10 m sections on L-lysin (Sigma) coated slides, by
cryostat. Slides were fixed with Carnoy‟s fixative. Carnoy‟s fixative was comprised of
10% acetic acid (Sigma), 30% chlorophorm (Sigma), and 60% absolute alcohol
(Pronalys, Biolab, Mulgrave, Victoria Australia).
Samples were then washed with tap water for 1 minute, and stained with Gill‟s
haematoxylin (VWR International Ltd, Poole, England) for 1 minute. Slides were rinsed
in tap water for a further minute, then in Scott‟s water for 30 seconds. Scott‟s water was
formulated from 1L of distilled water, 20g Sodium Bicarbonate (Chem-Supply,
Gillman, South Australia), and 7g of Magnesium Sulphate (Sigma).
After washing with Scott‟s water, samples were rinsed in tap water for another minute,
70% alcohol (Merck Pty Ltd, Kilsyth, Victoria Australia) for 30 seconds, and then 95%
alcohol (Merck) for 30 seconds. Slides were counterstained with Eosin (Gurr
Certistain®, BDH Laboratory Supplies, Pool, England) for 1 minute, and washed with
absolute alcohol (Pronalys, Biolab, Mulgrave, Victoria Australia) for 30 seconds each
time. Slides were cleaned three times with xylene (BDH), for one minute at a time.
- 70 -
Finally, slides were mounted in DPX (Scot Scientific, Welshpool, Western Australia),
air dried at room temperature, and examined by microscopy.
2 . 14 .2 . Resect ion specimens
To verify complete resection, pairs of animals undergoing surgery were randomly
selected for histology. The presence of a clear resection margin (i.e. healthy tissue),
located on superficial, lateral and deep aspects of the surgical specimen suggested
complete excision.
2 . 14 .3 . Kidneys
Mice with renal RencaHAM tumours were euthanased, and kidney specimens were
collected for histology. Four randomly selected specimens were processed as described
above.
2 . 14 .4 . Lymph nodes
Lymph node dissection was undertaken on two, randomly selected mice for each route
of RencaHAM transplantation (intravenous, intra-cardiac, intra-renal). Lymph nodes
included: cervical, axillary, brachial, inguinal, iliac, caudal, para-aortic, renal, celiac,
mesenteric and mediastinal. Samples were processed for histology, and examined for
the presence of tumour cells by an independent observer.
2 . 14 .5 . Lungs
Whole lungs were teased clear of the thoracic cavity by blunt dissection. Each trachea
was cannulated with a 25 gauge needle (BD PrecisionGlideTM
, BD, Singapore) and
injected with approximately 1mL of 50% OTC (Sakura Finetek)/50%PBS (Invitrogen).
Lung regions of interest (e.g. middle lobe) were incised and rapidly immersed in OTC
(Sakura Finetek) for freezing. H&E sections were subsequently processed, as previously
described.
- 71 -
2 .15 . Cul ture of necropsy spec imens
Lung and lymph nodes from RencaHAM bearing animals were assayed for the presence
of viable tumour cells by tissue culture. Pairs of mice with RencaHAM disease (day 21)
were randomly selected from groups of differently transplanted animals (intravenous,
intra-cardiac, intra-renal).
2 . 15 .1 . Lung
Lungs were collected into chilled R10, mechanically digested by glass slide, and
transferred into 75cm2 BD Falcon culture flasks (BD Biosciences) containing R10
supplemented with 400g/mL G418. Samples were incubated at 37°C and 5% CO2. 48
hours after culture was commenced, the flask culture surface was thoroughly rinsed
with warmed PBS (Invitrogen), and fresh medium was added. Flasks were inspected for
the presence of tumour cells, every 3 days. Medium was changed at the same time, as
per standard cell culture protocols. Once tumour cells were present at microscopy,
photographs were taken and the flasks were discarded. If no cells grew out after 6 weeks
of culture, the result was considered negative.
2 . 15 .2 . Lymph nodes
To determine whether viable tumour cells were present in the lymph nodes of
RencaHAM animals, total lymphadenectomy was performed on randomly selected pairs
of RencaHA bearing mice (intravenous, intra-cardiac, intra-renal). Samples were
collected into chilled R10 and cultured, as for 2.15.1. Once again, if no cells grew out
after 6 weeks of culture, the result was considered negative.
2 .16 . HA-speci f ic rea l t ime PCR
Resection beds and lymph nodes of RencaHAM or AB1HA tumour bearing and/or post-
operative animals were examined for HA positive cells (tumour cells). Genomic DNA
extraction and isolation were undertaken by standard methods, as below. HA specific
primers and real time PCR (RT-PCR) were used, as previously described.20
Preparations
of the relevant tumour cells (AB1HA and RencaHAM) were obtained from tissue
culture and used as positive controls. RNase free water, non-transfected cell lines (AB1,
Renca) and DNA extracted from healthy skin were used as negative controls.
- 72 -
2 . 16 .1 . Extract ion of DNA
Samples for DNA extraction were stored at -20°C in 1.5mL Eppendorf tubes (Sarstedt,
Ingle Farm, South Australia). Lysis buffer was formulated from 50mM Trizma ®
(Sigma-Aldrich), 0.4M NaCl (Sigma-Aldrich), 100mM EDTA (Sigma-Aldrich), 0.5%
SDS (Sigma-Aldrich) and sterile H2O. 600 L of lysis buffer was added to each sample,
along with 350 g of proteinase K (Promega, Annandale, NSW Australia).
Samples were incubated at 55°C and 200 rpm for one hour, or until completely
digested. 167 L of Saturated (6M) NaCl (Sigma-Aldrich) was added to each sample,
followed by a 15 second vortex. Samples were centrifuged, and the pellets were placed
in clean Eppendorfs (Sarstedt). 95% ethanol (Merck Pty Ltd) was added of
approximately equal volume to each pellet. Samples were inverted to precipitate DNA
and centrifuged for 30 minutes at 4°C . Pellets were washed with 70% ethanol (Merck
Pty Ltd) and dried at room temperature. Once the alcohol was gone, samples were re-
suspended in 200 L of RNase free H2O, in preparation for PCR.
2 . 16 .2 . PCR of DNA templates
The presence and absence of HA DNA was detected using HA specific forward and
reverse primers. A 30 L reaction mix was used, containing 10pmol of each primer,
12.5L of 2x QuantiTec SYBER Green PCR Master Mix (Qiagen, Doncaster, Victoria
Australia), 9.25L of RNase free water (Qiagen), 0.25L of 1/1000 Fluoroscein (Bio-
Rad, Hercules, California USA), and 5L of template DNA. HA was detected on the
Real Time PCR Machine (Bio-Rad) using the following protocol: 95°C for 15 minutes,
94°C for 15 seconds, 35 cycles of 58°C for 30 seconds, followed by one cycle of 72°C
for 30 seconds, 80 cycles of 55°C for 10 seconds. Amplified DNA was held at 10°C.
PCR amplification and melt graphs were obtained using the iCycler (Bio-Rad) and
associated software. Subject curves were compared to RNase free water, healthy skin,
non-transfected tumour, and HA transfectant tumour controls.
- 73 -
Chapter 3
- 74 -
3. Surgery and cross presentation
3 .1 . Introduct ion
Numerous immune therapies can be used in combination with surgery. Tumour
vaccines are a major class of such therapies, and as of 2003, there were some 200 – 300
tumour vaccines in phase II or phase III clinical trial.66
By definition, tumour vaccines
provide an exogenous source of tumour antigen and/or a mechanism of enhancing
antigen priming. The notion that antigen priming is limiting to the immune response is
implicit to the principle of tumour vaccination.
There are two possible sources of antigen in a tumour-bearing patient: the primary
tumour and secondary deposits (metastases). Primary tumours are usually the largest
and are examined first, but whether tumours in distal sites present antigen is also
investigated.
In the setting of bulky disease, when tumour antigen is abundant, it remains uncertain
whether antigen presentation is a limiting factor. Rather, it might be the quality of the
interaction between APC and CD8+ that is defective, and/or tumours immune
subversion mechanisms may operate. As evidence for this, tumour vaccination has been
almost universally disappointing in the setting of bulky disease.338
Even if antigen presentation is not a limiting factor with bulky tumour, it may become
limiting after surgery. Despite highly efficient antigen presentation, by removing
tumour mass, surgery may reduce antigen presentation to below stimulation threshold.
The effects of surgery on soluble tumour antigens are well characterised in the literature
(e.g. decline in CEA after colorectal carcinoma excision339
or the fall in PSA after
prostate resection340
) but post-operative tumour antigen presentation has not been
studied in vivo.
Understanding how surgery affects tumour antigen presentation is critical, because
tumour antigen provides the obligatory priming signal to the anti-tumour immune
response (see Figure 1.2) Thus, understanding the impact of surgery on tumour antigen
presentation could be important to planning the optimum type of immune therapy after
- 75 -
surgery. In particular, if tumour antigen presentation becomes limiting post-operatively,
this would indicate a role for tumour vaccines. In contrast, if tumour antigen
presentation remains sufficient after primary resection, it may be more appropriate to
enhance the quality of tumour antigen priming, or to boost the effector component of
the immune response (e.g. by adoptive immunotherapy).
Not only is it important to understand the effects of surgery on tumour antigen levels,
but also the impact of resection on the distribution of that antigen. For instance, surgery
is associated with neutrophil influx, vascular injury, and local protease activity.341
Such
phenomena might lead to the systemic efflux of tumour antigen and might have
implications for planning the mode of delivery for post-operative immune treatment.
More specifically, some immune therapies might work best in the vicinity of tumour
antigen.342
Thus while intra-tumoural or peritumoural treatments may be important
before surgery, intravenous therapy could be more useful if antigen becomes systemic
post-operatively.
In this chapter, the effects of surgery on the presentation of cell associated tumour
antigen are studied, aiming to facilitate a logical approach to planning the optimum
timing, type and mode of delivery for post-operative immune treatments. The effects of
surgery on the presentation of cell associated tumour antigen are examined using the
AB1HA mesothelioma tumour model.148
AB1HA expresses HA as a cell associated
antigen that is constitutively cross presented.343
Thus the emphasis of this investigation
is the effects of surgery on cross presented, cell-associated antigen. Given that CD8+
lymphocytes are the predominant anti-tumour effectors,344-346
and since continual high
level CD8+ lymphocyte effector function is closely related to continued tumour antigen
presentation,347
determining the impact of surgery on CTL function in vivo is logically
the second component to the study.
3 .2 . Resul t s
3 . 2 . 1 . AB1HA in wi ld type and immunodefic ient mice
Initially, the subcutaneous growth of AB1HA tumours was assessed in wild type and
immunodeficient mice. When 1x106
AB1HA cells were inoculated into wild type and
BALB/c nu-/-
, solid and vascularised tumours were the result (Figure 3.1B) Outgrowth
- 76 -
of AB1HA was slightly slower in wild type mice relative to immunodeficient BALB/c
(Figure 3.1A, P < 0.001, two way ANOVA) suggesting that AB1HA could evoke an
immune response. However, that immune response was of insufficient magnitude to be
protective because the tumour take rate was 100%, and all mice succumbed (data not
shown).
3 . 2 . 2 . HA-speci f ic presentat ion during AB1HA growth
The level of HA specific presentation in tumour draining lymph nodes (ipsilateral
axillary and inguinal), non draining nodes (contralateral inguinal, contralateral axillary,
brachial, cervical, iliac, caudal, para-aortic, celiac, mesenteric, mediastinal) and spleens
was measured using the Lyons Parish321
assay for days 4, 10, 16 and 21 of subcutaneous
AB1HA growth (representative data for day 21: Figure 3.2).
When background was defined as CL4 proliferation in the nodes and spleens of normal
(non tumour-bearing) animals, statistically significant proliferation (two tailed P < 0.01,
relative to normal controls) was seen in the draining lymph node at day 4 after tumour
inoculation (Figure 3.3). This corresponded to a mean tumour diameter of 0.85
0.11mm. Cross presentation increased in parallel to tumour size, reaching 51.96
6.93% by day 21 after inoculation (Figure 3.3A). In contrast, cross presentation was
not detectable in the non-draining nodes nor in the spleen, for any time point of tumour
growth (Figure 3.3B,C).
3 . 2 . 3 . Speci f ic i ty of CL4 prol i ferat ion
To ensure CL4 proliferation was specific for HA (i.e. not just due to the presence of a
tumour in the region) presentation was assayed in animals bearing established HA-
negative parental AB1 tumours. Proliferation was assessed on day 16 of AB1 tumour
growth, at which stage tumours were 3.86 0.29mm in diameter. No significant CL4
proliferation was detected in the draining lymph nodes of AB1 bearing animals on day
16 (P > 0.05 relative to background, student‟s t test). The lack of proliferation seen in
non-draining nodes and spleens of AB1HA tumour bearing mice was reinforced by the
statistical equivalence of these tissues to the non draining nodes and spleens of AB1
bearing animals (P = 0.050 and P = 0.080 respectively, student‟s t test).
- 77 -
3 . 2 . 4 . HA presentat ion af ter surg ery
To determine the effect of complete resection (Figure 3.5) on the kinetics of antigen
presentation, HA-specific presentation was assayed in groups of animals at various time
points before and after surgery. As was the case pre-operatively, cross presentation was
not seen in the non-draining lymph nodes nor the spleen for any post-operative
timepoint (data not shown). Within the draining lymph nodes, no immediate effect on
cross presentation was evident (Figure 3.6A). However, HA-specific presentation did
decline steadily after the day of surgery, until day 14 post-operatively, when antigen
presentation was no longer statistically significant (relative to matched sham surgery
controls), Figure 3.6B.
Figure 3.1. Growth kinetics of subcutaneous AB1HA in wild type and nude mice
A. AB1HA was grown subcutaneously in BALB/c and congenic BALB/c nu-/-
. Mean tumour diameter
SEM shown for each cohort. Data are shown from a single experiment (n = 10 for each group). *P <
0.001 (two way ANOVA). B. Subcutaneous tumour in BALB/c mouse at day 16.
- 78 -
Figure 3.2. Location of HA-specific presentation in AB1HA tumour bearing mice
A. Location of lymphoreticular tissues commonly assayed. B. Representative forward scatter (FSC)/side
scatter (SSC) plot showing inclusion gate (R1) used to detect proliferating lymphocytes. C.
Representative plots (from single experiment) showing region used to measure HA specific proliferation
in the draining (left), non-draining nodes (centre), and spleen (right). D. Representative histograms of
regions R2, showing HA specific proliferation “daughter” peaks in the draining nodes (left) but neither
non-draining nodes (centre) nor spleen (right).
- 79 -
Figure 3.3. CL4 proliferation during tumour growth
HA specific CD8+ proliferation was assessed in BALB/c mice during AB1HA tumour growth. Single
mice are indicated by points on the graph, mean CL4 proliferation is indicated by the bar for each cohort,
with a minimum of seven animals per cohort. A. CL4 proliferation in the draining nodes over time. B.
HA-specific CD8+ proliferation in the non-draining nodes. C. CL4 proliferation in the spleen. *Denotes
statistically significant proliferation relative to background (two tailed P < 0.05, student‟s t test).
- 80 -
Figure 3.4. Specificity of CL4 proliferation.
CL4 proliferation was assayed in animals bearing day 16 HA-negative parental AB1 tumours and
compared to BALB/c with established AB1HA tumours and naïve controls. Single mice are indicated by
points on the graph, mean proliferation is shown by bars. Data are shown from a single experiment with
at least five animals present per group. *Denotes two-tailed P < 0.05, relative to naïve controls (student‟s
t test). Key: LN = lymph node, SPL = spleen, DLN = draining lymph node, NDLN = non draining lymph
node.
3 . 2 . 5 . Completeness of resect ion
To determine if the persistence of tumour antigen presentation after surgery was due
to incomplete resection, surgical margins were examined by PCR to detect small
amounts of residual tumour cells. As previously described, this technique was
sensitive to as few as ten AB1HA tumour cells.20
Three pairs of animals undergoing
surgery were randomly selected for resection bed biopsy. Resection bed biopsies
were taken from on the day of surgery, 24 hours post-operatively, or 2 weeks after
surgery. Biopsies were examined for the presence of HA transgene by genomic PCR,
as per 2.14. HA transgene positive cells were absent from all biopsies of the surgical
site, on the day of surgery, 24 hours after surgery, and 2 weeks post-operatively
(Figure 3.7A,B). Additionally, surgical cure rates were assessed in congenic
BALB/c nu-/-
, who were athymic and had a markedly reduced capacity to
immunologically reject residual tumour. Tumours were grown in 10 nude BALB/c
- 81 -
until day 16 after inoculation (when tumour diameter averaged 5.75 0.61 mm) and
resection was undertaken. Animals remained free of recurrence for > 60 days after
surgery, and were thus cured by resection. Complete resection was also checked with
histological sections; clear resection margins were observed in three randomly
selected animals undergoing removal of AB1HA tumour (Figure 3.7C).
Figure 3.5. Surgery for AB1HA tumours
Subcutaneous AB1HA tumours were excised, as detailed in 2.3.9. A. Resection bed after ligature of
vascular pedicles and complete excision of AB1HA lesion plus adherent skin. B. Closure of tissue defect.
C. Appearance at two weeks after surgery.
- 82 -
Figure 3.6. CL4 proliferation before and after surgery
A. CL4 proliferation in the draining nodes before and after complete AB1HA resection. B. CL4
proliferation in naïve mice before and after sham surgery. Individual mice are shown by points, mean
proliferation indicated by bars. *P value < 0.05 for equivalent means between A and B at indicated time
points (two tailed student‟s t test). Key: Pre-op = pre-operative, DOS = day of surgery.
- 83 -
Figure 3.7. Completeness of resection.
A, B. Soft tissue and skin from resection bed biopsies and healthy mice were digested and subject to RT-
PCR analysis for HA transgene. Representative melt (A) and fluorescence (B) curves are shown.
Fluorescence seen from positive control sample (HA tumour, light blue) - corresponding melt curve peak
at 82°C. No fluorescence seen in negative controls (sham surgery, water, healthy skin) or in test samples:
24h postoperative (dark blue) and 2 weeks postoperative (red). C. Representative histology of AB1HA
tumour on removal. Keratinised epithelium with hair follicles visible at top of slide, subcutaneous
AB1HA tumour towards lower aspect of slide. Occasional vessels were seen in tumour (middle and lower
thirds of slide).
- 84 -
3 . 2 . 6 . HA presentat ion from recurrent AB1HA
The effects of local recurrence or post-operative metastasis on antigen presentation were
then investigated. To simulate local recurrence, BALB/c underwent incomplete
resection of AB1HA tumours by primary resection and immediate re-challenge with
AB1HA into the wound (n = 4). To simulate post-operative metastasis, mice with
AB1HA tumours were completely resected and then re-challenged into the healthy flank
on the day of surgery (n = 4). Locally recurrent tumours were clearly visible by day 16
after surgery in the 3 out of 4 animals in the enucleation group, measuring 8.05
0.75mm. “Metastatic” tumours were also detectable 3 out of 4 animals of the
“metastatic group”, measuring 2.95 0.51 mm at day 16.
In the locally recurrent cohort, antigen presentation was clearly detectable only in the
lymph nodes draining the locally recurrent tumour (Figure 3.8A). Average proliferation
rates of 74.62 5.65% were seen in that group, which was statistically comparable to
BALB/c with primary AB1HA tumours of similar size day 21 (51.96 6.93%) - Figure
3.8C. In animals with “metastatic tumour” after surgery, antigen presentation was seen
in the nodes draining the metastatic deposit, but was no longer detectable in the lymph
nodes of the post-operative site (Figure 3.8B). HA-specific proliferation from the
“metastatic deposit” was 52.98 5.61%, comparable to that normally seen from a
primary AB1HA tumour of similar size (day 10, 44.44 6.38%) - Figure 3.8C.
3 . 2 . 7 . Antigen presentat ion to CD4+ T cel l s pos t -op
HA presentation to the CD8+ T cell compartment was robust in animals with established
tumours, but was absent by two weeks post-operatively. However, CD4+ cells are
crucial to effective CTL generation and maintenance.224,226,230
To determine if tumour
antigen presentation to the CD4+ compartment was deficient compared with antigen
presentation to CD8+ cells, assays were repeated using HA CD4
+ epitope specific
(HNT) congenic T cells pre- and post-operatively (Figure 3.9A) Significant HNT
proliferation was detected on day 16 of tumour growth relative to naïve controls (Figure
3.9B, P < 0.005), although these levels were lower than CL4 proliferation at the same
stage of tumour growth (12.42 2.08% versus 45.96 7.28%, P = 0.006). As with CL4
proliferation, no HNT proliferation was observed two weeks after complete resection
(Figure 3.9A,B).
- 85 -
Figure 3.8. CL4 proliferation with locally recurrent or “metastatic” AB1HA.
BALB/c mice underwent incomplete primary resection, or complete primary resection with re-challenge
into the healthy flank on the day of surgery. Lyons Parish analyses were undertaken 16 days after surgery.
The experiment was performed once, with four animals per group. A. Lyons Parish analysis from local
recurrence group. B. Lyons Parish analysis from animal with metastatic tumour. C, Left: Proliferation
rates in the local recurrent bed compared to its nearest primary AB1HA equivalent (day 21). C, Right:
Proliferation rates of the metastatic nodes c.f. nearest primary AB1HA equivalent (day 10). Key: DLN =
draining lymph node, NDLN = non draining lymph node, Met = metastasis, Local Rec = local recurrence.
- 86 -
3 . 2 . 8 . Post -operat ive DC phenotype
Antigen presentation is governed by a number of factors including DC numbers,348
maturation,349,350
and subtype.350
It was therefore postulated that the decline in tumour
antigen presentation after surgery (3.2.4) may correlate with the disappearance or
emergence of a particular DC subtype. To evaluate this, changes in DC subtypes were
examined. Naïve and sham surgery animals were used as controls. Each sample
(draining and non draining nodes) was pooled from five animals.
DC were identified by CD11c positivity on side scatter/CD11c plots. High background
staining from the rat IgG2K-PE isotype required the use of stringent CD11c gating
(Figure 3.10A). Samples were divided into plasmacytoid (CD11c+CD45RA
+CD8
+/-),
CD8a+ (CD11c+CD45RA
-CD8
+) , and double negative (CD11c
+CD8a
-CD45RA
-)
populations as per accepted practice - Figure 3.10B.326,327
Surgery (sham or tumour) had
no impact on DC phenotypes, at either one week or two weeks post-operativelyA,B).
There was also no difference in DC phenotype between the non draining and draining
lymph nodes themselves, before and after surgery (Figure 3.11A,B)
3 . 2 . 9 . Cross presentat ion and in v ivo CTL funct ion
CD8+ lymphocytes require several signals to become continually activated, proliferate,
and differentiate into effectors.347,351
The first is antigen [peptide-MHC], the second a
co-stimulatory signal [e.g. CD80/86]214
and the third a cytokine signal [e.g. IL-12].352
Although antigen presentation declined after surgery (Figure 3.6), it was not certain that
a similar fall in CTL function would follow. To determine whether in vivo CTL function
decreased in parallel with antigen presentation, in vivo CTL assays were performed.
Assays were taken before surgery (day 16 tumours), 24 hours post-op, 1 week post-op,
or 2 weeks post-op. Inguinal, axillary, non-draining (e.g. contralateral inguinal/axillary)
and spleens were assessed in each animal. In vivo CTL lysis in subject animals was
compared to CL4 (positive) and naive (negative) controls.
HA-specific target lysis was routinely 40% in CL4 TCR transgenic mice (positive
controls), and absent from naïve animals (negative controls) - Figure 3.12B. Modest
endogenous responses (<10% killing) were seen in all tissues of tumour bearing mice
(<10%) except for the axillary node, where 23.77 2.45% killing was observed (Figure
3.12C). When targets were administered 24 hours post-op, killing in the inguinal node
markedly increased (24.00 4.56% post-op versus 6.13 0.35% pre-op, P = 0.0039) and
- 87 -
axillary node target lysis decreased (P = 0.0294). Thereafter, in vivo CTL function
declined gradually in all tissues, similar to the pattern seen in cross presentation (Figure
3.12C), indicating a close association between antigen and continued effector CTL
function.
Figure 3.9. HA-specific CD4+ proliferation before and after surgery.
HNT proliferation was assayed in BALB/c with established (day 16) AB1HA tumours, and two weeks
after surgery. Experiment was performed once, with five animals per group. A. Representative flow
cytometry showing HNT proliferation in the draining nodes of animal with day 16 tumour (left) but
absent two weeks after surgery (right). B. Single mice represented by points on the graph. Mean HNT
proliferation rates shown by bars. P values were two tailed and derived from the student‟s t test at the 5%
level of significance. Key: Pre-op = pre-operative (day 16 tumour), post-op = post-operative, 2w = two
weeks.
- 88 -
Figure 3.10. Flow cytometry for DC cell phenotyping.
Lymph nodes from animals after tumour resection or sham procedures were processed for analysis of
standard DC phenotypes.326,327
Compensation and thresholds were set using unstained, single stained, and
isotype controls. DCs were sub-gated from plots of CD11c and side scatter. The CD11c+ population was
then plotted for CD8 positivity (y-axis) and CD45RA positivity (x-axis). The experiment was performed
once, with five animals pooled from each group. Representative flow cytometry is shown from the
sentinel lymph node of a tumour bearing mouse. A: hamster PE isotype (left) and CD11c-PE mAb
staining (right). B: plasmacytoid (CD11c+CD45RA
+), CD8a-DC (CD11c
+CD8a
+CD45RA
-) and double
negative (CD11c+CD8a
-CD45RA
-) DC populations.
- 89 -
Figure 3.11. DC phenotype before and after surgery.
The proportions of DC that were CD8+, plasmacytoid, or double negative were quantified in the
draining and non-draining nodes. Assays taken pre-operatively and post-operatively. Data presented from
a single experiment, with pooled tissue from five animals in each group. A. DC phenotype in the draining
nodes. B. DC phenotype in the non-draining nodes. Key: pre-op = pre-operative, DOS = day of surgery,
2w = two weeks, post-op = post-operative.
- 90 -
Figure 3.12. Post-operative in vivo CTL
CFSE stained targets (CL4 peptide-pulsed and non-pulsed) were administered to BALB/c mice before
and after surgery. Representative flow cytometry and in vivo CTL readouts are shown for a single
experiment. Results are pooled from two separate experiments. At least 5 animals are present in each
group (range: 5 – 10). A. Gating of target and reference populations for in vivo CTL assay. B. Typical
profiles of in vivo CTL lysis from pre-injection, naïve, and CL4 mice. C. HA-specific in vivo CTL
function in lymphoreticular tissues before and after surgery. Mean SEM percentage killing of HA pulsed
targets shown for each tissue and timepoint, P values derived from student‟s t test at the 95% level of
significance. Key: (+) = pulsed, (-) = non-pulsed, DOS = day of surgery, 1w = 1 week, 2w = two weeks,
post-op = post-operative.
- 91 -
3 . 2 . 10 . Recurrent tumour and sys temic CTL responses
From 3.2.9, in vivo CTL responses correlated with antigen presentation. However, when
tumours emerge after surgery, they also present antigen (3.2.6). Hence they may impact
on existing effector and memory responses. To assess how the post-operative immune
system would function in the presence of metastatic tumour, CL4 peptide pulsed targets
were administered in the setting of post-operative tumour re-challenge. In vivo CTL was
measured for the following time points: day 16 of primary tumour growth, day of
surgery, and two weeks after surgery. CL4 pulsed and non-pulsed targets were
administered intravenously to all animals, four days after tumour re-challenge into the
healthy flank. As seen previously (Figure 3.12), in vivo CTL activity was predominantly
in the draining nodes before surgery. While there was no immediate change in draining
node CTL activity after surgery (P = 0.14), HA-specific killing did increase in the
draining nodes by two weeks post-op (P = 0.0011), Figure 3.13. The most striking
change occurred in the non-draining nodes and spleen, where minimal CTL was seen
pre-operatively (2 – 7%), but 12 – 19% killing was seen at two weeks post-operatively
(P ≤ 0.0162) - Figure 3.13.
3 .3 . Discussion
Tumour antigen presentation is the obligatory priming signal for a tumour-specific
immune response. Without sufficient antigen priming there can be no effector
differentiation, CD8+ proliferation, or memory development.
347,351 Therefore, the effects
of surgery on tumour antigen presentation were investigated using a non metastatic
tumour model20
bearing a membrane-bound model tumour neo antigen (HA) that is
constitutively cross presented.343
In the AB1HA model, HA presentation was previously found to be driven solely by
cross presentation in the tumour-draining lymph nodes.149,351
Consistent with these
previous findings, cross presentation of HA in this study was also found to be confined
to the draining lymph nodes, even after a more exhaustive examination of other
peripheral nodes (e.g. popliteal, brachial, para-aortic). The levels of HA cross
presentation were observed to be both constitutive and efficient, as previously
noted.149,351
Indeed, significant presentation was observed as early as four days post
implantation, when tumours were frequently too small to be visible. Extending on these
- 92 -
previous reports, the kinetics of tumour antigen presentation in vivo were found to be
directly proportional to tumour size, with no sign of plateau, even after 21 days of
tumour growth.
Figure 3.13. Primed in vivo CTL after surgery
In vivo CTL was assayed in animals four days after re-challenge, before and after surgery. Draining
nodes, non-draining nodes, and spleens were processed separately as shown. Experiment was performed
once, with a minimum of ten animals per group. Mean HA-specific lysis SEM shown for each group.
Cohorts compared by two sample student‟s t test at the 95% level of significance. *P = 0.0011, **P =
0.0079, ***P = 0.0162.
As hypothesised, complete surgery ablated antigen cross presentation. However,
intriguingly, tumour antigen presentation was not immediately ablated by surgery, but
persisted for at least a week post-operatively. This phenomenon was not attributable to
incomplete resection, as validated by the PCR, histology, and nude BALB/c data. The
decline in cross presentation was mirrored by changes in MHC II dependent antigen
presentation to CD4+ T cells. With only a single published abstract alluding to week-
- 93 -
long persistence of cross presentation after surgery, no substantive study had previously
defined this phenomenon.236
The finding that complete surgery did eventually ablate antigen presentation appeared at
odds with studies that suggest antigens are permanently presented through follicular
DCs.353,354
It has been postulated that permanent antigen presentation may be critical to
persistence of memory T355
and B356
cells. Indeed, “permanent” cross presentation has
been observed in the AB1HA model on one occasion. Marzo and colleagues found that
cross presentation could persist for 6 months despite the absence of visible tumour.20
Notably, this durable antigen presentation was in a rejection setting, rather than post-
operatively. The mechanism was also unclear, but the retained presentation may have
arisen from a permanent antigen reservoir on an accessory cell,20
or from tumour
dormancy. The latter concept refers to cancers that remain in small volumes, contained
by angiostatins,40
cell mediated immunity,357
or factors attributable to the tumour
microenvironment.40
Although it was not tested, dormant tumour might have been
detectable on PCR or immunohistochemistry in those experiments.40
As such, the
dormant tumour might have provided a continuous source of antigen.20
Numerous factors may have explained the loss of antigen presentation that was seen in
the data presented in this chapter. First, while permanent antigen presentation can occur
via follicular DCs,353,354
membrane bound antigens (like HA) are probably processed
through CD8+
- non-follicular DC.358
A second explanation could be attrition of the
DCs presenting HA in the draining lymph nodes. While the in vivo survival of different
DC subsets has yet to be fully elucidated,359
the half life of murine DC in general may
be in the order of 1.5 – 3 days.360
Accordingly, efforts were made to identify gross
changes in DC phenotype to correlate with the loss of antigen presentation. No change
in DC phenotype was detected after surgery, and so further efforts and time were not
devoted to that project. Other research in our laboratory361
suggests that CD11b+B7DC
+
cells may be important in processing tumour antigen. If those results are borne out in
further research, re-visiting the kinetics of DC phenotype (particularly the CD11bB7
DC population) after surgery might then be fruitful.
The final aspect of the work in this chapter is CD8+ lymphocyte function in vivo. With a
tumour in situ, in vivo CTL function is inextricably linked to antigen presentation, as
has been previously reported for the AB1HA model.155
An interesting pattern emerged
- 94 -
after surgery. Endogenous CTL function declined to background levels 14 days after
surgery, matching the decline in antigen presentation from the original tumour. When
there was recurrent disease after surgery, CTL function was “awakened” in the original
node bed and was also detectable systemically. This suggested that a form of effector
egress might occur after surgery, whereby primary resection releases trapped CD8+
from the draining nodes. Those effector cells may only be active in secondary nodes
when antigen is present, thus they are not visible on un-primed in vivo CTL assays.
If CD8+ effectors do egress from the draining nodes after surgery, it would be
consistent with the systemic appearance of memory CD4+ seen after surgery,48
and also
the egress of tumour-specific CD8+ with activating anti-CD40 mAb treatment.
155 One
concerning aspect was the pre-operative co-localisation of tumour antigen presentation
and tumour-specific in vivo CTL function. This suggested that node removal can
adversely affect the tumour-specific immune response, by removing the sites of priming
and CTL concentration. This hypothesis is tested in Chapter 5.
Also, the decline in cross presentation after surgery suggests patients with minimal
residual disease may become relatively depleted of antigen priming. Thus tumour
vaccines, which augment priming, may be of benefit in the eradication of minimal
residual disease. While the extent to which small recurrences and metastases engage the
immune system remained unclear, the efficacy of cross presentation (robust CL4
proliferation even when tumours were small) suggested local recurrences may interact
with the immune system at an early timepoint. Cross presentation of metastases was
also studied in this Chapter, the “metastasis” being created by inoculation of fresh
AB1HA cells into the healthy flank, rather being a true distant deposit from an
aggressive tumour. Metastases may be clonotypically and immunologically disparate
from the primary deposit, and it would be preferable to study cross presentation from an
orthotopic model that produces metastases ‟naturally‟. This issue is pursued in Chapter
6, where the orthotopic RencaHAM model is used.
3 .4 . Summary
In this Chapter, the kinetics of tumour antigen presentation before and after surgery
were described. This provides a useful principle in the planning of combined
surgery/immune therapy approaches. First, the localisation of tumour antigen to the
draining nodes suggested a place for locally delivered adjuvant therapy into the
- 95 -
resection bed, in the early post-operative phase. Second, patients with complete primary
resection and possible micrometastatic disease might benefit from additional immune
priming (e.g. tumour vaccination). Finally, given the efficiency of cross presentation,
patients who undergo debulking surgery for extensive disease probably have significant
reservoirs of tumour antigen. While they may benefit from strategies to improve the
quality of priming (e.g. DC preparations), it may be more appropriate to augment the
effector arm of the anti-tumour immune response (for example with adoptive immune
therapy).
In addition to the findings on cross presentation, surgery also had a profound impact on
endogenous tumour-specific CTL function. While in vivo CTL declined in parallel with
cross presentation after complete resection, recurrent tumours provided a fresh source of
antigen that evoked a powerful and systemic CTL response. This suggested surgery
enhanced CD8+
function overall, partially validating the practice of combining surgery
with immune therapy. With the effects of tumour resection on antigen presentation and
CTL function in mind, the impact of surgery on tumour resistance are now investigated
(Chapter 4).
- 96 -
Chapter 4
- 97 -
4. Sinecomitant immunity
4 .1 . Introduct ion
The effects of surgery on cross presentation and in vivo CTL function were examined in
the previous chapter. Surgery produced a gradual ablation of tumour antigen cross
presentation, but where antigen remained, surgery enhanced systemic CD8+
T cell
function. Based on those findings, it was hypothesised that surgery would enhance anti-
tumour immunity overall. To do so, tumour resistance after primary resection should
exceed the degree of tumour immunity when the primary remains in situ.
When primary tumours remain in situ, experimental animals show some degree of
resistance to re-challenge by the same tumour in a separate site. This phenomenon,
known as concomitant immunity, was first described by Ehrlich in 1906. Concomitant
immunity has previously been shown to be strong when the primary tumour is large362
and when the dosage of re-challenge is low.24
Concomitant immunity may arise from
immune mechanisms,363
but may also occur because of non-specific factors24
(e.g.
angiostatins released from the primary tumour).116,117
Concomitant immunity is
distinguishable from sinecomitant immunity, which is the tumour resistance seen in a
post-operative host. Numerous studies indicate that sinecomitant immunity is tumour-
specific rather than a non-specific (innate) phenomenon,24,25,106
but T cell dependency
has not been established. As with concomitant immunity, the extent of sinecomitant
immunity depends on numerous factors, including the antigenicity of the tumour, the
size of the primary tumour, the timing of re-challenge, and the strength of the re-
challenge inoculum.24,106,107
In this chapter, post-operative tumour resistance (sinecomitant immunity) is compared
to pre-operative tumour resistance (concomitant immunity) to establish the net effect of
tumour resection on tumour immunity in the AB1HA model. Extrapolating from the
data of Chapter 3, a number of immunological pre-requisites for post-operative tumour
resistance are identified, along with a several factors that can antagonise post-operative
tumour immunity. Finally, enhanced responses to immune therapy are seen in the post-
operative setting, reinforcing the hypothesis that surgery boosts tumour-specific
immunity.
- 98 -
4 .2 . Resul ts
4 . 2 . 1 . Concomitant immunity in the AB1HA m od el
To determine the degree of concomitant immunity in the AB1HA model, animals with
established AB1HA tumour (3.90 0.41 mm) were injected with a second inoculum of
AB1HA. Incidence of the secondary tumour was used in proxy of overall survival
(Figure 4.1B), because animals succumbed rapidly to the primary tumour. On average,
animals were culled 15.1 2.85 days after re-challenge (29.1 2.85 days from primary
AB1HA inoculation). Concomitant immunity was present, because a 3 – 6 day delay in
incidence of the secondary tumour was observed relative to naïve controls (P = 0.032,
Figure 4.1C). However, once tumours had emerged, their growth kinetics were
indistinguishable from naïve BALB/c (P = 0.401, Figure 4.1B). This was not surprising,
given that tumour-specific effector CTL responses to cross-presented tumour antigens
were weak and remained localized to the draining lymph node (3.2.9).
4 . 2 . 2 . Sinecomitant immunity in the AB1HA m odel
Post-operative improvements in CTL function (Chapter 3) suggest that surgery may be
beneficial to tumour immunity. To determine the overall effect of surgery on tumour
resistance, BALB/c mice were curatively resected of primary AB1HA tumour, and then
re-challenged with 1x106 cells of AB1HA into the opposite flank. Re-challenge inocula
were administered on the day of surgery, one week post-operatively, or two weeks after
surgery (Figure 4.2A). These time points were chosen because they corresponded,
respectively, to high antigen presentation from the original tumour, fading/low antigen
presentation from the original tumour, and absent cross presentation from the original
tumour.
Significant survival from rechallenge was seen in the healthy flank at all time points
after surgery (P < 0.0001 for equivalence to naïve controls, Figure 4.2B). Overall, cure
rates were 33 19.2% for the day of surgery group, 57.14 18.8% at one week after
surgery, and 66.67 12.2% for the two week post-operative cohort. In the animals
resistant to re-challenge, secondary tumours were neither visible nor palpable at any
time after inoculation.
- 99 -
Figure 4.1. Concomitant immunity in the AB1HA model.
BALB/c with established AB1HA tumours were challenged with 1x106 cells of AB1HA and compared to
naïve controls. All mice were monitored for tumour incidence and growth at the re-challenge site. Data
are pooled from two separate experiments. There is a minimum of ten animals per group. A. Timeline of
tumour inoculation and culls. B. Mean tumour diameter SEM shown for each timepoint after tumour
inoculation, *P = 0.401 (ANOVA). C. Tumour incidence in re-challenged animals and naïve controls,
**P = 0.0318 (Log Ranks).
4 . 2 . 3 . Sinecomitant immunity in the wounded f lank
Sinecomitant immunity was seen when animals were re-challenged into the healthy
flank post-operatively (4.2.2). However, tumours may recur locally after surgery. To
determine whether sinecomitant immunity occurred at the surgical site, BALB/c
underwent curative surgery and were then re-challenge into the wound site. Re-
- 100 -
challenges were administered on the day of surgery, one week after surgery, or two
weeks post-operatively (Figure 4.3A)
Significant resistance to re-challenge was observed in the surgical site (Figure 4.3B) for
all time points (P < 0.001). However, overall survival was <15% for all groups.
Moreover, there was no increase in tumour resistance when rechallenges were delayed
beyond the day of surgery (P > 0.90 for equivalent survival across cohorts).
Consequently, beyond one week post-operatively, a disparity in survival between the
surgical site and the healthy flank emerged (95% C.I. for H.R., 0.02 – 0.25).
Figure 4.2. Sinecomitant immunity in the AB1HA model.
Post-operative tumour resistance was assessed in BALB/c after surgery. Data are pooled from multiple
experiments, with a minimum of ten animals in each group. Kaplan Meier survival are shown for each
cohort. A. Timeline of surgery and rechallenge. B. Resistance to rechallenge over time. P values derived
from Log Ranks analysis.
4 . 2 . 4 . Sinecomitant immunity and re -chal lenge dose
Sinecomitant immunity depended on the location and timing of the re-challenge
(Sections 4.2.2, 4.2.3). To determine the relative strength of the sinecomitant response,
a log fold reduced re-challenge inoculum was used. Specifically, BALB/c underwent
- 101 -
curative resection, followed by rechallenge with 1x105 cells of AB1HA. Re-challenges
were administered into the surgical site, or the opposite (healthy) flank. AB1HA inocula
were given on the day of surgery, one week post-operatively, or two weeks after surgery
(Figure 4.4A). For all time points after surgery, a robust resistance to rechallenge was
seen in the healthy flank when an inoculum of 1x105 cells was used. Moreover, BALB/c
resisted tumour equivalently on the day of surgery and two weeks post-operatively (P =
0.10, Figure 4.4B) with an inoculum of 1x105 cells. When a log fold lower inoculum
was used into the surgical site, a robust resistance to rechallenge was observed. In fact,
survival from surgical site tumours approached equivalence to the healthy flank (95%
C.I. for H.R., 0.005 – 1.99), Figure 4.4C.
4 . 2 . 5 . Surgical t rauma and s inecomitant immunity
From Figure 4.3, sinecomitant immunity was significantly weaker in the surgical site.
To determine whether this was due to the effects of surgical wounding per se, two
different sham experiments were undertaken. In the first experiment, mice underwent
sham surgery alone in the left flank, followed by inoculations of 106 AB1HA cells into
either the wound, or into the opposite flank. Inoculations were administered on the day
of surgery. In this experiment, sham surgery had no effect on tumour growth rates
relative to naïve controls (Figure 4.5A). This was evident for both the site of the surgery
and for the opposite flank. Thus surgery had no effect on primary AB1HA growth, and
wound proximity was similarly unimportant to growth kinetics. Although wounding had
no impact on primary tumour growth, it did not exclude an effect on post-operative
tumour growth. To determine whether surgery caused a difference in tumour resistance
after surgery, a second sham experiment was undertaken (Figure 4.5B). In this case,
BALB/c underwent tumour resection as well as sham operations on the distant flank
(opposite the tumour site). Mice were then re-challenged with AB1HA at two weeks
after surgery, into the site of sham operation. Tumour resistance within the sham
wounded flank was still superior to tumour resistance within the original tumour site
(Figure 4.5C)
4 . 2 . 6 . HA in s inecomitant immunity to AB1HA
AB1HA has been transfected with the nominal neo-antigen, haemagglutinin (HA) of
PR8 influenza H1N1 (A/PR/8/34).148
However, it was not known whether HA was
required or redundant in the sinecomitant immune response. Indeed, there are presumed
to be many tumour neo-antigens in AB1 tumours independent of the model tumour neo-
- 102 -
antigen HA. To determine the relative importance of HA and non-HA antigens to
sinecomitant immunity, primary AB1HA was grown and resected in BALB/c mice.
Those animals were re-challenged into the healthy (non-surgical) flank at two weeks
after surgery, with AB1HA (as before) or AB1 (the parental line, lacking HA).
Alternatively, BALB/c underwent surgery for AB1 primary tumour, followed by re-
challenges with AB1, at two weeks post-operatively. Survival was equivalent in all
cohorts (P > 0.05, Figure 4.6) indicating that HA was redundant in the sinecomitant
immune response (i.e. a true, “spy” antigen). The minor role of HA in the immune
response accorded with the low in vivo CTL activity seen previously (Chapter 3), and
the low frequency of endogenous HA-specific effectors on pentamer staining (0).
However, the minor role for HA might also be observed if sinecomitant immunity was
an innate phenomenon, rather than an antigen dependent phenomenon (see next section,
4.2.7).
Figure 4.3. Sinecomitant immunity in the surgical site.
BALB/c underwent curative primary resection, followed by re-challenge into the surgical site on the day
of surgery, one week post-operatively, or two weeks after surgery. Data are pooled from multiple
experiments, with a minimum of ten animals present per group. A. Timeline of surgery and re-challenge.
B. Kaplan Meier survival shown for each cohort. P values derived from the Log Ranks test.
- 103 -
Figure 4.4. Sinecomitant immunity: significance of re-challenge dosage.
BALB/c underwent curative primary resection, followed by re-challenge with 1x105 AB1HA cells. Re-
challenges were administered into the surgical site or into the opposite (healthy flank). AB1HA was
administered on the day of surgery or two weeks post-operatively. Data are pooled from two experiments,
with a minimum of ten animals per group. A. Timeline of surgery and re-challenge. B. Kaplan Meier
survival from re-challenge into the surgical site. C. Kaplan Meier survival from re-challenge into the
healthy flank. P values derived from Log Ranks analysis.
4 . 2 . 7 . T cel l dependence of s inecomitant immunity
To determine whether sinecomitant immunity was antigen specific, congenic BALB/c
nu-/-
mice were used to assess the T cell dependency of post-operative tumour
resistance. Nude mice were curatively resected of primary AB1HA, and then re-
challenged at two weeks after surgery (when the sinecomitant response was usually
strong in wild type mice). There was no significant difference in survival between post-
- 104 -
operative BALB/c nu-/-
and naïve BALB/c nu-/-
controls (Figure 4.7), suggesting that the
sinecomitant immunity seen in wild type mice was T-cell dependent.
To further characterise the T cell dependency of sinecomitant immunity, YTS 169 mAb
and GK1.5 mAb were used to selectively deplete for CD8+ and CD4
+ cells respectively.
In each instance, (Figure 4.8A) depleting antibody was administered one day prior to
tumour re-challenge (14 days post-operatively) and continued for two weeks.
Depletions were verified by flow cytometry on whole blood, and rat IgG2a isotype was
given to a control group of animals. Depletions were successful (>95% CD4+ or CD8
+
depletion) in all animals treated with depleting mAb, and normal T cell profiles were
seen in the controls (data not shown).
Post-operative mice treated with CD8 depleting antibody had comparable survival to
naïve controls, indicating complete ablation of the sinecomitant immune response
(Figure 4.8B). In contrast, animals treated with isotype had strong capacity to resist
tumour re-challenge (60 20.91% cure), consistent with preserved sinecomitant
immunity. CD4+ depletion significantly reduced survival compared to isotype control
(Figure 4.8B), but this was less marked than depletion for CD8+ cells. Indeed, some
30.77 12.8% of BALB/c were able to resist tumour re-challenge in the absence of
CD4+ cells.
4 . 2 . 8 . Pers is tent tumour and s inecomitant immunity
Complete resection evoked sinecomitant immunity (Section 4.2.2 and Figure 4.2). To
determine if sinecomitant immunity also occurred when resection was incomplete,
sinecomitant immunity was tested in the setting of persistent primary AB1HA tumour.
For this experiment, tumour resistance at two weeks after surgery was tested in cohorts
of BALB/c with or without recurrent primary AB1HA tumours.
Given the progressive growth of the recurrent primary AB1HA tumours, the period of
follow-up for this experiment was short. One mouse of the “recurrent primary” AB1HA
cohort did resist rechallenge, but had to be euthanased at just 10 days of follow-up. This
animal had to be considered a “survivor” despite the short follow-up. Notably, the
failure of tumour to emerge by ten days did lie beyond the usual 7 day timepoint for
AB1HA emergence after surgery (95% C.I., 4.52 - 9.48), n = 18.
- 105 -
Figure 4.5. Effect of surgical wounding on sinecomitant immunity.
Both sham surgery experiments (A and B/C) were performed once, with a minimum of five animals per
group. A. BALB/c underwent sham surgery and re-challenge with AB1HA into the surgical site, or the
opposite flank. Mean tumour diameter SEM shown for each timepoint, P values derived from two way
ANOVA. B. Timing and location of re-challenge in post-operative sham surgery experiment. C. Kaplan
Meier survival of post-operative sham surgery experiment cohorts. P values derived from Log Ranks
analysis.
- 106 -
Figure 4.6. HA specific immunity does not dominate the sinecomitant response
BALB/c underwent surgery for AB1 or AB1HA primary tumours, followed by re-challenge at two weeks
after surgery. Re-challenge inoculums contained AB1 or AB1HA, as indicated above. Data are pooled
from two separate experiments, with a minimum of seven animals per group. Kaplan Meier survival
shown for each cohort, P values derived from Log Rank analysis.
Figure 4.7. Sinecomitant immunity in BALB/c nu-/-
Nude BALB/c underwent resection for AB1HA, followed by re-challenge with AB1HA at two weeks
after surgery. Tumour resistance was compared to naïve BALB/c nu-/-
controls. Kaplan Meier survival
Data are shown for a single experiment, with at least five animals per group. P value was derived from
the Log Ranks test.
- 107 -
Figure 4.8. The effect of T cell depletion on sinecomitant immunity
BALB/c mice were treated with CD4+ or CD8
+ depleting antibody, and re-challenged with AB1HA two
weeks after surgery. Reference cohorts were isotype treated and naïve controls. Data are shown from a
single experiment, with at least five animals per group. A. Time-line of surgery, re-challenge, mAb
treatment, and monitoring of depletion. B. Kaplan Meier survival for each cohort. P values derived from
Log Ranks analysis.
4 . 2 . 9 . Pers is tent ant igen and s inecomitant immunity
Incomplete resection ablated sinecomitant immunity (4.2.8). This could be due to the
persistence of non-specific tumour associated factors (e.g. TGF, VEGF). Alternatively,
incompletely resected tumours might cause antigen persistence (Chapter 3), paralysing
the tumour-specific response.287,292
To dissect the relative importance of tumour factors
and tumour antigens in antagonising sinecomitant immunity, tumour resistance was
tested when there was persistent tumour and/or persistent tumour antigens after surgery.
- 108 -
Firstly, sinecomitant immunity was measured in the presence of persistent HA antigen,
without tumour. HA could be provided as purified peptide, PR8 virus, or RencaHAM
(see Chapter 6). The third option was chosen, as purified protein was unavailable and
PR8 virus contains viral-associated immunostimulatory agents (e.g. viral RNA) that
could confound results. Accordingly, BALB/c mice underwent primary surgery for
AB1HA, followed by re-challenge into the surgical site with RencaHAM. In this
setting, there was no persisting tumour in the surgical bed, but HA antigen persisted
post-operatively (Chapter 6). Tumour resistance to AB1HA was impaired in the
presence of RencaHAM, indicating that persistent tumour antigen alone (even without
persistent tumour) was sufficient to antagonise the sinecomitant immune response.
To investigate whether other mesothelioma associated antigens could similarly
antagonise the sinecomitant response (even without persisting tumour), BALB/c were
curatively resected for AB1HA and inoculated with AB1 into the surgical site, before
re-challenge with AB1HA. AB1 failed to grow in any of the animals and in doing so,
presumably, it provided a source of AB1 antigen that persisted until tumour re-
challenge (similar to RencaHAM). While this could not be verified, because there are
no other “spy antigens” in the AB1 model, reduced sinecomitant immunity was
observed. Sinecomitant immunity was also assessed in the presence of persisting
AB1HA tumour. As such, persistent AB1HA tumour should present the full suite of
AB1HA tumour antigens and tumour associated suppressive factors. In that situation,
sinecomitant immunity was nearly ablated. That impairment in sinecomitant immunity
was attributable (at least in part) to the persistence of antigens rather than other tumour
associated factors, because if a highly aggressive364
but antigenically irrelevant murine
malignancy persisted in the surgical site (RencaWT) there was no impairment of the
sinecomitant immune response.
4 . 2 . 10 . Distr ibut ion of HA speci f ic ef fectors p os t -op
In Figure 4.10 and Figure 4.8, sinecomitant immunity was antigen sensitive and CD8+
dependent. Moreover, in vivo CTL function co-located with antigen presentation before
surgery (Figure 3.12) but became systemic post-operatively (Figure 3.13). This
suggested effector cells were present in the draining nodes pre-operatively, but may
have egressed systemically after surgery. In turn, this phenomenon may have
contributed to the delayed sinecomitant response of the healthy flank.
- 109 -
Endogenous HA-specific effectors should form part of the sinecomitant immune
response to AB1HA, and thus the kinetics of HA-specific CD8+ should be
representative of the tumour-specific repertoire. HA specific CD8+ were examined in
the draining (axillary), non draining (contralateral axillary) and spleens from normal
animals, CL4 mice, tumour bearing BALB/c, and postoperative animals (day of surgery,
one week post-op, two weeks post-op). Nodes were examined by flow cytometry, using
fluorescently labelled IYSTVASSL-MHC pentameric complexes (Pro5® MHC
Pentamer, ProImmune) and CD8a antibody (Figure 4.11, A). Due to the high cost of
pentamer reagent, this experiment was performed only once, with four animals present
in each group. CD8+Pentamer
+ cells were quantified as a proportion of lymph node
CD8+ cells in each tissue, and compared between groups as continuous data. Consistent
with the CTL data shown previously (Chapter 3), HA-specific CD8+ cells were
disproportionately represented in the draining node relative to the non draining node
(0.66 0.17% versus 0.12 0.01%). As previously published,155
the relative numbers of
HA-specific effectors in the AB1HA system was low. Despite the small numbers of
HA-specific CD8+, a decline in Pentamer
+ cells was still detectable in the draining
nodes within 24 hours after surgery (“day of surgery” timepoint), 0.65 0.17% to 0.34
0.09% (P = 0.0026). However, no corresponding increase in HA-specific effectors in
the non draining node or spleen (P >0.88) was detected.
Figure 4.9. Incomplete surgery did not protect against new tumour challenges.
Sinecomitant immunity was assessed in mice after curative and non-curative resection for AB1HA. Data
are shown from a single experiment, with at least 5 animals per group. Kaplan Meier tumour incidence
shown for all cohorts. Continued primary AB1HA growth necessitated the truncated follow-up of the
“persisting primary AB1HA” group. *P = 0.044, **P = 0.029 (Log Ranks).
- 110 -
Figure 4.10. Tumour antigen persistence partially ablated sinecomitant immunity
A. AB1HA tumours were grown and then resected, for each cohort to this experiment. On the day of
surgery, animals were re-challenged into the surgical site with AB1 (parental to AB1HA, lacking the HA
antigen – A), RencaHAM (shares no known antigens with AB1HA except for HA – B), AB1HA (C),
RencaWT (shares no known antigens with AB1HA but is a highly aggressive tumour – D), or saline (E).
Each cohort was rechallenged with 1x106
AB1HA tumour cells into the healthy (non-surgical) flank at
two weeks post AB1HA resection. Experiment was performed once, with five animals present in each
group. B. Kaplan Meier survival shown for each cohort. P values derived from Log Ranks.
4 . 2 . 11 . Suppress ion a nd s inecomitant immunity
Surgery enhanced tumour-specific immunity, but tumour resistance was relatively
impaired on the day of surgery and at the original tumour site. Surgical wounding and
tumour antigen kinetics may affect the topography and timing of tumour-specific
immunity (see 4.2.5). However, cancers have also been shown to hamper tumour-
specific immunity by numerous other mechanisms, including Treg366
and MSCs.27
To determine whether post-operative tumour immunity correlated with the
accumulation and distribution of Treg, regulatory T cells were quantified (as a proportion
of lymph node CD4+ cells) in naïve, tumour bearing, and post-operative animals (Figure
4.15). Treg were compared between the surgical flank (lastingly vulnerable to tumour)
- 111 -
and the healthy flank (resists tumour robustly at two weeks after surgery). There was no
significant accumulation of Treg in the nodes with mice bearing AB1HA tumour, the
percentages of CD4+CD25
HiFoxp3
+ did not change after surgery, and Treg were not
over-represented in the region of the original tumour relative to the healthy flank
(Figure 4.16). While Treg did not accumulate in the draining nodes, this cell type might
still hamper post-operative tumour immunity. To determine the impact of Treg on
sinecomitant immunity, PC61 mAb (Monoclonal Antibody Facility) Treg depletions
were performed. Depletions were undertaken when resistance to re-challenge was
weakest (on the day of surgery, with re-challenge into the surgical site).335
Depletions
were verified on whole blood and lymph node biopsy (Figure 4.18A).
While CD25+ cells were reduced with anti-CD25 mAb, the FoxP3
HICD4
HI population
were not significantly depleted (Figure 4.17). This was consistent with a recent
publication reporting that Treg are not removed by PC61,336
but rather CD25 expression
is down-regulated. Nevertheless, PC61-induced downregulation of Il-2R does impair
Treg function.336
Thus PC61 treatment would still reduce Treg function.
CD25 expression was ablated by PC61 at 24 hours after treatment (Figure 4.17A). At 7
days after PC61 mAb injection, CD25 ablation was maintained in 4 out of 5 animals
(Figure 4.18B,C). At that time point, tumour was emerging in the animal that was
beginning to express CD25 (tumour diameter 1.41mm). A single treatment of PC61
mAb delayed the onset of tumours for approximately seven days, significantly
improving survival relative to saline controls (Figure 4.19).
Thus Treg depletion boosted the sinecomitant immune response but this was not
complete. Another factor was additionally investigated, specifically, the relationship
between MSC and sinecomitant immunity. Spleens were collected from BALB/c in the
absence of tumour, with established (day 16 tumour), on the day of surgery, and two
weeks post-operatively.
Splenic MSC were identified by flow cytometry and the characteristic markers of
CD11b and Gr-1.246
As it is unknown which cell type of the MSC population is
responsible for immune suppression,246
the group was quantified as a whole.
Quantification was derived by dividing the number of Gr-1+CD11b
+ cells by the number
of viable, non red cell splenocytes collected over the same period - Figure 4.20A.281
- 112 -
No statistically significant increase in splenic MSC occurred with tumour growth (4.96
0.48% versus 3.87 0.41% seen in naïve animals), P = 0.097 - Figure 4.20B. There
appeared to be a small decrease in splenic MSC from tumour bearing animals relative to
the two weeks post-op group (4.96 0.48% versus 3.59 0.34%), but this was not
statistically significant (P = 0.0728).
Figure 4.11. Distribution of HA-specific CD8+ T cells.
HA-specific CD8+ cells were identified using IYSTVASSL-MHC pentameric complexes (Pro5
® MHC
Pentamer, ProImmune). Due to the high cost of pentamer reagent and the quantities required to detect
HA-specific CD8+ cells, this experiment size had to be restricted. Of the tumour draining nodes, the
axillary nodes were chosen because they contained higher counts of pentamer positive cells, compared to
inguinal nodes. Only four animals were present in each group, and the experiment was performed once.
A. Representative flow cytometry from CL4 nodes, axillary nodes, non-draining nodes, and spleen. B.
Mean % CD8+ cells expressing IYSTVASSL-specific TCR for each cohort, + SEM for each mean value.
*P < 0.05 (student‟s t test).
- 113 -
Figure 4.12. CD127 and CD44 analysis of CD8+Pentamer
+ cells
Representative plots of CD127 and CD44 on pentamer+ and pentamer
- cell populations. A. isotype
staining for CD44 and CD127 mAb. B. Representative flow cytometry for CD44 and CD127 expression
of Pentamer+ and Pentamer
- populations.
- 114 -
Figure 4.13. Expression of CD44+ in Pentamer+ and Pentamer
- CD8
+
Pentamer+ and Pentamer
- CD8
+ cells were assessed for expression of CD44. Mean values +SEM shown
for each cohort. Experiment was performed once, with four animals per group. P values were two tailed
at the 5% level of significance, derived from the student‟s t test.
- 115 -
Figure 4.14. Expression of CD127 in CD8+CD44
+ populations
CD8+CD44
+Pentamer
+ and CD8
+CD44
+Pentamer
- cells were assessed for expression of CD127. Mean
values +SEM shown for each cohort. Experiment was performed once, with four animals per group. P
values were two tailed at the 5% level of significance, derived from the student‟s t test.
- 116 -
Figure 4.15. Representative flow cytometry for Treg quantification.
Treg were quantified in the draining and non-draining lymph nodes of tumour bearing BALB/c, normal
(naïve mice), and post-operative animals. A, B. CD4+ cells were sub-gated from the lymphocyte
population on the forward scatter and side scatter plots. C. CD4+ lymphocytes were plotted for Foxp3
positivity (y-axis) against CD25 positivity (x-axis). CD4+CD25
HIFoxp3
+ cells were selected as the natural
Treg population and quantified as a proportion of CD4+ cells in each tissue. Percentages of
CD4+CD25
HIFoxp3
+ cells were compared between subject groups, as continuous variables.
- 117 -
Figure 4.16. Treg frequency pre- and post-operatively
CD4+CD25
HiFoxp3
+ cells were quantified proportionately in the draining and non-draining nodes, before
and after surgery. Data are pooled from two separate experiments, with a minimum of four animals per
cohort (range: 4 – 8). Individual mouse lymph nodes shown. Mean %CD4+CD25
HiFoxp3
+ indicated by
bars. All groups compared using the student‟s t test, and no statistically significant difference was found
(two tailed P > 0.05).
- 118 -
Figure 4.17. Treg remained despite PC61 mAb.
The presence of CD4+Foxp3
+ cells was assessed in PC61 and saline treated animals, for the Treg depletion
experiment. Each experiment was performed once, with five animals per group. Flow cytometry was
performed on three separate occasions. Representative flow cytometry shown from the 24 hours post-
treatment timepoint. A,B. Whole blood CD25 and Foxp3 expression for saline and PC61 treated animals.
C. Mean %CD4+Foxp3
+ cells +SEM shown for depleted BALB/c and controls. P value derived from the
student‟s t test.
- 119 -
Figure 4.18. Correlation of Treg depletion with tumour emergence
BALB/c were curatively resected of AB1HA, and then re-challenged into the surgical site on the day of
surgery. Concurrently, these mice were depleted for CD25 using PC61 mAb, and followed for emergence
of the locally recurrent tumours. A. Timeline of surgery, PC61 mAb depletion, and monitoring for Treg
depletion. B. Flow cytometry on lymph node biopsy specimens, one week after PC61 mAb/saline
treatment. Depleted animals shown in the top panel, saline controls in the bottom panel. Mouse with
recovering CD25 expression (highlighted) had emerging locally recurrent tumour at the time of biopsy. C.
CD25 expression levels at the time of biopsy (data from B). P value derived from the student‟s t test.
- 120 -
Figure 4.19. Effect of Treg depletion on local recurrence.
BALB/c underwent Treg depletion and re-challenge into the site of AB1HA resection, as for Figure 4.18.
Experiment was performed once, with five animals per group. Kaplan Meier tumour-free survival for
PC61 and saline treated BALB/c. P value derived from Log Ranks analysis.
4 . 2 . 12 . Sinecomitant immunity and immune therapy
If surgery boosted the anti-tumour immune response (4.2.2), immune therapies could be
more effective in treating post-operative recurrence than primary disease. To determine
whether immune therapy was more effective for post-operative recurrence than primary
disease, BALB/c with primary AB1HA or post-operative recurrent AB1HA were
treated with immune therapy. Post-operative recurrent AB1HA was generated by re-
challenge with fresh tumour cells in the healthy flank, on the day of primary AB1HA
resection. In all animals, treatment was initiated when tumours were first palpable
(1mm in diameter). Antigen-based therapy (tumour vaccines) were avoided, given the
apparent importance of antigen decline (4.2.9) to the sinecomitant immune response.
Response to therapy was assessed by the outgrowth of treated tumours and overall
survival. Therapies were chosen for which dosage regimens were previously optimised.
The intra-tumoural route of administration was chosen because it theoretically offered
the highest drug concentration within the tumour, and a lower systemic toxicity.367
Specific treatments were murine activating anti-CD40 mAb (FGK45, Monoclonal
Antibody Facility), CpG-ODN 1668 (Tib-Molbiol), poly I:C (InvivoGen), and 3M019
(3M Pharmaceuticals).
- 121 -
Figure 4.20. MSC in tumour bearing and post-operative mice.
MSC were quantified proportionate to non-erythrocyte splenocytes in BALB/c spleens before and after
surgery. Data are shown for single experiment. A. Representative flow cytometry from naïve, tumour
bearing and post-operative animals (day of surgery and two weeks post-operative). B. Percentages of
CD11b+Gr-1
+splenocytes shown for each cohort. Individual mice shown, along with mean values (bar).
No statistically significant difference was observed between the groups (student‟s t test).
- 122 -
CpG 1668 was equally able to cure all animals with either primary AB1HA or post-
operative AB1HA disease (data not shown). Each of the remaining therapies (FGK45,
3M019, poly I:C) improved survival in the setting of primary AB1HA disease (P < 0.05
relative to saline controls), but cure rates ranged from just 8.33 7.98% (FGK45) to 40.00
21.90% (poly I:C) - Figure 4.21 and Figure 4.22. For each of those treatments, survival
was more remarkable in the post-operative setting (71.43 12.08% to 100.00%) and
significantly improved relative to primary disease (P < 0.05) - Figure 4.21A,B and
Figure 4.22A,B. Cure rates were less impressive for tumours sited in the surgical flank
relative to the healthy flank, but still superior to treatment for primary disease (data
shown for FGK45 - Figure 4.22A,B).
4 .3 . Discussion
Surgery has been much maligned as an immune suppressive treatment.31-33,95,96
However, if surgery is immune suppressive, this seems at odds with a number of key
clinical and empirical findings. First, patients have residual disease more frequently
than predicted from post-operative recurrence and metastasis rates. Indeed, modern
techniques of PCR and flow cytometry indicate that a significant proportion of patients
with clinically localised epithelial malignancy have circulating tumour cells both before
and after surgery. 35,37,38,40
Thus dissemination of tumour cells might occur when as few
as 106 cells are present, several log scales before the earliest tumours are detected.
40 In
breast cancer for instance, at least a subset of such patients never present with
metastasis or recurrence after surgery.40
Such a phenomenon would seem unlikely if
surgery suppressed immune function.
Second, it has been shown that immune therapies are more effective in combination
with surgery than without surgery in renal cancer.12,14,15,112
Once again, if surgery
depressed anti-tumour immunity rather than boosted it, such a finding would be
improbable. Finally, recent empirical publications suggest that surgery can re-set several
important parameters of the anti-tumour immune response, with beneficial effects on
immune suppressive cytokine levels,140
MSC networks,46,285
CD4+ memory phenotype
and trafficking,48
and general cell mediated immunity.285
- 123 -
Figure 4.21. Response to immune therapy after surgery.
BALB/c with primary or post-operative AB1HA tumours were treated with poly I:C, 3M019, or saline.
Post-operative tumours were sited in the healthy flank. All tumours were treated intra-tumourally, on
emergence (tumour diameter 1 mm). Kaplan Meier survival shown for each cohort, P values derived from
the Log Ranks test. Data are shown for a single experiment, with at least five animals present per group.
A. Poly I:C treated mice received a dosage of 10 g every day, for a maximum of six treatments. B. Mice
undergoing 3M019 therapy received 50 g every second day, for a maximum of six treatments.
- 124 -
Figure 4.22. Response to immune therapy in the healthy flank and surgical site.
BALB/c with primary or post-operative AB1HA tumours were treated with FGK45 or saline. Post-
operative tumours were sited in the healthy flank (A) or the surgical site (B). All tumours were treated
intra-tumourally, on emergence (tumour diameter 1 mm). Mice received a dosage of 40 g, up to every
second day, for a maximum of six treatments. Data are pooled from three separate experiments, with a
minimum of 5 animals per group. Kaplan Meier survival shown for each cohort, P values derived from
the Log Ranks test. A. FGK45 versus saline for the treatment of primary AB1HA tumours, or post-
operative tumours in the healthy flank. B. FGK45 versus saline for the treatment of primary AB1HA
tumours, or post-operative tumours in the healthy flank .
- 125 -
In this chapter, tumour resistance was compared between post-operative animals and
mice with untreated primary AB1HA. Animals bearing primary AB1HA did display
somewhat reduced growth of a second AB1HA challenge (concomitant immunity),23,24
but this was a weak phenomenon. In contrast, animals robustly resisted tumour
rechallenge after surgery. That phenomenon, previously dubbed “operation immunity”
or “sinecomitant immunity”,25
would indicate that surgery could boost tumour
immunity, providing a useful platform for post-operative immune therapy. It might also
explain why immune therapy has been more successful after surgery than
preoperatively, as discussed above.
Sinecomitant immunity was never 100%, suggesting that sinecomitant immunity was
variable across individual mice. Numerous factors have been reported to contribute to
such variation, including: tumour size at surgery, duration of primary tumour growth,
and dosage of inoculum.25
Additional factors that may have been operant included:
blood loss, procedure times, pain, anaesthesia dosages, and so on.25,106
To reduce bias,
every effort was made to ensure comparison groups were matched for tumour size, time
of tumour growth, and mouse age. Other variables were more difficult to control but
assumed to be comparable, given that a single surgeon (the author) administered all
anaesthesia and performed all procedures.
A number of factors seemed to affect the strength of the sinecomitant immune response.
In particular and in accordance with the preceding literature, sinecomitant immunity
was affected by the dosage and timing of the re-challenge.110,111
However, sinecomitant
immunity was also affected by the location of the re-challenge. This had not been seen
previously, possibly because preceding publications have used smaller dosages for re-
challenge (105 cells or less).
106-108 As seen in this chapter, dosages of 10
5 could be
resisted reliably irrespective of site of injection. It was not until dosages of 106 cells
were used that the difference in post-operative tumour resistance between the surgical
site and a distant site was observed.
Having established the extent of sinecomitant immunity in the AB1HA model,
numerous immunological correlates of successful and unsuccessful sinecomitant
immunity were sought. Looking at the healthy flank, successful resistance to
rechallenge occurred only at two weeks after surgery. This pointed to a remarkable
parallel between the wane of tumour antigen cross presentation (seen in Chapter 2), and
- 126 -
the emergence of sinecomitant immunity. Indeed, the reduction in tumour antigen, or
“antigen holiday”, seemed critical to sinecomitant immunity, since antigen persistence,
even in the absence of tumour persistence (e.g. rechallenge with AB1 or RencaHA),
could abrogate sinecomitant immunity. Moreover, sinecomitant immunity against
AB1HA was preserved when aggressive364
but antigenically irrelevant tumour
(RencaWT) persisted in the surgical site.
The inverse relationship between sinecomitant immunity and tumour persistence and/or
tumour antigen persistence had been noted twice previously. Gorelick and colleagues
found that primary resection had to be complete for sinecomitant immunity to
develop,24
suggesting that the removal of tumour suppressive factors and/or removal of
tumour antigen en masse could be important to sinecomitant immunity. Further, using
the methylcholanthrene induced fibrosarcoma model (C3H/HeJ inbred mice), Kahan
and colleagues described a series of surgical experiments in which tumour antigen
vaccines were given in the first two weeks after surgery.106
Under these conditions, the
usual resistance to re-challenge was impaired. This led them to conclude that
sinecomitant immunity and (tumour) antigen therapy are “mutually antagonistic”,106
as
was found to be the case in this Chapter.
Given the significance of tumour antigen in sinecomitant immunity, it was not
surprising that T cells were required: congenic BALB/c nu-/-
did not display post-
operative tumour resistance. Depletion studies using YTS169 confirmed this was the
case, with ablation of sinecomitant immunity occurring when CD8+ cells were absent.
Thus CD8+ function seemed integral to the sinecomitant immune response, as with the
anti-tumour immune response more generally.344-346
CD4+ cells were not essential to the sinecomitant response, at least when they were
depleted after surgery. However, CD4+ cells may be more important in the “APC
licensing” phase of the anti-tumour immune response, when effectors are being primed
for the first time.224,225,227,229
Thus significant reductions in sinecomitant immunity may
ultimately have been observed if CD4+ cells were depleted during tumour growth (the
initial priming and licensing phases). Additionally, Treg express CD4 and these were
probably depleted along with helper T cells when GK1.5 CD4+ depleting antibody was
used.363,368
Since Treg depletion enhanced tumour immunity (4.2.11), a reduction in Treg
- 127 -
may have offset any detrimental effects of reduced helper T cells during CD4+
depletion.
A disparity in tumour resistance was seen between the early post-operative phase and
the late post-operative phase, as well as the healthy flank and the surgical site. One
potential explanation was some immune suppressive effect from surgical trauma, and/or
the inherent vulnerability of surgical wounds to tumour growth.96,369,370
To determine
whether surgical wounding contributed to the topography and timing of sinecomitant
immunity, sham experiments were performed. Sham surgery did not accelerate tumour
growth, and only partially impaired the robust tumour immunity of the healthy flank.
Thus surgical wounding was an improbable factor in the distribution and time
dependency of sinecomitant immunity; other mechanisms were more likely to be
operant.
One such potential mechanism was the selective accumulation of Treg in the draining
lymph nodes of tumour bearing mice. CD4+CD25
++Foxp3
+ could be identified in the
lymph nodes of normal animals, and these accorded with previously published values
for both mouse371,372
and man.372,373
Treg did not accumulate in the draining nodes nor
did they decline significantly after surgery. This was consistent with a recent study by
Needham and colleagues in the AE17 C57BL/6J mesothelioma model, who found that
CD4+CD25
+ cells do not accumulate in the nodes, but rather within the tumour itself.
259
The fact that Treg did not decline after surgery suggested they may be impeding post-
operative tumour immunity. As sinecomitant immunity was most impaired in the site of
the surgery and on the day of surgery, the role of Treg was investigated in that setting.
Accordingly, Treg depletion with PC61 could ameliorate tumour vulnerability in the
operated flank. Not only could local recurrence be prevented during the phase of Treg
depletion, but the return of Treg correlated with the emergence of local recurrence. Thus
Treg may be a critical impediment to sinecomitant immunity, and may be a major factor
in the vulnerability of resection beds to local recurrence.
Enhanced sinecomitant immunity after PC61 depletion suggested that Treg were indeed
dampening sinecomitant immunity. Treg have been previously found to limit both
concomitant immunity363
and sinecomitant immunity.374
Combining Treg depletion with
surgery might therefore have considerable benefit374
and numerous treatment strategies
- 128 -
may be used to boost sinecomitant immunity, including: anti-CTLA4 antibody,375,376
cyclophosphamide (a conventional chemotherapy that reduces CD4+CD25+ cells),377
intra-tumoural Treg depleting antibody,259
denileukin diftitox (Ontak®, Ligand
Pharmaceuticals, San Diego, California USA),372,378,379
cyclosporine/tacrolimus
(transplantation drugs that suppress Il-2 production and signalling and could abrogate
Treg function)372
and anti-glucocorticoid induced tumour necrosis factor receptor related
protein antibody (anti-GITR mAb).372,380
As well as identifying Treg impediments to sinecomitant immunity, the candidate
attempted to investigate the relationship between sinecomitant immunity and MSCs.
MSCs, also known as “inhibitory macrophages” 250,251
and “early myeloid cells”249,250
may accumulate in the lymphoreticular system253
and in tumours of cancer-bearing
mice.27
Those MSCs may deplete arginine in the tumour microenvironment, impairing
CD8+ proliferation and maintenance of the CD3 chain.
256 Moreover, MSC produce
peroxynitrites, which induce CD8+ apoptosis
246 and tumour nutritive polyamines
(through the L-ornithine pathway).282
MSC may also directly produce T cell tolerance,
at least to soluble tumour antigens, by uptake and presentation to T cells in a tolerogenic
manner.254
In this chapter, MSCs accounted for 1.98 0.41% of splenocytes in healthy mice,
consistent with previously published values.381
MSCs appeared to accumulate in the
spleens of tumour bearing animals, but the increase was minimal over naïve mice, and it
was not statistically significant. The increase in MSCs for this system was less than
previously reported in other BALB/c mesothelioma models250
where around 28.5% of
splenocytes had Gr-1 and CD11b positivity during tumour growth.
A number of explanations may underpin that difference, including mouse strain
(C57Bl6 versus BALB/c), tumour model (AB1HA versus TC-1), and technical issues
(antibody choice, isotypes, compensation). However, the most important factor was
possibly tumour size (Albeda and colleagues used tumours 6 – 8 times larger than
studied herein).250
As MSCs are known to accumulate more or less in parallel with
tumour burden,253
a lesser MSC accumulation would be predicted with the smaller
tumour sizes used in this chapter.
- 129 -
Reductions in MSCs after surgery have been reported in previous
publications.46,281,285,382
In this Chapter, no statistically significant decline in MSC levels
was present. If larger AB1HA tumours were grown and/or a large series was
undertaken, a post-operative decline in MSCs might be validated to statistical
significance.
Given the low accumulation of MSCs, it would seem doubtful they were of significance
in impairing sinecomitant immunity. To assess this more rigorously, additional
depletions of MSCs might be fruitful. For example, one could compare sinecomitant
immunity in MSC depleted and non-depleted animals, to determine whether there was a
difference. Techniques that could be used include: all-trans retinoic acid (which induces
MSC differentiation) 383
and gemcitabine (which reduce splenic MSCs).250
In Chapter 3, it was found that systemic CTL activity increased after surgery, and this
correlated with a decline in cross presentation within the sentinel nodes. It was therefore
hypothesised that surgery induced a systemic egress of HA specific CD8+, and that
those cells would have a central memory phenotype; similar to recently published data
for the CD4+ compartment.
48 The robust antigen dependent proliferation of Chapter 3
suggests it would be easy to track the endogenous HA specific CD8+ repertoire in the
AB1HA model. Unfortunately, as has been published previously for this model, only a
few HA specific CD8+ are visible on HA epitope–MHC-fluorochrome flow
cytometry.155
Even with high grade reagent (IYSTVASSL Pro5® MHC Pentamer,
ProImmune), detection of HA specific effectors was extremely difficult.
Nevertheless, it was shown that tumour-specific CD8+, like in vivo CTL function, were
disproportionately represented in the draining lymph nodes of the tumour. After
surgery, there was a reduction in those tumour-specific CD8+ in the regional nodes, as
was recently reported for the CD4+ compartment.
48 This decline in CD8
+ cells was
consistent with an egress of CD8+ from the draining lymph node to the systemic lymph
nodes and spleen. However, again similar to the findings of Benigni and colleagues,48
there was no corresponding increase in CD8+ cells within systemic nodes to correspond
to the reduction in draining node CD8+ cells. Unfortunately, due to the small cell
numbers involved, flow cytometry was unlikely to detect a difference. While apoptosis
would be an alternative explanation for the reduction in tumour-specific CD8+ in the
- 130 -
sentinel node, more sensitive techniques (e.g. quantitative PCR) might subsequently
support or refute an egress hypothesis.
An attempt was made to determine the kinetics of memory phenotype markers in
tumour-specific effectors after resection. CD44 was used to identify the proportion of
CD8+ lymphocytes that were memory cells. A second memory marker, Il-7 receptor
(C127), was also used. Expression of Il-7R receptor (CD127) is thought to indicate a
true “central memory” cell that is capable of homeostatic proliferation, via Il-7 and the
STAT-5/Bcl-2 mechanism of anti-apoptosis.365
Notably, studies of viral infection have
suggested that with antigen persistence, Il-7R is downregulated and that such cells
display an anergic memory profile.365
In contrast, when infection can be controlled, Il-
7R is upregulated, homeostatic proliferation occurs, and cells are again capable of
producing IFN.365
The persistence of tumour antigen during tumour growth was thought to parallel chronic
viral infection, where antigen burden is high and CD8+ memory cells have an anergic
phenotype (including low Il-7R expression). After surgery, antigen loads are reduced
(Section 3.2.4) and as with acute viral infection, cells might then upregulate Il-7R
expression and the capacity to produce IFN may be recovered.
In this chapter, surgery did not upregulate CD127 expression in the tumour-specific
memory pool. However, assessment of Il-7R expression was extremely difficult in this
system. The problem of low HA specific pentamer+ cell counts was compounded by
high background rat IgG2aK-APC isotype binding and technical difficulties with four
colour FACS compensation. No rigorous Il-7R specific signal was possible to analyse,
and the results obtained (no change in Il-7R) were considered tentative, at best.
Alternative experiments may be helpful to address the effects of surgery on CD127
expression. One method is to adoptively transfer naïve tumour-specific CD8+ cells from
congenic mice, prior to tumour growth. This approach has been used extensively by
other researchers in tumour or viral models298,299,307
and effectively would amplify the
number of tumour-specific CD8+ cells available for antigen encounter and memory
differentiation. This approach was not used by the author, because the required murine
strains were unavailable and because adoptive transfer of CD8+ could fundamentally
modify the dynamics of the immune response. Specifically, the ratio of CD8+ and CD4
+
- 131 -
cells to the APC could be disrupted, artificially imprinting patterns of memory
development307
that would not otherwise be seen in the endogenous repertoire.
Applying the concepts of this chapter, the finding that surgery enhanced tumour-
specific immunity was encouraging. It was postulated that surgery may induce a decline
in tumour related suppressive factors, including tumour antigen itself. While the role of
immune therapy after surgical resection had yet to be proven,27
the early post-operative
period after surgery was thought to present a “window of opportunity” for effective
immune therapy. In the clinical setting, patients may be optimally responsive to immune
therapy within the early and intermediate post-operative phases: tumour associated
suppressive factors are at their lowest,27
and tumour antigen presentation is declining.
Beyond that period, the growth of locally recurrent tumour or metastases may again
suppress the immune system.27
To delay treatment beyond the early post-operative
phase may therefore miss the optimum time for immunotherapy.384,385
It is not clear how long the “window of opportunity” remains open. In the murine model
of mesothelioma, tumours emerged on 8.14 0.33 days after surgery, i.e. two days
longer than naïve controls. This suggested the immune system of post-operative animals
was competent for 2 more days than a naïve mouse. In humans, the window of
opportunity may be open for longer. Indeed, patients may hold residual cancer cells in
check (tumour dormancy) for years or even decades after surgery.3,26,40,357,386
That
period of immune competence probably varies from patient to patient. Differences may
relate to numerous (tumour histology, disease burden, sites affected, extent of
cytoreduction) and host factors e.g. co-morbidities. With renal cancer, the average
period of immune competence may be several years on average: most patients who
present with recurrence do so within 3 – 5 years post resection.387
For mesothelioma,
the window of opportunity may be shorter – most patients have presented with recurrent
disease by 2 years after surgery.388
In this chapter, the window of opportunity was explored using numerous immune
therapies that target the nexus between CD4+ T cells, DC, and CD8
+ lymphocytes (see
Chapter 1). Given the importance of a decline in tumour antigen to sinecomitant
immunity, exogenous antigen delivery (tumour vaccination) was avoided. The therapies
used were activating anti-CD40 antibody (FGK45, Perth Monoclonal Antibody
- 132 -
Facility), 3M019 (an imidazoquiniline, 3M), poly I:C (InvivoGen) and 1668 CpG-ODN
(Tib-Molbiol).
CD40 activation immunotherapy has previously been combined with the conventional
therapies of radiotherapy389
and chemotherapy,333
but has not previously been used in
combination with surgery. Similarly, there was no preceding data on the use of locally
delivered poly I:C and 3M019 in the post-operative setting. For each therapy tested
(excepting CpG 1668), responses to immune therapy were much better in animals after
surgery than they were in the setting of de novo (primary) AB1HA disease.
The local recurrence site responded poorly in each instance, which was predicted from
the weak improvements in tumour-specific immunity seen in the surgical flank (4.2.3).
As discussed earlier, the factors underlying that poor response might be the inherent
vulnerability of scar tissue to tumourigenesis369
and/or some form of suppression
imprint from the tumour, specifically sited in the draining nodes (e.g. IDO+
plasmacytoid DC258
or Treg366
). Thus future immune strategies for local recurrence may
have to consider this problem, perhaps by a combined approach of immune stimulation
and Treg depletion.
4 .4 . Summary
Surgery has been postulated to boost tumour-specific immunity by the disruption of
immune suppressive networks, but few publications have addressed this hypothesis. In
this chapter, concomitant immunity (the resistance of a host to a second tumour, by
virtue of a primary lesion being present)25,105
was found to be a weak phenomenon. In
contrast, animals could robustly resist tumour re-challenge after primary resection. This
phenomenon, known as “sinecomitant immunity”,25
supports the concept that surgery
can boost the tumour immune response. As such, surgery could provide a powerful
platform for effective immune therapy. In this chapter, it was demonstrated that primary
resection could synergise with several TLR ligands and activate anti-CD40 antibody to
eradicate locally recurrent and metastatic solid tumours.
Sinecomitant immunity could be hampered by Treg and was absolutely dependent on
cytototoxic lymphocytes. Sinecomitant immunity was also strongest temporally and
spatially disparate from the original tumour site. The cause of the disparity was unclear,
- 133 -
but there were no significant differences in absolute numbers of MSC, Treg and DC
phenotype to explain that trend. Surprisingly, sinecomitant immunity required the
decline in tumour antigen presentation seen after surgery (reported in Chapter 3) and
tumour antigen persistence (even when the tumour itself was not persistent) could
antagonise post-operative tumour resistance.
Taken together, these findings would support the practice of cytoreduction surgery
(maximal antigen ablation) and suggest tumour vaccines could be contraindicated in the
early post-operative phase. The importance of CD8+ and Treg was also highlighted,
indicating that therapies which target Treg and/or boost CTLs may be beneficial after
surgery.
Despite these insights, the role of sentinel lymph nodes (tumour draining nodes) in the
sinecomitant immune response remained unclear. Certainly the tumour draining nodes
were the local reservoir of antigen presentation after tumour resection, and it seemed
that antigen presentation was inversely correlated with sinecomitant immunity. It was
therefore hypothesised that sentinel node biopsy could accelerate the decline in tumour
antigen presentation after surgery, and enhance tumour resistance in the early post-
operative phase. The next chapter tests this hypothesis, investigating the effects of
sentinel node removal on antigen presentation kinetics and sinecomitant immunity.
- 134 -
Chapter 5
- 135 -
5. Tumour immunity & sentinel nodes
5 .1 . Introduct ion
Sentinel lymph nodes are defined as the first lymph nodes situated along the line of
afferent lymphatics that drain a particular body region.129
The concept of sentinel node
biopsy has become popular because the tumour status of sentinel nodes predicts the
status of the remaining nodes in the regional basin. This obviates the need for extensive
lymphadenectomy for prognostication. 129
The use of sentinel node biopsy has become increasingly frequent in the surgical
management of melanoma,135
squamous cell carcinoma,132,136
and breast cancer.134
The
sentinel node biopsy concept is theoretically translatable to a large number of solid
malignancies,132
and may be increasingly utilised into the future. Despite the increasing
prevalence of sentinel node biopsy, very little is known about how the procedure
impacts on anti-tumour immunity in vivo.
In Chapter 3, the tumour draining lymph nodes (axillary and inguinal nodes) of the
BALB/c flank subcutis were the solitary sites of cross presentation. It had been
postulated that lymph node dissection may dilute antigen delivery from tumours to a
level that falls below immunogenic thresholds (through passage into the vasculature),151
completely ablate antigen presentation,152
or force antigen presentation to secondary
lymph nodes.153
However, these hypotheses had never been confirmed or refuted.
It had also been suggested that numerous factors could affect the contribution of
sentinel nodes to tumour immunity. Several groups have reported that tumour proximity
reduced node function, lowered CD8+ mitogenicity,
171 reduced CD4
+:CD8
+ ratios,
172,173
increased Treg accumulation,174
and reduced tumour reactivity.175
It had additionally
been postulated that tumour invasion of the regional nodes could impact on their
capacity to participate in the immune response against tumour. Previous research had
indicated that nodal metastasis was associated with impaired tumour immune function,
including reduced cytokine production and T lymphocyte anergy.137,145,390
However, the
- 136 -
effects of tumour proximity and nodal invasion on cross presentation and in vivo CTL
function had not previously been assessed.
Also, in Chapter 3, it was identified that surgery improved systemic CTL function, but
that change took time to mature (Chapter 3). This was hypothesised to reflect a systemic
egress of effectors from the draining nodes after surgery, although this was not
demonstrated (Chapter 4). Given the central role of tumour draining lymph nodes in
presenting tumour antigens, and the critical changes occurring within the lymph node
after surgery (e.g. the decline in cross presentation), sentinel node removal seemed
unlikely to be a null event. The effects of sentinel node biopsy on post-operative tumour
immunity (sinecomitant immunity) were therefore investigated.
As the model used (AB1HA) is non-metastatic to the lymph nodes, and given time
constraints, analysis was restricted to the usual scenario – the effect of removing a
tumour negative (healthy) sentinel node on sinecomitant immunity. In future studies
using the RencaHAM model, the impact of removing a tumour-invaded sentinel node
on sinecomitant immunity will be investigated (see also, 7.3).
5 .2 . Resul ts
5 . 2 . 1 . Ident i f icat ion of sent inel nodes
Methylene blue was formulated at a concentration of 10mg/mL and 50 was injected
into a series of BALB/c. Injections were sited in the caudal flank region and serial
photography of the lymph nodes was undertaken at five timepoints after injection (range
5 minutes to 24 hours).
At 5 minutes and 15 minutes after inoculation of methylene blue, afferent lymphatics
were visible, Figure 5.1. Those lymphatics ran parallel to the lateral thoracic and
inferior epigastric veins. In each instance, they led to the axillary and inguinal nodes
respectively. Those nodes invariably appeared blue, with the axillary node tending to
darken more noticeably than the inguinal node (Figure 5.1). The axillary and inguinal
- 137 -
nodes were therefore identified as sentinel nodes for the caudal BALB/c flank subcutis.
Similar results were obtained when a tumour was in situ (data not shown).
At later dissections (1 hour and beyond) the afferent lymphatics were no longer visible,
and the nodes faded. In its place, the kidneys were darkened in appearance (30 minutes
to one hour) and the urine was blue. At 24 hours after inoculation, no dye was visible in
the animals at any location.
Figure 5.1. Transit of methylene blue dye into the sentinel nodes.
50L of 10mg/mL methylene blue was administered into the caudal flank subcutis of 10 BALB/c mice.
Animals were systemically dissected and photographed at 5 minutes, 15 minutes, 30 minutes, 1 hour and
24 hours after inoculation (n = 2 for each timepoint). Afferent lymphatics were visible within 15 minutes
after injection (above left) and the inguinal and axillary nodes took up blue dye (above right).
5 . 2 . 2 . Dendri t ic t racking and the sent inel nodes
Methylene blue showed the transit of soluble factors into sentinel nodes (5.2.1).
However, HA is cross presented by DCs, which requires cellular traffic. To determine
whether the traffic of DC was similar to that of soluble factors, DCs were tracked from
the BALB/c flank using GM-CSF, CFSE, and DC markers (CD11c, CD11b, DEC-205).
- 138 -
The di-acetate form of carboxyfluoroscein succinimidyl ester (CFDASE) is a non-toxic,
non-fluorescent molecule that diffuses passively into cells.322
Once inside the plasma
membrane of live cells, cellular esterases cleave the acetyl groups of CFDASE to form
the active fluorophore carboxyfluoroscein succinimidyl ester (CFSE). CFSE forms dye-
protein adducts that are retained within or at the cell surface. It emits and absorbs light
at wavelengths characteristic of its fluoroscein moiety. Since CFSE is only formed
intracellularly, CFSE fluorescence on flow cytometry can only be present from cells or
debris of cells that encountered CFSE. As such, CFSE can only track cells or cellular
debris from the site where it was inoculated. Thus CFSE is a useful fluorochrome to
mark cellular traffic.
GM-CSF was used in combination with CFSE to amplify signal. GM-CSF is a growth
factor for Langerhans DCs, the major cutaneous antigen presenting cell type.391
Moreover, GM-CSF conditioning of a skin site can increase the trafficking of antigen
presenting cells from the skin site to the regional lymphatics.391
Protocol details were provided in Chapter 2, and outlined in Figure 5.2. By this
approach, CFSE signal was only identified in combination with the DC marker
(CD11c). Langerhans-like DCs, bearing CFSE from the flank, were identified in the
inguinal and axillary nodes (Figure 5.2). No CFSE signal was found in any other node
groups (e.g. popliteal, brachial, cervical, para-aortic, iliac) nor the spleen. Moreover, the
number of CFSE+CD11c
+CD11b
+DEC-205
+ cells, as a proportion of CD11c
+ cells, was
equivalent between the axillary (2.49%) and inguinal (2.25%) nodes.
5 . 2 . 3 . Tumour proximi ty and node funct ion
A transient increase in CTL function and cross presentation was apparent on the day of
surgery (Chapter 3). This suggested a “rebound” phenomenon, whereby nodes were
impaired in cross presentation and CTL function with a tumour in situ, and then
“released” when the tumour was excised. To delineate the “rebound” of cross
presentation and in vivo CTL more precisely, inguinal and axillary nodes were
processed separately in pre-operative and post-operative mice. In each instance, the
AB1HA tumour was located superficial to the inguinal node, and distant to the axillary
node.
- 139 -
Figure 5.2. Traffic of DC to the sentinel nodes
DC were tracked from the BALB/c caudal flank subcutis. Tracking sites were pre-conditioned with 10g
rGM-CSF (ProSpec-Tany Technogene Ltd), and then inoculated with 100L of 10m CFSE (Molecular
Probes). 24 hours after inoculation, cells from the nearby nodes (cervical, brachial, axillary, inguinal,
popliteal, etc.) were stained with CD45RA-FITC (BD PharMingen), 1:50 CD11c-PE (eBioscience),
CD11b-APC (BioLegend), and CD8-PECy5 (BioLegend). Samples were then examined by flow
cytometry. The experiment was performed once, with three animals studied. Results were similar in each
animal. Representative flow cytometry shown for the axillary node. A. Selection of cells from the
“lymphocyte shoulder” on forward scatter/side scatter. B. Identification of CFSE positive DC. C.
Expression of Langerhans DC markers by the CFSE positive and CFSE negative DC of the axillary node.
- 140 -
In terms of cross presentation, the inguinal node displayed poor CL4 proliferation
(17.90 11.16%) relative to the axillary node (66.16 2.48%) before surgery (P =
0.003). The inguinal node presented HA robustly (39.44 6.24%) after surgery,
significantly enhanced relative to its pre-operative value (P < 0.001). However, cross
presentation was unchanged within the distant (axillary) node (P = 0.283). A similar
pattern was present for in vivo CTL function. With the tumour in situ, inguinal node in
vivo CTL was poor in the inguinal node (6.30 0.54%), although comparable to the
axillary node P = 0.160. After surgery, in vivo CTL improved in the inguinal node
(24.46 4.03%) P = 0.007. By comparison, in vivo CTL was robust in the more distant
sentinel node (axillary node) on the day of surgery (20.28% 8.34%), and remained
similarly robust after surgery (P = 0.381).
Since the inguinal node (close to the tumour) rebounded strongly after surgery but the
axillary (distant) node changed little, a proximity-related suppression phenomenon was
suggested. To test whether tumour proximity affected in vivo CTL and antigen
presentation, these parameters were compared between BALB/c with caudal flank or
rostral flank tumours (Figure 5.5A). The two groups were matched for tumour size,
since this factor affects antigen presentation and in vivo CTL (see 3.2.9) and would
otherwise confound the results.
AB1HA grew faster in the upper flank compared to the lower flank, so for tumour size
equivalence, in vivo CTL and antigen presentation was tested on day 13 for upper flank
tumours and day 16 for lower flank tumours. When tumours were present in the lower
flank, both antigen presentation and in-vivo CTL were strong within the axillary node.
When equivalently sized AB1HA tumours were sited in the rostral flank position
instead (close to the axillary node), axillary nodal antigen presentation (Figure 5.4B)
and in vivo CTL function (Figure 5.4C) were reduced. In vivo CTL and antigen
presentation in the inguinal nodes were close to background levels once tumours were
sited in the rostral flank. This is because the inguinal node did not drain the upper flank,
but instead the brachial node exhibited in vivo CTL killing and HA cross presentation
(data not shown). These differences in antigen presentation and in vivo CTL function
(between inguinal and axillary nodes) were not explained by artefacts in cell viability
(Figure 5.5A) nor inequalities in penetrance of the transferred assay cells (Figure 5.5B).
- 141 -
5 . 2 . 4 . Tumour invasion and nodal funct ion
Tumour proximity was a factor that affected cross presentation and in vivo CTL in
sentinel nodes (5.2.3). These parameters may also be affected by tumour invasion.
Antigen presentation and in vivo CTL function were therefore assayed in BALB/c with
intra-nodal tumours, and compared to mice with extra-nodal tumours of a similar size
and location. Tumours were grown directly within the axillary node (see Chapter 2)
because it was larger than the inguinal node, and therefore easier to inject intra-nodally.
In vivo CTL and antigen presentation for those mice were compared to BALB/c with
similar size tumours, abutting the axillary fossa. When tumours were sited near the
axillary node, but not involving the axillary node, low amounts of in vivo CTL were
seen (4.59 2.09%). In vivo CTL was comparable and poor when the tumour invaded
the axillary node (6.37 2.39%), P = 0.31, Figure 5.6B. For cross presentation, when
tumours were located near the axillary node, proliferation rates of 34.42 9.11% were
seen. As with in vivo CTL, antigen presentation was equivalent when the tumour had
invaded the node itself (25.46 7.71%, P = 0.13) - Figure 5.6C.
5 . 2 . 5 . Surgical d issect ion of the sent inel nodes
The anatomical locations of the two sentinel nodes necessitated distinct surgical
approaches. Steps of the operation were provided in Chapter 2 (see also, Figure 5.7).
Various combinations of sentinel node surgery were explored. Bleeding from the
axillary vein occurred in 2 out of 150 animals and those animals were euthanased.
Another animal developed ischaemia of the forefoot at 24 hours post-operatively, and
was euthanased. No other complications (reduced mobility/contractures, lymphoedema,
infection, etc) occurred.
5 . 2 . 6 . Antigen ablat ion and sent inel node excis ion
Cross presentation from subcutaneous BALB/c flank tumours was confined to the
axillary and inguinal nodes (Sections 3.2.2, 3.2.4). By inference, if the tumour was
removed along with the sentinel nodes, it was hypothesised that cross presentation
would be ablated, i.e. it would not immediately appear in other non-resected nodes or
spleen. To test this, animals underwent primary resection with sentinel node excision,
on day 16 after inoculation. Antigen presentation was assessed on the day of surgery,
and day 5 after sentinel node resection. As before, antigen presentation was quantified
in the spleen and the following nodes: popliteal, brachial, contralateral axillary,
contralateral inguinal, iliac, para-aortic, celiac, mesenteric, renal, mediastinal,
- 142 -
cervical/jugular, and facial. Cross presentation was not detectable in the spleen, or in
any node group after tumour resection and sentinel node biopsy. Thus cross presentation
was immediately and completely ablated by primary resection and sentinel node biopsy
(Figure 5.8).
Figure 5.3. Cross presentation and in vivo CTL function after surgery.
BALB/c mice underwent assays for cross presentation and in vivo CTL function on day 16 of AB1HA
tumour growth, or 24 hours after resection. Inguinal and axillary nodes were processed separately.
Individual mice were depicted by points on the graphs, mean values were shown by bars. P values were
derived from the student‟s t test. A. Antigen presentation before and after surgery, for inguinal and
axillary nodes. B. In vivo CTL function pre- and post-operatively, for inguinal and axillary nodes.
- 143 -
Figure 5.4. Tumour proximity and nodal function.
Antigen presentation and in vivo CTL were quantified in the axillary and inguinal nodes for BALB/c mice
with caudal flank (A, Left) or rostral flank (A, Right) tumours of equivalent size. Individual mice were
shown by points on the graphs, mean values were denoted by bars. B. Antigen presentation in the axillary
and inguinal nodes. C. In vivo CTL for axillary and inguinal nodes. *P = 0.019, **P = 0.013 (student‟s t
test).
- 144 -
Figure 5.5. Viability and assay cell penetrance in the axillary and inguinal nodes.
Lymphocyte viability (% trypan blue exclusion) and CL4 penetrance (% lymphocytes with CFSE signal
at Lyons Parish analysis) were assessed in BALB/c inguinal and axillary nodes. Both experiments were
performed once, although measurements were taken in triplicate. Points on graph were from individual
mice, and a minimum of 5 animals was in each cohort. Mean values were shown by the bars and
compared by student‟s t test. P values were two tailed at the 5% significance level. A. Viability of
lymphocytes from inguinal and axillary node preparations. B. CL4 penetrance into axillary and inguinal
nodes.
- 145 -
Figure 5.6. Nodal invasion: effects on antigen presentation and in vivo CTL.
In vivo CTL and antigen presentation were quantified in the axillary node. Tumours were involving the
node itself (intra-nodal), or located nearby (extra-nodal). All tumours were of similar size. Data was
pooled from two separate experiments with a minimum of four animals per group. Individual mice were
shown as points, mean values were depicted by bars. P values were derived from student‟s t test. A.
Typical appearance of intra-nodal tumour (left). Comparison between nodally tumour-invaded and a
normal axillary node (right). B. In vivo CTL function for axillary nodes with nodal invasion (intra-nodal)
and those with tumour nearby (extra-nodal). C. Antigen presentation for axillary nodes with intra-nodal
tumour (intra-nodal) or tumours nearby the axillary node (extra-nodal).
- 146 -
Figure 5.7. Primary resection with sentinel node excision.
The axillary fossa was exposed via oblique incision (A), and the lower border of pectoralis major was
retracted superiorly (B). The axillary node was dissected clear of the axillary vein (C). Inguinal nodes
were excised en bloc with the tumour and vascular pedicle (D), or via a separate inguinal incision (not
shown). Wounds were closed with interrupted suture (E). Animals healed without complication (F)
5 . 2 . 7 . Tumour ant igen presentat ion af ter node removal
Complete primary resection plus sentinel node biopsy ablated cross presentation (5.2.6).
However, sentinel node biopsy is commonly undertaken for aggressive tumours (e.g.
advanced melanoma) that may not be completely resected. To determine the impact of
sentinel node biopsy on cross presentation from incompletely resected tumours, animals
had sentinel node biopsy in the setting of persistent tumour (i.e. without resection). CL4
were transferred on the day of surgery, or five days after surgery.
When CL4 were transferred on the day of sentinel biopsy, no cross presentation was
visible in any nodal location (Figure 5.9A). This was consistent in all animals. When
CL4 were transferred five days after sentinel biopsy, cross presentation was detected in
distant locations (Figure 5.9B). There was no clear pattern in cross presentation after
sentinel node biopsy, but the ipsilateral brachial node (6/8 animals) and the mediastinal
nodes displayed cross presentation most frequently (5/8 animals) - Figure 5.9C.
Importantly, this systemic cross presentation pattern did not reflect metastases at these
locations, as BALB/c that undergo primary resection remained tumour-free for at least
six months post-operatively.
- 147 -
Figure 5.8. Antigen presentation after resection and sentinel node biopsy.
Antigen presentation was quantified in BALB/c mice after primary resection plus sentinel node biopsy.
Lyons Parish assays were undertaken on the day of surgery and five days post-operatively. Individual
mice were shown as points on the graph, and mean proliferation was indicated by the bar.
5 . 2 . 8 . Sent inel node removal and re -chal lenge
To determine the effect of sentinel node removal on sinecomitant immunity, animals
underwent sentinel node removal and tumour excision, followed by re-challenge with
AB1HA. (Figure 5.10A). As before, when animals were re-challenged with AB1HA on
the day of surgery, there was a modest sinecomitant immune response (approximately
20% survival) seen in the surgical site and the opposite (healthy) flank. Sentinel node
biopsy did not alter this effect (Figure 5.10B,C).
If BALB/c were re-challenged at two weeks after sentinel node biopsy plus tumour
resection instead (Figure 5.10A) a significant decline in survival was seen for re-
challenges into the healthy flank (Figure 5.10E). However, sentinel node removal did
not impact on survival from re-challenge into the surgical site (Figure 5.10D).
- 148 -
Figure 5.9. Cross presentation from local recurrence, after sentinel node removal
BALB/c mice with AB1HA tumours underwent sentinel lymphadenectomy. Antigen presentation was
assessed on the day of surgery (A) or 5 days post-operatively (B). Representative flow cytometry shown
from node specimens in each instance (clockwise from lower left): ipsilateral popliteal, abdominal
(mesenteric, para-aortic, iliac), ipsilateral brachial, cervical, mediastinal, contralateral axillary, spleen,
contralateral inguinal). C. Proportion of mouse lymph node specimens with >10% CL4 proliferation, five
days post sentinel node excision.
- 149 -
5 . 2 . 9 . Sent inel sampl ing and s taged lymphadenectomy
Two strategies were attempted to reduce the “harm” of sentinel node biopsy on post-
operative tumour resistance, for both the surgical site and the healthy flank. In the first
instance, the sentinel nodes were sampled (axillary or inguinal lymphadenectomy)
rather than both nodes being removed. In the second experiment, both sentinel nodes
were removed, but staged at two weeks after tumour resection. For both experiments,
the readout was survival from re-challenge into the surgical site or the opposite
(healthy) flank, at two weeks post resection.
As before, sentinel node biopsy was a null event for sinecomitant immunity into the
surgical site. Removing one or other of the sentinel nodes, or delaying sentinel node
removal was also a null event (data not shown). Importantly, survival from re-challenge
into the healthy flank was equivalent between animals that had sentinel node sampling,
and those with intact sentinel nodes (Figure 5.11B). Similarly, when sentinel node
removal was staged at two weeks after surgery, survival from re-challenge was similar
to animals that had no sentinel node procedure (Figure 5.11C).
5 .3 . Discussion
The role of lymph node surgery has evolved considerably over the years. In recent times
there has been a waning of radical lymph node dissection206
in favour of incomplete
dissections or targeted biopsy (sentinel node biopsy). Nevertheless, lymph nodes yield
useful prognostic information and are frequently invaded by tumour.21
Therefore
lymphadenectomy will probably remain a component of cancer surgery.
Identification of sentinel nodes for the BALB/c flank subcutis had previously been
reported using vital dyes (e.g. isosulfan blue392
or Evans blue393
), radio-isotope
imaging,394
and even micro-magnetic resonance lymphangiography.395
The first of the
techniques (vital dye) was chosen because it was inexpensive, presented few
occupational hazards, was easily formulated from standard ingredients, and could be
readily transported to sites of experimentation. Using the methylene blue dye, inguinal
and axillary nodes were identified as the sentinel nodes for the BALB/c flank subcutis.
Those nodes were not only the site of first transit for soluble factors, but also for DCs.
- 150 -
Figure 5.10. Effect of sentinel node removal on survival from re-challenge
Data were pooled from two separate experiments, with a minimum of ten animals present in each group.
All animals were matched for pre-operative tumour size. Surgical impost was approximately equivalent
across cohorts (extra incision when axillary lymphadenectomy performed). Kaplan Meier survival was
shown for each cohort. P values were derived from the Log Ranks test. A. Timeline of tumour resection,
sentinel node removal, and re-challenges. B. Survival from re-challenge into the surgical site on the day
of surgery sentinel node removal. C. Survival from re-challenge into the healthy (opposite) flank on the
day of surgery sentinel node removal. D. Survival from re-challenge into the surgical site at two weeks
after tumour resection sentinel node removal. E. Survival from re-challenge into the healthy (opposite)
flank at two weeks after tumour resection sentinel node removal.
- 151 -
Figure 5.11. Re-challenge after sentinel node sampling or delayed biopsy.
BALB/c underwent tumour resection on day 16 after AB1HA inoculation, and re-challenge into the
healthy flank at two weeks after tumour removal. Tumour sizes were matched across cohorts, and surgical
impost was similar. Data were pooled from two separate experiments. At least 10 animals were present in
each group. Kaplan Meier survival shown for each cohort. P values were derived from the Log Ranks
test. A. Timeline for sentinel node sampling and delayed sentinel node removal. B. Survival from re-
challenge into the healthy flank, after sentinel node sampling (axillary or inguinal lymphadenectomy) at
the time of tumour resection. C. Survival from re-challenge into the healthy flank, when sentinel nodes
(inguinal and axillary) were removed at two weeks post tumour resection.
- 152 -
In Chapter 1, the importance of the interaction between the APC, the CD8+ and the
CD4+ cells was identified. That interaction probably spans only a few cell diameters,
and to occur efficiently it must take place in an architecturally and functionally
optimised environment.366
That optimised environment is the lymph node.366
In Chapter
3 it was found that cross presentation was confined to the axillary and inguinal lymph
nodes: identified as sentinel nodes in this chapter.
Given the importance of the sentinel nodes to DC tracking and APC-T cell interaction,
lymphadenectomy could have a profound effect on tumour antigen priming. It had been
postulated previously that lymph node dissection may dilute antigen below
immunogenic thresholds (through passage into the vasculature),151
completely ablate
antigen presentation,152
or force antigen presentation to secondary lymph nodes.153
This
chapter indicated that the third hypothesis was correct. Cross presentation was absent
for three days after node removal, and then shifted by five days post lymph node
removal. The mechanism by which cross presentation shifted was unknown, but the
new sites of cross presentation were not from metastases (no tumour outgrowth
occurred in corresponding locations).
It was possible that new lymphatic channels were created to explain the shift in cross
presentation. Little is known about how rapidly lymphangiogenesis occurs, but
lymphatic endothelium migrates in culture at only a few micrometres per day. 396
Moreover, if lymphangiogenesis explained the new sites of cross presentation, it would
be likely that axillary and inguinal nodes would be replaced by the next most proximal
nodes i.e. “second-tier nodes” (brachial, popliteal, para-aortic). However, as cross
presentation was frequently found in distant sites (e.g. spleen and contralateral axillary
node), this suggested lymphangiogenesis was not the mechanism. It has also been
identified that lymphatico-lymphatic and lymphatico-venous connections exist. 123,397
As a corollary, when a main lymphatic vessel has been disrupted by surgery, antigen
bearing presenting cells could be shunted into adjacent lymph channels and away from
the dissected sites.
Thus when a lymph node is removed there may be open vessels left behind in the
resection bed,397
which may uptake the DC traffic and shunt it through to systemic sites.
Indeed, angiogenesis is promoted because of pro-angiogenic signals from the hypoxic
- 153 -
wound environment.120,369,398
Those new vessels, tend to be highly permeable397
which
may further enhance the uptake and systemic intravascular trafficking of DCs.
As an alternative to shifting cross presentation to systemic sites, sentinel node biopsy
could ablate cross presentation when it was combined with complete primary resection.
This accorded with Chapter 3 and the findings of others,20,148,149
where cross
presentation has been confined to draining nodes. Also, since node dissection and
primary excision only left DC in transit to cross present (i.e. DC between the regional
node and the tumour site). This insinuated that DC had a lifecycle of less than five days
(the usual time taken for cross presentation to appear in new node), consistent with the
work of Kamath et al., who reported the DC life cycle to be between 1.5 and 3 days.360
Despite the effects of sentinel node excision on cross presentation, the overall impact of
lymphadenectomy on tumour immunity was ambiguous. A number of factors may
complicate the contribution of sentinel nodes to immunity, including whether those
nodes are invaded by tumour, and how close the tumour is to the nodes.
The data presented in this chapter, and the findings of numerous others,171,173,175,399,22
hint at a topography of lymph node function, based on tumour proximity. Specifically,
those nodes closest to a tumour may be exposed to the greatest tumour stimulation but
also the strongest tumour derived suppression. Those nodes further away will react
poorly also, because they are too far away to be engaged by the tumour. Thus it is the
intermediate zone nodes which function best – encountering antigen, but relatively
spared form tumour suppression.
In the flank tumour model described herein, the inguinal node was sited very close to
the tumour itself. However, the axillary node, which also drained the tumour, was more
distant. With an established tumour in situ, the inguinal node always performed poorly
in terms CL4 proliferation and in vivo CTL function. That difference in function was
not explained by a difference in viability of cells between the two nodes on assay, nor
was it attributable to preferential DC trafficking to the axillary node. When the tumour
was moved close to the axillary node, in vivo CTL and cross presentation declined. That
was despite an equivalent volume of tumour, similar volumes of CL4, and/or equivalent
proportions of tumour-specific targets.
- 154 -
A mechanism to account for the topography of node function was not identified.
However, the proximity effect indicated a soluble factor may have been involved.
Numerous soluble factors have been identified previously, including: VEGF,400
Il-10,401
and TGF402
Given the importance of Treg in this system (see depletion studies, Chapter
4), tumour chemokine CCL22 (which recruits and expands Treg cells) may be
significant.264
A second complicating factor was that nodes could be invaded by tumour. By replacing
the space normally occupied by APC and T cells, tumours may tip the balance away
from cross presentation and towards direct presentation. In addition, previous groups
have found invaded nodes to have higher “suppressor” T cell counts,174
anergic T
cells,177-179
and ablated concomitant immunity.180
In this chapter, robust cross
presentation and in vivo CTL function were seen in tumour-invaded axillary nodes. That
level of function was comparable to animals with equivalent size tumours, not involving
the node. Nevertheless, while tumour-invaded nodes may contribute to immunity, they
will likely succumb to increasing tumour growth390
and would be a source of
locoregional recurrence. Hence, it is not advocated that tumour invaded nodes should be
left in situ.
Despite the factors complicating the contribution of sentinel nodes to tumour immunity,
sentinel nodes predominantly suppress rather than activate anti-tumour immunity.
Indeed, the tumour draining nodes may be suppressed within 4 – 5 days by tumour
growth.258
Munn and Mellor suggest the tumour draining nodes are a powerful “factory”
for generating local and systemic tolerance to tumour antigens, however that was not a
major factor in our model.366
In Chapter 4, tumour antigen presentation frustrated post-
operative tumour resistance. By extension, removing sentinel lymph nodes might have
assisted immune function – removing the “factory” of suppression and re-setting the
immune response. Accordingly, in one approach for colorectal cancer, dubbed “immune
corrective surgery”, (ICSTM
, Biocrystal, Columbus Ohio USA), investigators have
attempted to resect all lymph nodes where tumour antigen is found.154
The authors
reported an improvement in survival with this technique in a phase 1 study,154
but a
larger study has not been published.
In this thesis, a somewhat different view is supported. While tumour proximity impaired
cross presentation and in vivo CTL function, that impairment could be alleviated by
- 155 -
surgery. Improved nodal function after surgery would correlate with the findings of Lee
and colleagues, who reported that sentinel lymph node IL-10 levels declined after
surgery140
- presumably enhancing T cell activation. In Chapter 4, the importance of a
decline in cross presentation after surgery was reported. That signal was presumably
integrated in the node. It was further seen that tumour-specific effectors and in vivo
CTL cluster in the draining node, correlating with the findings of others.155
Therefore, the combination of improved nodal function after surgery, the importance of
a decline in cross presentation in the draining nodes, and the localisation of effectors
and CTL to the tumour nodes led to a postulate that sentinel node biopsy would
adversely impact on tumour immunity. The impact of sentinel node biopsy was difficult
to assess in the surgical flank, where sinecomitant immunity was weak (see Chapter 4).
However, sentinel node biopsy had a clear impact on sinecomitant immunity in the
healthy flank. Specifically, only 10% of animals survived re-challenge after sentinel
node biopsy (compared to 50% survival with intact nodes).
While sentinel node surgery seemed to be detrimental to post-operative immunity,
sinecomitant immunity could be preserved by altering the extent and timing of sentinel
node excision. Specifically, leaving one or other of the sentinel nodes intact preserved
tumour resistance (Figure 5.11B) – suggesting a degree of redundancy in the system.
Additionally, it was postulated that surgery induces an effector egress of CD8+ from the
sentinel nodes in parallel with the decline in cross presentation (see pentamer staining of
Chapter 4, and primed in vivo CTL of Chapter 3). It was therefore hypothesised that
immediate sentinel lymph node excision could be detrimental (Figure 5.13) but delayed
sentinel node biopsy may leave sinecomitant immunity intact (Figure 5.14). Indeed,
delaying sentinel excision did improve survival relative to sentinel lymphadenectomy
concurrent with surgery, and approached similar levels to BALB/c with intact sentinel
nodes (Figure 5.11C).
Incomplete sentinel node biopsy may not be practical, because sentinel nodes may need
to be removed en bloc as part of the local clearance procedure. Delayed sentinel node
biopsy may similarly be problematic, because this would require re-operation in close
proximity to the previous procedure – where tissue planes have been distorted.
Therefore, an alternate strategy to reduce the “immunological harm” of sentinel node
- 156 -
biopsy may be preferable. Activating anti-CD40 antibody may be one such strategy,
because FGK45 has been shown to “push” effectors out of the tumour draining nodes
and into systemic sites.155,156
In short, pre-operative activating anti-CD40 antibody
might allow the effector cells to refuge in systemic sites by the day of surgery, so that
sentinel node excision does not deplete the patient of effector cells156
(Figure 5.15).
Although there was insufficient time to test the pre-operative CD40 hypothesis during
this thesis, it would be an attractive hypothesis to test in future pre-clinical work.
Importantly, the effect of sentinel node biopsy on sinecomitant immunity was restricted
to the removal of healthy (non tumour-invaded) nodes. Although in this chapter (section
5.2.4) antigen presentation and CTL function were preserved in the tumour-invaded
nodes, numerous publications have reported that node function is impaired when they
are positive for tumour.137,145,390
It is therefore possible that removing tumour-invaded
sentinel nodes is beneficial to tumour immunity overall, rather than detrimental.
Moreover, lymph nodes that are invaded with tumour may produce local recurrence
and/or metastases,403-405
quite independent of their tumour immune effects. Such
metastases may produce considerable morbidity and mortality. Consequently, it is
difficult to argue for the preservation of sentinel nodes (or even regional nodes) that
bear tumour metastasis, and determining the effect of removing tumour-positive sentinel
nodes on sinecomitant immunity is a priority for future work (see 7.3). The RencaHAM
model (described in Chapter 6) may prove to be a good tool for investigating this area.
- 157 -
Figure 5.12. The post-operative CD8+ effector egress postulate.
In Chapter 3, cross presentation waned after surgery and was associated with enhanced, systemic in vivo
CTL function. In Chapter 4, pentamer staining was confined to the sentinel nodes before surgery and
declined after surgery. In this chapter, removing the sentinel nodes on the day of surgery ablated
sinecomitant immunity, but delaying node excision preserved it. Taken together, these data suggested an
egress of tumour effectors from the draining nodes, possibly relating to the loss of cross presentation
signal in those nodes. The following schema was proposed. Kinetics of cross presentation shown in the
top of the figure: increasing until surgery and then decreasing after surgery. The distribution of tumour-
specific effectors (from pentamer and CTL data) shown on the diagram also: initially confined to the
sentinel nodes (dark green) and becoming systemic (light green) as cross presentation wanes.
Sinecomitant immunity was successful in the majority of animals at two weeks after surgery (indicated by
the star).
- 158 -
Figure 5.13. Predicted implications of sentinel biopsy: scenario 1.
From the postulate indicated in Figure 5.12, if sentinel node biopsy was undertaken on the day of surgery,
then cross presentation would be ablated immediately and effectors would be removed. Thus sinecomitant
immunity would be poor at the two week challenge test-point. Indeed, sinecomitant immunity was more
than halved by sentinel node biopsy concurrent with surgery (Figure 5.10). Sinecomitant was not ablated
completely however, possibly because HA specific CD8+ were representative only of anti-tumour CD8
+
directed against cell-associated antigens. The kinetics of CD8+ directed against soluble tumour antigens
may be different – possibly they are located systemically.406
Secondly, there may be some (lower than
detection threshold) egress of tumour-specific CD8+ during tumour growth i.e. before the day of surgery.
- 159 -
Figure 5.14. Predicted implications of sentinel biopsy: scenario 2.
Consistent with the postulate of Figure 5.12, if sentinel node biopsy were to be undertaken at two weeks
after surgery, it was predicted that sinecomitant immunity would be preserved. In this instance, effector
egress would be complete (dark green) and antigen presentation would have waned (dark blue).
Empirically, this was the case (Figure 5.11C and Figure 5.10).
- 160 -
Figure 5.15. Predicted implications of sentinel biopsy: scenario 3.
As discussed in this section and as shown in Figure 5.14, sentinel node biopsy may be harmful to post-
operative tumour resistance. That harm could be alleviated by sentinel node sampling (Figure 5.11B) or
delaying sentinel node biopsy (Figure 5.11C). Another alternative would be to force tumour-specific
effectors out of the sentinel nodes, prior to surgery. In the diagram above, it is proposed that pre-operative
activating anti-C40 antibody induces systemic spread of effectors from the sentinel nodes, so that when
the sentinel nodes are removed, strong sinecomitant responses are still seen (dark green) with successful
tumour immunity (yellow). This will be investigated in future research.
- 161 -
5 .4 . Summary
Sentinel node biopsy is increasingly undertaken during cancer surgery, but the
immunological impact of this procedure is unknown. The inguinal and axillary nodes
were identified as sentinel nodes for the BALB/c flank subcutis using methylene blue.
A rapidly reproducible technique of sentinel node surgery was then provided. As
predicted from the cross presentation, in vivo CTL and pentamer data of Chapters 3 and
4, sentinel node removal had a profound effect on tumour antigen processing and
overall anti-tumour immunity. While sentinel node removal might reduce antigen load
and potentially boost tumour immunity on the one hand, it ablated the pool of tumour-
specific effectors on the other.
It may therefore be important to consider the impact of sentinel node biopsy when
planning combined immune therapy/surgery strategies. Where practical, consideration
might be given to sparing the sentinel nodes or staging sentinel node biopsy beyond the
early post-operative phase. Alternatively, pre-operative immune therapy may prove
valuable, as a means of “flushing” effector CTL from the sentinel nodes before surgery.
The final experiment of this thesis is now presented. In this experiment, the derivation
and properties of the RencaHAM model are described. It is hoped that this model will
provide a useful tool for studying the effects of surgery on tumour immunity, and for
the development of combined surgery/immune therapy strategies for renal cancer.
- 162 -
Chapter 6
- 163 -
6. RencaHA
6 .1 . Introduct ion
As a Urological surgeon scientist, my initial interest was to develop more effective
immune therapy strategies for renal cancer. Renal cell carcinoma accounts for 3% of
cancers in adults and is the sixth most common cause of cancer death overall.7,407,408
Due to the widespread use of abdominal imaging,409
the incidence of renal cancer has
been increasing, due to increased detection. Overall, about 2000 new cases are
diagnosed each year in Australia and 1100 patients die annually of this disease.410
Renal cell carcinoma is probably the prototypical target for immune therapy because of
its proven sensitivity to biological treatments and its poor response to conventional
strategies (chemotherapy411
and radiotherapy7). Numerous renal cell carcinoma antigens
have been identified and substantial lymphocytic infiltrates have been observed in
deposits of kidney cancer.412,413
Strikingly, metastatic disease spontaneously regresses
in up to 1% of cases with primary resection alone.16,42
There is also some evidence that
more immunogenic renal cancers have better prognosis, and conversely that patients
with high B7-H1 expression fare poorer.414
The underlying objective of this thesis was to develop a rationale for combining surgery
and immune therapy more effectively. As such, renal cancer is the first malignancy for
which a benefit for combining surgery and immune therapy has been demonstrated in
randomised clinical trials. Specifically, primary resection plus IFN therapy is more
effective than IFN alone in the setting of advanced disease,14,16,415
and tumour
vaccines improve disease-free survival and overall survival for patients who undergo
clinically complete primary resection.12,13,237,416
To study combined surgery/immune therapy strategies and tumour-specific immune
responses in renal cancer pre-clinically, a murine renal cancer line with a trackable neo-
antigen was required. The RencaHA cell line seemed ideal, having been previously
developed by Dr Linda Sherman and colleagues.318
Accordingly, RencaHA was sought
and kindly donated by Drs Sotomayer and Cheng at the H Lee Moffitt Cancer Centre
(Tampa, Florida USA).
- 164 -
A number of unforseen difficulties with the RencaHA line were encountered, and thus a
new sub-clone was derived (RencaHAM). While time did not allow extensive work
with the renal cancer model, different routes of administration for RencaHAM were
explored, including the biologically most valid method (intra-renal implantation).
Having described the RencaHAM subclone and its properties, hopefully this will be
useful to future empirical studies of combined surgery/immune therapy in renal cancer.
Already, a Master of Surgery student at The University of Western Australia has begun
a project based on RencaHAM, and several other researchers at the Tumour
Immunology Group (Sir Charles Gairdner Hospital) are using RencaHAM in their
work.
6 .2 . Resul ts
6 . 2 . 1 . Ini t ia l exper ience wi th RencaHA
After a number of failed attempts to obtain RencaHA by freight, the candidate visited
the H Lee Moffitt Cancer centre in person and was kindly provided with a fresh sample
of cells by Dr Cheng. After a transit time of approximately 24 hours, culture was
commenced in the Tumour Immunology Group laboratory. On arrival, the cell line was
lightly contaminated and mycoplasma positive. The culture was treated with
amphotericin, penicillin and gentamicin, followed by ciprofloxacin. After four or five
passages, the culture was viable and negative for mycoplasma on three consecutive PCR
examinations. While initial experiments have suggested that RencaHA is too
immunogenic to grow subcutaneously,318
subcutaneous transplantation was attempted in
20 mice with 1x105
RencaHA cells (n = 5), 1x106
cells of RencaHA (n = 10), and 2x106
RencaHA cells (n = 5). Subcutaneous tumours grew in 4 out of 5 mice with 1x105
RencaHA, 8 out of ten mice with 1x106 RencaHA, and 2 out of 5 mice with 2x10
6
RencaHA, (data not shown). The lowest inoculum concentration (1x105 cells of
RencaHA) grew significantly slower than the stronger inoculums, but there was no
difference in growth rate between injections of 1x106 and 2x10
6 cells of RencaHA.
Five animals with day 31 RencaHA (1x106 group) were euthanased and the tumours
were prepared into five sterile cultures. After two in vitro passages, those cell lines were
stained for HA expression using H18 antibody and examined by flow cytometry using
- 165 -
FACScan (Becton Dickinson) and associated Cell Quest V3.1 (Becton Dickinson)
software. HA expression was absent from four of the five samples, and positive in the
remaining culture (Figure 6.1A). The broad HA expression of that culture represented
cells that retained RencaHA expression in vivo. This cell culture was passaged in vitro
and FACS sorted thrice for HA expression, using the FACS Vantage Cell Sorter and
with the assistance of Dr Kathy Heel (BIAF, University of Western Australia, Perth,
Western Australia). Three populations resulted: RencaHALOW
, RencaHAMEDIUM
, and
RencaHAHIGH
. These three subpopulations were quite different with respect to in vitro
growth characteristics: both RencaHALOW
and RencaHAHIGH
grew slowly and sparsely.
By comparison, RencaHAMEDIUM
formed confluent monolayers (Figure 6.1C) and grew
prolifically (tripling approximately every 24 hours). After successive sorting, the
RencaHAMEDIUM
sub-clone was denoted “RencaHAM” and selected for experimentation
because of its favourable HA expression profile and stable in vitro growth
characteristics. RencaHAM fluorescence was at least a log shift higher than background
staining (RencaWT) and unstained controls (Figure 6.1B).
6 . 2 . 2 . Subcutaneous RencaHAM
1x106 cells of RencaHAM line were inoculated subcutaneously into 20 BALB/c and
followed for growth kinetics. However, no tumours were present by day 27 after
inoculation. To determine whether RencaHAM failed to transplant because of
immunological rejection, 1x106 RencaHAM was inoculated into congenic BALB/c nu
-/-
(BALB/c background but athymic). Tumours were successfully transplanted in 100% of
nude BALB/c, with lesions reaching 9.58 0.40 mm by day 31 after inoculation (Figure
6.2). Growth of RencaHAM in nude BALB/c was significantly slower than for
RencaWT in wild type BALB/c. To determine whether HA antigen was presented
despite the absence of tumour in wild type mice, antigen presentation assays were
performed on days 4, 10, 16, 21, and 27 after tumour cell inoculation. As with
subcutaneous AB1HA, class I restricted HA presentation was robust and solely
confined to the sentinel lymph nodes (Figure 6.3A). Antigen presentation was strongly
visible at the day four time point, and declined gradually to background by day 27
(Figure 6.3B).
- 166 -
Figure 6.1. Derivation of RencaHA sub-clone.
A. Subcutaneous RencaHA tumours were harvested and stained for HA expression using H18 mAb. A
single culture retained HA expression after subcutaneous passage, and was subsequently sorted by flow
cytometry as shown. B. Representative flow cytometry from HA staining of RencaHAMEDIUM
(RencaHAM) sub-clone relative to unstained and H18 treated RencaWT controls. C. Appearance of
RencaHAM in vitro.
- 167 -
Figure 6.2. RencaWT and RencaHAM in wild type and congenic BALB/c nu-/-
RencaHAM and RencaWT were inoculated into wild type and congenic BALB/c nu-/-
mice. Mean tumour
diameter SEM was shown for each cohort. Data was shown for a single experiment, with ten animals per
group. Points of statistically significant difference in mean tumour diameter shown by the asterisk (two
tailed P < 0.05 on student‟s t test).
6 . 2 . 3 . Intravenous RencaHAM
Since RencaHAM did not grow subcutaneously (6.2.2), other routes of transplantation
were investigated. Intravenous injection was studied first, as this route of administration
has previously been described.198,318
To verify tumour kinetics of RencaHAM, 1x106
cells of RencaHAM were injected intravenously into 32 mice. The lateral tail vein was
used to inject most of the mice, but due to technical difficulties, eight mice received
tumour cells by direct intra-cardiac injection. While the results of intra-cardiac injection
were reported, the procedure itself was performed as a “last resort” and did not
constitute a formal component of the investigation. Mice were culled serially for
autopsy and Lyons Parish analysis. In the tail vein group, all animals appeared healthy
at day 21, but two culls were necessitated between day 21 and day 34. By day 34,
remaining animals were cachectic and lethargic. At autopsy of mice with day 21
tumour, the thorax was grossly normal - with the exception of enlarged mediastinal
nodes. Macroscopically, the animals appeared free of tumour. At day 28, pleural
plaques appeared and small solid tumours were visible in the lung. By day 34,
pulmonary and pleural tumours were extensive, and occasionally extended onto the
pericardium (Figure 6.4A). On Lyons Parish analysis at day 21 after inoculation, HA
presentation was robust in the mediastinal nodes (approximately 80% proliferation) and
- 168 -
low levels of presentation were found in the spleen and the para-aortic nodes. No other
nodes (renal, cervical, axillary, inguinal, brachial, iliac, popliteal etc.) demonstrated HA
specific presentation (Figure 6.5).
Figure 6.3. Presentation of HA from subcutaneous RencaHAM.
Class I restricted presentation of HA was assayed in BALB/c after subcutaneous inoculation of
RencaHAM. Data was shown from a single experiment with a minimum of four animals per group. A.
representative flow cytometry from BALB/c at day four after inoculation of RencaHAM. B. Antigen
presentation in cohorts of BALB/c culled serially after inoculation of RencaHAM. Individual mice were
shown as points, mean proliferation depicted with bars. Statistically significant proliferation (relative to
background controls on student‟s t test, P < 0.05). were denoted by asterisks.
- 169 -
Disease progression was more rapid in the animals that underwent intra-cardiac
injection of RencaHAM. By day 14, one cull was required and half the mice looked
ruffled and sluggish (unwell). All were mildly cachectic. At day 21, one of four animals
autopsied had adrenal enlargement (probable metastases) and all had mild pleural and
pericardial infiltration. By day 28, all animals that received intra-cardiac RencaHAM
were euthanased. Injection site tumour was visible in each of the three mice with day 28
tumour, and extensive pulmonary and pleural nodules were present. The adrenals were
enlarged in all three, with two mice showing obvious renal lesions. Most lymph nodes
were grossly enlarged, with a chalky to haemorrhagic appearance, suggestive of tumour
invasion. On Lyons Parish analysis at day 17 after injection, cross presentation was
visible in all node groups (Figure 6.6).
At histology, both intra-cardiac and tail vein administered RencaHAM produced
subpleural tumour and intra-parenchymal deposits. The intra-parenchymal deposits
were more frequent with intra-cardiac RencaHAM (Figure 6.4E,F), but the subpleural
tumours seemed more prominent in tail vein injected RencaHAM (Figure 6.4C,D). In
short, whether RencaHAM was given by tail vein injection or intra-cardiac injection, the
main sequelae were subpleural and intraparenchymal tumour deposits. Animals given
intra-cardiac tumour tended to have accelerated disease progression, with euthanasia
required about one week earlier. Also, there was clinical evidence of lymph node
invasion in animals given intravenous RencaHAM via intra-cardiac injection. The issue
of lymph node invasion was formally investigated in 6.2.5.
6 . 2 . 4 . Orthotopic ( intra -renal) RencaHAM
To study post-operative tumour-specific immunity, a biologically valid and surgically
resectable form of RencaHAM transplantation was required. While RencaHAM can be
given by intravenous injection, those deposits are not surgically resectable. Moreover,
Renca is known to behave differently when transplanted orthotopically (i.e. into the
kidney) as opposed to ectopic growth (e.g. subcutaneous inoculation).317
For this reason,
the intra-renal injection of RencaHAM was studied, for which there was no previously
published data. To establish the tumour kinetics of intra-renal RencaHAM, 16 animals
were administered 1x106 RencaHAM cells into the right kidney (Figure 6.7A). Renal
RencaHAM grew in a subcapsular manner on histological section, and this was
irrespective of whether the cells were originally injected subcapsularly or
intraparenchymally (Figure 6.7C,D). By 21 days after surgery, right flank tumours were
- 170 -
palpable in all animals (Figure 6.7B). Euthanasia was obligatory in one in three animals
by this stage, due to cachexia and/or lethargic behaviour. By 28 days after injection, all
remaining animals were euthanased because of the onset of cachexia and indicators of
distress (particularly sluggish behaviour and ruffled fur). At necropsy, very large renal
tumours were present with extensive peritoneal deposits, and significant metastatic
disease (Figure 6.7E,F). Metastatic sites included spleen, liver, contralateral kidney and
the lung. When BALB/c were assayed for class I – restricted HA presentation at 21 days
after intra-renal injection of RencaHAM, multiple sites of HA-specific CD8+
proliferation were seen. In all five animals, HA presentation was seen in the spleen,
renal, para-aortic and mediastinal nodes. Importantly, HA presentation within the
mediastinal nodes correlated with the presence of “micrometastatic disease” in the lung
(Figure 6.9A,B) and the absence of mediastinal node invasion on histology (6.2.5). This
suggested the immune system was not ignorant of orthotopic, micrometastatic disease
and that cross presentation was important to the immune recognition of
micrometastasis.
6 . 2 . 5 . Lymph node m etas tases from RencaHAM
HA was presented robustly after intra-cardiac, intra-renal, and tail vein injection of
RencaHAM. However, it remained unclear whether direct or cross presentation was
important in this system. To determine whether HA presentation could occur
independently of lymph node tumour, the extent of lymph node invasion in the
RencaHAM system was determined. Pairs of mice at day 21 after intravenous or intra-
renal RencaHAM were selected and examined for nodal invasion. Specifically, renal,
para-aortic and mediastinal nodes were sampled separately for histology. Additionally,
lymph nodes from the individual mice were assayed en masse (i.e. total lymph node
dissection) for PCR and culture (Figure 6.10E) Intravenous RencaHAM was found to
metastasise to the lymph nodes on histology, culture, and PCR (Figure 6.10A,B,D).
While tumour cells were only identified histologically in mediastinal nodes, diffuse
lymph node invasion was present clinically at later time points (e.g. day 28, 6.2.3); all
nodes were markedly enlarged, chalky, and haemorrhagic in appearance. Orthotopic
RencaHAM also metastasised to the lymph nodes. On histology, tumour was identified
in the renal (Figure 6.10C) and para-aortic nodes. However, RencaHAM tumour was
not seen in the mediastinal nodes of the orthotopic system. This suggested that
mediastinal node CL4 proliferation in animals with micrometastases (Figure 6.8) was
cross-presentation.
- 171 -
Figure 6.4 Pulmonary morphology and histology post intravenous RencaHAM
RencaHAM was administered intravenously into BALB/c mice at a dosage of 1x106 cells per mouse.
Tumour was administered via tail vein or intra-cardiac injection. A. Thick subpleural tumour rind in
BALB/c at day 34 after intravenous injection of RencaHAM by tail vein. B. Multiple pleural nodules,
pericardial plaque (with healed right ventricular scar) of BALB/c at day 28 post intra-cardiac injection of
RencaHAM. C. Histological section (x20) of lung surface from BALB/c at day 34 after tail vein injection
of RencaHAM. D. Histological section (x20) of deep lung from BALB/c. day 34 after tail vein injection
of RencaHAM. E. Histological section (x20) of lung surface from BALB/c, day 28 after intra-cardiac
injection of RencaHAM. F. Histological section (x10) of deep lung from BALB/c at day 34 after intra-
cardiac injection of RencaHAM.
- 172 -
Figure 6.5. Antigen presentation from i.v. RencaHAM.
BALB/c were assayed for class I restricted HA presentation at day 21 after tail vein injection of
RencaHAM. The histograms were representative of CD8+CFSE
high cell populations at flow cytometry.
Data were shown for a single animal, representative of eight BALB/c assayed with similar results. The
proportion of CFSEhigh
cells with lower fluorochrome intensity than parental was shown for each lymph
node group. Clockwise from left: axillary and inguinal nodes, cervical, mediastinal, spleen, renal, and
para-aortic nodes.
- 173 -
Figure 6.6. Antigen presentation from intra-cardiac RencaHAM.
BALB/c were assayed for class I restricted HA presentation at day 17 after intra-cardiac injection of
RencaHAM. The histograms were representative of CD8+CFSE
high cell populations, at flow cytometry.
Data were shown for a single animal, representative of five animals assayed with similar results. The
proportion of CFSEhigh
cells with lower fluorochrome intensity than parental was shown for each lymph
node group. Clockwise from left: axillary and inguinal nodes, cervical, mediastinal, spleen, renal, and
para-aortic nodes.
- 174 -
Figure 6.7. Gross morphology and histology of orthotopic RencaHAM.
A. Orthotopic transplantation of RencaHAM cells via operative exposure and subcapsular injection. B.
Flank tumour of typical BALB/c mouse, day 21 after intra-renal inoculation of RencaHAM. C. Kidney at
21 days after inoculation of RencaHAM (above) relative to normal kidney (below). D. Histological
section (H&E, x10) of renal capsule and kidney with RencaHAM at day 21 after injection. E. Kidney at
day 28 after intra-renal injection of RencaHAM. F. left sagittal view of BALB/c coelom, day 28 after
intra-renal injection of RencaHAM to the right kidney.
- 175 -
Figure 6.8. HA presentation from orthotopic RencaHAM.
BALB/c were assayed for class I restricted HA presentation at day 17 after intra-renal injection of
RencaHAM. Representative histograms of CD8+CFSE
high cell populations at flow cytometry. Data were
shown for a single animal, representative of five animals assayed with similar results. Proportions of
CFSEhigh
cells with sub-parental fluorochrome intensity shown for each lymph node group. Clockwise
from left: axillary and inguinal nodes, cervical, mediastinal, spleen, renal, and para-aortic nodes.
- 176 -
Figure 6.9. Pulmonary micrometastases from orthotopic RencaHAM.
A. Macroscopic appearance of BALB/c lungs at 21 days after intra-renal injection of RencaHAM.
Puncture site from adoptive transfer seen at cardiac apex. B. Histological section of lung parenchyma
(H&E, x20) from the same mouse. Experiment was repeated on two additional mice, with similar results.
6 .3 . Discussion
In this chapter, the derivation of a new RencaHA clone (RencaHAM) was explained,
together with the properties of that clone. The characteristics of Renca, RencaHA, and
RencaHAM afforded numerous insights into the workings of the immune response
against solid tumour. When RencaHA was grown subcutaneously, 80% of mice lost HA
expression. That finding was consistent with immune escape,245
where CD8+ T cells
effectively target antigenic clones and negatively select against those clones, producing
antigen-negative tumour outgrowth. Moreover, when immune escape could not occur
(i.e. when a stable HA expressing RencaHAM cell line was inoculated), tumours could
not be transplanted. That failure to transplant was not explained by physical (e.g. poor
viability) or nutritional (e.g. poor blood supply) factors because transplantation was
successful in athymic BALB/c mice.
While RencaHAM did not grow subcutaneously, it did cross present HA for somewhere
between 3 and 4 weeks. As such, subcutaneous RencaHAM provided a means of
administering HA antigen without inducing a tumour or requiring the use of virus. In
this regard, in Chapter 4 of this thesis, subcutaneous RencaHAM was used to provide
HA antigen persistence without tumour persistence in studies of sinecomitant immunity.
- 177 -
Figure 6.10. Evidence of nodal invasion from RencaHAM.
A. mediastinal lymph node from a mouse 21 days after tail vein injection of RencaHAM (H&E section,
x10). B. Culture of total node pool of BALB/c mice, 21 days after tail vein injection of RencaHAM
(phase microscopy, x10). C. Renal lymph node of BALB/c mouse, 21 days after intra-renal injection of
RencaHAM (H&E section, x10). D. RT-PCR for HA transgene in total node pools of BALB/c mice at 21
days after transplantation of RencaHAM via intra-cardiac, intra-renal, or tail vein injection. Fluorescence
shown with reference to positive (RencaHAM and AB1HA culture) and negative (RencaWT, AB1, and
RNase free H2O) controls. Results were similar in both mice processed for each group (intra-cardiac, tail
vein, and intra-renal). E. Summary of PCR, histology and culture evidence of lymph node metastases with
RencaHAM. Culture and PCR was performed on total pooled nodes of each assayed animal (pools
included: mediastinal, renal, para-aortic, inguinal, axillary, cervical), whereas histology was performed on
nodes separately (separate nodes assayed: mediastinal, renal, para-aortic).
- 178 -
RencaHAM could also be used to model disseminated renal cancer. When RencaHAM
was administered by intravenous injection (tail vein or intra-cardiac), rapidly
progressive, diffusely metastatic disease was apparent. Lymph node invasion was
included in the metastatic profile of intravenous RencaHAM, providing the opportunity
to examine the immune function of lymph nodes when invaded by tumour. However,
intravenous RencaHAM disease was not amenable to surgery – multiple pulmonary
deposits and extensive seeding are not resectable.
For this reason, orthotopic (intra-renal) RencaHAM was probably the real niche of
RencaHAM. In this chapter, RencaHAM could be reliably transplanted intrarenally,
producing contralateral renal, splenic, hepatic and pulmonary metastasis. In future
studies, the tumour-specific response to RencaHAM could be tracked in vivo and
disease burden could be quantified by PCR for HA transgene.
There were differences in RencaHAM growth patterns across the various methods of
transplantation. This different behaviour may have reflected not only the difference in
blood and nutrient supply of each implantation site, but also that the tumour may have
expressed different phenotypes according to its location (e.g. type IV collagenase and
EGFR expression).317
The central question of whether small metastases engage the immune system was also
be addressed in the orthotopic RencaHAM system. As discussed in Chapter 3, tumour
antigen presentation is the obligatory priming signal to the anti-tumour immune
response. Without sufficient antigen priming, there can be no effector differentiation,
CD8+ proliferation, nor effective memory development.
347,351 As a corollary, active
immune therapies, which require an endogenous immune response against the tumour,
will be unsuccessful if priming is inadequate. In renal cancer, one third of patients who
undergo „curative‟ surgery still ultimately progress.12
This suggests a proportion of
patients have microscopic residual tumour after clinically complete resection in renal
cancer. To date, it remained unclear whether those patients had sufficient antigen, or
whether antigen priming was a limiting factor in micrometastatic disease.
In the past, others have proposed that the immune system is not ignorant of small
metastases.198
Indeed, in Chapter 3 of this thesis, cross presentation was identified at
four days after subcutaneous inoculation, where tumours were frequently too small to be
- 179 -
seen or palpated. However, to determine whether micrometastases cross present antigen
definitively, true micrometastases must be studied. Tumours that metastasise normally
go through a cascade of phenotypic changes that enable them to detach from one
another,417,418
invade the extracellular matrix,419
access blood vessels,419
attach to
vessels at distant sites, extravasate, and acquire necessary properties to establish a new
colony.420
In parallel with those phenotypic changes may be the downregulation of antigen
presentation and other immunological changes. Tumour injections into the skin or
tumours that embolise from intravenous injection do not transit through many of these
steps. Therefore, the immunological characteristics of those tumour models may not
relate to the properties of a true metastasis.
In this chapter, RencaHAM was injected into the kidney, which was the orthotopic
location for this tumour. Left to grow in its natural environment, RencaHAM
metastasised to the renal and para-aortic nodes, the spleen, contralateral kidney and
lungs. Those tumours represented true secondaries, whose biology should parallel
metastases from primary renal cancer in humans. As such, at 21 days after intra-renal
inoculation, micrometastases were present in the lung (proven on histology). In that
setting, micrometastases were associated with robust antigen presentation in the
mediastinal lymph nodes. Whether that antigen was cross presented or directly
presented remained uncertain, although the apparent absence of mediastinal node
invasion suggested the former. To prove cross presentation was the mechanism, it
would be helpful to perform PCR surveys of the mediastinal nodes to exclude
metastases to those sites. Similarly, a bone marrow chimera study would be useful.
There was insufficient time for the candidate to undertake such experiments.
Furthermore, while the data of this chapter suggested antigen presentation was highly
efficient from micrometastatic disease, work was limited to the study of a proliferative
response in tumour-specific CD8+ to cross presentation. This is only one component of
priming. It is still possible those CD8+ cells, while proliferating, were not getting the
necessary second signals and the cytokine milieu to enable them to form capable
effectors. Thus while micrometastases do present antigen adequately, the quality of
priming might be limiting. By extension, while patients may not require additional
quantities of antigen after surgery, they may benefit from strategies that augment the
- 180 -
quality of that antigen priming (e.g. tumour cell-DC hybridomas,421
gene-modified
tumour vaccines,422
and HSP-antigen complexes etc.).423,424
While micrometastastic disease may insufficiently prime the immune response, efficient
antigen presentation from micrometastases might impair sinecomitant immunity. As
seen in Section 4.2.9, enhanced post-operative tumour resistance requires a fall in
antigen presentation, or “antigen holiday”. If micrometastases present antigen
efficiently, this may lessen the immune benefit of cancer resection. It is still possible
macroscopically complete resection may provide an adequate decline in antigen
presentation, even in the presence of tumour antigen presentation from occult
metastases. To answer this question, the RencaHAM model may prove useful (see also,
7.3).
In future studies, nephrectomies of orthotopic RencaHAM could be undertaken to
render animals with “minimal residual disease” renal cancer. In that setting, the question
as to whether tumour immunity improves despite microscopic disease could be tested.
IYSTVASSL-MHC pentameric staining (Pro5®, ProImmune), Lyons Parish analysis321
and in vivo CTL assays323
could be used to assess tumour-specific immunity in vivo and
metastatic burden could be objectively quantified using RT-PCR for the HA transgene.
6 .4 . Summary
In this chapter, the properties of a stable HA-expressing RencaHAM clone were
expounded. This model may prove useful in future studies of combined surgery/immune
therapy strategies for renal cancer. Using RencaHAM, objective tracking of the tumour-
specific CD8+ response, cross presentation, and metastatic burden would be possible. In
addition, this model has provided three key insights into the workings of the anti-
tumour immune response. Firstly, loss of HA expression with subcutaneous growth of
RencaHAM suggested the immune system can “edit” tumours to select antigenically
negative variants. This issue adds weight to the importance of using whole cell vaccines
rather than single peptide preparations (Chapters 1 and 7), because boosting the
response to one antigen may be associated with loss of that antigen and resistance to
therapy.
- 181 -
Secondly, antigen presentation was preserved, even when nodes were invaded with
tumour. This was consistent with Chapter 5, where invaded lymph nodes had
comparable antigen presentation and in vivo CTL function to intact nodes. Finally, it
was found that antigen presentation was robust, even with minimal residual
“micrometastatic” disease. Thus patients with micrometastases may not benefit from
additional antigen per se. Rather strategies to improve the quality of priming (e.g. DC-
tumour cell hybridomas421
) or boosting the effector arm of the immune response (e.g.
cytokine therapy) may be more appropriate.
The key findings of this thesis are now summarised. Based on these findings, an
empirical framework for the combination of surgery with immune therapy is
synthesised.
- 182 -
Chapter 7
- 183 -
7. Thesis Summary
Patients with locally aggressive and metastatic tumours continue to fare poorly,369
and
cancer remains the second most common cause of death in western nations.425
This
thesis advocates and facilitates a new treatment paradigm for malignancy: combined
surgery/immune therapy.3
The concept of combining surgery with immune therapy is not without danger, because
surgery can expose the patient to morbidity and mortality in itself. Indeed, operative
morbidity may preclude that patient from the conventional treatments or immune
therapy they would otherwise receive.112,113,415
A second difficulty with combining
surgery and immune therapy is the prevailing dogma that surgery is immune
suppressive.29,30,73
As such, surgery could impair the response to immune therapy rather
than improve it.
While it is acknowledged that surgery may be generally immune suppressive, in this
thesis it has been argued that surgery can boost the tumour-specific component of
immunity. The concept of sinecomitant immunity was highlighted, a phenomenon that
has been absent from the literature for some thirty years. As such, the benefit of tumour
resection was identified as the excess of sinecomitant immunity (post-operative tumour
resistance)25,106,107
relative to concomitant immunity (tumour resistance without primary
resection).24,25,105
Difficulties aside, a central factor that limits the uptake of combined surgery/immune
therapy strategies is the lack of understanding about how surgery impacts on tumour-
specific immunity. Until these effects can be elucidated, the logic for planning
combined surgery/immune therapy strategies will remain unclear. This thesis has
rectified some of the defects in existing knowledge about surgery and tumour immunity,
by providing insights into the effects of surgery on antigen processing, CTLs, and
overall tumour resistance. It is hoped those insights will aid in the planning of combined
surgery/immune therapy strategies in the future, ultimately improving results for
patients.
- 184 -
7 .1 . Principal Findings
7 . 1 . 1 . Effects of surgery on ant igen presentat ion
The pre-operative and post-operative kinetics of tumour antigen presentation were
examined in the RencaHAM and/or AB1HA tumour models. It was demonstrated that
HA antigen is presented to CTLs in a highly efficient manner. In particular, HA
presentation was detectable in association with tiny primary lesions and
micrometastases. This implied that the immune system was not ignorant of small
primary tumours and micrometastases.
HA presentation was also confined to the sentinel lymph nodes for all stages of tumour
growth, and increased in parallel with tumour burden. Conversely after surgery, there
was a decline in HA presentation, until it was no longer detectable at two weeks post-
operatively. As was the case pre-operatively, HA presentation was confined to the
tumour draining nodes for all time points after surgery. The localisation of tumour
antigen presentation suggested a role for locally delivered immune strategies, exploiting
the sentinel node reservoir of antigen in the early post-operative phase.
7 . 1 . 2 . Surgery & tumour -speci f ic CTLs
Given the dependence of CTLs on tumour antigen processing, it was unsurprising that
HA specific CTL would co-localise with antigen presentation. HA-specific CTL and
HA-specific in vivo CTL activity were disproportionately represented in the sentinel
lymph glands of tumour bearing hosts. After surgery, there was a decline in antigen
presentation in the tumour draining lymph nodes, and this was associated with enhanced
HA-specific in vivo CTL and systemic egress of that CTL activity. Conceptually, it was
postulated that chemokine release and/or the continuous stream of antigen from the
tumour might “tether” tumour-specific CTL into the draining lymph gland. Surgery
could cut that “tether”, enabling improved and systemic tumour-specific CTL function.
Attempts were made to characterise the phenotype of HA specific CTL before and after
surgery. However, there were no obvious differences in memory characteristics of the
pre-operative and post-operative HA-specific CD8+ repertoire.
- 185 -
7 . 1 . 3 . Sent inel lymph nodes & ant i - tumour immunity
The sentinel lymph nodes provided a pool of tumour-specific CD8+. They were also the
site where the decline in tumour antigen presentation was seen after surgery (7.1.1).
Accordingly, when the sentinel lymph glands were removed concurrent with the
tumour, tumour antigen presentation was completely and permanently ablated. Given
the localisation of tumour-specific effectors in the sentinel node, it was predicted that
sentinel node biopsy would be detrimental to tumour-specific immunity. Indeed,
removing the sentinel nodes on the day of surgery almost completely ablated the anti-
tumour immune benefit of primary resection. It was envisaged that sentinel node
sampling and/or delaying the sentinel node procedure might preserve the pool of
tumour-specific CTL, as these cells were thought to gradually egress from the sentinel
nodes after surgery. This was the case.
7 . 1 . 4 . Propert ies of s inecomitant immunity
Sinecomitant immunity seemed most successful when the tumour re-challenge was
temporally and spatially distant to the cancer surgery itself. Unfortunately, patients that
have incomplete resection will have tumour “re-challenges” occurring on the same day
as the surgery. Hence strategies that boost sinecomitant immunity in the early post-
operative phase are desirable (including TLR ligands and activating anti-CD40 mAb, as
demonstrated in this thesis).
The inherent susceptibility of the surgical site to recurrence was worrying. Poor tumour
resistance at the surgical site was seen repeatedly, and inferior responses to immune
therapy were also observed in that location. Surgical wounding could partly explain that
phenomenon, and wounds have previously been reported to contain significant
quantities of immune suppressive cytokines.120
However, local tumour suppression
vulnerability may also relate to the accumulation of specific cells. In the AB1HA
model, Treg had an active role in impairing tumour resistance of the surgical site, but
there was no difference in plasmacytoid DC concentrations between tumour draining
and systemic nodes.
Fundamentally, sinecomitant immunity was dependent on the three, logically linked
factors outlined above: the decline in tumour antigen presentation (7.1.1), CTLs (7.1.2)
and the sentinel lymph nodes (7.1.3). If any of these factors was missing, then
sinecomitant immunity was poor. These findings introduce a tone of caution for the
- 186 -
practice of sentinel node biopsy, and raise the possibility that tumour vaccines (which
boost tumour antigen) may be contraindicated in the early post-operative phase. The
role of CTLs in tumour-specific immunity was also highlighted, indicating that CD8+-
targeted therapy might be fruitful.
7 .2 . Conclus ions
Provided the appropriate requirements are met, surgery may provide a post-operative
“window of opportunity” for effective immune treatments (Figure 7.1). In the early
post-operative phase, CD8+ targeted therapies may be most rewarding – given the
critical role of these cells in tumour resistance. Such therapies might be delivered
locally (i.e. into the tumour bed) where they can be absorbed into the lymphatics and
exert activity on the sentinel nodes (where CTL are initially located).
In the intermediate post-operative period, in vivo CTL function is found systemically.
Thus systemic modes of administration for CD8+ boosting therapies might be best. Such
therapies could be combined with Treg depleting treatments (e.g. cyclophosphamide)372
since Treg actively hamper tumour immunity. In addition, while tumour antigen decline
is initially required for enhancements in CTL function, it might be that tumour antigen
becomes limiting in the adaptive immune response. It may then be necessary to
administer a tumour vaccine, augmenting priming and boosting CD8+ immunity.
At delayed time points after surgery, patients may present with bulky recurrent primary
disease and/or metastases. At that stage, it is likely tumours have re-constituted their
immune suppressive networks,27
and strategies to disrupt those networks (e.g.
cytoreduction surgery) may be paramount. Additional treatments, such as anti-VEGF
antibody (e.g. Bevacizumab, AvastinTM
, Genentech Inc, South San Francisco, California
USA) might be helpful, because these could inhibit tumour associated suppressive
signals at the molecular level. The immune intervention itself may also need to be
complex at this stage.28
- 187 -
Figure 7.1. The window of opportunity for post-operative immune therapy.
Surgery decreased tumour antigen presentation (see Chapter 3) and presumably, tumour derived soluble factors. In addition, CTL were confined to the sentinel nodes before surgery
but increased and spread systemically post resection (Chapter 3). CTL were integral to tumour immunity and post-operative changes correlated with enhanced tumour resistance
(Chapter 4). These findings suggest a role for locally delivered, CTL boosting therapies in the early post-operative phase (as demonstrated in Chapter 4) and a systemic approach in
the intermediate post-operative phase. In the clinical situation, patients may develop post-operative recurrence and metastases, with re-constitution of tumour antigen burden
suppression networks. In that setting, it may be necessary to again disrupt the immune suppressive network (e.g. by surgery or chemotherapy) and/or use a multi-faceted immune
intervention.
- 188 -
7 .3 . Future Direc t ions
In this thesis, the in vivo effects of primary resection and sentinel node biopsy upon
tumour immunity were described. It is hoped these findings will provide a logical
framework for the combination of surgery and immune therapy in the treatment of
malignancy. As such, it is anticipated that combined surgery/immune therapy strategies
(examples in Chapter 4) will become standard practice into the future. Indeed, there is
already clinical trial evidence for the combination of surgery and immune therapy in the
treatment of both clinically localised12,237
and advanced renal cancer.14
A major emphasis of this project was to correlate primary resection with certain
endpoints including: tumour resistance, CTL function, and antigen presentation (see
Chapters 3 and 4). In this situation, complete tumour clearance ablated antigen
presentation and correlated with an improvement in CTL function in vivo, and tumour
resistance. Another clinically important question would be whether antigen presentation
is sufficiently ablated after incomplete primary resection (i.e. debulking surgery), and/or
in the setting of residual micrometastases (i.e. “minimal residual disease”) to improve
tumour immunity. The RencaHAM model (Chapter 6) should be a useful tool to address
this question.
A second focus of this thesis was the sentinel lymph nodes and their contribution to
tumour immunity in vivo (Chapter 5). It was suggested that sentinel nodes contribute
positively to tumour immunity in vivo, and that removing the sentinel nodes could be
detrimental to tumour resistance. However, sentinel nodes may be removed because
they have intermediate or high probability of being involved with tumour, thus their
prognostic value. The experiments of Chapter Five would suggest tumour-invaded
nodes can function well in the proliferation of tumour-specific CD8+ and in the
clustering of tumour-specific CTL lysis. However, it remains unknown whether
removing a sentinel node is detrimental to tumour immunity, if that node is positive for
tumour. In future studies, it would be possible to assess the effect of removing a tumour
invaded sentinel node on post-operative tumour resistance, using the intra-nodal
injection method in AB1HA (see 2.3.5, 5.2.4) or with the RencaHAM model (6.2.4),
which metastasises to the nodes.
- 189 -
Additionally, if sentinel lymphadenectomy is detrimental to tumour resistance,
investigating mechanisms of avoiding that harm would be desirable. A previous study
has shown that activating anti-CD40 mAb may flush tumour-specific CD8+ from
tumour draining nodes155
and into systemic lymph nodes (Figure 5.15). Determining
whether anti-CD40 mAb can lessen the “immune harm” of sentinel lymphadenectomy
would therefore be of clinical interest.
Finally, there is clearly a complex relationship between tumour antigen presentation and
CD8+ function (Chapters 3 and 4). Based on findings from virology, it is possible that
tumour antigen persistence leads to the upregulation of PD-1, and CD8+ anergy.
297,300
Certain tumours (e.g. renal cancer)414
may compound this anergy, by expressing B7-H1
and thereby directly ligating CD8+ PD-1. It is postulated that PD-1/PD-1L signalling
will be important in tumour immunology, and that surgery might provide a mechanism
of lessening PD-1 activation. Surgery may also prove synergistic with PD-1/PD-1L
blockade in recovering tumour-specific CD8+ function in vivo. A Master of Surgery
student at The University of Western Australia is now testing this hypothesis, using
RencaHAM (described in this thesis – Chapter 6).
- 190 -
Appendix A: References
1. Hewitt M, Breen N, Devesa S. Cancer prevalence and survivorship issues:
analyses of the 1992 National Health Interview Survey. J Natl Cancer Inst 1999;
91: 1480-6
2. Bailar J, Gornik H. Cancer undefeated. N Engl J Med 1997; 336: 1569-74
3. Morton DL, Ollila DW, Hsueh EC, Essner R, Gupta RK. Cytoreductive
surgery and adjuvant immunotherapy: a new management paradigm for
metastatic melanoma. CA-Cancer J Clin 1999; 49: 101-16, 65
4. Boutin C, Nussbaum E, Monnet I, Bignon J, Vanderschueren R, Guerin JC,
Menard O, Mignot P, Dabouis G, Douillard JY. Intrapleural treatment with
recombinant gamma-interferon in early stage malignant pleural mesothelioma.
Cancer 1994; 74: 2460-7
5. Robinson BW, Mukherjee SA, Davidson A, Morey S, Musk AW, Ramshaw
I, Smith D, Lake R, Haenel T, Garlepp M, Marley J, Leong C, Caminschi I,
Scott B. Cytokine gene therapy or infusion as treatment for solid human cancer.
J Immunother 1998; 21: 211-7
6. Powell A, Creaney J, Broomfield S, Van Bruggen I, Robinson B.
Recombinant GM-CSF plus autologous tumor cells as a vaccine for patients
with mesothelioma. Lung Cancer 2006; 52: 189-97
7. Curti B. Renal cell carcinoma. JAMA 2004; 292
8. Fossa S. Interferon in metastatic renal cell carcinoma. Sem Oncol 2000; 27: 187-
93
9. Figlin R, Thompson J, Bukowski R, Vogelzang N, Novic A, Lange P,
Steinberg G, Belldegrun A. Multicenter, randomized, phase III trial of CD8(+)
tumor-infiltrating lymphocytes in combination with recombinant interleukin-2 in
metastatic renal cell carcinoma. J Clin Oncol 1999; 17: 2521-9
10. Mukherjee S, Nelson D, Loh S, van Bruggen I, Palmer LJ, Leong C,
Garlepp MJ, Robinson BW. The immune anti-tumor effects of GM-CSF and
B7-1 gene transfection are enhanced by surgical debulking of tumor. Cancer
Gene Ther 2001; 8: 580-8
11. Broomfield S, Currie A, van der Most RG, Brown M, van Bruggen I,
Robinson BW, Lake RA. Partial, but not complete, tumor-debulking surgery
promotes protective antitumor memory when combined with chemotherapy and
adjuvant immunotherapy. Cancer Res 2005; 65: 7580-4
12. Repmann R, Goldschmidt A, Richter A. Adjuvant therapy of renal cell
carcinoma patients with an autologous tumor cell lysate vaccine: a 5-year
follow-up analysis. Anticancer Res 2003; 23: 969-74
13. Repmann R, Wagner S, Richter A. Adjuvant therapy of renal cell carcinoma
with active-specific-immunotherapy (ASI) using autologous tumour vaccine.
Anticancer Res 1997; 17: 2879-82
14. Flanigan R, Mickisch G, Sylvester R, Tangen C, Van Poppel H, Crawford
D. Cytoreductive nephrectomy in patients with metastatic renal cancer: a
combined analysis. J Urol 2004; 171: 1071 - 6
15. Bromwich E, Hendry D, Aitchison M. Cytoreductive nephrectomy: is it a
realistic option in patients with renal cancer? Br J Urol 2002; 89: 523-5
16. Flanigan R, Salmon M, Blumenstein B, Bearman S, Roy V, McGrath P,
Caton J, Munshi N, Crawford D. Nephrectomy followed by interferon alfa-2b
alone for metastatic renal cell cancer. N Engl J Med 2001; 345: 1655 - 9
- 191 -
17. Fallick M, McDermott D, LaRock D, Long J, Atkins M. Nephrectomy before
interleukin-2 therapy for patients wtih metastatic renal carcinoma. J Urol 1997;
158: 1691 - 5
18. Walther M, Yang J, Pass H, Linehan W, Rosenberg S. Cytoreductive surgery
before high dose Il-2 based therapy in patients with metastatic renal cancer. J
Urol 1997; 158: 1675-8
19. Weigel BJ, Rodeberg DA, Krieg AM, Blazar BR. CpG oligodeoxynucleotides
potentiate the antitumor effects of chemotherapy or tumor resection in an
orthotopic murine model of rhabdomyosarcoma. Clin Cancer Res 2003; 9: 3105-
14
20. Marzo A, Lake R, Lo D, Sherman L, McWilliam A, Nelson D, Robinson B,
Scott B. Tumor antigens are constitutively expressed in the draining lymph
nodes. J Immunol 1999; 162: 5838 - 45
21. Cochran AJ. Melanoma metastases through the lymphatic system. Surg Clin
North Am 2000; 80: 1683-93
22. Cochran AJ, Huang RR, Lee J, Itakura E, Leong SP, Essner R. Tumour-
induced immune modulation of sentinel lymph nodes. Nat Rev Immunol 2006; 6:
659-70
23. Woglom W. Immunity to transplantable tumors. The Cancer Review 1929; 4:
129-214
24. Gorelik E. Concomitant tumor immunity and the resistance to a second tumor
challenge. Adv Cancer Res 1983; 39: 71-120
25. Fisher B, Saffer E, Fisher E. Comparison of concomitant and sinecomitant
immunity. Proc Soc Exp Biol Med 1970; 135: 68-71
26. Ben-Eliyahu S. The promotion of tumor metastasis by surgery and stress:
immunological basis and implications for psychoneuroimmunology. Brain
Behav Immun 2003; 17 Suppl 1: S27-36
27. Kim R, Emi M, Tanabe K, Arihiro K. Tumor-driven evolution of
immunosuppressive networks during malignant progression. Cancer Res 2006;
66: 5527-36
28. Gattinoni L, Finkelstein SE, Klebanoff CA, Antony PA, Palmer DC, Spiess
PJ, Hwang LN, Yu Z, Wrzesinski C, Heimann DM, Surh CD, Rosenberg
SA, Restifo NP. Removal of homeostatic cytokine sinks by lymphodepletion
enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J
Exp Med 2005; 202: 907-12
29. Vittimberga FJ, Jr., Foley DP, Meyers WC, Callery MP. Laparoscopic
surgery and the systemic immune response. Ann Surg 1998; 227: 326-34
30. Hackam DJ, Rotstein OD. Host response to laparoscopic surgery: mechanisms
and clinical correlates. Can J Surg 1998; 41: 103-11
31. Shiromizu A, Suematsu T, Yamaguchi K, Shiraishi N, Adachi Y, Kitano S.
Effect of laparotomy and laparoscopy on the establishment of lung metastasis in
a murine model. Surgery 2000; 128: 799-805
32. Da Costa ML, Redmond P, Bouchier-Hayes DJ. The effect of laparotomy and
laparoscopy on the establishment of spontaneous tumor metastases. Surgery
1998; 124: 516-25
33. Lundy J, Lovett EJ, 3rd, Wolinsky SM, Conran P. Immune impairment and
metastatic tumor growth: the need for an immunorestorative drug as an adjunct
to surgery. Cancer 1979; 43: 945-51
34. Yamaguchi K, Takagi Y, Aoki S, Futamura M, Saji S. Significant detection
of circulating cancer cells in the blood by reverse transcriptase-polymerase chain
reaction during colorectal cancer resection. Ann Surg 2000; 232: 58-65
- 192 -
35. Braun S, Pantel K, Muller P, Janni W, Hepp F, Kentenich CR, Gastroph S,
Wischnik A, Dimpfl T, Kindermann G, Riethmuller G, Schlimok G.
Cytokeratin-positive cells in the bone marrow and survival of patients with stage
I, II, or III breast cancer. N Engl J Med 2000; 342: 525-33
36. Terhune M, Swanson N, Johnson M. Use of chest radiography in the initial
evaluation of patients with localized melanoma. Arch Dermatol 1998; 134: 569-
72
37. Israeli R, Miller W, Su S, Samadi D, Powell C, Heston W, Wise G, Fair W.
Sensitive detection of prostatic hematogenous tumor cell dissemination using
prostate specific antigen and prostate specific membrane-derived primers in
polymerase chain reaction. J Urol 1995; 153: 573-7
38. Loric S, Dumas F, Eschwege P, Blanchet P, Benoit G, Jardin A, Lacour B.
Enhanced detection of hematogenous circulating prostatic cells in patients with
prostate adenocarcinoma by using nested reverse transcription polymerase chain
reaction assay based on prostate-specific membrane antigen. Clin Chem 1995;
41: 1698-704
39. Jakóbisiak M, Lasek W, Golab J. Natural mechanisms protecting against
cancer. Immunol Lett 2003; 90: 103-22
40. Uhr J, Scheuermann R, Street N, Vitetta E. Cancer dormancy: opportunities
for new therapeutic approaches. Nat Med 1997; 3: 505-9
41. Karrison T, Ferguson D, Meier P. Dormancy of mammary carcinoma after
mastectomy. J Natl Cancer Inst 1999; 91: 80-5
42. Montie J, Stewart B, Straffon R, Banowsky L, Hewitt C, Montague D. The
role of adjunctive nephrectomy in patients with metastatic renal cell carcinoma.
J Urol 1977; 117: 272-5
43. Chang W. Complete spontaneous regression of cancer: four case reports, review
of literature, and discussion of possible mechanisms involved. Hawaii Med J
2000; 59: 379-87
44. Rosenberg SA, Fox E, Churchill W. Spontaneous regression of hepatic
metastases from gastric carcinoma. Cancer 1972; 29: 472-4
45. Robinson BW, Robinson C, Lake RA. Localised spontaneous regression in
mesothelioma -- possible immunological mechanism. Lung Cancer 2001; 32:
197-201
46. Sinha P, Clements VK, Ostrand-Rosenberg S. Reduction of myeloid-derived
suppressor cells and induction of M1 macrophages facilitate the rejection of
established metastatic disease. J Immunol 2005; 174: 636-45
47. Almand B, Resser JR, Lindman B, Nadaf S, Clark JI, Kwon ED, Carbone
DP, Gabrilovich DI. Clinical significance of defective dendritic cell
differentiation in cancer. Clin Cancer Res 2000; 6: 1755-66
48. Benigni F, Zimmermann VS, Hugues S, Caserta S, Basso V, Rivino L,
Ingulli E, Malherbe L, Glaichenhaus N, Mondino A. Phenotype and homing
of CD4 tumor-specific T cells is modulated by tumor bulk. J Immunol 2005;
175: 739-48
49. Hoption Cann S, van Netten J, van Netten C, Glover D. Spontaneous
regression: a hidden treasure buried in time. Med Hypotheses 2002; 58: 115-9
50. Verneuil A. Inoculation of erysipelas as a means of cure. Union Med (Paris)
1886; 41: 19-22, 217-22
51. Heckelsmiller K, Beck S, Rall K, Sipos B, Schlamp A, Tuma E,
Rothenfusser S, Endres S, Hartmann G. Combined dendritic cell- and CpG
oligonucleotide-based immune therapy cures large murine tumors that resist
chemotherapy. Eur J Immunol 2002; 32: 3235-45
- 193 -
52. Hoption Cann S, Van Netten J, Van Netten C. Dr William Coley and tumour
regression: a place in history or in the future. PMJ 2003; 79: 672-80
53. Heckelsmiller K, Rall K, Beck S, Schlamp A, Seiderer J, Jahrsdorfer B,
Krug A, Rothenfusser S, Endres S, Hartmann G. Peritumoral CpG DNA
elicits a coordinated response of CD8 T cells and innate effectors to cure
established tumors in a murine colon carcinoma model. J Immunol 2002; 169:
3892-9
54. Hersey P, Coates A, McCarthy W, Thompson JF, Sillar R, McLeod R, Gill
P, Coventry BJ, McMullen A, Dillon H, Simes R. Adjuvant immunotherapy
of patients with high-risk melanoma using vaccinia viral lysates of melanoma:
results of a randomized trial. J Clin Oncol 2002; 20: 4181-90
55. Sauer G, Kurzeder C, Heilmann V, Kreienberg R, Deissler H.
Immunotherapy and cancer vaccines in the management of cancer. Curr Pharm
Des 2005; 11: 3475-83
56. Harris J, Ryan L, Hoover H, Stuart R, Oken MM, Bensen Ar, Mansour E,
Haller D, Manola J, Hanna MJ. Adjuvant active specific immunotherapy for
stage II and III colon cancer with an autologous tumor cell vaccine: Eastern
Cooperative Oncology Group Study E5283. J Clin Oncol 2000; 18: 148-57
57. Kwon ED, Foster B, Hurwitz A, Madias C, Allison JP, Greenberg N, Burg
M. Elimination of residual metastatic prostate cancer after surgery and
adjunctive cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade
immunotherapy. Immunology 1999; 96: 15074-9
58. Hirschowitz E, Foody T, Krysco R, Dickson L, Sturgill J, Yannelli J.
Autologous dendritic cell vaccines for non-small-cell lung cancer. J Clin Oncol
2004; 22: 2808-15
59. Ascierto P, Palmieri G, Strazzullo M, Daponte A, Botti G, Satriano S, Motti
M, Mozillo N, Castello G. Low doses interferon-a in the treatment of high-risk
cutaneous melanoma. Ann Oncol 2000; 11: 487-90
60. Astoul P, Picat-Joossen D, Viallat JR, Boutin C. Intrapleural administration of
interleukin-2 for the treatment of patients with malignant pleural mesothelioma:
a Phase II study. Cancer 1998; 83: 2099-104
61. Bier J, Rapp H, Borsos T, Zbar B, Kleinschuster S, Wagner H, Röllinghoff
M. Randomized clinical study on intratumoral BCG-cell wall preparation
(CWP) therapy in patients with squamous cell carcinoma in the head and neck
region. Cancer Immunol Immunother 1995; 12: 71-9
62. Bakker W, Nijhuis-Heddes J, van der Velde E. Post-operative intrapleural
BCG in lung cancer: a 5-year follow-up report. Cancer Immunol Immunother
1986; 22: 155-9
63. Mitchell M, Abrams J, Thompson J, Kashani-Sabet M, DeConti R, Hwu W,
Atkins M, Whitman E, Ernstoff M, Haluska FG, Jakowatz J, Das Gupta T,
Richards JM, Samlowski W, Costanzi J, Aronson F, Deisseroth A, Dudek
A, Jones V. Randomized trial of allogeneic melanoma lysate vaccine with low-
dose interferon Alfa-2b compared with high-dose interferon Alfa-2b for resected
state III cutaneous melanoma. J Clin Oncol 2007; 25: 2078-85
64. Brivio F, Lissoni P, Alderi G, Barni S, Lavorato F, Fumagalli L.
Preoperative interleukin-2 subcutaneous immunotherapy may prolong the
survival time in advanced colorectal cancer patients. Oncology 1996; 53: 263-8
65. Vermoken J, Claessen A, van Tinteren H, Gall H, Ezinger R, Meijer S,
Scheper R, Meijer C, Bloemena E, Ransom J, Hanna M, Pinedo H. Active
specific immunotherapy for stage II and stage III human colon cancer: a
randomised trial. Lancet 1999; 353: 345 - 50
- 194 -
66. Berzofsky J, Terabe M, Oh S, Belyakov I, Ahlers J, Janik J, Morris J.
Progress on new vaccine strategies for the immunotherapy and prevention of
cancer. J Clin Invest 2004; 113: 1515 - 25
67. O'Donnell M. Practical applications of intravesical chemotherapy and
immunotherapy in high-risk patients with superficial bladder cancer. Urol Clin
North Am 2005; 32: 121-31
68. Davies MG, Hagen PO. Systemic inflammatory response syndrome. Br J Surg
1997; 84: 920-35
69. Hunter J. Effects of anaesthesia on the human immune system. Hosp Med
1999; 60: 658-63
70. Burrows L, Tartter P. Effect of blood transfusions on colonic malignancy
recurrent rate. Lancet 1982; 2: 662
71. Tartter PI, Burrows L, Kirschner P. Perioperative blood transfusion adversely
affects prognosis after resection of Stage I (subset N0) non-oat cell lung cancer.
J Thorac Cardiovasc Surg 1984; 88: 659-62
72. Tartter PI, Burrows L, Papatestas AE, Lesnick G, Aufses AH, Jr.
Perioperative blood transfusion has prognostic significance for breast cancer.
Surgery 1985; 97: 225-30
73. Novitsky YW, Litwin DE, Callery MP. The net immunologic advantage of
laparoscopic surgery. Surg Endosc 2004; 18: 1411-9
74. Chambrier C, Chassard D, Bienvenu J, Saudin F, Paturel B, Garrigue C,
Barbier Y, Bouletreau P. Cytokine and hormonal changes after
cholecystectomy. Effect of ibuprofen pretreatment. Ann Surg 1996; 224: 178-82
75. Elenkov IJ, Wilder RL, Chrousos GP, Vizi ES. The sympathetic nerve - an
integrative interface between two supersystems: the brain and the immune
system. Pharmacol Rev 2000; 52: 595-638
76. Bodner G, Ho A, Kreek MJ. Effect of endogenous cortisol levels on natural
killer cell activity in healthy humans. Brain Behav Immun 1998; 12: 285-96
77. Biffl WL, Moore EE, Moore FA, Peterson VM. Interleukin-6 in the injured
patient. Marker of injury or mediator of inflammation? Ann Surg 1996; 224:
647-64
78. Baumann H, Gauldie J. The acute phase response. Immunol Today 1994; 15:
74-80
79. Kolsen-Petersen JA, Nielsen JO, Tonnesen EM. Effect of hypertonic saline
infusion on postoperative cellular immune function: a randomized controlled
clinical trial. Anesthesiology 2004; 100: 1108-18
80. Wakefield CH, Carey PD, Foulds S, Monson JR, Guillou PJ.
Polymorphonuclear leukocyte activation. An early marker of the postsurgical
sepsis response. Arch Surg 1993; 128: 390-5
81. Yakar I, Melamed R, Shakhar G, Shakhar K, Rosenne E, Abudarham N,
Page GG, Ben-Eliyahu S. Prostaglandin e(2) suppresses NK activity in vivo
and promotes postoperative tumor metastasis in rats. Ann Surg Oncol 2003; 10:
469-79
82. Cristaldi M, Rovati M, Elli M, Gerlinzani S, Lesma A, Balzarotti L,
Taschieri AM. Lymphocytic subpopulation changes after open and laparoscopic
cholecystectomy: a prospective and comparative study on 38 patients. Surg
Laparosc Endosc 1997; 7: 255-61
83. Allendorf JD, Bessler M, Whelan RL, Trokel M, Laird DA, Terry MB,
Treat MR. Postoperative immune function varies inversely with the degree of
surgical trauma in a murine model. Surg Endosc 1997; 11: 427-30
- 195 -
84. Trokel MJ, Bessler M, Treat MR, Whelan RL, Nowygrod R. Preservation of
immune response after laparoscopy. Surg Endosc 1994; 8: 1385-7; discussion 7-
8
85. Lennard TW, Shenton BK, Borzotta A, Donnelly PK, White M, Gerrie LM,
Proud G, Taylor RM. The influence of surgical operations on components of
the human immune system. Br J Surg 1985; 72: 771-6
86. Akiyoshi T, Koba F, Arinaga S, Miyazaki S, Wada T, Tsuji H. Impaired
production of interleukin-2 after surgery. Clin Exp Immunol 1985; 59: 45-9
87. Lee SW, Feingold DL, Carter JJ, Zhai C, Stapleton G, Gleason N, Whelan
RL. Peritoneal macrophage and blood monocyte functions after open and
laparoscopic-assisted cecectomy in rats. Surg Endosc 2003; 17: 1996-2002
88. Schwenk W, Jacobi C, Mansmann U, Bohm B, Muller JM. Inflammatory
response after laparoscopic and conventional colorectal resections - results of a
prospective randomized trial. Langenbecks Arch Surg 2000; 385: 2-9
89. De AK, Laudanski K, Miller-Graziano CL. Failure of monocytes of trauma
patients to convert to immature dendritic cells is related to preferential
macrophage-colony-stimulating factor-driven macrophage differentiation. J
Immunol 2003; 170: 6355-62
90. Ayala A, Ertel W, Chaudry IH. Trauma-induced suppression of antigen
presentation and expression of major histocompatibility class II antigen complex
in leukocytes. Shock 1996; 5: 79-90
91. Decker D, Schondorf M, Bidlingmaier F, Hirner A, von Ruecker AA.
Surgical stress induces a shift in the type-1/type-2 T-helper cell balance,
suggesting down-regulation of cell-mediated and up-regulation of antibody-
mediated immunity commensurate to the trauma. Surgery 1996; 119: 316-25
92. Munford RS, Pugin J. Normal responses to injury prevent systemic
inflammation and can be immunosuppressive. Am J Respir Crit Care Med 2001;
163: 316-21
93. Faist E, Schinkel C, Zimmer S. Update on the mechanisms of immune
suppression of injury and immune modulation. World J Surg 1996; 20: 454-9
94. Lin E, Calvano SE, Lowry SF. Inflammatory cytokines and cell response in
surgery. Surgery 2000; 127: 117-26
95. Goshima H, Saji S, Furuta T, Taneumura H, Takao H, Kida H, Takahashi
H. Experimental study on preventive effects of lung metastases using LAK cells
induced from various lymphocytes--special references to enhancement of lung
metastasis after laparotomy stress. Nippon Geka Gakkai Zasshi 1989; 90: 1245-
50
96. Eggermont AM, Steller EP, Marquet RL, Jeekel J, Sugarbaker PH. Local
regional promotion of tumor growth after abdominal surgery is dominant over
immunotherapy with interleukin-2 and lymphokine activated killer cells. Cancer
Detect Prev 1988; 12: 421-9
97. Oka M, Hazama S, Suzuki M, Wang F, Shimoda K, Iizuka N, Wadamori K,
Suzuki T, Attwood S. Depression of cytotoxicity of nonparenchymal cells in
the liver after surgery. Surgery 1994; 116: 877-82
98. Allendorf JD, Bessler M, Kayton ML, Oesterling SD, Treat MR, Nowygrod
R, Whelan RL. Increased tumor establishment and growth after laparotomy vs
laparoscopy in a murine model. Arch Surg 1995; 130: 649-53
99. Gill GV, Prudhoe K, Cook DB, Latner AL. Effect of surgical trauma on
plasma concentrations of cyclic AMP and cortisol. Br J Surg 1975; 62: 441-3
- 196 -
100. Rosenne E, Melamed R, Abudarham N, Ben-Eliyahu S. Attenuation of the
immunosuppressive and metastasis-promoting effects of surgery by the
combined use of b-adrenergic and prostaglandin antagonists (abstract). Brain
Behav Immun 2001; 15: 180
101. Deguchi M, Isobe Y, Matsukawa S, Yamaguchi A, Nakagawara G.
Usefulness of metyrapone treatment to suppress cancer metastasis facilitated by
surgical stress. Surgery 1998; 123: 440-9
102. Koltun WA, Bloomer MM, Tilberg AF, Seaton JF, Ilahi O, Rung G, Gifford
RM, Kauffman GL, Jr. Awake epidural anesthesia is associated with improved
natural killer cell cytotoxicity and a reduced stress response. Am J Surg 1996;
171: 68-72; discussion -3
103. Allendorf JD, Bessler M, Horvath KD, Marvin MR, Laird DA, Whelan RL.
Increased tumor establishment and growth after open vs laparoscopic surgery in
mice may be related to differences in postoperative T-cell function. Surg Endosc
1999; 13: 233-5
104. Carter JJ, Feingold DL, Kirman I, Oh A, Wildbrett P, Asi Z, Fowler R,
Huang E, Whelan RL. Laparoscopic-assisted cecectomy is associated with
decreased formation of postoperative pulmonary metastases compared with open
cecectomy in a murine model. Surgery 2003; 134: 432-6
105. Bashford E, Russell B. Further evidence on the homogeneity of the resistance
to the implantation of malignant new growths. Proc R Soc Lond [Biol] 1910;
182: 298-306
106. Inaba S, Pellis NR, Kahan BD. The effect of immunotherapy with extracted
tumor antigens on sinecomitant immunity. Cancer 1983; 52: 64-9
107. Nomi S, Pellis NR, Kahan BD. Antigen-specific therapy of experimental
metastases. Cancer 1985; 55: 1296-302
108. Klein G, Sjögren H, Klein E. Demonstration of host resistance against
isotransplantation of lymphomas induced by Gross agent. Cancer Res 1962; 22:
955-61
109. Old LJ, Boyse E, Clarke D, Carswell E. Antigenic properties of chemically
induced tumors. Ann NY Acad Sci 1962; 101: 80-106
110. Riggins R, Pilch Y. Immunity to spontaneous and methylcholanthrene induced
tumors in in-bred mice. Cancer Res 1964; 24: 1994-6
111. Stjernsward J. Immune status of the primary host toward its own
methylcholanthrene-induced sarcomas. J Natl Cancer Inst 1968; 40: 13-22
112. Mickisch GH, Garin A, van Poppel H, de Prijck L, Sylvester R. Radical
nephrectomy plus interferon-alfa-based immunotherapy compared with
interferon alfa alone in metastatic renal-cell carcinoma: a randomised trial.
Lancet 2001; 358: 966-70
113. Mosharafa A, Koch M, Shalhav A, Gardner T, Logan T, Bihrle R, Foster R.
Nephrectomy for metastatic renal cell carcinoma: Indiana University experience.
Urology 2003; 62: 636-40
114. Schwartz MW, Woods SC, Porte D, Jr., Seeley RJ, Baskin DG. Central
nervous system control of food intake. Nature 2000; 404: 661-71
115. Abcouwer SF. Effects of glutamine on immune cells. Nutrition 2000; 16: 67-9
116. Folkman J. What is the evidence that tumors are angiogenesis dependent? J
Natl Cancer Inst 1990; 82: 4-6
117. Zetter BR. Angiogenesis and tumor metastasis. Annu Rev Med 1998; 49: 407-
24
118. Roberts S, Long L, Jonasson O, Mc GR, Mc GE, Cole WH. The isolation of
cancer cells from the blood stream during uterine curettage. Surg Gynecol Obstet
1960; 111: 3-11
- 197 -
119. Eschwege P, Dumas F, Blanchet P, Le Maire V, Benoit G, Jardin A, Lacour
B, Loric S. Haematogenous dissemination of prostatic epithelial cells during
radical prostatectomy. Lancet 1995; 346: 1528-30
120. Hofer SO, Shrayer D, Reichner JS, Hoekstra HJ, Wanebo HJ. Wound-
induced tumor progression: a probable role in recurrence after tumor resection.
Arch Surg 1998; 133: 383-9
121. Hayward OS. The history of oncology. I. Early oncology and the literature of
discovery. Surgery 1965; 58: 460-8
122. Santin AD, Parham GP. Routine lymph node dissection in the treatment of
early stage cancer: are we doing the right thing? Gynecol Oncol 1998; 68: 1-3
123. Cady B. Lymph node metastases. Indicators, but not governors of survival. Arch
Surg 1984; 119: 1067-72
124. Foster R, Bihrle R. Current status of retroperitoneal lymph node dissection and
testicular cancer: when to operate. Cancer Control 2002; 9: 277 - 83
125. Hughes T, Thomas J. Combined inguinal and pelvic lymph node dissection for
stage III melanoma. Br J Surg 1999; 86: 1493-8
126. Lopes A, Rossi B, Fonseca F, Morini S. Unreliability of modified inguinal
lymphadenectomy for clinical staging of penile carcinoma. Cancer 1996; 77:
2099-102
127. Johnson D, Lo R. Management of regional lymph nodes in penile carcinoma.
Five year results following therapeutic groin dissections. Urology 1984; 24: 308-
11
128. Cabanas RM. An approach for the treatment of penile carcinoma. Cancer 1977;
39: 456-66
129. Treseler P, Tauchi P. Pathologic analysis of the sentinel lymph node. Surg Clin
North Am 2000; 80: 1695-719
130. Uren R, Howman-Giles R, Thompson JF. Patterns of lymphatic drainage from
the skin in patients with melanoma. J Nucl Med 2003; 44: 570-82
131. Morton DL, Cochran AJ, Thompson JF, Elashoff R, Essner R, Glass EC,
Mozzillo N, Nieweg OE, Roses DF, Hoekstra HJ, Karakousis CP, Reintgen
DS, Coventry BJ, Wang HJ. Sentinel node biopsy for early-stage melanoma:
accuracy and morbidity in MSLT-I, an international multicenter trial. Ann Surg
2005; 242: 302-11; discussion 11-3
132. Lim R, Wong J. Sentinel lymphadenectomy in gynecologic and solid
malignancies other than melanoma and breast cancer. Surg Clin North Am 2000;
80: 1787-98
133. Kim T, Giuliano AE, Lyman GH. Lymphatic mapping and sentinel lymph
node biopsy in early-stage breast carcinoma: a metaanalysis. Cancer 2006; 106:
4-16
134. Kelley MC, Hansen N, McMasters KM. Lymphatic mapping and sentinel
lymphadenectomy for breast cancer. Am J Surg 2004; 188: 49-61
135. Leong SP. Selective sentinel lymphadenectomy for malignant melanoma,
Merkel cell carcinoma, and squamous cell carcinoma. Cancer Treat Res 2005;
127: 39-76
136. Weisberg NK, Bertagnolli MM, Becker DS. Combined sentinel
lymphadenectomy and mohs micrographic surgery for high-risk cutaneous
squamous cell carcinoma. J Am Acad Dermatol 2000; 43: 483-8
137. Leong S, Wong J. Future perspectives on selective sentinel lymphadenectomy.
Surg Clin North Am 2000; 80: 1839-44
138. Leong SP, Peng M, Zhou YM, Vaquerano JE, Chang JW. Cytokine profiles
of sentinel lymph nodes draining the primary melanoma. Ann Surg Oncol 2002;
9: 82-7
- 198 -
139. Schule J, Bergkvist L, Hakansson L, Gustafsson B, Hakansson A. Down-
regulation of the CD3-zeta chain in sentinel lymph node biopsies from breast
cancer patients. Breast Cancer Res Treat 2002; 74: 33-40
140. Lee JH, Torisu-Itakara H, Cochran AJ, Kadison A, Huynh Y, Morton DL,
Essner R. Quantitative analysis of melanoma-induced cytokine-mediated
immunosuppression in melanoma sentinel nodes. Clin Cancer Res 2005; 11:
107-12
141. Botella-Estrada R, Dasi F, Ramos D, Nagore E, Herrero MJ, Gimenez J,
Fuster C, Sanmartin O, Guillen C, Alino S. Cytokine expression and dendritic
cell density in melanoma sentinel nodes. Melanoma Res 2005; 15: 99-106
142. Steinbrink K, Jonuleit H, Muller G, Schuler G, Knop J, Enk AH.
Interleukin-10-treated human dendritic cells induce a melanoma-antigen-specific
anergy in CD8(+) T cells resulting in a failure to lyse tumor cells. Blood 1999;
93: 1634-42
143. Steinbrink K, Graulich E, Kubsch S, Knop J, Enk AH. CD4(+) and CD8(+)
anergic T cells induced by interleukin-10-treated human dendritic cells display
antigen-specific suppressor activity. Blood 2002; 99: 2468-76
144. Huang RR, Wen DR, Guo J, Giuliano AE, Nguyen M, Offodile R, Stern S,
Turner R, Cochran AJ. Selective modulation of paracortical dendritic cells and
T-lymphocytes in breast cancer sentinel lymph nodes. Breast J 2000; 6: 225-32
145. Poindexter NJ, Sahin A, Hunt KK, Grimm EA. Analysis of dendritic cells in
tumor-free and tumor-containing sentinel lymph nodes from patients with breast
cancer. Breast Cancer Res 2004; 6: R408-15
146. Munn DH, Sharma MD, Lee JR, Jhaver KG, Johnson TS, Keskin DB,
Marshall B, Chandler P, Antonia SJ, Burgess R, Slingluff CL, Jr., Mellor
AL. Potential regulatory function of human dendritic cells expressing
indoleamine 2,3-dioxygenase. Science 2002; 297: 1867-70
147. Frumento G, Rotondo R, Tonetti M, Damonte G, Benatti U, Ferrara GB.
Tryptophan-derived catabolites are responsible for inhibition of T and natural
killer cell proliferation induced by indoleamine 2,3-dioxygenase. J Exp Med
2002; 196: 459-68
148. Marzo A, Lake R, Robinson B. T-cell receptor transgenic analysis of tumor-
specific CD8 and CD4 responses in the eradication of solid tumors. Cancer Res
1999; 59: 1071-9
149. Robinson B, Scott B, Lake R, Stumbles P, Nelson D, Fisher S, Marzo A.
Lack of ignorance to tumor antigens: evaluation using nominal antigen
transfection and T-cell receptor transgenic lymphocytes in Lyons-Parish analysis
- implications for tumor tolerance. Clin Cancer Res 2001; 7(s): 811s - 7s
150. Robinson B, Lake R, Nelson D, Scott B, Marzo A. Cross-presentation of
tumour antigens: evaluation of threshold, duration, distribution and regulation.
Immunol Cell Biol 1999; 77: 552 - 8
151. Fisher B, Fisher E. Studies concerning the regional lymph node in cancer I.
Initiation of immunity. Cancer 1971; 27: 1001-4
152. Pilch Y, Bard D, Ramming K. The role of the regional lymph nodes in the
development of host immunological response to tumors. Am J Roentgenol 1971;
111: 48-55
153. Bard D, Hammond W, Pilch Y. The role of the regional lymph nodes in the
immunity to a chemically induced sarcoma in C3H mice. Cancer Res 1969; 29:
1379-84
154. Barbera-Guillem E, Arnold MW, Nelson MB, Martin EW, Jr. First results
for resetting the antitumor immune response by immune corrective surgery in
colon cancer. Am J Surg 1998; 176: 339-43
- 199 -
155. Stumbles PA, Himbeck R, Frelinger JA, Collins EJ, Lake RA, Robinson
BW. Cutting edge: tumor-specific CTL are constitutively cross-armed in
draining lymph nodes and transiently disseminate to mediate tumor regression
following systemic CD40 activation. J Immunol 2004; 173: 5923-8
156. van Mierlo GJ, den Boer AT, Medema JP, van der Voort EI, Fransen MF,
Offringa R, Melief CJ, Toes RE. CD40 stimulation leads to effective therapy
of CD40(-) tumors through induction of strong systemic cytotoxic T lymphocyte
immunity. Proc Natl Acad Sci U S A 2002; 99: 5561-6
157. Hammond W, Rolley R. Retained regional lymph nodes: effect on metastases
and recurrence after tumor removal. Cancer 1970; 25: 368-72
158. Obhrai JS, Oberbarnscheidt MH, Hand TW, Diggs L, Chalasani G, Lakkis
FG. Effector T cell differentiation and memory T cell maintenance outside
secondary lymphoid organs. J Immunol 2006; 176: 4051-8
159. Gardner G, Rosen R. The effect of lymphadenectomy on tumor immunity.
Surg Gynecol Obstet 1967; 125: 351-4
160. Kanayama H, Izumi A, Osaki Y, Hamazoe R, Karino T, Shimizu N, Maeta
M, Koga S. Effect of regional or distant lymph node removal and of
splenectomy on immunologic responses in ehrlich tumor-bearing mice. Jpn J
Surg 1982; 12: 381-6
161. Fisher B, Fisher E. Studies concerning the regional lymph node in cancer. II.
Maintenance of immunity. Cancer 1972; 29: 1496-501
162. Perez C, Stewart C, Palmer-Hanes L, Powers W. Role of the regional lymph
nodes in the cure of a murine lymphosarcoma. Cancer 1973; 32: 562-72
163. Crile G, Jr. The effect on metastasis of removing or irradiating regional nodes
of mice. Surg Gynecol Obstet 1968; 126: 1270-2
164. Serpell JW, Carne PW, Bailey M. Radical lymph node dissection for
melanoma. ANZ J Surg 2003; 73: 294-9
165. Stephenson AJ, Sheinfeld J. Management of patients with low-stage
nonseminomatous germ cell testicular cancer. Curr Treat Options Oncol 2005;
6: 367-77
166. Fisher B, Montague E, Redmond C, Barton B, Borland D, Fisher ER,
Deutsch M, Schwarz G, Margolese R, Donegan W, Volk H, Konvolinka C,
Gardner B, Cohn I, Jr., Lesnick G, Cruz AB, Lawrence W, Nealon T,
Butcher H, Lawton R. Comparison of radical mastectomy with alternative
treatments for primary breast cancer. A first report of results from a prospective
randomized clinical trial. Cancer 1977; 39: 2827-39
167. Veronesi U, Adamus J, Bandiera DC, Brennhovd IO, Caceres E, Cascinelli
N, Claudio F, Ikonopisov RL, Javorskj VV, Kirov S, Kulakowski A, Lacoub
J, Lejeune F, Mechl Z, Morabito A, Rode I, Sergeev S, van Slooten E,
Szcygiel K, Trapeznikov NN. Inefficacy of immediate node dissection in stage
1 melanoma of the limbs. N Engl J Med 1977; 297: 627-30
168. Sim FH, Taylor WF, Ivins JC, Pritchard DJ, Soule EH. A prospective
randomized study of the efficacy of routine elective lymphadenectomy in
management of malignant melanoma. Preliminary results. Cancer 1978; 41:
948-56
169. Fisher B, Redmond C, Fisher ER. The contribution of recent NSABP clinical
trials of primary breast cancer therapy to an understanding of tumor biology - an
overview of findings. Cancer 1980; 46: 1009-25
170. Tachibana T, Yoshida K. Role of the regional lymph node in cancer
metastasis. Cancer Metast Rev 1986; 5: 55-66
- 200 -
171. Wen DR, Hoon DS, Chang C, Cochran AJ. Variations in lymphokine
generation by individual lymph nodes draining human malignant tumors.
Cancer Immunol Immunother 1989; 30: 277-82
172. Farzad Z, Cochran AJ, McBride WH, Gray JD, Wong V, Morton DL.
Lymphocyte subset alterations in nodes regional to human melanoma. Cancer
Res 1990; 50: 3585-8
173. Morton BA, Ramey WG, Paderon H, Miller RE. Monoclonal antibody-
defined phenotypes of regional lymph node and peripheral blood lymphocyte
subpopulations in early breast cancer. Cancer Res 1986; 46: 2121-6
174. Hoon DS, Bowker RJ, Cochran AJ. Suppressor cell activity in melanoma-
draining lymph nodes. Cancer Res 1987; 47: 1529-33
175. Farzad Z, McBride WH, Ogbechi H, Asnong-Holthoff C, Morton DL,
Cochran AJ. Lymphocytes from lymph nodes at different distances from
human melanoma vary in their capacity to inhibit/enhance tumor cell growth in
vitro. Melanoma Res 1997; 7 Suppl 2: S59-65
176. Yang G, Mizuno MT, Hellstrom KE, Chen L. B7-negative versus B7-positive
P815 tumor: differential requirements for priming of an antitumor immune
response in lymph nodes. J Immunol 1997; 158: 851-8
177. Sakai K, Chang AE, Shu SY. Phenotype analyses and cellular mechanisms of
the pre-effector T-lymphocyte response to a progressive syngeneic murine
sarcoma. Cancer Res 1990; 50: 4371-6
178. Matsumura T, Sussman JJ, Krinock RA, Chang AE, Shu S. Characteristics
and in vivo homing of long-term T-cell lines and clones derived from tumor-
draining lymph nodes. Cancer Res 1994; 54: 2744-50
179. Mukherji B, Nashed AL, Guha A, Ergin MT. Regulation of cellular immune
response against autologous human melanoma. II. Mechanism of induction and
specificity of suppression. J Immunol 1986; 136: 1893-8
180. Finlay-Jones JJ, Bartholomaeus WN, Fimmel PJ, Keast D, Stanley NF.
Biologic and immunologic studies on a murine model of regional lymph node
metastasis. J Natl Cancer Inst 1980; 64: 1363-72
181. Harada M, Tamada K, Abe K, Li T, Onoe Y, Tada H, Takenoyama M,
Yasumoto K, Kimura G, Nomoto K. Systemic administration of interleukin-12
can restore the anti-tumor potential of B16 melanoma-draining lymph node cells
impaired at a late tumor-bearing state. Int J Cancer 1998; 75: 400-5
182. Cortesina G, De Stefani A, Giovarelli M, Barioglio M, Carvallo G, Jemma
C, Forni G. Treatment of recurrent squamous cell carcinoma of the head and
neck with low doses of interleukin-2 injected perilymphatically. Cancer 1998;
62: 2482-5
183. Greenberg A, Greene M. Non-adaptive rejection of small tumour inocula as a
model of immune surveillance. Nature 1976; 264: 356-9
184. Street S, Hayakawa Y, Zhan Y, Lew AM, Macgregor D, Jamieson A,
Diefenbach A, Yagita H, Godfrey DI, Smyth MJ. Innate immune surveillance
of spontaneous B cell lymphomas by natural killer cells and T cells. J Exp
Med 2004; 199: 879 - 84
185. Medzhitov R, Janeway CA, Jr. Innate immunity: impact on the adaptive
immune response. Curr Opin Immunol 1997; 9: 4 - 9
186. Shu S, Plautz GE, Krauss JC, Chang AE. Tumor immunology. JAMA 1997;
278: 1972-81
187. Rosenberg SA. Progress in human tumour immunology and immunotherapy.
Nature 2001; 411: 380-4
188. Rosenberg SA. Progress in the development of immunotherapy for the
treatment of patients with cancer. J Intern Med 2001; 250: 462-75
- 201 -
189. Kupelian P, Katcher J, Levin H, Zippe C, Klein E. Correlation of clinical and
pathologic factors with rising prostate-specific antigen profiles after radical
prostatectomy alone for clinically localised prostate cancer. Urology 1996; 48:
249-60
190. Irvine T, Scott M, Clark C. A small rise in CEA is sensitive for recurrence
after surgery for colorectal cancer. Colorectal Dis 2007; 9: 527-31
191. Rock K, Shen L. Cross-presentation: underlying mechanisms and role in
immune surveillance. Immunol Rev 2005; 207: 166-83
192. Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D,
Goldberg AL. Inhibitors of the proteasome block the degradation of most cell
proteins and the generation of peptides presented on MHC class I molecules.
Cell 1994; 78: 761-71
193. Goldberg AL, Rock KL. Proteolysis, proteasomes and antigen presentation.
Nature 1992; 357: 375-9
194. Rock KL, Goldberg AL. Degradation of cell proteins and the generation of
MHC class I-presented peptides. Annu Rev Immunol 1999; 17: 739-79
195. Kisselev AF, Akopian TN, Woo KM, Goldberg AL. The sizes of peptides
generated from protein by mammalian 26 and 20 S proteasomes. Implications
for understanding the degradative mechanism and antigen presentation. J Biol
Chem 1999; 274: 3363-71
196. Serwold T, Gonzalez F, Kim J, Jacob R, Shastri N. ERAAP customizes
peptides for MHC class I molecules in the endoplasmic reticulum. Nature 2002;
419: 480-3
197. Goldberg AL, Cascio P, Saric T, Rock KL. The importance of the proteasome
and subsequent proteolytic steps in the generation of antigenic peptides. Mol
Immunol 2002; 39: 147-64
198. Cuenca A, Cheng F, Wang H, Brayer J, Horna P, Gu L, Bien H, Borrello
IM, Levitsky HI, Sotomayor EM. Extra-lymphatic solid tumor growth is not
immunologically ignored and results in early induction of antigen-specific T-cell
anergy: dominant role of cross-tolerance to tumor antigens. Cancer Res 2003;
63: 9007-15
199. Jenkinson S, Williams N, Morgan D. The role of intercellular adhesion
molecule-1/LFA-1 interactions in the generation of tumor-specific CD8+ T cell
responses. J Immunol 2005; 174: 3401-7
200. Spiotto MT, Yu P, Rowley DA, Nishimura M, Meredith S, Gajewski T, Fu
YX, Schreiber H. Increasing tumor antigen expression overcomes "ignorance"
to solid tumors via crosspresentation by bone marrow-derived stromal cells.
Immunity 2002; 17: 737-47
201. Abken H, Hombach A, Heuser C, Kronfeld K, Seliger B. Tuning tumor-
specific T-cell activation: a matter of costimulation? Trends Immunol 2002; 23:
240-5
202. Garrido F, Ruiz-Cabello F. MHC expression on human tumors - its relevance
for local tumor growth and metastasis. Semin Cancer Biol 1991; 2: 3-10
203. Rock KL, Rothstein L, Gamble S, Fleischacker C. Characterization of
antigen-presenting cells that present exogenous antigens in association with
class I MHC molecules. J Immunol 1993; 150: 438-46
204. Grant EP, Rock KL. MHC class I-restricted presentation of exogenous antigen
by thymic antigen-presenting cells in vitro and in vivo. J Immunol 1992; 148:
13-8
205. Huang AY, Bruce AT, Pardoll DM, Levitsky HI. In vivo cross-priming of
MHC class I-restricted antigens requires the TAP transporter. Immunity 1996; 4:
349-55
- 202 -
206. Santin AD. Lymph node metastases: the importance of the microenvironment.
Cancer 2000; 88: 175-9
207. Heath W, Belz G, Behrens G, Smith C, Forehan S, Parish I, Davey G,
Wilson N, Carbone F, Villadangos J. Cross-presentation, dendritic cell
subsets, and the generation of immunity to cellular antigens. Immunol Rev 2004;
199: 9-26
208. Ke Y, Kapp JA. Exogenous antigens gain access to the major
histocompatibility complex class I processing pathway in B cells by receptor-
mediated uptake. J Exp Med 1996; 184: 1179-84
209. Limmer A, Ohl J, Kurts C, Ljunggren HG, Reiss Y, Groettrup M,
Momburg F, Arnold B, Knolle PA. Efficient presentation of exogenous
antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-
cell tolerance. Nat Med 2000; 6: 1348-54
210. Savinov AY, Wong FS, Stonebraker AC, Chervonsky AV. Presentation of
antigen by endothelial cells and chemoattraction are required for homing of
insulin-specific CD8+ T cells. J Exp Med 2003; 197: 643-56
211. Kovacsovics-Bankowski M, Clark K, Benacerraf B, Rock KL. Efficient
major histocompatibility complex class I presentation of exogenous antigen
upon phagocytosis by macrophages. Proc Natl Acad Sci U S A 1993; 90: 4942-6
212. Debrick JE, Campbell PA, Staerz UD. Macrophages as accessory cells for
class I MHC-restricted immune responses. J Immunol 1991; 147: 2846-51
213. Heath WR, Kurts C, Miller JFAP, Carbone FR. Cross-tolerance: a pathway
for inducing tolerance to peripheral tissue antigens. J Exp Med 1998; 187: 1549-
53
214. Banchereau J, Steinman RM. Dendritic cells and the control of immunity.
Nature 1998; 392: 245-52
215. Mellman I, Steinman RM. Dendritic cells: specialized and regulated antigen
processing machines. Cell 2001; 106: 255-8
216. Medzhitov R, Janeway CA, Jr. Innate immune recognition and control of
adaptive immune responses. Semin Immunol 1998; 10: 351-3
217. Rock KL, Hearn A, Chen CJ, Shi Y. Natural endogenous adjuvants. Springer
Semin Immunopathol 2005; 26: 231-46
218. Shi Y, Zheng W, Rock KL. Cell injury releases endogenous adjuvants that
stimulate cytotoxic T cell responses. Proc Natl Acad Sci U S A 2000;97:14590-5
219. Shi Y, Evans JE, Rock KL. Molecular identification of a danger signal that
alerts the immune system to dying cells. Nature 2003; 425: 516-21
220. Harshyne LA, Watkins SC, Gambotto A, Barratt-Boyes SM. Dendritic cells
acquire antigens from live cells for cross-presentation to CTL. J Immunol 2001;
166: 3717-23
221. Young JW, Inaba K. Dendritic cells as adjuvants for class I major
histocompatibility complex-restricted antitumor immunity. J Exp Med 1996;
183: 7-11
222. Igney FH, Krammer PH. Tumor counterattack: fact or fiction? Cancer
Immunol Immunother 2005; 54: 1127-36
223. Osborne BA. Apoptosis and the maintenance of homoeostasis in the immune
system. Curr Opin Immunol 1996; 8: 245-54
224. Schoenberger SP, Toes RE, van der Voort EI, Offringa R, Melief CJ. T-cell
help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions.
Nature 1998; 393: 480-3
225. Bennett SR, Carbone FR, Karamalis F, Flavell RA, Miller JF, Heath WR.
Help for cytotoxic-T-cell responses is mediated by CD40 signaling. Nature
1998; 393: 478-80
- 203 -
226. Lu Z, Yuan L, Zhou X, Sotomayor E, Levitsky HI, Pardoll DM. CD40-
independent pathways of T cell help for priming of CD8(+) cytotoxic T
lymphocytes. J Exp Med 2000; 191: 541-50
227. Mackey MF, Gunn JR, Maliszewsky C, Kikutani H, Noelle RJ, Barth RJ,
Jr. Dendritic cells require maturation via CD40 to generate protective antitumor
immunity. J Immunol 1998; 161: 2094-8
228. Shinde S, Wu Y, Guo Y, Niu Q, Xu J, Grewal IS, Flavell R, Liu Y. CD40L is
important for induction of, but not response to, costimulatory activity. ICAM-1
as the second costimulatory molecule rapidly up-regulated by CD40L. J
Immunol 1996; 157: 2764-8
229. Koch F, Stanzl U, Jennewein P, Janke K, Heufler C, Kampgen E, Romani
N, Schuler G. High level IL-12 production by murine dendritic cells:
upregulation via MHC class II and CD40 molecules and downregulation by IL-4
and IL-10. J Exp Med 1996; 184: 741-6
230. Dumortier H, van Mierlo GJ, Egan D, van Ewijk W, Toes RE, Offringa R,
Melief CJ. Antigen presentation by an immature myeloid dendritic cell line
does not cause CTL deletion in vivo, but generates CD8+ central memory-like T
cells that can be rescued for full effector function. J Immunol 2005; 175: 855-63
231. Janssen EM, Lemmens EE, Wolfe T, Christen U, von Herrath MG,
Schoenberger SP. CD4+ T cells are required for secondary expansion and
memory in CD8+ T lymphocytes. Nature 2003; 421: 852-6
232. Tite JP, Janeway CA, Jr. Cloned helper T cells can kill B lymphoma cells in
the presence of specific antigen: Ia restriction and cognate vs. noncognate
interactions in cytolysis. Eur J Immunol 1984; 14: 878-86
233. Ozaki S, York-Jolley J, Kawamura H, Berzofsky JA. Cloned protein antigen-
specific, Ia-restricted T cells with both helper and cytolytic activities:
mechanisms of activation and killing. Cell Immunol 1987; 105: 301-16
234. Echchakir H, Bagot M, Dorothee G, Martinvalet D, Le Gouvello S,
Boumsell L, Chouaib S, Bensussan A, Mami-Chouaib F. Cutaneous T cell
lymphoma reactive CD4+ cytotoxic T lymphocyte clones display a Th1 cytokine
profile and use a fas-independent pathway for specific tumor cell lysis. J Invest
Dermatol 2000; 115: 74-80
235. Robinson BW, Lake RA, Nelson DJ, Scott BA, Marzo AL. Cross-
presentation of tumour antigens: evaluation of threshold, duration, distribution
and regulation. Immunol Cell Biol 1999; 77: 552-8
236. Kurts C, Heath WR, Carbone FR, Kosaka H, Miller JF. Cross-presentation
of self antigens to CD8+ T cells: the balance between tolerance and
autoimmunity. Novartis Found Symp 1998; 215: 172-81; discussion 81-90
237. Jocham D, Richter A, Hoffmann L, Iwig K, Fahlenkamp D, Zakrzewski G,
Schmitt E, Dannenberg T, Lehmacher W, Wietersheim Jv, Doehn C.
Adjuvant autologous renal tumour cell vaccine and risk of tumour progression in
patients with renal-cell carcinoma after radical nephrectomy: phase III,
randomised controlled trial. Lancet 2004; 363: 594-9
238. Lake RA, van der Most RG. A better way for a cancer cell to die. N Engl J
Med 2006; 354: 2503-4
239. Ioannides CG, Whiteside TL. T cell recognition of human tumors:
implications for molecular immunotherapy of cancer. Clin Immunol
Immunopathol 1993; 66: 91-106
240. North RJ. Down-regulation of the antitumor immune response. Adv Cancer Res
1985; 45: 1-43
- 204 -
241. Wick M, Dubey P, Koeppen H, Siegel CT, Fields PE, Chen L, Bluestone JA,
Schreiber H. Antigenic cancer cells grow progressively in immune hosts
without evidence for T cell exhaustion or systemic anergy. J Exp Med 1997;
186: 229-38
242. Speiser DE, Miranda R, Zakarian A, Bachmann MF, McKall-Faienza K,
Odermatt B, Hanahan D, Zinkernagel RM, Ohashi PS. Self antigens
expressed by solid tumors Do not efficiently stimulate naive or activated T cells:
implications for immunotherapy. J Exp Med 1997; 186: 645-53
243. Melero I, Singhal MC, McGowan P, Haugen HS, Blake J, Hellstrom KE,
Yang G, Clegg CH, Chen L. Immunological ignorance of an E7-encoded
cytolytic T-lymphocyte epitope in transgenic mice expressing the E7 and E6
oncogenes of human papillomavirus type 16. J Virol 1997; 71: 3998-4004
244. Baxter AG, Hodgkin PD. Activation rules: the two-signal theories of immune
activation. Nat Rev Immunol 2002; 2: 439-46
245. Igney FH, Krammer PH. Immune escape of tumors: apoptosis resistance and
tumor counterattack. J Leukoc Biol 2002; 71: 907-20
246. Serafini P, De Santo C, Marigo I, Cingarlini S, Dolcetti L, Gallina G,
Zanovello P, Bronte V. Derangement of immune responses by myeloid
suppressor cells. Cancer Immunol Immunother 2004; 53: 64-72
247. Kobayashi M, Kobayashi H, Pollard RB, Suzuki F. A pathogenic role of Th2
cells and their cytokine products on the pulmonary metastasis of murine B16
melanoma. J Immunol 1998; 160: 5869-73
248. Tatsumi T, Kierstead LS, Ranieri E, Gesualdo L, Schena FP, Finke JH,
Bukowski RM, Mueller-Berghaus J, Kirkwood JM, Kwok WW, Storkus
WJ. Disease-associated bias in T helper type 1 (Th1)/Th2 CD4(+) T cell
responses against MAGE-6 in HLA-DRB10401(+) patients with renal cell
carcinoma or melanoma. J Exp Med 2002; 196: 619-28
249. Pelaez B, Campillo J, Lopez-Asenjo J, Subiza J. Cyclophosphamide induces
the development of early myeloid cells suppressing tumor cell growth by a nitric
oxide-dependent mechanism. J Immunol 2001; 166: 6608-15
250. Suzuki E, Kapoor V, Jassar A, Kaiser LR, Albelda SM. Gemcitabine
selectively eliminates splenic Gr-1+/CD11b+ myeloid suppressor cells in tumor-
bearing animals and enhances antitumor immune activity. Clin Cancer Res
2005; 11: 6713-21
251. Bronte V, Apolloni E, Cabrelle A, Ronca R, Serafini P, Zamboni P, Restifo
NP, Zanovello P. Identification of a CD11b+/Gr-1+/CD31+ myeloid progenitor
capable of activating or suppressing CD8+ T cells. Blood 2000; 96: 3838-46
252. Bronte V, Zanovello P. Regulation of immune responses by L-arginine
metabolism. Nat Rev Immunol 2005; 5: 641-54
253. Melani C, Chiodoni C, Forni G, Colombo MP. Myeloid cell expansion
elicited by the progression of spontaneous mammary carcinomas in c-erbB-2
transgenic BALB/c mice suppresses immune reactivity. Blood 2003; 102: 2138-
45
254. Kusmartsev S, Nagaraj S, Gabrilovich DI. Tumor-associated CD8+ T cell
tolerance induced by bone marrow-derived immature myeloid cells. J Immunol
2005; 175: 4583-92
255. Liu Y, Van Ginderachter JA, Brys L, De Baetselier P, Raes G, Geldhof AB.
Nitric oxide-independent CTL suppression during tumor progression:
association with arginase-producing (M2) myeloid cells. J Immunol 2003; 170:
5064-74
- 205 -
256. Ochoa JB, Strange J, Kearney P, Gellin G, Endean E, Fitzpatrick E. Effects
of L-arginine on the proliferation of T lymphocyte subpopulations. JPEN J
Parenter Enteral Nutr 2001; 25: 23-9
257. Murata Y, Shimamura T, Hamuro J. The polarization of Th1/Th2 balance is
dependent on the intracellular thiol redox status of macrophages due to the
distinctive cytokine production. Int Immunol 2002; 14: 201 - 12
258. Munn DH, Sharma MD, Hou D, Baban B, Lee JR, Antonia SJ, Messina JL,
Chandler P, Koni PA, Mellor AL. Expression of indoleamine 2,3-dioxygenase
by plasmacytoid dendritic cells in tumor-draining lymph nodes. J Clin Invest
2004; 114: 280-90
259. Needham DJ, Lee JX, Beilharz MW. Intra-tumoural regulatory T cells: a
potential new target in cancer immunotherapy. Biochem Biophys Res Commun
2006; 343: 684-91
260. Somasundaram R, Jacob L, Swoboda R, Caputo L, Song H, Basak S,
Monos D, Peritt D, Marincola F, Cai D, Birebent B, Bloome E, Kim J,
Berencsi K, Mastrangelo M, Herlyn D. Inhibition of cytolytic T lymphocyte
proliferation by autologous CD4+/CD25+ regulatory T cells in a colorectal
carcinoma patient is mediated by transforming growth factor-beta. Cancer Res
2002; 62: 5267-72
261. He X, Stuart JM. Prostaglandin E2 selectively inhibits human CD4+ T cells
secreting low amounts of both IL-2 and IL-4. J Immunol 1999; 163: 6173-9
262. Nolte-'t Hoen EN, Wagenaar-Hilbers JP, Boot EP, Lin CH, Arkesteijn GJ,
van Eden W, Taams LS, Wauben MH. Identification of a CD4+CD25+ T cell
subset committed in vivo to suppress antigen-specific T cell responses without
additional stimulation. Eur J Immunol 2004; 34: 3016-27
263. Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl
SM. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+
regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp
Med 2003; 198: 1875-86
264. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, Evdemon-
Hogan M, Conejo-Garcia JR, Zhang L, Burow M, Zhu Y, Wei S, Kryczek I,
Daniel B, Gordon A, Myers L, Lackner A, Disis ML, Knutson KL, Chen L,
Zou W. Specific recruitment of regulatory T cells in ovarian carcinoma fosters
immune privilege and predicts reduced survival. Nat Med 2004; 10: 942-9
265. Ichihara F, Kono K, Takahashi A, Kawaida H, Sugai H, Fujii H. Increased
populations of regulatory T cells in peripheral blood and tumor-infiltrating
lymphocytes in patients with gastric and esophageal cancers. Clin Cancer Res
2003; 9: 4404-8
266. Liyanage UK, Moore TT, Joo HG, Tanaka Y, Herrmann V, Doherty G,
Drebin JA, Strasberg SM, Eberlein TJ, Goedegebuure PS, Linehan DC.
Prevalence of regulatory T cells is increased in peripheral blood and tumor
microenvironment of patients with pancreas or breast adenocarcinoma. J
Immunol 2002; 169: 2756-61
267. Alderson MR, Tough TW, Davis-Smith T, Braddy S, Falk B, Schooley KA,
Goodwin RG, Smith CA, Ramsdell F, Lynch DH. Fas ligand mediates
activation-induced cell death in human T lymphocytes. J Exp Med 1995; 181:
71-7
268. O'Connell J, Bennett MW, O'Sullivan GC, O'Callaghan J, Collins JK,
Shanahan F. Expression of Fas (CD95/APO-1) ligand by human breast cancers:
significance for tumor immune privilege. Clin Diagn Lab Immunol 1999; 6:
457-63
- 206 -
269. Bennett MW, O'Connell J, O'Sullivan G C, Roche D, Brady C, Kelly J,
Collins JK, Shanahan F. Expression of Fas ligand by human gastric
adenocarcinomas: a potential mechanism of immune escape in stomach cancer.
Gut 1999; 44: 156-62
270. Bennett MW, O'Connell J, O'Sullivan GC, Brady C, Roche D, Collins JK,
Shanahan F. The Fas counterattack in vivo: apoptotic depletion of tumor-
infiltrating lymphocytes associated with Fas ligand expression by human
esophageal carcinoma. J Immunol 1998; 160: 5669-75
271. Ganss R, Ryschich E, Klar E, Arnold B, Hämmerling GJ. Combination of T-
cell therapy and trigger of inflammation induces remodeling of the vasculature
and tumor eradication. Cancer Res 2002; 62: 1462-70
272. Onrust SV, Hartl PM, Rosen SD, Hanahan D. Modulation of L-selectin
ligand expression during an immune response accompanying tumorigenesis in
transgenic mice. J Clin Invest 1996; 97: 54-64
273. Hersey P. Impediments to successful immunotherapy. Pharmacol Therapeut
1999; 81: 111-9
274. Singh S, Ross SR, Acena M, Rowley DA, Schreiber H. Stroma is critical for
preventing or permitting immunological destruction of antigenic cancer cells. J
Exp Med 1992; 175: 139-46
275. Raffaghello L, Prigione I, Airoldi I, Camoriano M, Levreri I, Gambini C,
Pende D, Steinle A, Ferrone S, Pistoia V. Downregulation and/or release of
NKG2D ligands as immune evasion strategy of human neuroblastoma.
Neoplasia 2004; 6: 558-68
276. Roth W, Isenmann S, Nakamura M, Platten M, Wick W, Kleihues P, Bahr
M, Ohgaki H, Ashkenazi A, Weller M. Soluble decoy receptor 3 is expressed
by malignant gliomas and suppresses CD95 ligand-induced apoptosis and
chemotaxis. Cancer Res 2001; 61: 2759-65
277. Whiteside TL. Tumor-induced death of immune cells: its mechanisms and
consequences. Semin Cancer Biol 2002; 12: 43-50
278. Gorter A, Meri S. Immune evasion of tumor cells using membrane-bound
complement regulatory proteins. Immunol Today 1999; 20: 576-82
279. Whiteside TL. Signaling defects in T lymphocytes of patients with malignancy.
Cancer Immunol Immunother 1999; 48: 346-52
280. Apolloni E, Bronte V, Mazzoni A, Serafini P, Cabrelle A, Segal DM, Young
HA, Zanovello P. Immortalized myeloid suppressor cells trigger apoptosis in
antigen-activated T lymphocytes. J Immunol 2000; 165: 6723-30
281. Sinha P, Clements V, Ostrand-Rosenberg S. Interleukin-13-regulated M2
macrophages in combination with myeloid suppressor cells block immune
surveillance against metastases. Cancer Res 2005; 65: 11743-51
282. Wu G, Morris SM, Jr. Arginine metabolism: nitric oxide and beyond. Biochem
J 1998; 336 (Pt 1): 1-17
283. Sun B, Zhang S, Ni C, Zhang D, Liu Y, Zhang W, Zhao X, Zhao C, Shi M.
Correlation between melanoma angiogenesis and the mesenchymal stem cells
and endothelial progenitor cells derived from bone marrow. Stem Cells Dev
2005; 14: 292-8
284. Almand B, Clark JI, Nikitina E, van Beynen J, English NR, Knight SC,
Carbone DP, Gabrilovich DI. Increased production of immature myeloid cells
in cancer patients: a mechanism of immunosuppression in cancer. J Immunol
2001; 166: 678-89
285. Salvadori S, Martinelli G, Zier K. Resection of solid tumors reverses T cell
defects and restores protective immunity. J Immunol 2000; 164: 2214-20
- 207 -
286. Rocha B, von Boehmer H. Peripheral selection of the T cell repertoire. Science
1991; 251: 1225-8
287. Rocha B, Grandien A, Freitas AA. Anergy and exhaustion are independent
mechanisms of peripheral T cell tolerance. J Exp Med 1995; 181: 993-1003
288. Aichele P, Brduscha-Riem K, Zinkernagel RM, Hengartner H, Pircher H. T
cell priming versus T cell tolerance induced by synthetic peptides. J Exp Med
1995; 182: 261-6
289. Zinkernagel RM. Localization dose and time of antigens determine immune
reactivity. Semin Immunol 2000; 12: 163-71; discussion 257-344
290. Zajac AJ, Blattman JN, Murali-Krishna K, Sourdive DJ, Suresh M, Altman
JD, Ahmed R. Viral immune evasion due to persistence of activated T cells
without effector function. J Exp Med 1998; 188: 2205-13
291. Redmond WL, Marincek BC, Sherman LA. Distinct requirements for
deletion versus anergy during CD8 T cell peripheral tolerance in vivo. J
Immunol 2005; 174: 2046-53
292. Rocha B, Tanchot C, Von Boehmer H. Clonal anergy blocks in vivo growth of
mature T cells and can be reversed in the absence of antigen. J Exp Med 1993;
177: 1517-21
293. Ramsdell F, Fowlkes BJ. Maintenance of in vivo tolerance by persistence of
antigen. Science 1992; 257: 1130-4
294. Dubois PM, Pihlgren M, Tomkowiak M, Van Mechelen M, Marvel J.
Tolerant CD8 T cells induced by multiple injections of peptide antigen show
impaired TCR signaling and altered proliferative responses in vitro and in vivo.
J Immunol 1998; 161: 5260-7
295. Schonrich G, Kalinke U, Momburg F, Malissen M, Schmitt-Verhulst AM,
Malissen B, Hammerling GJ, Arnold B. Down-regulation of T cell receptors
on self-reactive T cells as a novel mechanism for extrathymic tolerance
induction. Cell 1991; 65: 293-304
296. Hawiger D, Masilamani RF, Bettelli E, Kuchroo VK, Nussenzweig MC.
Immunological unresponsiveness characterized by increased expression of CD5
on peripheral T cells induced by dendritic cells in vivo. Immunity 2004; 20: 695-
705
297. Barber DL, Wherry EJ, Masopust D, Zhu B, Allison JP, Sharpe AH,
Freeman GJ, Ahmed R. Restoring function in exhausted CD8 T cells during
chronic viral infection. Nature 2006; 439: 682-7
298. Wherry EJ, Barber DL, Kaech SM, Blattman JN, Ahmed R. Antigen-
independent memory CD8 T cells do not develop during chronic viral infection.
Proc Natl Acad Sci USA 2004; 101: 16004-9
299. Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, Antia R,
von Andrian UH, Ahmed R. Lineage relationship and protective immunity of
memory CD8 T cell subsets. Nat Immunol 2003; 4: 225-34
300. Blattman JN, Greenberg PD. PD-1 blockade: rescue from a near-death
experience. Nat Immunol 2006; 7: 227-8
301. Carter L, Fouser LA, Jussif J, Fitz L, Deng B, Wood CR, Collins M, Honjo
T, Freeman GJ, Carreno BM. PD-1:PD-L inhibitory pathway affects both
CD4(+) and CD8(+) T cells and is overcome by IL-2. Eur J Immunol 2002; 32:
634-43
302. den Boer AT, van Mierlo GJ, Fransen MF, Melief CJ, Offringa R, Toes RE.
The tumoricidal activity of memory CD8+ T cells is hampered by persistent
systemic antigen, but full functional capacity is regained in an antigen-free
environment. J Immunol 2004; 172: 6074-9
- 208 -
303. Murali-Krishna K, Altman JD, Suresh M, Sourdive DJ, Zajac AJ, Miller
JD, Slansky J, Ahmed R. Counting antigen-specific CD8 T cells: a
reevaluation of bystander activation during viral infection. Immunity 1998; 8:
177-87
304. Slifka MK, Whitton JL. Functional avidity maturation of CD8(+) T cells
without selection of higher affinity TCR. Nat Immunol 2001; 2: 711-7
305. Veiga-Fernandes H, Walter U, Bourgeois C, McLean A, Rocha B. Response
of naive and memory CD8+ T cells to antigen stimulation in vivo. Nat Immunol
2000; 1: 47-53
306. Harty JT, Tvinnereim AR, White DW. CD8+ T cell effector mechanisms in
resistance to infection. Annu Rev Immunol 2000; 18: 275-308
307. Marzo AL, Klonowski KD, Le Bon A, Borrow P, Tough DF, Lefrancois L.
Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat
Immunol 2005; 6: 793-9
308. Lefrancois L, Marzo AL. The descent of memory T-cell subsets. Nat Rev
Immunol 2006; 6: 618-23
309. Wu CY, Kirman JR, Rotte MJ, Davey DF, Perfetto SP, Rhee EG, Freidag
BL, Hill BJ, Douek DC, Seder RA. Distinct lineages of T(H)1 cells have
differential capacities for memory cell generation in vivo. Nat Immunol 2002; 3:
852-8
310. Lefrancois L, Marzo A, Williams K. Sustained response initiation is required
for T cell clonal expansion but not for effector or memory development in vivo.
J Immunol 2003; 171: 2832-9
311. Northrop JK, Shen H. CD8+ T-cell memory: only the good ones last. Curr
Opin Immunol 2004; 16: 451-5
312. Alexander P, Hall J. The role of immunoblasts in host resistance and
immunotherapy of primary sarcomata. Adv Cancer Res 1970; 13: 1-37
313. de Visser KE, Coussens LM. The inflammatory tumor microenvironment and
its impact on cancer development. Contrib Microbiol 2006; 13: 118-37
314. Chamoto K, Wakita D, Narita Y, Zhang Y, Noguchi D, Ohnishi H, Iguchi
T, Sakai T, Ikeda H, Nishimura T. An essential role of antigen-presenting
cell/T-helper type 1 cell-cell interactions in draining lymph node during
complete eradication of class II-negative tumor tissue by T-helper type 1 cell
therapy. Cancer Res 2006; 66: 1809-17
315. Davis M, Manning L, Whitaker D, Garlepp M, Robinson B. Establishment
of a murine model of malignant mesothelioma. Int J Cancer 1992: 881 - 6
316. Murphy G, Hrushesky W. A murine renal cell carcinoma. J Natl Cancer Inst
1973; 50: 1013 - 25
317. Ahn K, Jung Y, Kim J, Lee H, Yoon S. Behaviour of murine renal carcinoma
cells grown in ectopic or orthotopic sites in syngeneic mice. Tumor Biol 2001;
22: 146-53
318. Morgan DJ, Kreuwel HT, Fleck S, Levitsky HI, Pardoll DM, Sherman LA.
Activation of low avidity CTL specific for a self epitope results in tumor
rejection but not autoimmunity. J Immunol 1998; 160: 643-51
319. Morgan D, Liblau R, Scott B, Fleck S, McDevitt H, Sarvetnick N, Lo D,
Sherman L. CD8+ T cell-mediated spontaneous diabetes in neonatal mice. J
Immunol 1996; 157: 978
320. Scott B, Liblau R, Degermann S, Marconi A, Ogata L, Caton A, McDevitt
H, Lo D. A role for non-mhc genetic polymorphism in susceptibility to
spontaneous autoimmunity. Immunity 1994; 1: 73-82
321. Lyons A, Parish C. Determination of lymphocyte division by flow cytometry. J
Immunol Methods 1994; 171: 131 - 7
- 209 -
322. Weston S, Parish C. New fluorescent dyes for lymphocyte migration studies. J
Immunol Methods 1990; 133: 87 - 97
323. Oehen S, Brduscha-Riem K. Differentiation of naive CTL to effector and
memory CTL: correlation of effector function with phenotype and cell division.
J Immunol 1998; 161: 5338-46
324. Vremec D, Zorbas M, Scollay R, Saunders DJ, Ardavin CF, Wu L,
Shortman K. The surface phenotype of dendritic cells purified from mouse
thymus and spleen: investigation of the CD8 expression by a subpopulation of
dendritic cells. J Exp Med 1992; 176: 47-58
325. Winkel K, Sotzik F, Vremec D, Cameron PU, Shortman K. CD4 and CD8
expression by human and mouse thymic dendritic cells. Immunol Lett 1994; 40:
93-9
326. Smith CM, Belz GT, Wilson NS, Villadangos JA, Shortman K, Carbone FR,
Heath WR. Cutting edge: conventional CD8 alpha+ dendritic cells are
preferentially involved in CTL priming after footpad infection with herpes
simplex virus-1. J Immunol 2003; 170: 4437-40
327. Belz GT, Smith CM, Eichner D, Shortman K, Karupiah G, Carbone FR,
Heath WR. Cutting edge: conventional CD8 alpha+ dendritic cells are generally
involved in priming CTL immunity to viruses. J Immunol 2004; 172: 1996-2000
328. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by
the transcription factor Foxp3. Science 2003; 299: 1057-61
329. Fontenot JD, Rudensky AY. A well adapted regulatory contrivance: regulatory
T cell development and the forkhead family transcription factor Foxp3. Nat
Immunol 2005; 6: 331-7
330. Budd RC, Cerottini JC, MacDonald HR. Phenotypic identification of memory
cytolytic T lymphocytes in a subset of Lyt-2+ cells. J Immunol 1987; 138: 1009-
13
331. Salazar A, Levy H, Ondra S, Kende M, Scherokman B, Brown D, Mena H,
Martin N. Long-term treatment of malignant gliomas with intramuscularly
administered polyinosinic-polyctidylic acid stabilised with polylysine and
carboxymethylcellulose: an open pilot study. Neurosurgery 1996; 38: 1096-104
332. Ma Z, Li J, Yang L, Mu Y, Xie W, Pitt B, Li S. Inhibition of LPS and CpG
DNA-induced TNF-alpha response by oxidized phospholipids. Am J Physiol
Lung Cell Mol Physiol 2004; 286: L808-L16
333. Nowak AK, Robinson BW, Lake RA. Synergy between chemotherapy and
immunotherapy in the treatment of established murine solid tumors. Cancer Res
2003; 63: 4490-6
334. Gerloni M, Xiong S, Mukerjee S, Schoenberger SP, Croft M, Zanetti M.
Functional cooperation between T helper cell determinants. Proc Natl Acad Sci
U S A 2000; 97: 13269-74
335. McHugh RS, Shevach EM. Cutting edge: depletion of CD4+CD25+ regulatory
T cells is necessary, but not sufficient, for induction of organ-specific
autoimmune disease. J Immunol 2002; 168: 5979-83
336. Kohm AP, McMahon JS, Podojil JR, Begolka WS, DeGutes M, Kasprowicz
DJ, Ziegler SF, Miller SD. Cutting edge: anti-CD25 monoclonal antibody
injection results in the functional inactivation, not depletion, of CD4+CD25+ T
regulatory cells. J Immunol 2006; 176: 3301-5
337. Jiang W, Swiggard WJ, Heufler C, Peng M, Mirza A, Steinman RM,
Nussenzweig MC. The receptor DEC-205 expressed by dendritic cells and
thymic epithelial cells is involved in antigen processing. Nature 1995; 375:151-5
338. Sotomayor EM, Borrello I, Levitsky HI. Tolerance and cancer: a critical issue
in tumor immunology. Crit Rev Oncog 1996; 7: 433-56
- 210 -
339. Wichman M, Lau-Werner U, Muller C, Hornung H, Stieber P, Schildberg
F. Carcinoembryonic antigen for the detection of recurrent disease following
curative resection of colorectal cancer. Anticancer Res 2000; 20: 4953-5
340. Lange P, Ercole C, Lightner D, Fraley E, Vessella R. The value of serum
prostate specific antigen determinations before and after radical prostatectomy. J
Urol 1989; 141: 873-9
341. Szpaderska AM, DiPietro LA. Inflammation in surgical wound healing: friend
or foe? Surgery 2005; 137: 571-3
342. Klinman DM, Barnhart KM, Conover J. CpG motifs as immune adjuvants.
Vaccine 1999; 17: 19-25
343. Nowak A, Lake R, Marzo A, Scott B, Heath WR, Collins E, Frelinger J,
Robinson BW. T-cell receptor transgenic analysis of tumor-specific CD8 and
CD4 responses in the eradication of solid tumours. Cancer Res 1999; 59: 1071-9
344. Melief C. Tumor eradication by adoptive transfer of cytotoxic T lymphocytes.
Adv Cancer Res 1992; 58: 143-75
345. Kast W, Bronkhorst A, de Waal L, Melief C. Cooperation between cytotoxic
and helper T lymphocytes in protection against lethal Sendai virus infection.
Protection by T cells is MHC restricted and MHC-regulated; a model for MHC-
disease associations. J Exp Med 1986; 164: 723-38
346. Rodolfo M, Zilocchi P, Accornero P, Cappetti B, Airioli I, Colombo M. Il-4
transuced tumor cell vaccine induces immunoregulatory type 2 CD8 T
lymphocytes that cure lung metastases upon adoptive transfer. J Immunol 1999;
163: 1923-8
347. Cordaro TA, de Visser KE, Tirion FH, Graus YM, Haanen JB, Kioussis D,
Kruisbeek AM. Tumor size at the time of adoptive transfer determines whether
tumor rejection occurs. Eur J Immunol 2000; 30: 1297-307
348. Gabrilovich DI, Corak J, Ciernik IF, Kavanaugh D, Carbone DP. Decreased
antigen presentation by dendritic cells in patients with breast cancer. Clin
Cancer Res 1997; 3: 483-90
349. Rescigno M, Winzler C, Delia D, Mutini C, Lutz M, Ricciardi-Castagnoli P.
Dendritic cell maturation is required for initiation of the immune response. J
Leukoc Biol 1997; 61: 415-21
350. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu
Rev Immunol 1991; 9: 271-96
351. Heath W, Carbone F. Cytotoxic T lymphocyte activation by cross-priming.
Curr Opin Immunol 1999; 11: 314 - 8
352. Romagnani S. The Th1/Th2 paradigm. Immunol Today 1997; 18: 263-6
353. Tew JG, Phipps RP, Mandel TE. The maintenance and regulation of the
humoral immune response: persisting antigen and the role of follicular antigen-
binding dendritic cells as accessory cells. Immunol Rev 1980; 53: 175-201
354. Mandel TE, Phipps RP, Abbot A, Tew JG. The follicular dendritic cell: long
term antigen retention during immunity. Immunol Rev 1980; 53: 29-59
355. Gray D, Matzinger P. T cell memory is short-lived in the absence of antigen. J
Exp Med 1991; 174: 969-74
356. Schittek B, Rajewsky K. Maintenance of B-cell memory by long-lived cells
generated from proliferating precursors. Nature 1990; 346: 749-51
357. Wheelock E, Weinhold K, Levich J. The tumor dormant state. Adv Cancer Res
1981; 34: 107-40
- 211 -
358. Schnorrer P, Behrens GM, Wilson NS, Pooley JL, Smith CM, El-Sukkari D,
Davey G, Kupresanin F, Li M, Maraskovsky E, Belz GT, Carbone FR,
Shortman K, Heath WR, Villadangos JA. The dominant role of CD8+
dendritic cells in cross-presentation is not dictated by antigen capture. Proc Natl
Acad Sci U S A 2006; 103: 10729-34
359. Ronchese F, Hermans IF. Killing of dendritic cells: a life cut short or a
purposeful death? J Exp Med 2001; 194: F23-6
360. Kamath AT, Pooley J, O'Keeffe MA, Vremec D, Zhan Y, Lew AM,
D'Amico A, Wu L, Tough DF, Shortman K. The development, maturation,
and turnover rate of mouse spleen dendritic cell populations. J Immunol 2000;
165: 6762-70
361. Smith A: Unpublished data: personal communication., 2007.
362. Vaage J, Agarwal S. Stimulation or inhibition of immune resistance against
metastatic or local growth of a C3H mammary carcinoma. Cancer Res 1976; 36:
1831-6
363. Turk MJ, Guevara-Patino JA, Rizzuto GA, Engelhorn ME, Sakaguchi S,
Houghton AN. Concomitant tumor immunity to a poorly immunogenic
melanoma is prevented by regulatory T cells. J Exp Med 2004; 200: 771-82
364. Chagnon F, Tanguay S, Ozdal OL, Guan M, Ozen ZZ, Ripeau JS,
Chevrette M, Elhilali MM, Thompson-Snipes LA. Potentiation of a dendritic
cell vaccine for murine renal cell carcinoma by CpG oligonucleotides. Clin
Cancer Res 2005; 11: 1302-11
365. Lang K, Recher M, Navarini A, Harris N, Löhning M, Junt T, Probst H,
Hengartner H, Zinkernagel RM. Inverse correlation between Il-7 receptor
expression and CD8 T cell exhaustion during persistent antigen stimulation. Eur
J Immunol 2005; 35: 738-45
366. Munn DH, Mellor AL. The tumor-draining lymph node as an immune-
privileged site. Immunol Rev 2006; 213: 146-58
367. van Herpen C, De Mulder P. Locoregional immunotherapy in cancer patients:
Review of clinical studies. Ann Oncol 2004; 11: 1229 - 39
368. Yu P, Lee Y, Liu W, Krausz T, Chong A, Schreiber H, Fu YX. Intratumoral
depletion of CD4+ cells unmasks tumor immunogenicity leading to the rejection
of late-stage tumors. J Exp Med 2005; 201: 779-91
369. Hockel M, Dornhofer N. The hydra phenomenon of cancer: why tumors recur
locally after microscopically complete resection. Cancer Res 2005; 65: 2997-
3002
370. Baker DG, Masterson TM, Pace R, Constable WC, Wanebo H. The
influence of the surgical wound on local tumor recurrence. Surgery 1989; 106:
525-32
371. Kaparakis M, Laurie KL, Wijburg O, Pedersen J, Pearse M, van Driel IR,
Gleeson PA, Strugnell RA. CD4+ CD25+ regulatory T cells modulate the T-
cell and antibody responses in helicobacter-infected BALB/c mice. Infect Immun
2006; 74: 3519-29
372. Zou W. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev
Immunol 2006; 6: 295-307
373. Battaglia A, Ferrandina G, Buzzonetti A, Malinconico P, Legge F, Salutari
V, Scambia G, Fattorossi A. Lymphocyte populations in human lymph nodes.
Alterations in CD4+ CD25+ T regulatory cell phenotype and T-cell receptor
Vbeta repertoire. Immunology 2003; 110: 304-12
374. Simova J, Bubenik J, Bieblova J, Rosalia RA, Fric J, Reinis M. Depletion of
T(reg) cells inhibits minimal residual disease after surgery of HPV16-associated
tumours. Int J Oncol 2006; 29: 1567-71
- 212 -
375. Phan GQ, Yang JC, Sherry RM, Hwu P, Topalian SL, Schwartzentruber
DJ, Restifo NP, Haworth LR, Seipp CA, Freezer LJ, Morton KE,
Mavroukakis SA, Duray PH, Steinberg SM, Allison JP, Davis TA,
Rosenberg SA. Cancer regression and autoimmunity induced by cytotoxic T
lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma.
Proc Natl Acad Sci U S A 2003; 100: 8372-7
376. Hodi FS, Mihm MC, Soiffer RJ, Haluska FG, Butler M, Seiden MV, Davis
T, Henry-Spires R, MacRae S, Willman A, Padera R, Jaklitsch MT,
Shankar S, Chen TC, Korman A, Allison JP, Dranoff G. Biologic activity of
cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously
vaccinated metastatic melanoma and ovarian carcinoma patients. Proc Natl Acad
Sci U S A 2003; 100: 4712-7
377. Zhang H, Chua KS, Guimond M, Kapoor V, Brown MV, Fleisher TA, Long
LM, Bernstein D, Hill BJ, Douek DC, Berzofsky JA, Carter CS, Read EJ,
Helman LJ, Mackall CL. Lymphopenia and interleukin-2 therapy alter
homeostasis of CD4+CD25+ regulatory T cells. Nat Med 2005; 11: 1238-43
378. Foss FM. DAB(389)IL-2 (ONTAK): a novel fusion toxin therapy for
lymphoma. Clin Lymphoma 2000; 1: 110-6; discussion 7
379. Attia P, Maker AV, Haworth LR, Rogers-Freezer L, Rosenberg SA.
Inability of a fusion protein of IL-2 and diphtheria toxin (Denileukin Diftitox,
DAB389IL-2, ONTAK) to eliminate regulatory T lymphocytes in patients with
melanoma. J Immunother 2005; 28: 582-92
380. Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S. Stimulation of
CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-
tolerance. Nat Immunol 2002; 3: 135-42
381. Kusmartsev S, Gabrilovich DI. Inhibition of myeloid cell differentiation in
cancer: the role of reactive oxygen species. J Leukoc Biol 2003; 74: 186-96
382. Danna E, Sinha P, Glibert M, Clements V, Pulaski B, Ostrand-Rosenerg S.
Surgical removal of primary tumor reverses tumor-induced immunosuppression
despite the presence of metastatic disease. Cancer Res 2004; 64: 2205 - 11
383. Kusmartsev S, Gabrilovich D. Immature myeloid cells and cancer-associated
immune suppression. Cancer Immunol Immunother: 2002; 51: 293-8
384. Carter JJ, Whelan RL. The immunologic consequences of laparoscopy in
oncology. Surg Oncol Clin N Am 2001; 10: 655-77
385. Whelan RL. Open versus laparoscopy assisted colectomy. Lancet 2003; 361:
75; author reply -6
386. McCoy JL, Rucker R, Petros JA. Cell-mediated immunity to tumor-associated
antigens is a better predictor of survival in early stage breast cancer than stage,
grade or lymph node status. Breast Cancer Res Treat 2000; 60: 227-34
387. Itano N, Blute M, Spotts B, Zincke H. Outcome of isolated renal cell
carcinoma fossa recurrence after nephrectomy. J Urol 2000; 164: 322-5
388. Sugarbaker DJ, Flores RM, Jaklitsch MT, Richards WG, Strauss GM,
Corson JM, DeCamp MM, Jr., Swanson SJ, Bueno R, Lukanich JM,
Baldini EH, Mentzer SJ. Resection margins, extrapleural nodal status, and cell
type determine postoperative long-term survival in trimodality therapy of
malignant pleural mesothelioma: results in 183 patients. J Thorac Cardiovasc
Surg 1999; 117: 54-63; discussion -5
389. Honeychurch J, Glennie MJ, Johnson PW, Illidge TM. Anti-CD40
monoclonal antibody therapy in combination with irradiation results in a CD8 T-
cell-dependent immunity to B-cell lymphoma. Blood 2003; 102: 1449-57
- 213 -
390. Nagata H, Arai T, Soejima Y, Suzuki H, Ishii H, Hibi T. Limited capability
of regional lymph nodes to eradicate metastatic cancer cells. Cancer Res 2004;
64: 8239-48
391. Disis ML, Bernhard H, Shiota FM, Hand SL, Gralow JR, Huseby ES, Gillis
S, Cheever MA. Granulocyte-macrophage colony-stimulating factor: an
effective adjuvant for protein and peptide-based vaccines. Blood 1996; 88: 202-
10
392. Chin CS, Bear HD. Sentinel node mapping identifies vaccine-draining lymph
nodes with tumor-specific immunological activity. Ann Surg Oncol 2002; 9: 94-
103
393. Qian CN, Berghuis B, Tsarfaty G, Bruch M, Kort EJ, Ditlev J, Tsarfaty I,
Hudson E, Jackson DG, Petillo D, Chen J, Resau JH, Teh BT. Preparing the
"soil": the primary tumor induces vasculature reorganization in the sentinel
lymph node before the arrival of metastatic cancer cells. Cancer Res 2006; 66:
10365-76
394. Edreira MM, Colombo LL, Perez JH, Sajaroff EO, de Castiglia SG. In vivo
evaluation of three different 99mTc-labelled radiopharmaceuticals for sentinel
lymph node identification. Nucl Med Commun 2001; 22: 499-504
395. Kobayashi H, Kawamoto S, Sakai Y, Choyke PL, Star RA, Brechbiel MW,
Sato N, Tagaya Y, Morris JC, Waldmann TA. Lymphatic drainage imaging
of breast cancer in mice by micro-magnetic resonance lymphangiography using
a nano-size paramagnetic contrast agent. J Natl Cancer Inst 2004; 96: 703-8
396. Shao X, Liu C. Influence of IFN- alpha and IFN- gamma on
lymphangiogenesis. J Interferon Cytokine Res 2006; 26: 568-74
397. Lewis RJ. Is radical lymphadenectomy a valid oncologic procedure? Eur J
Cardiothorac Surg 1999; 16 Suppl 1: S11-2
398. Sieweke MH, Thompson NL, Sporn MB, Bissell MJ. Mediation of wound-
related Rous sarcoma virus tumorigenesis by TGF-beta. Science 1990; 248:
1656-60
399. Hoon DS, Korn EL, Cochran AJ. Variations in functional immunocompetence
of individual tumor-draining lymph nodes in humans. Cancer Res 1987; 47:
1740-4
400. Gabrilovich D. Mechanisms and functional significance of tumour-induced
dendritic-cell defects. Nat Rev Immunol 2004; 4: 941-52
401. Halak BK, Maguire HC, Jr., Lattime EC. Tumor-induced interleukin-10
inhibits type 1 immune responses directed at a tumor antigen as well as a non-
tumor antigen present at the tumor site. Cancer Res 1999; 59: 911-7
402. Gorelik L, Flavell RA. Transforming growth factor-beta in T-cell biology. Nat
Rev Immunol 2002; 2: 46-53
403. Morton D, Thompson J, Cochran A, Mozillo N, Elashoff R, Essner R,
Nieweg OE, Roses DF, Hoekstra HJ, Karakousis CP, Reintgen DS,
Coventry BJ, Glass EC, Wang H. Sentinel-node biopsy or nodal observation
in melanoma. N Engl J Med 2006; 355: 1307-17
404. Essner R, Lee J, Wanek L, Itakura H, Morton D. Contemporary surgical
treatment of advanced-stage melanoma. Arch Surg 2004; 139: 961-7
405. Lee J, Essner R, Wanek L, Morton DL. Sentinel lymphadenectomy guided
complete lymph node dissection improves loco-regional disease control in early-
stage head and neck melanoma. J Clin Oncol 2004; 22: 723s
406. Nelson D, Bundell C, Robinson B. In vivo cross-presentation of a soluble
protein antigen: kinetics, distribution, and generation of effector CTL
recognizing dominant and subdominant epitopes. J Immunol 2000; 165: 6123-32
- 214 -
407. Landis S, Murray T, Bolden S, Wingo P. Cancer statistics. CA-Cancer J Clin
1999; 49: 8-31
408. Chow W, Devesa S, Warren J, Fraumeni J. Rising incidence of renal cell
cancer in the United States. JAMA 1999; 281: 1628-31
409. Pantuck A, Zisman A, Belldegrun A. The changing natural history of renal
cell carcinoma. J Urol 2001; 166: 1611-23
410. Registries AIoHaWAAoC: Cancer survival in Australia, 2001: Part 1: National
Summary (Cancer Series No. 18). Canberra, Australian Institute of Health and
Welfare, 2001, pp 74.
411. Mertens W, Eisenhauer E, Moore M, Venner P, Stewart D, Muldal A,
Wong D. Gemcitabine in advanced renal cell carcinoma. A phase II study of the
National Cancer Institute of Canada Clinical Trials Group. Ann Oncol 1993; 4:
331-2
412. Finke J, Tubbs R, Connelly B, Pontes E, Montie J. Tumor-infiltrating
lymphocytes in patients with renal-cell carcinoma. Ann NY Acad Sci 1988; 532:
387-94
413. Neumann E, Engelsberg A, Decker J, Storkel S, Jaeger E, Huber C, Seliger
B. Heterogenous expression of the tumor-associated antigens RAGE-1,
PRAME, and glycoprotein 75 in human renal cell carcinoma: candidates for T-
cell based immunotherapies? Cancer Res 1998; 58: 4090-5
414. Thompson R, Kuntz S, Leibovich B, Dong H, Lohse C, Webster W,
Sengupta S, Frank I, Parker A, Zincke H, Blute M, Sebo T, Cheville J,
Kwon E. Tumor B7-H1 is associated with poor prognosis in renal cell
carcinoma patients with long-term followup. Cancer Res 2006; 66: 3381-5
415. Flanigan R. Debulking nephrectomy in metastatic renal cancer. Clin Cancer
Res 2004; 10: 6335s - 41s
416. Galligioni E, Quaia M, Merlo A, Carbone A, Spada A, Favaro D, Santarosa
M, Sacco C, Talamini R. Adjuvant immunotherapy treatment of renal
carcinoma patients with autologous tumor cells and bacillus Calmette-Guerin:
five-year results of a prospective randomized study. Cancer 1996; 77: 2560-6
417. Albelda SM. Role of integrins and other cell adhesion molecules in tumor
progression and metastasis. Lab Invest 1993; 68: 4-17
418. Dorudi S, Sheffield JP, Poulsom R, Northover JM, Hart IR. E-cadherin
expression in colorectal cancer. An immunocytochemical and in situ
hybridization study. Am J Pathol 1993; 142: 981-6
419. Cioce V, Castronovo V, Shmookler BM, Garbisa S, Grigioni WF, Liotta
LA, Sobel ME. Increased expression of the laminin receptor in human colon
cancer. J Natl Cancer Inst 1991; 83: 29-36
420. Mareel MM, Bracke ME, Boghaert ER. Tumour invasion and metastasis:
therapeutic implications? Radiother Oncol 1986; 6: 135-42
421. Kugler A, Stuhler G, Walden P, Zoller G, Zobywalski A, Brossart P,
Trefzer U, Ullrich S, Muller CA, Becker V, Gross AJ, Hemmerlein B, Kanz
L, Muller GA, Ringert RH. Regression of human metastatic renal cell
carcinoma after vaccination with tumor cell-dendritic cell hybrids. Nat Med
2000; 6: 332-6
422. Ribas A, Butterfield LH, Glaspy JA, Economou JS. Current developments in
cancer vaccines and cellular immunotherapy. J Clin Oncol 2003; 21: 2415-32
423. Tamura Y, Peng P, Liu K, Daou M, Srivastava PK. Immunotherapy of
tumors with autologous tumor-derived heat shock protein preparations. Science
1997; 278: 117-20
- 215 -
424. Srivastava PK, Menoret A, Basu S, Binder RJ, McQuade KL. Heat shock
proteins come of age: primitive functions acquire new roles in an adaptive
world. Immunity 1998; 8: 657-65
425. Stewart B, Kleihues P (eds.): World Cancer Report. Lyon, IARC Press, 2003.
- 216 -
Appendix B: Abbrevia t ions
Ab Antibody. An immune protein targeting a particular structure e.g.
peptide.
AB1 A murine mesothelioma cell line, developed by immortalising tumour
from the peritoneum of asbestos injected BALB/c mice.
AB1HA AB1 mesothelioma that was transfected with genes for
Haemagglutinin and neomycin resistance.
ANOVA “ANalysis Of VAriance”. A statistical method used to test for
differences between three or more independent groups.
APC Antigen presenting cell. A cell capable of antigen uptake and
presentation to CTLs and helper T cells.
BCG Bacillus Calmette-Guérin. An attenuated form of Mycobacterium
bovis. Used as an active non-specific immunotherapy.
BSA Bovine serum albumin. A protein used in general laboratory work,
including the supplementation of cell culture medium.
CD Cluster determinant. An antigen cluster with which antibodies react.
Characteristic CD patterns assist in identifying cell type.
CD4+ Helper T cells. Secrete cytokines and augment the immune response.
CD8+ Widely used to denote CTLs.
CFSE 5,6-carboxyfluoroscein succinimidyl ester. An intracellular dye used
to stain lymphocytes.
CL4 Clone Four transgenic mouse. Homozygous for CD8+ cells with
specificity for the 1-Ad restricted IYSTVASSL epitope of
haemagglutinin.
CTL CTL. CD8+ T lymphocytes capable of lysing target cells.
95% CI 95% confidence interval. A range of values that, with a probability of
0.95, contains the population mean.
DLN Draining lymph node. Becomes synonymous with “sentinel lymph
node” in this thesis.
DC Dendritic cell. Characteristically CD11c+. Thought to have a critical
role in tumour antigen presentation.
DNA Deoxyribonucleic acid. Contains the genetic code. Certain fragments
of bacterial or viral DNA can be immune stimulatory.
DOS Day of surgery. Occurring within 24 hours of tumour resection.
- 217 -
DTH Delayed type hypersensitivity. Evoked when macrophages present
antigen to CTLs and helper T cells. Involved in allergic responses,
transplant rejection, and the immune response to mycobacterial
infection.
FasL Fas ligand. Also known as apoptosis antigen ligand 1, or CD95
ligand. Trimerises Fas Receptors of the target cell membrane, leading
to apoptosis of that cell.
FCS Foetal calf serum. A protein used in general laboratory work,
including the supplementation of cell culture medium.
FGK-45 A monoclonal antibody that ligates and activates the CD40 receptor.
Foxp3 A forkhead/winged helix transcription factor, critical to the
development and function of Treg.
GK1.5 Rat monoclonal antibody with specificity for murine CD4+, used to
deplete CD4+ cells in vivo.
GL113 Rat isotype antibody, used as control for depletion and FGK45
therapy experiments.
GM-CSF Granulocyte macrophage – colony stimulating factor. A protein that
stimulates bone marrow to produce myeloid cells. Also attracts DC
and causes proliferation of the same.
HA Haemagglutinin. A characteristic antigen of PR8 influenza that was
transfected into the AB1 and Renca cell lines.
HEPES N-2-hydroxyethylpiperazine-N‟-2-ethanesulfonic acid. A buffer used
in cell culture medium.
HNT Transgenic mouse with high frequency of CD4+ cells with specificity
for the H-2Kd restricted HNTNGVTAACSHE epitope of
haemagglutinin.
HR Hazard ratio. A statistical measure of survival in one group relative to
another.
HSPs Heat shock proteins. A family of chaperone proteins that form
complexes with intracellular peptides in response to cellular stress.
Provide a danger signal.
IFN Interferon alpha. A glycoprotein with immune stimulatory and anti-
angiogenic characteristics. Used in metastatic renal cancer.
- 218 -
IFN Interferon gamma. A dimeric glycoprotein cytokine that enhances
antigen presentation, activates macrophages, and up-regulates TH1
function.
Il-2 Interleukin two. A T cell growth factor cytokine. Also activates NK
cells. Used in metastatic renal cancer.
Il-3 Interleukin three. Stimulates proliferation of haematopoietic
pluripotent progenitor cells, as well as T cell growth and
differentiation.
Il-4 Interleukin four. A cytokine that activates B cells on numerous levels,
including the promotion of immunoglobulin class switching and
upregulation of MHC class II expression.
Il-5 Interleukin five. A cytokine that enhances eosinophil function,
stimulates B cell growth, and promotes plasma cell immunoglobulin
class switching.
Il-6 Interleukin six. Released by macrophages and T cells in response to
trauma. Involved in fever and the inflammatory response to trauma.
Il-10 Interleukin ten. Sometimes referred to as human cytokine synthesis
inhibitory factor. Impairs production of pro-inflammatory cytokines
and stimulates B cells.
Il-12 Interleukin twelve. Involved in the differentiation of naïve helper T
cells into the TH1 subset. Activates CTLs and natural killer cells and
stimulates IFN production.
iNOS Inducible nitric oxide synthase. Produces nitric oxide, a free radical.
i.p. Intraperitoneal. Used to denote the intra-coelomic route of
administration e.g. for monoclonal antibody.
i.v. Intravenous. Used to denote intravascular administration of a drug or
agent e.g. FGK-45.
LN Lymph node. A highly specialised aggregate of lymphoid tissue that
filters and processes incoming lymph from the tissues.
M1 A subset of macrophages that produce iNOS and are associated with
tumour immunity.
M2 A subset of macrophages that produce arginase, impair tumour
immunity, and foster cancer growth.
mAb Monoclonal antibody. Antibodies identical in specificity, produced by
a single clone of cells.
- 219 -
2-ME 2-mercaptoethanol. An anti-oxidant used to supplement cell culture
medium.
MHC Major histocompatibility complex. A set of cell surface molecules
involved in self recognition and antigen presentation.
MSC Myeloid-derived Suppressor Cells. A heterogenous population of
immature myeloid cells that accumulate during tumour growth and
suppress anti-tumour immunity.
NDLN Non draining lymph node. Lymph nodes which are not situated on the
afferent lymphatics of the tumour site (e.g. mediastinal nodes).
NK Natural Killer cells. Innate cells with cytotoxic capability, able to
recognize tumors or viral infected cells, plus the absence of MHC
Class I. Characteristically express CD16 and CD56 in humans, or
NK1.1/NK1.2 in mice.
NKT Natural killer T cells. A heterogenous group of cells that have
properties of both NK and T cells. They recognize non-polymorphic
CD1d.
P The probability that the observed or a greater difference in means (t
test) or medians (Mann Whitney test) for two samples could be seen
by chance, if the samples were drawn from the same population.
PBS Phosphate buffered saline. A ubiquitous laboratory solution, helpful
for processing cell samples.
PC61 Monoclonal antibody for the Il-2 receptor alpha (CD25). Can be used
to deplete for Treg in vivo.
PCR Polymerase chain reaction. A technique to amplify DNA enabling the
sensitive detection of minute quantities of a gene of interest (e.g.
haemagglutinin gene).
PgE2 Prostaglandin E2. A lipid derived autocrine and paracrine mediator,
involved in vasodilatation, inflammation, and fever.
Post-op Post-operative. Occurring after surgery.
RCC Renal cell carcinoma. A malignant neoplasm of the renal cortex.
S.C. Subcutaneous. Used to denote the subcuticular route of administration
or location.
SD Standard deviation. Calculated as the square root of the variance.
Measures variation in values about the mean.
- 220 -
SEM Standard error of measurement. Indicates whether the mean value
observed is a reliable estimate of the population mean. Calculated by
dividing the standard deviation of the sample by the square root of n
(the number of values).
sPS Soluble phosphatidylserine. A tumour derived soluble factor that has
anti-inflammatory properties.
TAM Tumour associated macrophages. Population of tumour infiltrative
macrophages that may develop from MSC and exert immune
suppressive properties within the tumour microenvironment.
TGF Transforming growth factor beta. Affects function, proliferation and
activation of numerous cell types. Sometimes released by tumours.
TH1 T helper cell type one. A subset of CD4+ helper T cells where
cytokines foster CTL function.
TH2 T helper cell type two. A subset of CD4+ helper T cells where
cytokines limit CTL function, but enhance IgE mediated immunity.
TIL Tumour infiltrating lymphocytes. Lymphocytes found within the
tumour, sometimes harvested and cultured for adoptive
immunotherapy.
TLR Toll like receptor. A group of pattern recognition receptors that react
to generic inflammatory signals (e.g. bacterial DNA).
TNF Tumour necrosis factor alpha. A cytokine released by white blood
cells and blood vessels in response to trauma. Chemoattractant to
neutrophils. Pro-inflammatory. Also involved with fever and cachexia
(hence its name).
Treg CD4+CD25
+ regulatory T cells that functionally suppress other cell
types.
TS Suppressor T cells. The former name for CD4+CD25
+ regulatory T
cells.
VEGF Vascular endothelial growth factor. Released in association with
hypoxia. Stimulates vasculogenesis and angiogenesis. Also associated
with the accumulation of MSC.
YTS169 Rat monoclonal antibody with specificity for murine CD8+, used to
deplete CD8+ in vivo.
Recommended