Characterization of a Novel Dendritic Cell Population · 2013-10-17 · 1 CHAPTER 1 Introduction Dendritic cells comprise a heterogeneous population of cells that are specialized

Characterization of a Novel Dendritic Cell Population

by

Anastassia Mikhailova

A thesis submitted in conformity with the requirements for the degree of Master of Science

Institute of Medical Science University of Toronto

© Copyright by Anastassia Mikhailova 2012

ii

Characterization of a Novel Dendritic Cell Poluation

Anastassia Mikhailova

Master of Science

Institute of Medical Science University of Toronto

2012

Abstract

Conventional DC (cDC) arise from circulating immediate precursors (pre-cDC), and are

currently thought to be terminally differentiated. Here we show that cDC are capable of

generating progeny that lost all characteristic features of cDC and aquired regulatory properties.

Sorted bone marrow pre-cDCs were cultured on a stromal monolayer in the presence or absence

of granulocyte-macrophage colony stimulating factor (GM-CSF). In the absence of GM-CSF,

pre-cDC derived DCs gave rise to a homogeneous population of CD11clow MHClow cells (DC-

regs) on day 8-10 of culture. DC-regs failed to up-regulate major histocompatibility complex

class II (MHCII) and co-stimulatory molecules in response to DC maturation stimuli, were poor

stimulators in T cell proliferation assays and suppressed T cell proliferation in cultures

containing immuno-stimulatory DC. Co-transfer of DC-regs with DCs in vivo did not inhibit

proliferation of T cells. These findings reveal the potential of DCs to generate a regulatory DC

population with immunosuppressive properties.

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Acknowledgments

I would like to thank my supervisor, Dr. Mark Cattral for providing me with the opportunity to

learn, grow, and evolve under his mentorship. I am extremely appreciative of all his support,

guidance, and advice.

I would also like to thank my co-supervisor, Dr. Reginal Gorczynski for his support, wisdom,

constructive criticism and feedback on my work as well as inspiration. I am very lucky to have

such wise superiors as supervisors.

I would like to thank Jun Diao and Jun Zhao for training in logic, design of scientific

experiments and technical training. I would not achieve what I have without your support.

Also, I would like to thank all members of Gorczynski Lab for ongoing support, conversations

and humor.

Thank you also to my family for their support and understanding.

Thank you also to Heart and Stroke Foundation and Canadian Institute for Health and Research

for their financial contribution that allowed this research to take place.

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Table of Contents

Abstract .......................................................................................................................................... ii

Acknowledgments ........................................................................................................................ iii

List of Figures .............................................................................................................................. vii

CHAPTER 1 .................................................................................................................................. 1

1 Types of DCs .......................................................................................................................... 1

1.1 Conventional DCs ........................................................................................................... 1 1.2 Plasmacytoid DCs ........................................................................................................... 2 1.3 Lymphoid tissue resident DCs ........................................................................................ 3 1.4 Peripheral tissue DCs ...................................................................................................... 5 1.5 Intestinal DCs.................................................................................................................. 7 1.6 Thymic DCs .................................................................................................................... 7

2 Current model of DC ontogeny ........................................................................................... 8

2.1 Generation of DC from monocytes ................................................................................. 9 2.2 In vitro methods of dendritic cell generation ................................................................ 11

3 Dendritic cell activation of immunity ................................................................................ 11

4 Dendritic cell activation of tolerance ................................................................................ 12

4.1 Regulatory dendritic cells ............................................................................................. 12 DCs in central tolerance ....................................................................................................... 12

DCs in peripheral tolerance ................................................................................................. 13

5 Mechanisms of immunosuppression ................................................................................. 14

5.1 PD-L1/PD-L2 ................................................................................................................ 14 5.2 Arginase and nitric oxide synthase (NOS).................................................................... 15 5.3 Indoleamine 2,3-dioxygenase (IDO) ............................................................................ 17 5.4 IL-10 ............................................................................................................................. 18

6 Antigen presenting cells in cancer immunology ............................................................... 19

6.1 Tumor-derived dendritic cells ....................................................................................... 19 6.2 Myeloid derived suppressor cells (MDSC)................................................................... 20 6.3 Tumor associated macrophages (TAMs) ...................................................................... 21

CHAPTER 2 ................................................................................................................................ 23

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1 Preliminary observations ................................................................................................... 23

2 Hypothesis ............................................................................................................................ 24

3 Objective .............................................................................................................................. 24

4 Specific aims ........................................................................................................................ 24

CHAPTER 3 Methodology ........................................................................................................ 25

1 Mice ...................................................................................................................................... 25

2 Primary skin stromal cell preparation .............................................................................. 25

3 Cell Isolation ........................................................................................................................ 25

4 Flow cytometry .................................................................................................................... 26

5 Mixed lymphocyte reactions .............................................................................................. 26

6 CFSE labelling ..................................................................................................................... 27

7 Reverse transcriptase PCR ................................................................................................ 27

8 Arginase and iNOS activity assays .................................................................................... 28

9 Adoptive transfer studies ................................................................................................... 28

10 Statistics ............................................................................................................................... 29

CHAPTER 4 Results .................................................................................................................. 30

1 CD11clow MHCIIlow cells exhibit potent immuno-suppressive properties in vitro ......... 30

2 Mechanisms of immuno-suppression mediated by CD11clow MHCIIlow DC-regs ......... 32

3 Immunosuppressive activity of CD11clow MHCIIlow cells in vivo .................................... 35

CHAPTER 5 Discussion ............................................................................................................. 37

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CHAPTER 6 ................................................................................................................................ 46

1 Conclusion ........................................................................................................................... 46

2 Future directions ................................................................................................................. 46

References .................................................................................................................................... 73

vii

List of Figures

Figure 1. Dendritic cell subtypes grouped based on physiological location .................................. 4

Figure 2. Schematic representation of DC ontogeny. .................................................................. 10

Figure 3. Differentiation and proliferation of pre-cDC on stroma .............................................. 50

Figure 4. CD11clow MHCII low arise from CD11c+ MHC II+cDC ................................................ 51

Figure 5. Immunophenotype of cDC-derived CD11clow MHCII low cells ..................................... 52

Figure 6. DC-regs fail to up-regulate co-stimulatory molecules in response to maturation stimuli........................................................................................................................................................ 53

Figure 7. CD11clow MHCII low cells are poor stimulators of allogeneic T cell lymphocytes ........ 54

Figure 8. CD11clow MHCII low DC-derived cells have increased phagocytic capacity. ............... 55

Figure 9. CD11clow MHCII low suppress T cell proliferation in allogeneic mixed lymphocyte reaction. ......................................................................................................................................... 56

Figure 10. DC-regs suppress effector function of allogeneic T cells in mixed lymphocyte cultures .......................................................................................................................................... 57

Figure 11. DC-regs suppress OT-II T cell proliferation in response to OVA-pulsed DCs .......... 58

Figure 12. DC-regs suppress OT-II T cell proliferation .............................................................. 59

Figure 13. DC-regs do not suppress OT-II T cell cytokine release ............................................. 60

Figure 14. Expression of CD25 and CD44 by T cells in allogeneic mixed lymphocyte reaction in the presence or absence of DC-regs .............................................................................................. 61

Figure 15. DC-regs do not induce T cell death in mixed lymphocyte reactions .......................... 62

Figure 16. DC-regs do not induce Foxp3+ Tregs in mixed lymphocyte cultures ........................ 63

Figure 17. Mechanism of DC-reg-mediated immuno-suppression involves both soluble and contact-dependent factors ............................................................................................................. 64

Figure 18. RT-PCR expression of candidate molecules responsible for observed in vitro immuno-suppression ..................................................................................................................... 65

Figure 19. DC-regs express high levels of arginase1 and iNOS (reflected by nitrite production) activity when stimulated with LPS ............................................................................................... 66

viii

Figure 20. Both DCs and DC-regs express high levels of PD-L1. 1×106 DC or DC-regs were pulsed with 2 µg/mL LPS overnight. ............................................................................................ 67

Figure 21. LPS-pulsed DC-regs express high levels of IL-10. Freshly isolated spleen DCs or DC-regs were pulsed with 2 µg/mL LPS overnight ..................................................................... 68

Figure 22. DC-regs suppress T cell proliferation through an iNOS-dependent mechanism ....... 69

Figure 23. DC-regs do not suppress T cell proliferation and activation in vivo .......................... 70

Figure 24. DC-regs fail to suppress T cell proliferation and activation in vivo ........................... 71

Figure 25. DC-regs induce OT-II cell activation in vivo ............................................................. 72

1

CHAPTER 1

Introduction

Dendritic cells comprise a heterogeneous population of cells that are specialized in antigen

uptake, processing, and presentation, and play a key role in linking innate and adapitive immune

responses. Dendritic cells were first discovered in mouse spleen by Steinman in 1973 1, and

were named dendritic cells because of their numerous motile cellular processes - dendrites.

Subsequent studies revealed that DCs were potent stimulators of allogeneic T cells in mixed

lymphocyte reaction 2 and their potency exceeded that of other “professional” antigen presenting

cells (i.e. macrophages and B cells).

1 Types of DCs

DCs are currently divided into two major categories: 1) interferon-producing plasmacytoid

(pDCs); and 2) conventional DCs (cDCs). DC can be further divided based on their location

(lymphoid, migratory), expression of cell-surface markers, and functional attributes. Lymphoid

resident DCs reside within lymphoid tissues throughout their life cycle, whereas migratory DCs

migrate from peripheral tissues to the lymph nodes. DC migration occurs continously during

steady-state conditions, and increases with inflammation. DCs can also be classified based on

their ability to polarize differentially T cell responses in tolerance and immunity.

1.1 Conventional DCs

Conventional DCs (cDCs) have a typical heterogeneous morphology with abundant cytoplasm,

multiple dendrites and irregular nucleus 5. These cells are widely distributed in lymphoid and

peripheral tissues (Figure 1). cDCs are superior to macrophages and B cells in Ag presentation

because of their higher capacity to capture and process Ag 6. Upon encounter of foreign Ag,

cDCs undergo a process of maturation where they up-regulate surface expression of MHCII and

co-stimulatory molecules (CD80, CD86, CD40) and activate naïve Ag-specific T cells. Three

major subtypes of cDCs exist: CD4+ CD8-, CD4- CD8+, CD4- CD8-, all of which are found

2

within multiple physiological compartments in the body and differ in their ability to induce

differential T cell responses. These subsets are described in detail below.

Physiological DC localization is driven by expression of chemokine receptors. Immature cDCs

express receptors for inflammatory chemokines including CXCR1, CCR1, CCR2 and CCR5 17,

which drive DC migration towards lymphoid organs. Chemokines produced by freshly isolated

splenic cDCs were identified as macrophage inflammatory protein 1 alpha (MIP-1α or CCL3),

MIP-1β or CCL4 and regulated upon activation, normal T cell expressed and secreted (RANTES

or CCL5). Moreover, it was observed that different subsets of splenic cDCs express these

chemokines in different proportions with all three (CCL3, CCL4 and CCL5) expressed highest

on CD4+ cDCs 18. During maturation, when cDCs encounter foreign Ag and inflammatory

stimuli, they upregulate MHCII, co-stimulatory molecules and their migratory capacity. Mature

cDCs migrate in response to chemokines to lymph nodes via afferent lymphatics and localize in

T cell areas of LN. DC migration to peripheral lymphatic vessels is guided by the

chemoattractant gradient of CCL19 and CCL21, which bind to chemokine receptor CCR7 found

on cDCs 19, 20. CCR7 is upregulated after DC encounter maturation stimuli 21. It was

demonstrated that CCR7 KO mice not only have deficient DC and T cell migration to LN, but

also fail to mount primary immune response 19. CXCR4 was also observed to be upregulated on

mature cDCs 20. Additionally, sensitivity of DCs to CCL3, CCL4 and CCL5 22 as well as

expression of CCR1 and CCR5 21 upon maturation is dramatically reduced.

1.2 Plasmacytoid DCs

Plasmacytoid DCs (pDCs) were first identified in humans and were later shown to exist in mice 45 as lin- CD11cint CD11b- Ly6C+ B220+ cells. pDCs were identified in lymphoid organs, bone

marrow, lung, liver, blood and skin 5, 46. Poor in vitro survival is observed when cells are

cultured in liquid medium alone. Survival is moderately enhanced when medium is

supplemented with GM-CSF alone or in combination with IL-3 47. Further survival and

maturation are induced with addition of IFN-α, influenza virus, CpG or CpG and GM-CSF 5, 45.

The in vivo life span of pDCs was determined to be about two weeks 48.

Immature pDCs have a round shape, smooth surface and eccentric nucleus and acquire dendritic

cell-like morphology upon activation with CD40L or CpG. Similarly to cDCs, pDCs upregulate

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surface MHCII as well as CD40 and CD86 expression. In contrast to cDCs, freshly isolated

pDCs fail to stimulate T cell proliferation in allogeneic mixed lymphocyte reaction and only do

so upon maturation 45. When activated with viral stimuli pDCs produce large amounts of type I

IFNs (IFNα, IFNβ) and moderate levels of IL-12 5. They migrate to inflamed LN and cluster

around high endothelial venules 47. In contrast to pDCs, cDCs and monocyte migrate from non-

lymphoid tissues to T cell rich areas of lymph nodes through afferent lymphatics 48. pDCs

express TLR7, TLR8, which recognize imidazoquinolins and ssRNA; and TLR9, which

recognizes bacterial DNA 49. pDCs, however, lack TLR2, 3, 4, 5 and, therefore, do not respond

to microbial stimuli such as LPS or poly I:C 15, 46. Immature pDCs were shown to induce IL-10

production in CD4+ T cells 50. It was also suggested that immature pDCs are able to induce Treg

cells in vitro 51. Activation of pDCs leads to rapid activation of NK cells and CD8+ T cell, IFN-γ

production and Th1 differentiation leading to anti-viral responses in both humans 50 and mice 15,

52. Moreover, they promote differentiation and maturation of cDCs 52 and stimulate B cells 46.

1.3 Lymphoid tissue resident DCs

Spleen contains about 20% pDCs and 80% cDCs 6 (Figure 1). Three major populations of cDCs

can be subdivided based on CD4 and CD8 staining: CD4+ CD8α- (60% of total); CD4- CD8α+

(20%); CD4- CD8α- (20%) 7 8. Other cell surface markers segregate with CD4 and CD8. For

example, CD8α+ cDC are CD11b- DEC-205+, whereas CD8α- cDC are CD11b+ and DEC-205-.

Both CD4+ CD8α- and CD4- CD8α- DCs exist mainly in the marginal zone in the steady state

and move to T cell zones upon maturation. The marginal zone is located between the red pulp,

which filters the blood of damaged red blood cells and other debris, and the white pulp, which

mainly contains lymphocytes. By contrast, CD8α+ cDCs are located in T cell zones within the

white pulp in the steady state 6. LNs contain all DC subsets seen in spleen along with additional

migratory DC subtypes (dermal DCs and epidermal Langerhans cells). CD8aint and CD8αlow

DCs are also present in LN, but not in spleen 9. All splenic cDC subpopulations showed half-life

kinetics of about 1.5 days, with all cells replenished by day 3 10.

4

CD8a- CD4+

CD8a+ CD4-

CD8a- CD4-

pDC

cDC

Lymph nodes

pDC

cDC

CD8a- CD4+

CD8a+ CD4-

CD8a- CD4-

CD8alow

CD8ahi

Dermal DC

Langerin+ LC

Peripheral

tissues

Langerin+ LC

CD8+ Langerinlow DC

CD8- Langerinhi DC

Resident

Migratory

Intestine:

Peyer Patches

CD11b= CD103+ CX3CR1=

CD11b+ CD103- CX3CR1+

Intestine:

Lamina Propria

CD11b+ CD8-

CD11b- CD8+

CD11b- CD8-

cDC

Thymus

pDC

CD11b- CD8+ CD172- cDC

CD11b+ CD8- CD172+ cDC

cDC

LEGEND

DENDRITIC CELL SUBTYPES

Spleen

Figure 1 Dendritic cell subtypes grouped based on physiological location. pDC, plasmacytoid DC; cDC, conventional DC; LC, langerhan cell

5

Although all splenic cDC subtypes capture antigens effectively, each have specialized properties

in inducing Ag-specific responses 10. For example, CD8+ DCs possess a potent capacity for

cross-presentation of soluble and cell-associated Ag to CD8+ T cells 11. CD8a+ DCs also release

high levels of IL-12 and preferentially induce Th1 responses. By contrast, CD8- DCs show

superior priming of CD4+ T cell 12, and preferentially induce Th2 responses 13, 14. However, it is

now recognized that the microenvironment plays a large role in directing how DCs skew Th

responses. It appears that different subtypes of DCs are specialized to recognize a particular type

of microbial stimulus and release a defined array of cytokines, which directs T cell

differentiation from Th0 into Th1 or Th2. For example, microbial molecules such as soluble

tachyzoite Ag (STAg) and CpG trigger cDCs to pomote Th1 differentiation, whereas nematode

antigens or yeast toxin trigger cDCs to drive Th2 differentiation 13, 15.

Microbial structural units or pathogen associated molecular patterns (PAMPs) are recognized by

Toll-like receptors expressed by DC. TLR expression on cDCs appears to be fairly ubiquitous,

with some exceptions. TLR3, which recognizes viral double stranded RNA, is expressed highest

on CD8α+ cDC and lowest on CD4+ cDC and vice versa for TLR5, which recognizes bacterial

flagellin. Additionally, TLR7, which recognizes endosomal single stranded RNA, has low

expression on CD8α+ cDC but is expressed on the other cDC subtypes 16. Therefore, ligation of

different TLRs determines the subtype of DC activated, which subsequently drives appropriate T

cell response. It has also been suggested that Ag dose affects CD4+ T cell response directed by

cDCs. Both CD8a+ and CD8a- cDCs were observed to induce Th1 response at high Ag doses and

Th2 response at low Ag doses 15.

1.4 Peripheral tissue DCs

Langerhan cells (LCs) or epidermal DCs and dermal DCs reside in the periphery and sample Ag

from skin and mucosal body surfaces. LCs account for 3-5% of all nucleated cells in the

epidermis and form a cellular network that provides the first immunological barrier to

environmental insults 23, 24. LCs express langerin (CD207), which is also expressed on some

dermal DC subtypes 25, 26. LC are also distinguished by expression of CD45, CD11c, CD11b,

F4/80, DEC-205, high expression of MHCII, absence of CD103 and the presence of Birbeck

granules in the cytoplasm 27, 23. The formation of Birbeck granules, which is associated with Ag

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capture, is a consequence of langerin expression 24. Upon Ag capture, langerin associates with

Birbeck granules and facilitates transport of captured ligands into a non-classical Ag-processing

pathway 25. Upon capture of Ag, LCs migrate via dermal lymphatics to skin draining LN 6. The

rate of migration increases during inflammation. Using a model of allergic contact dermatitis it

was demonstrated that LCs mediate immunity to cutaneous Ag 28. However, another study

showed that LCs are unable to prime CD8+ T cells in epidermal infection with Herpes Simplex

virus 218. The precise role of LCs in cutaneous immunity, therefore, appears to depend on the

type of Ag.

LC in subcutaneous LN can be distinguished from other DC subtypes by their larger size, higher

expression level of MHCII, low CD11b expression and intermediate CD8 expression 9. In the

steady state, LCs arise from a local pool of radioresistant hematopoietic precursors 25, 29. During

inflammatory processes, circulating monocytes appear to have a role in replenishing LCs31.

TGF-β is required for LC differentiation or maintenance 30 and mice lacking M-CSFR also lack

LCs 31. LCs turnover is slower than for other DC subtypes, as shown by only 50% of the cells

staining positive for BrdU at day 2132.

Another migratory DC subtype found in the skin, cutaneous and mesenteric lymph nodes

displays the phenotype of langerin+ CD11c+ MHCII+ CD11b+ CD205+ F4/80low CD103- 26

(Figure 1). Other langerin+ DC subtypes exist. For example, langerin positive dermal DCs 26, 33

divide into CD8+ langerinlow and CD8- langerinhi subsets 25. Langerin+ DCs also express high

levels of CD11c and MHCII 26. However, they can be distinguished from LCs by the presence

of CD103 marker 33, which is not expressed on LCs, and by the absence of F4/80 expression and

Birbeck granules 23. Additionally, LCs express higher levels of CD11b and the adhesion

molecule EpCAM 33. Dermal DCs were observed in the dermis when LCs were conditionally

ablated 26, 33. The life time of dermal langerin+ DCs is much shorter than that of epidermal LCs

and is marked by rapid repopulation of these cells with conditional ablation. Both dermal DC

populations repopulate dermis within 5 days (for CD8+ langerinlow DCs) and within 14 days (for

CD8- langerinhi subset) from bone marrow precursors migrating from the blood, long before LCs

repopulate epidermis 25, 26. BrdU labelling studies also demonstrated that langerin+ dermal DCs

proliferate at a higher rate than LCs 26. The kinetics of dermal DCs proliferation is similar to

7

those of spleen and LN DCs. Moreover, development of these cells seems to be independent of

TGFβ and M-CSFR.

1.5 Intestinal DCs

In the gut, DCs are found in the lamina propria (LP) of the small and large intestine (loose

connective tissue underlying gut epithelium) as well as in gut associated lymphoid organs such

as Peyer’s patches (PPs), mesenteric lymph nodes (MLN) and isolated lymphoid follicles. LP

DCs directly sample the luminal environment of the gut by penetrating epithelial tight junctions 37. Two main DC subtypes in the LP are defined by cell surface markers: CD11b- CD103+

CX3CR1- and CD11b+ CD103- CX3CR1+ 34, 35 (Figure 1). CD11b+ DCs originate from

monocytes 34, whereas the CD11b- subset originates from pre-cDC 34. CD11b+ DCs appear to

promote inflammaton because ablation of CD11b- DCs exacerbated colitis in a murine model.

Secretion of TNF-α 34 and induction of a Th17 response was associated with the development of

colitis 35, 36. At low APC:T cell ratios, all LP DC subsets can induce generation of FoxP3+ Tregs 35.

The main DC subsets in PP are CD11b+ CD8a-, CD11b- CD8a+ and CD11b- CD8a- 38 (Figure 1).

CD11b+ CD8a- DCs were found to be located in the subepithelial dome of PP where they pick up

Ag transported across intestinal epithelium by M cells. A CD11b- CD8a+ fraction was detected

exclusively in the interfollicular region where it likely activates naïve T cells 38. Here, CD11b+

CD8a- DCs were shown to induce differentiation of IL-10 and IL-4 producing Th2 cells 39 40.

CD11b- CD8a+ and CD11b- CD8a- DCs were shown to produce IL-12 and induce Th1 type

responses 39. CD11b- CD8a- DCs are located in both PP compartments. Gut DCs were shown to

be involved in tolerance induction towards oral Ag and commensal bacteria.

1.6 Thymic DCs

DCs in the thymus constitute only 0.5% of total cell number 41 and localize almost exclusively to

the thymic medulla 42. Three thymic DC subsets have been identified. Two are of the cDC

phenotype: CD11chi CD11b- CD8a+ CD172- and CD11chi CD11b+ CD8a- CD172+ 43 (Figure 1).

These cells displayed all markers typical of mature DCs and were similar to CD8a+ DCs in

spleen 7. About 35% of total thymic DCs are pDCs, which stain CD11cint MHCII low CD45RAhi

8

41. It has been demonstrated recently that only the CD11chi CD11b- CD8a+ subset is of

intrathymic origin and that CD11chi CD11b+ CD8a- and pDC subsets are emigrants into the

thymus 43. Migratory cDC and pDC take up circulating soluble and particulate Ag and transport

it to the thymus 43. Shortly after emigration into the thymus, migratory DCs mature and

upregulate both MHCII and co-stimulatory molecules. However, most thymic DCs are present in

an immature state with low expression of co-stimulatory molecules and moderate expression of

surface MHCII 44.

2 Current model of DC ontogeny

DCs develop from bone marrow derived lin- Sca+ c-kithi hematopoietic stem cells (HSC) 2

(Figure 2). Early studies suggested that DCs belong to myeloid lineage and arise from a common

myeloid progenitor (CMP) 53. More recent evidence suggests developmental flexibility exist in

the DC lineage. Early stages of differentiation can occur from both a common lymphoid

progenitor (CLP) and CMP. A CMP gives rise to the myeloid lineage of immune cells including

macrophages, monocytes, megakaryocytes and erythrocytes. CLP gives rise to lymphoid lineage

cells such as NK cells, T and B cells.

The first evidence for a possible lymphoid origin of DC came from the observation that early T

cell precursors in the thymus can generate thymic DC 54. Later it was observed that CD8a+ DCs

in both spleen and thymus differentiate from thymic T cell progenitors 55. For three years, CD8a+

DCs were considered to be of lymphoid origin and CD8a- of myeloid origin. In 2000, it was

reported that CD8a+ and CD8a- DCs were both capable of differentiating from CMP 56 and that

both DC types differentiated from the same CD4low lymphoid precursor population 57. Finally,

both CMP and CLP were observed to give rise to DC with similar efficiency 58 59. A CMP was

defined as lin- FcRyII/IIIslow CD34+ c-kit+ Sca-1- IL7Ra- and a CLP as lin- c-kitint Sca-1int IL7Ra+

Thy1.1- 59. Accordingly, the concept of myeloid vs lymphoid DC was abandoned.

The next steps in DC differentiation after CMP and CLP involve a sequential series of

precursors: common macrophage DC progenitors (MDP; lin- c-kit+ CX3CR1+ CD115+ Flt3+)60;

common DC precursors (CPDs) 61; and CD11c+ MHCII - B220- pre-cDCs and B220+ pre-pDCs,

9

which give rise exclusively to cDCs and pDCs respectively 62, 63. CD24- pre-cDC are committed

to CD8a- cDCs and CD24+ pre-cDCs are pre-committed to CD8a+ cDCs 64.

The distinguishing feature of early stage precursors that give rise to the DC lineage is the

expression of fms-like tyrosine kinase 3 (Flt3) 65. In particular, the majority of CLP and some

CMP express Flt3 65, 66. Flt3 is then progressively down-regulated in granulocyte macrophage

progenitor (GMP) downstream of CMP and is completely absent in cells committed to both T

and B lineages as well as to megakaryocyte/erythrocyte lineage 66. Flt3 expression is also absent

in mature cells of hematopoietic lineage but present on most subtypes of DCs. Moreover, mice

lacking Flt3L have deficient DC lineage haematopoiesis 67 whereas treatment with Flt3L

dramatically increases dendritic cell population in spleen, lymph nodes, blood and other organs 68. By contrast, administration of GM-CSF alone or GM-CSF and IL-4 – cytokines that are

commonly used to generate DC in vitro –have little effect on DC generation in vivo 68.

Furthermore, mice lacking GM-CSF have normal DC numbers in lymphoid tissues 69.

Initial experiments evaluating DC proliferation showed very low numbers of BrdU+ DCs after 2

hours of labelling. This finding led to the conclusion that peripheral DC were replenished solely

by migrating non-replicating precursor populations 10, 32. Kinetic studies revealed a short half-

life of about 1.5 days for splenic cDCs 32. Subsequent studies, however, challenged this view

when it was established that 4% of spleen DCs and 3.6% of bone marrow DCs were in the

S/G2/M phases of the cell cycle 63 70. It is now generally accepted that in situ DC proliferation

plays a key role in maintaining the peripheral DC pool. In addition, dividing DCs can pass on

Ag to their progeny, thereby prolonging and expanding the potential for antigen presentation.

2.1 Generation of DC from monocytes

Monocytes are heterogeneous cells of the mononuclear phagocyte system that constitute less

than 2% of peripheral blood cells. Their phenotype is CD11b+ CD115+ F4/80+. They arise in

bone marrow and are released into blood to give rise to macrophages and dendritic cells in

tissues. Two monocyte subtypes exist: inflammatory monocytes are CCR2+ CD62L+ CXCR3-

GR-1+ (Ly6C+) and resident monocytes are CCR2- CD62L- CXCR3+ GR1- (Ly6C-) 72. During

inflammation GR-1+ inflammatory monocytes migrate into inflamed tissue and differentiate into

macrophages and into DCs in draining lymph nodes 72, 73. GR-1- resident monocytes home to the

10

non-inflammed tissues in the steady state to give rise to macrophages and DCs 72, 74. Bone

marrow derived monocytes were shown to replenish DC populations in peripheral tissues but not

in spleen 74. However, the monocyte lineage and monocyte-derived DCs were shown to be

distinct from DC lineage 64. In the steady state, pre-cDCs have a far superior ability to generate

spleen DCs when compared to monocytes, which were only 2% as effective 64. Flt3 is expressed

only on myeloid and lymphoid progenitor derived DC but not on monocyte-derived DC 66.

Systemic administration of Flt3L but not GM-CSF, which is a monocyte growth factor

responsible for DC differentiation from monocytes, expands the DC pool in vivo 68.

CMP/CLP

pDC

cDC

Lymphoid tissues

HSC

Pre-pDC

Pre-cDC

CDP

(pro-DC)

MDP

Bone Marrow

monocytes

Non-lymphoid tissues

Inflammatory DC

Steady state DC

\\

Figure 2 Schematic representation of DC ontogeny. HSC, hematopoietic stem cell (Lin- Sca+ c-kithi); CMP, common myeloid progenitor (Lin- FcRγII/IIIs low CD34+ c-kit+ Sca-1- IL7Ra- Flt3+); CLP, common lymphoid progenitor (Lin- c-kitint Sca-1int IL7Ra+ Thy1.1- Flt3+); MDP, monocyte dendritic cell progenitor (Lin- c-kit+ CX3CR1+ CD115+ Flt3+); CDP, common DC progenitor (c-kit low CD115+ Flt3+); pre-cDC, immediate precursor of cDC (CD11c+ MHCII - B220-); pre-pDC, immediate precursor of pDC (CD11c+ MHCII - B220+); cDC, conventional DC; pDC, plasmacytoid DC.

11

2.2 In vitro methods of dendritic cell generation

When generated from bone marrow or peripheral blood, DCs are non-adherent or loosely

adherent cells which can be characterized based on expression of CD11c and MHC class II and

lack of lineage (lin) markers (CD3, CD19, B220, CD49b) 3, 4.

Inaba et al 75 was the first to describe a method for generating DC from cultured blood cells.

Cells suspensions were depleted of red blood cells and cultured overnight in GM-CSF. Non-

adherent cells were then removed and cultured for 10 more days and supplemented with GM-

CSF for last 3-4 days. Dendritic cell aggregates attached to an adherent monolayer were

harvested and cultured for further 4-10 days in the presence of GM-CSF 75. It was then

demonstrated that DCs could be obtained by culturing bone marrow supplemented with GM-CSF

alone or in combination with IL-4 for 6-10 days. Loosely attached cells are then harvested and

matured for 1-2 days in the presence of GM-CSF and TNFα or LPS 3, 4. This method generates

monocyte-drived DC similar to that which arise from monocytes in vivo during inflammatory

conditions. Alternatively, DCs can be generated by culturing bone marrow in liquid with human

Flt3L (100 ng/mL) for 9 days. These cultures generate both pDC and cDC and appear to

recapitulate DC generation under steady-state conditions. Non-adherent and loosely adherent

cells are harvested and matured in the presence of GM-CSF as well as IFN-γ or LPS for 24

hours76. Another method of generating DCs first described in Cattral’s laboratory involves

culturing immediate DC precursor, pre-cDC, on a stromal monolayer in the presence of GM-CSF

for 12 days 62. Mimicking physiologic conditions, this method generates immature DCs, which

can be matured overnight with LPS or TNFα to produce mature DCs. Culture of DC precursors

on a stromal monolayer generates a highly pure, homogeneous DC population, which is not

possible with bone marrrow culture. Moreover, the stromal monolayer allows for greater

expansion and longer survival of DCs as compared to liquid culture systems 212.

3 Dendritic cell activation of immunity

In the immunogenic model of DC activation, DC maturation is triggered by ligation of pattern

recognition receptors (PRRs) like TLRs, NODs, RIG-I-like, and c-type lectin receptors 6 and by

other pro-inflammatory signals such as cytokines that indicate injury or inflammation. Upon

12

activation, DC migrate from peripheral tissues into draining lymph nodes where they present Ag

and subsequently activate naïve T cells. Inflammatory cytokines secreted by Th1 and Th2 cells,

as well as ligation of CD40 on DC surface by primed T cells also provides activating signals.

During maturation, DCs upregulate surface expression of MHCII and co-stimulatory molecules

CD80 and CD86, which bind CD28 on T cell surface. They also release pro-inflammatory

cytokines such as IL-12, which triggers T cell activation and proliferation.

4 Dendritic cell activation of tolerance

Tolerogenic DCs generally have a distinct phenotype from that of mature stimulatory DCs. They

express low levels of surface MHCII and co-stimulatory molecules CD80, CD86 and CD40, do

not mature in response to classical DC activation stimuli (such as LPS, TNFa) and have low

capacity to prime T cells 77.

In the steady state in lymphoid organs DCs are present in immature state. It has been shown that

these DCs are able to sample and present Ag in the context of MHCI and MHCII without

maturation. For example, DCs sample apoptotic debris or self Ag and present these to naïve T

cells. Injection of immature DCs loaded with Ag renders T cells non-responsive to Ag (anergy)

and triggers Ag-specific T cell deletion or development of Tregs. Several factors can render DCs

tolerogenic. Both innate and adaptive immune systems can create local tolerogenic environment

dominated by immuno-suppressive cytokines such as IL-10 or TGFβ. Apoptotic debris can also

provide tolerogenic signals to DCs. Treg subsets such as FoxP3+ CD4+ and Tr1 cells can induce

DC tolerance.

4.1 Regulatory dendritic cells

DCs in central tolerance

Involvement of DCs in central tolerance was first mentioned in 1985 78. Four years later

Matzinger showed that when fetal thymuses were incubated with splenic DCs, donor-specific

tolerance developed 79. The role of thymic dendritic cells in central tolerance was later

demonstrated by studies of targeted expression of MHCII on DCs. It was demonstrated that these

13

cells mediate negative selection but not positive selection 80, 81. In one such study 80, the MHC

class II I-E transgene was expressed in DCs under a CD11c promoter in a C57BL/6 mouse. I-E-

specific T cells were deleted in this mouse. The frequency of I-E-specific T cells was much

lower then that of the wild type animal but equivalent to that of animals expressing MHC class II

I-E in all tissues. These results indicate that I-E expressing DCs mediated negative selection of I-

E-specific thymocytes. It was then shown that thymic DCs are able to pick up tissue-specific Ag

from medulary thymic epithelial cells and delete autoreactive T cells via cross-presentation 82.

Thymic DCs were also directly shown to delete Ag-specific single positive thymocytes in vivo 83.

Recently it was demonstrated that thymic CD11b+ CD8a- CD172+ DCs are also capable of

inducing natural Tregs 84.

DCs in peripheral tolerance

DCs are strategically positioned in the periphery (eg. skin, airway, and intestine) to capture Ag

and present Ag to T cells in draining LN. In the steady state, these DCs mediate peripheral cell

tolerance to harmless environmental Ag (ingested 85 or inhaled 86) or self-Ag. When exogenous

soluble Ag are fed to mice or introduced by inhalation, Tregs or Tr1 cells are induced via IL-10

or TGFβ. High levels of MHCII expression bound to self-Ag was observed on LN DCs. These

DCs were able to induce apoptosis of Ag-reactive T cells 86. Peripheral tolerance induction has

also been noted in DCs that present non-self Ag in immature state 87, 88, 89. In this situation, TCR

stimulation is not accompanied by co-stimulatory signal and anergy or deletion of peripheral T

cell takes place 87, 88, 90. Induction of IL-10-producing Tr1 cells has also been reported 89. In

addition, naïve T cells may be converted into CD4+ FoxP3+ Tregs or IL-10-producing Tr1 cells 91. CD103+ migratory DC in the gut 92, 93 and CD103- migratory DC in the skin 94 have been

observed to transport Ag to mesenteric LN and induce naïve CD4+ T cells to become Tregs via

TGFβ and retinoic acid dependent pathway.

Splenic CD8a+ DCs have been observed in multiple studies to be capable of tolerance induction.

In an airway hypersensitivity model, CD8a+ splenic DCs were able to inhibit Th2 cytokine

response and reverse airway hyper-responsiveness in vivo 95. Another study demonstrated that

spleen CD8a+ CD205+ DCs can convert naïve CD4+ T cells into Tregs via secretion of TGFβ 91.

Moreover, targeting OVA to DEC-205 – a scavenging receptor expressed on DC surface – has

14

been shown to induce tolerance of T cells to OVA. In one such study, adoptively transferred OT-

I cells exhibited defective cytokine production and were deleted from the system 90. Similarly,

anergy induction of Ag-specific T cells occurred in mice previously targeted with DEC-205-Ag

conjugates 87. When DCs are pulsed with apoptotic debris containing OVA, tolerance usually

ensues to OVA 88. It is important to note that in all of these studies, T cell proliferation was

evident at day 3, where as at time points beyond 10 days these T cells were anergic or deleted.

Numerous attempts to manipulate DCs to acquire stable tolerogenic properties have also been

undertaken. The rational for this approach is to avoid the use of immature DCs as these have an

unstable phenotype and can be converted to stimulatory DCs easily by exposure to inflammatory

conditions 77. Several groups have been able to convert immature DC into tolerogenic DCs by

using variety of culture conditions including low levels of GM-CSF, IL-10 96, 97 , or the

combination of IL-10 and TGFβ 77. For example, CD45RB+ CD11clow IL-10-producing

regulatory DCs were generated from c-kit+ progenitors by culturing them on spleen stromal

monolayer 98 99. Moreover, these cells could induce conversion of CD4+ T cells into FoxP3+

Tregs 96, induce IL-10-producing Tr1 cells, and induce T cell anergy 77, 97, 98. In vitro generated

tolerogenic DC have been used in adoptive transfer therapy to induce peripheral tolerance in

vivo, prolong allograft survival, and prevent GVHD 77.

Certain pathogens have also been observed to induce DC reprogramming towards tolerogenic

type. In particular, fungal morphocyte haphae induces DCs to activate Treg cells. S. masoni

conditions DCs through TLR2 signalling to induce Tregs. Filamentous hemagglutinin from

bacteria Bordetella Petrussis induces DCs to secrete IL-10 and prime Tr1 cells 100.

5 Mechanisms of immunosuppression

5.1 PD-L1/PD-L2

The B7 family of ligands includes co-stimulatory molecules (CD80 (B7-1) and CD86 (B7-2))

and two in inhibitory molecules (programmed death-1 ligand (PD-L1) or B7-H1 and PD-L2 or

B7-H2). In humans, IFN-γ induces PD-L1 and PD-L2 expression on PBMCs. When human

monocytes are cultured in the presence of IFN-γ, both PD-L1 and PD-L2 expression are induced

15

101, 102. PD-L1 expression was also high on both mature and immature human monocyte-derived

DCs 101 and was further increased upon stimulation with IFN-γ and LPS 102. The expression

pattern was similar on murine DC counterparts 102. PD-L1 was also expressed on human CD4+

and CD8+ T cells that were activated with anti-CD3 Ab. Moreover, PD-L1 expression was

observed on cells of non-hematopoietic lineage, such as vascular endothelial cells 103.

Both PD-L1 and PD-L2 were shown to inhibit T cell proliferation and cytokine production

through engagement of a PD-1 receptor 102, 104. PD-1 is expressed on activated B, T and myeloid

cells 105, 106. Engagement of PD-1 results in T cell cycle arrest 104 by limiting the production of

IL-2 105. When T cells were stimulated with immature DCs in allogeneic MLR in the presence of

anti-PD-L2 Ab, both proliferation and IFN-γ production were increased. The same result was

observed when a combination of anti-PD-L1 and anti-PD-L2 Abs were used, but not anti-PD-L1

alone 101. The same pattern was observed in an Ag-specific system. When CD4+ DO11.10 cells

were stimulated with DCs pulsed with OVA in the presence of anti-PD-L1, anti-PD-L2 or both

Abs, cytokine secretion was significantly increased. Moreover, PD-L1/PD-L2-/- NOD mice had

rapid onset of autoimmune disease, significantly earlier than their NOD counterparts 103.

5.2 Arginase and nitric oxide synthase (NOS)

Arginase metabolizes L-arginine to produce urea and L-orthinine, whereas NOS metabolizes L-

arginine to produce nitric oxide (NO) and L-citrulline 107. When NO combines with oxygen it

produces anions (NO2-, NO3

-) and peroxynitrites (ONOO-), which damage cellular lipid, protein

and DNA 108.

Arginase 1 (ARG1) is constitutively expressed in the cytosol of hepatocytes and is also induced

in myeloid cells in response to various stimuli such as Th2 cytokines and TGF-β 109, 110.

Induction of ARG1 by Th2 cytokines was also observed in bone marrow-derived DCs 111.

Arginase2 is a mitochondrial enzyme with wide tissue distribution. It is expressed in kidney,

lactating mammory gland, prostate, brain and small intestine. Constitutive expression of ARG2

was also observed in bone marrow derived macrophages, but was not up-regulated by ARG1

inducers 111.

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NOS has three isoforms, two of which are constitutively expressed (neuronal and endothelial

NOS) and one which is inducible (iNOS). Here, only iNOS is described. iNOS is expressed in

cells of the immune system (including macrophages and DCs) 112 upon induction with Th1

cytokines, IFN-β and TNF 109-111. Both ARG1 and iNOS are induced by LPS 109, 110. In addition

to reciprocal regulation of iNOS and ARG1 by Th1 and Th2 cytokines respectively, these two

enzymes also negatively cross-regulate each other 109, 110. NG-hydroxy-L-arginine (NOHA)

released as a by-product of iNOS enzymatic activity during L-arginine metabolism inhibits ARG

1 and stimulates surrounding immune cells to produce NO by iNOS. This differential regulation

and enzyme production has been used to differentiate classically and alternatively activated

macrophages. Classically activated macrophages release pro-inflammatory cytokines, such as IL-

1, IL-6 and TNF as well as reactive oxygen and nitrogen species as byproducts of iNOS activity,

which, in turn, leads to its anti-microbial action 110. On the other hand, alternatively activated

macrophages secrete IL-10 and up-regulate ARG1 activity. These cells are responsible for tissue

repair and fibrosis.

The effect of NO, which is produced by iNOS, is microbicidal 107. iNOS KO mice were shown to

be more susceptible to L. major bacterial infection than heterozygous and WT mice 113. iNOS

activity also has potent immuno-suppressive effects. Although iNOS consumes L-arginine, its

immunosuppressive properties have been shown to be unrelated to L-arginine starvation of T

cells123. Rather, it has been noted that by-products of iNOS activity such as reactive nitrogen

species suppress T cell proliferation 114, 115. T cells from iNOS KO mice displayed higher levels

of proliferation and IFN-γ production but less IL-4 production in response to Leishmania Ag or

concanavalin A 113. The mechanism of suppression involved T cell cycle arrest through

impairment of IL-2R signalling (inhibited phosphorylation of STAT5, JAK3, Erk1/2 and Akt),

although IL-2R chain expression remained normal 116, 117. T cell suppression was reversible only

during first 24 hours of culture but not at later time points. iNOS was observed to be induced by

IFN-γ. However, blocking IFN-γ reversed suppression by about half, suggesting that other

mechanisms are involved in iNOS upregulation. Suppression of T cell responses also required

cell contact 116, 117. Induction of cell death was not observed in co-cultures 116, 117. Moreover, it

was observed that alveolar macrophages were able to suppress the stimulatory capacity of DC

via NO production by iNOS 118. The effect was reversed by inhibiting iNOS.

17

The role of NO in tumour killing has been recognized 107, 108. It was first observed that primary

mouse macrophages could kill mouse tumour cell lines 119, an effect that was mimicked by

addition of NO and abolished with the addition of an iNOS inhibitor and in iNOS KO

macrophages 108 119. An anti-tumour effect of microbe-induced NO production was also

observed in vivo 120. Mice that received bacillus Calmette-Guerin (BCG) bacterium ip, had

reduced number of ovarian tumour cells transplanted ip and no evident ascites in the peritoneum

as compared to mice that did not receive the bacterium injection. The effect was completely

reversed with co-administration of iNOS inhibitor and anti-IFN-γ Ab 121.

High ARG1 activity has been observed in patients with various malignancies 122. ARG1 activity

in tumor-associated macrophages (TAMs), MDSC and some types of tolerogenic DCs was also

shown to be immuno-suppressive in multiple studies. Tumor-derived mature myeloid cells

(identified as macrophages) were observed to be the source of ARG1 in 3LL murine lung

carcinoma. These cells also produced IL-10, IL-1 and IL-6 122 and suppressed T cell proliferation

in vitro by down-regulating the CD3ζ chain of the T cell receptor complex 123, 124. Another study

demonstrated down-regulation of both CD3ε and CD3ζ chains and inhibition of ARG1 in TAMs

restored T cell proliferation 122. An alternative mechanism of suppression mediated by ARG1 is

thought to be associated with inhibition of cell cycle progression and absence of cyclin D3 and

cdk4 expression – enzymes responsible for cell cycle progression 125.

Paradoxically, arginase and iNOS seem to be co-upregulated in some cell types 107. Several

studies have shown that tumour-derived MDSC 126 and certain types of DC-regs suppress T cell

proliferation in vitro via both arginase and iNOS-mediated L-arginine depletion or reactive

oxygen species generated as a by-product of these two enzyme activities. The relevance of iNOS

and arginase activity in reactive oxygen species production can also be appreciated in the context

of resolution of the immune response. In this setting, contraction of T cell response takes place,

possibly due to the mechanisms mentioned above 107.

5.3 Indoleamine 2,3-dioxygenase (IDO)

IDO is an intracellular enzyme expressed in many tissues. A role of IDO has been described in

tumor progression 128, T cell tolerance to tumors, inhibition of T cell proliferation both in vitro

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127, 133 and in vivo 133 and as negative regulator of immune disorders. It was also observed that

over-expression of IDO results in immuno-suppression and tolerance 130.

IDO is part of the innate immune defence against pathogens. IDO metabolizes tryptophan to

yield its degradation by-products known as kynurenines. Some microorganisms depend on

exogenous tryptophan as a source of this essential amino acid. Limiting trypthophan in the

microenvironment by induction of IDO, therefore, acts as a microbicidal strategy of the innate

immune system 130. Constitutive IDO expression occurs at maternal-fetal interface as well as in

mouse gut, lymph nodes, spleen, thymus and gut130. Moreover, IDO expression exists in multiple

primary human tumors 128. Myeloid-lineage cells such as monocytes, macrophages and DCs

express IDO after exposure to IFN-γ 129, LPS 130 and CD40L or to a combination of these

molecules 127. Only certain DC subsets seem to be able to express IDO. These subsets include

CD8a+ cDCs and B220+ pDCs 131 and are termed ‘IDO competent DCs’. IDO-expressing pDCs

were identified in tumor-draining LN 132. By contrast, pro-inflammatory signals that trigger DC

maturation also down-regulate IDO production, whereas certain tolerogenic signals (eg. ligation

of CD80/CD86 by inhibitory receptor CTLA-4 131) up-regulate IDO in DCs.

The immuno-regulatory role of IDO was demonstrated in vitro when it inhibited proliferation of

tumor cells by consumption of amino acid tryptophan 129. This effect was mediated through

kinase GCN2, which triggers cell cycle arrest 133. Furthermore, one study found that T cell

unresponsiveness could be induced in response to the metabolites L-kynurenine and picolinic

acid produced as a by-product of IDO activity 134.

5.4 IL-10

IL-10 is an immuno-regulatory cytokine that prevents auto-immune and other inflammatory

pathologies. IL-10 or IL-10R KO mice do not develop systemic auto-immune disease but

develop colitis in the presence of microorganisms 135. IL-10 is widely expressed by cells of the

immune system. Its expression has been demonstrated in multiple subsets of T cells (such as

Th1, Th2, Th17, Tregs) as well as DCs, macrophages, mast cells, eosinophils, NK cells and

neutrophils 135. In macrophages and DCs, IL-10 can be induced by TLR ligands including

TLR2, TLR4, and TLR9 136. DC-SIGN and Dectin-1, which ligate other PRR, can also stimulate

IL-10 release from DCs 137.

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IL-10 inhibits IFN-γ production by Th1 cells and drives T cell responses toward a Th2

phenotype 138. In another report it was demonstrated that IL-10 inhibits both proliferation and

cytokine production by both Th1 and Th2 cells 139 140. Moreover, when CD4+ T cells are

activated in vitro, IL-10 causes them to convert into regulatory Tr1 phenotype 140. The inhibitory

effect of IL-10 on T cell proliferation is mediated partly though inhibition of APC function 139.

IL-10 was demonstrated to suppress APC function of monocytes by reducing expression of

MHC 141 and co-stimulatory molecules 142, as well as reduce the release of pro-inflammatory

cytokines such as IL-12 143, TNF, IL-1β, IL-6 and GM-CSF 144. Additionally, IL-10 enhances

the release of soluble TNF-αR and IL1-βR antagonist that act in an anti-inflammatory fashion 145.

IL-10 can inhibit monocyte differentiation into DC and promote their differentiation into

macrophages 146. The immunosuppressive function of IL-10 has been shown to be mediated, at

least partly, by STAT3 signalling downstream of IL-10R 147.

6 Antigen presenting cells in cancer immunology

In addition to their importance in the maintenance of tolerance in the steady state, DCs, together

with other types of APCs, contribute to poor immune responses in various pathological

conditions. In cancer, it is thought that an immunosuppressive tumor microenvironment drives T

cell hyporesponsiveness and tolerance towards tumor Ag. Below, I describe key tumor-

associated APCs and their relevance to tumor tolerance.

6.1 Tumor-derived dendritic cells

Immune recognition of tumor antigens is thought to be mediated by tumor DCs that have

migrated into secondary lymphoid tissues. There, tumor DCs prime T cells, which then return to

the tumor to kill tumor cells. 148. For most patients, however, this process appears to be

ineffective, in part because of DC dysfunction at various levels. The first evidence for the

impairment of DC function in cancer came from observations that patients with advanced tumors

have reduced number of DCs in their blood 149. Decreased numbers of mature DCs were also

observed in spleen, lymph nodes and tumor in tumor-bearing mice 148. Similarly, DC

recruitment to tumors was impaired in a wide variety of primary tumors 150, 151, which correlated

with poor patient prognosis 152. Tumor-infiltrating DC expressed low levels of MHCII and co-

20

stimulatory molecules 149 153 154, failed to respond to maturation stimuli 155, and primed T cells

poorly 149 153. Increased numbers of immature DCs have been detected in peripheral blood of

cancer patients, indicating that DC dysfunction can be systemic149. DC maturation was also

inhibited when DCs were cultured with tumor-conditioned medium in vitro 149 153 156,157.

Immature tumor DCs suppress T cell responses and lead to unresponsiveness toward tumor Ag 158. In other reports, an increased rate of DCs apoptosis was detected in tumors 159.

Collectively, these factors result in reduced activation or direct suppression of T cell responses.

Tumor DCs arise from pre-cDCs that migrate into the tumor via a CCL3-dependent mechanism 160. Once in the tumor, DC differentiation is influenced by the intra-tumoral inflammatory

milieu, which has been shown to alter DC differentiation 161. Studies in our laboratory describe a

high proportion of GR-1+ DCs both in the tumor itself (up to 35% of total tumor DCs), in

draining lymph nodes and in spleen of tumor-bearing mice 161. Moreover, the frequency of GR-

1+ DCs in lymphoid tissues correlated directly with tumor size 161. These cells were defective in

priming T cells in allogeneic MLRs and had a reduced expression of MHCII and CD86 after

maturation. IL-10 was implicated in the defective T cell stimulation ability.

A variety of molecules in the tumor microenvironment have been implicated in the impairment

of tumor DC including IL-10, IL-6, VEGF, M-CSF and prostanoids 146. IL-6 inhibits DC

differentiation both in vitro and in vivo 162. VEGF has been associated with reduction in DC

number and accumulation of immature myeloid cells in tumor bearing mice and patients156 163.

Anti-VEGF Ab treatment increased the number of DC in spleen and LN of tumor-bearing mice

and increased the ability of the DCs to prime T cells156 163. A recent study suggested that lipid

accumulation in DC may contribute to DC dysfuntion in cancer patients 164.

6.2 Myeloid derived suppressor cells (MDSC)

MDSC are a heterogenous population of neutrophils, monocytes, and primitive myeloid cells that

increase in frequency in bone marrow, blood, and lymphoid tissues of tumor-bearing mice and

patients149 165 169. In mice, they are typically defined by the expression of CD11b and GR-1 107;

however, these markers are non-specific and cell populations expressing CD11b and GR-1 occur

in normal mice albeit at much lower frequencies (less than 1% of total circulating cells; 2-4% of

spleen cells; and up to 50% of bone marrow cells) 148 166. Further, these cells differentiate into

21

mature functional myeloid cells in normal mice 167. Recent studies in tumor-bearing mice

suggest that MDSC can be subdivided into CD11b+ Ly6c+ Ly6Glow monocyte-like and CD11b+

Ly6Clow Ly6G+ granulocyte-like cells that suppress immune responses through different

mechanisms 170 169. Monocytic MDSC produce higher levels of NO and induced substantially

elevated levels of tyrosine nitrosylation than granulocytic MDSC, which produce increased

levels of ROS 169. In other studies, the expresson of CD115 (M-CSFR) and CD124 (IL-4-Ra)

has been used to define MDSC subsets 169.

Immature myeloid CD11b+ cells or MDSC isolated from tumors were shown to be potent

suppressors of Ag-specific T cell proliferation 168 and function 171 both in vitro and in vivo 172.

MDSC from peripheral organs suppress T cell responses in an Ag-specific manner, whereas

MDSC from tumor sites mediate Ag-non-specific suppression 126. However, MDSC isolated

from the spleen of tumor-bearing mice showed a mixed suppressive activity 173 and were not

suppressive in some studies 126. It was observed that MDSC did not engage TCR or activate T

cells 171 but caused dissociation of TCR complex with the CD3ζ chain and with CD8 in OTI

cells 171 176. MDSC have also been shown to inhibit IL-12 production by macrophages in an IL-

10-dependent fashion 174.

Most studies have reported that the suppressive effects of MDSCs are cell-contact dependent.

Activation of MDSC results in upregulation of ARG1 and iNOS enzymatic activity and

increased production of ROS and NO via STAT3175. High ARG1 and iNOS activity are

considered the main mechanisms by which MDSC suppress immune functions 126 177 116. In

particular, production of peroxynitrite, a byproduce of iNOS, results in nitration of the TCR and

CD8, which renders T cells unresponsive to Ag-specific stimulation 171. Some reports found

suppression to be IFN-γ-dependent 116, 170.

6.3 Tumor associated macrophages (TAMs)

Evidence for the role of macrophages in tumor progression has accumulated over decades.

TAMs are derived from blood circulating monocytes that are recruited to tumors by CCL2 as

well as CCL5, CCL7, CCL8, CXCL12, VEGF, PDGF and M-CSF produced within tumor

microenvironment 178 179 180. Multiple studies have reported that high numbers of TAMs

correlates with poor outcome in many human cancers 178. M-CFS is the main growth factor

22

responsible for survival, proliferation, differentiation and chemotaxis of cells of mononuclear

phagocyte system. High levels of M-CSF correlate with poor prognosis in patients with cancer 181.

In established tumors, TAMs have a characteristic M2 macrophage phenotype 182. Under normal

physiological conditions, M2 cells promote wound healing, tissue remodelling, angiogenesis, and

suppression of immune responses. In tumors, these cells produce low amount of IL-12 and high

amounts of IL-10, TGF-β and arginase, which act in immunosuppressive fashion. TAMs

preferentially recruit naïve T lymphocytes devoid of cytotoxic function as well as Th2 and Treg

cells to tumors via CCL18 183, CCL17 and CCL22 respectively 184 185. Direct T cell immuno-

suppressive function of TAMs was reported to involve PD-L1 ligand 186.

Analogous to the wound interior, tumors exhibit a highly hypoxic microenvironment. Hypoxia

up-regulates HIF-1α 187 and HIF-2α 188 expression in TAMS resulting in increased VEGF

production, which stmulates tumor angiogenesis 189. M-CFS has also been shown to stimulate

VEGF release from TAMs 190. TAMs produce other pro-angiogenic factors such as PDGF, TNF,

and CXCL8 178. TAMs can also be found in vascular areas inside the tumor 191, where they

enhance nutrient and oxygen supply for tumor growth 192. The production of metalloproteases

(MMP2, MMP9) by macrophages, a component of tissue remodelling, promotes cancer invasion

and metastasis 193.

23

CHAPTER 2

Statement of the Problem

1 Preliminary observations

Conventional DCs (cDCs) arise from pre-cDCs, an immediate precursor population originally

identified in bone marrow by Diao et al 62. When placed on a stromal monolayer in the presence

of GM-CSF, pre-cDCs differentiate into a homogenous population of proliferating cDCs that

continue to divide over 10-12 days (Figure 3A). In the absence of GM-CSF, pre-cDCs also

generate cDCs initially, but by day 9 the cells lose surface expression of both CD11c and

MHCII, the classic hallmarks of cDCs (Figure 3B). The morphology of the cells also changes

(Figure 3c): cDCs appear as loosely adherent clusters with cells displaying motile denditic

processes; cells generated in the absence of GM-CSF are round with few dendrites and appear

tightly adherent or embedded in the monolayer.

The development of CD11c- MHC II- cells from CD11c+ MHCII - cDC was unexpected as it

is generally believed that cDCs are terminally differentiated. To further confirm that CD11c-

MHCII - cells arose from proliferating cDCs, CD11c-Cre+ Rosa26-EGFP transgenic mice and

their CD11c-Cre- littermate controls were used to trace the life history of CD11c- cells back to

CD11c+ progenitors. Cre recombinase, driven by the CD11c promoter, deletes the stop codon

for ROSA-GFP. Cells that express CD11c are permanently tagged by GFP irrespective of

subsequent CD11c expression levels in the progeny. Before culture, GFP could be detected in

about 10-20% of pre-cDC from CD11c-Cre+/- Rosa26-EGFP transgenic mice (Figure 4). Cre+/-

pre-cDCs differentiated into MHCII+ cDC at day 3 concurrent with the up-regulation of GFP

expression. At day 10, when the Cre+/- cells had lost CD11c and MHCII expression, GFP

expression persisted.

Phenotypic characterization of CD11clow MHCII low cells revealed absence of most

phenotypic markers characteristic of DC subsets (Figure 5). The cells stain negative for CD11c,

MHCII, CD4, CD8, CD103 and GR-1. However, the cells express high levels of CD11b and

CD172a. Moreover, contrary to cDCs, these cells are resistant to maturation stimuli such as LPS

24

and fail to up-regulate MHCII and co-stimulatory molecules (Figure 6). Additionally, these cells

were observed to be poor stimulators of T cell proliferation in allogeneic mixed lymphocyte

reaction (Figure 7). Although these cells are poor T cell stimulators, they demonstrated an Ag

uptake capacity that was comparable to that of DCs (Figure 8).

2 Hypothesis

Previous studies suggest that DC with low expession levels of MHCII and co-stimulatory

molecules are immunosuppressive and promote immunologic tolerance77. Based on preliminary

investigations of pre-cDC-derived CD11clow MHCII low cells, we hypothesize that they have

immuno-suppressive properties in vitro and in vivo.

3 Objective

To characterize the functional properties of DC-derived CD11clow MHCII low cells

4 Specific aims

Aim 1: To investigate the functional and immunosuppresive properties of CD11clow

MHCII low cells in vitro.

Aim 2: To define the mechanisms by which CD11clow MHCII low cells promote unresponsiveness.

Aim 3: To evaluate the in vivo effects of CD11clow MHCII low.

25

CHAPTER 3

Methodology

1 Mice

C57BL/6, Balb/c, C57BL/6.SJL congenic and OT-II OVA (323-339)-specific TCR transgenic

mice (B6.Cg-Tg(TcraTcrb)425Cbn/J) were purchased from the Jackson Laboratory. Mice were

maintained in pathogen-free conditions in accordance with institutional guidelines, and used at 6-

8 weeks of age.

2 Primary skin stromal cell preparation

New born skin stroma was prepared from newborn C57BL/6 mice. The skin of new born mice

was minced and cultured at 37ºC in 10 cm plates containing DMEM medium supplemented with

10% FBS, penicillin (50 U/mL) and streptomycin (50 ug/mL). After 1 week, when the cells had

formed a confluent monolayer, the cells were treated with 0.25% trypsin/1mM EDTA, split, and

passaged three times. Immediately prior to co-culture with pre-cDCs, the confluent monolayer

was irradiated (25 Gy).

3 Cell Isolation

DCs were isolated from spleens of C57BL/6 mice, which were inoculated previously with a Flt3

ligand-producing B16 melanoma cell line (B16-Flt3L). Spleens were minced and digested with

collagenase D and DNase for 0.5 hours at 37ºC. Cells were passed through a 0.42 µm nylon

mesh and subjected to density gradient centrifugation using Nycodenz 194. Nycodenz was

prepared by using 7:7:16 v/v/v Nycoprep (Cederlane Laboratories), tricine and PBS with 5%

FBS and 2mM EDTA (binding buffer). Dendritic cells were further enriched for CD11c+ cells

by positive selection using MACS (Milteniy Biotech) CD11c+ immuno-magnetic beads. Cells

were washed with binding buffer during all steps. Cells were subsequently cultured overnight

before use at a density of 1×106 cells/mL in RPMI supplemented with 10% FBS, 50 uM 2-ME,

26

1mM sodium pyruvate, 10 mM non-essential amino acids, 50 U/mL penicillin and 50 ug/mL

streptomycin (complete medium) in the presence of GM-CSF (4 ng/mL; BD Pharmingen).

Pre-cDCs were isolated from bone marrow of B16-Ftl3L melanoma treated C57BL/6 mice.

Femurs and tibia were flushed with binding buffer and subjected to Lympholyte-M (Cederlane

Laboratories) density gradient centrifugation. Cell suspensions were layered on top of

Lympholyte-M at ratio of 2:1 v/v. CD11c+ bone marrow precursors were further enriched by

MACS (Milteniy Biotech) CD11c+ immuno-magnetic beads. Cells retained in the column were

eluted and labelled with anti-I-Ab-PE, anti-CD11c-APC and anti-lineage makers (anti-CD3-,

anti-CD19-, anti-B220-, anti-CD49b-FITC) mAbs. Lin- CD11c+ MHCII - pre-cDC were isolated

using MoFlo High Speed Cell Sorter using Summit acquisition and analysis software

(DakoCytomation). The purity of the pre-cDCs population used was routinely ≥99% based on

reanalyzed samples.

4 Flow cytometry

Flow cytometry was performed on Beckman FC500 using CXP Analysis software (Beckman

Coulter). Prior to staining, cell suspensions were pre-incubated with anti-CD16/32 in binding

buffer to block FcRs for 25 min at 4ºC. Cells were then washed with binding buffer and stained

with mAb conjugates for 25 min at 4ºC in a final volume of 100 ul of binding buffer with cell

density of not more than 5×106/100 ul. Appropriate isotype controls were included.

For intracellular staining, cells were pre-incubated with anti-CD16/32 in binding buffer for 25

min at 4ºC, surface stained as above, fixed and permeabilized using BD Cytofix/Cytoperm kit

(BD Biosciences) according to manufacturer’s instructions. Anti-IL-2-, anti-IL-4-, anti-IFNγ-PE

mAbs were used for intracellular staining.

5 Mixed lymphocyte reactions

CD4+ OT-II T cells or CD8+ OT-I T cells were isolated from the spleen and lymph nodes of OT-

II or OT-I transgenic mice, respectively. Tissues were minced, passed through a 42 µm nylon

mesh and subjected to density gradient centrifugation with Lympholyte-M. CD4+ or CD8+

27

lymphocytes were enriched using MACS CD4+ or CD8+ immuno-magnetic beads. Graded

numbers of stimulator cells (splenic DCs) cultured overnight with GM-CSF and/or suppressor

cells (CD11clow MHCII low cells derived from pre-cDC) were seeded in triplicate in 96-well U-

bottom plates (BD Biosciences). Responder spleen cells (1×105/well) from BALB/c mice, CD4+

OT-II or CD8+ OT-I cells were added to the wells in total volume of 200 µl of RPMI 1640

complete medium. 10 µg/ml IL-10R Ab, 500 µM N ω-hydroxy-nor-Arginine (Nor-NOHA) and

200 µM N6-(1-iminoethyl)-L-lysine, dihydrochloride (L-NIL) were used to block IL-10R,

arginase I and iNOS, respectively. Cells were cultured in humidified atmosphere of 5% CO2 in

air at 37 ºC. Cultures were pulsed with 1 µCi of [H3]-thymidine (Amersham) 16 hours before

harvest and collected into glass fiber filters (Millipore). [H3]-thymidine incorporation was

quantified using Beckman scintillation counter. Results are expressed as mean cpm of triplicate

cultures.

6 CFSE labelling

Isolated cells were washed twice with PBS and stained with 1uM CFSE (Molecular Probes) for

15 min at 37 ºC. Cells were then washed twice with PBS.

7 Reverse transcriptase PCR

Total RNA was extracted from DCs and DC-regs with TRIzol (Invitrogen Life Technologies) as

per the manufacturer’s instructions. RNA was resuspended with RNase free water and treated

with DNase I (Invitrogen Life Technologies) to remove contaminating genomic DNA. RNA was

then reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen Life Technologies)

and amplified by PCR using the following primers: murine PD-L1, sense: 5’-

GTGAAACCCTGAGTCTTATCC-3’, anti-sense: 5’-GACCATTCTGAGACAATTCC-3’; IDO,

sense: 5’-GTACATCACCATGGCGTATG-3’, anti-sense: 5’-

GCTTTCGTCAAGTCTTCATTG-3’; arginase1, sense: 5’-

CAGAGTATGACGTGAGAGACCAC-3’, anti-sense: 5’-

CAGCTTGTCTACTTCAGTCATGGAG-3’; iNOS, sense: 5’-

AGCTTCTGGCACTGAGTAAAGATAA-3’, anti-sense: 5’-TTCTCTGCTCTCAGCTCCAAG-

28

3’; FasL, sense: 5’-AACCCCAGTACACCCTCTGAAA-3’, anti-sense: 5’-

GGTTCCATATGTGTCTTCCCATTC-3’; TGFβ2, sense: 5’-

TGGCCGCCTGGAGCAAGAAA-3’, anti-sense: 5’-AAGCGGCTGGGGGATGAC-3’; IL-10,

sense: 5’-GGATCTTAGCTAACGGAAACAACT-3’, anti-sense: 5’-

AAGCGGCTGGGGGATGAC-3’; ICOS-L, sense: 5’-CTTGGTCTGTTCTTGCTGCTG-3’,

anti-sense: 5’- GGCTATTGTCCGTTGTGTTG-3’.

8 Arginase and iNOS activity assays

To determine nitrite production by DC-regs and DCs, 1×106 cells were pulsed with LPS (2

µg/mL) overnight at 37 ºC in RPMI 1640 complete medium and supernatants harvested. Nitrites

were quantified in supernatants using Griess Reagent Kit for Nitrite Determination (Invitrogen),

according to manufacturer’s instructions.

Arginase activity was measured in cell lysates of cells previously pulsed with LPS. After

overnight culture with LPS, cells were digested with 0.25% trypsin/2mM EDTA and washed

twice with PBS. Cells were lysed in 100 uL 0.1% Triton X-100 126. To 100 uL of protein lysate,

100 uL of 25 mM Tris-HCl and 10 uL of 10 mM MnCl2 were added and enzyme was activated

by heating for 10 min at 56ºC. Arginine hydrolysis was conducted by incubating the lysate with

200 uL of 0.5 mM L-arginine, pH 9.7, at 37ºC for 60 min. The reaction was stopped with 900 uL

of H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v). 40 uL of 9% beta- isonitrosopropiophenone

dissolved in 100% ethanol was then added and the reaction mixture was incubated at 95ºC for 25

min. Urea concentration was determined by measuring absorbance at 562 nm. One unit of

enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol urea

per minute.

9 Adoptive transfer studies

OVA-pulsed CFSE-labelled cDCs or DC-regs (1×106) were injected i.p. into mice that had

received 1×106 CFSE-labelled OT-II CD4+ cells 24 hours earlier. Spleen and lymph nodes were

collected 3 and 5 days later. T cell proliferation was assessed by CFSE dilution. T cell function

29

was assessed by pulsing spleen and lymph node cells with OVA peptide in vitro and assessing

cytokine production 12 hours later.

10 Statistics

Continuous variables are expressed as mean±SE and were analyzed by two-tailed Student t test.

A P value below 0.05 was considered statistically significant.

30

CHAPTER 4

Results

1 CD11clow MHCIIlow cells exhibit potent immuno-suppressive properties in vitro

It was demonstrated previously that pre-cDCs generate proliferating cDC 62. In this study, pre-

cDCs were placed on a stromal monolayer and their proliferation was observed. On stroma

supplemented with DC growth factor GM-CSF, pre-cDCs generated a highly pure population of

cDCs by day 3. In the absence of GM-CSF, pre-cDCs also generated cDCs by day 3 (Figure

3A). However, when cDCs were cultured further in the absence of GM-CSF (up to day 14), loss

of both CD11c and MHCII expression, which normally characterize DCs, was observed.

Moreover, the cells acquired distinct morphology. In contrast to DCs, which appeared as small

irregular-shape cell clusters loosely attached to the monolayer, these cells were evenly

distributed through the stroma. The new cells appeared much larger than DC and had smooth

round shape without dendrites (Figure 3B).

Incubation of CD11clow MHCII low cells with LPS failed to upregulate co-stimulatory molecules

and MHCII (Figure 6), indicating that these cells were resistant to maturation. CD11chi MHCIIhi

cDC cells generated in the presence of GM-CSF efficiently stimulated T cell proliferation in

allogeneic MLR. In contrast, CD11clow MHCII low displayed very poor T cell stimulatory

capacity even when pulsed with TNF-α, another maturation stimulus (Figure 7).

DC with low expression of co-stimulatory molecules and poor T cell priming capacity in

allogeneic MLR were previously shown by Sato et al to have T cell regulatory activity. 77.

Svensson et al. showed that CD11clow CD45RB+ DCregs cells generated from bone marrow

progenitors on stroma in the absence of GM-CSF also had poor T cell priming capacity and had

potent immuno-suppressive properties98. It was, therefore, hypothesized that

CD11clow MHCII low cells generated from pre-cDC have the ability to actively suppress T cell

responses.

31

To test whether CD11clow MHCII low cells have suppressive properties, various numbers (0.25-

2x104) of CD11clow MHCII low cells were added to allogeneic mixed lymphocyte cultures

containing 1×105 BALB/c spleen cells and 1×104 CD57BL6 spleen cDCs. Addition of

CD11clow MHCII low cells suppressed T cell proliferation in a dose-dependent fashion (Figure 9).

When added at a ratio of 1:1 with stimulatory DCs, CD11clow MHCII low cells were able to

suppress T cell response by 75%. T cell proliferation was decreased to baseline when

CD11clow MHCII low cells were added at higher ratios. Because of this potent suppressive

property, it was decided to refer to CD11clow MHCII low as DC-regs.

Although DC-regs suppressed T cell proliferation, it was still possible that T cells were activated

and capable of mounting an effector response212. Therefore, the ability of T cells previously

primed with allogeneic DC-regs to produce cytokines after secondary re-stimulation was tested.

T cells were recovered from primary cultures with DC-regs and re-stimulated with anti-CD3 and

anti-CD28 Abs. Intracellular cytokine production was measured by flow cytometry. T cells that

were pre-incubated with DC-regs with or without DCs did not produce IL-2, IFN-γ or IL-4

(Figure 10). These results demonstrate that DC-regs are able to inhibit both allogeneic T cell

proliferation and activation, which was not reversed despite the absence of DC-regs in the

secondary MLR.

To determine if DC-regs suppressed Ag-specific T cell responses, TCR-transgenic CD4+ T cells

purified from spleens of OT-II mice, which are specific for OVA 323-339 peptide, were used as

responders. Splenic DCs pulsed with OVA protein and matured with GM-CSF overnight were

mixed at 1:1 ratio with DC-regs that were or were not pulsed with OVA overnight. OT-II cells

were then incubated with DC-OVA ± DC-reg / DC-reg-OVA or DC-reg / DC-reg-OVA alone.

The number of OT-II cells was measured on day 3 by flow cytometry (Figure 11). As expected,

DC-OVA stimulated proliferation and expansion of OT-II T cells. DC-regs alone did not induce

T cell proliferation regardless of whether they were pulsed with OVA or not. When OT-II cells

were pulsed with a combination of DC-OVA and DC-regs or DC-regs-OVA, T cell proliferation

was suppressed almost to baseline.

32

It was possible that the low T cell number observed was due to excessive proliferation and cell

death rather than suppressed proliferation. To exclude this possibility, CFSE-labelled OT-II cells

were analyzed after stimulation with various combinations of OVA-pulsed and control-pulsed

DC and DC-regs. These studies confirmed that DC-reg inhibited OT-II cell proliferation (Figure

12). Stronger suppression of proliferation occurred when DC-regs were not pulsed with OVA.

In contrast to the allogeneic system, DC-regs failed to suppress cytokine production in OT-II

primed with DC-OVA (Figure 13). In these studies, T cells were cultured for 7 days99 before re-

stimulation with anti-CD3 and anti-CD28 mAbs.

2 Mechanisms of immuno-suppression mediated by CD11clow

MHCIIlow DC-regs

There are several possible mechanisms that could account for suppression of T cell responses by

DC-regs:

1) DC-regs may induce production of Tregs. Previous studies have shown that peripheral

tolerance induction involves DC-regs or immature DCs and their ability to promote

generation of Tr1 and FoxP3+ Tregs 36, 92, 94, 99.

2) DC-regs may inhibit T cell cyling through the release of various metabolites. Inhibition

of T cell responses has been observed previously in conditions of L-arginine starvation

induced by arginase1-expressing cells including tumor-derived DCs, TAMs and MDSC.

High expression of arginase1 results in down-regulation of one of the TCR chains

(CD3ε122 or CD3ζ123, 124) which can be reversed with arginase1 inibition or addition of

excess L-arginine to the culture medium. Moreover, arginase1 was also reported to

inhibit cell cycle progression via suppression of cyclin D3 and cdk4 expression125.

Tryptophan starvation as a result of high IDO expression by certain subsets of DCs has

also been reported to inhibit T cell proliferation131, 133. Moreover, high levels of reactive

nitrogen species produced as a by-product of iNOS activity can induce cell cycle arrest

via inhibition of IL-2R signalling 116, 117. Suppression of T cell proliferation mediated by

iNOS was reported to be independent of its L-arginine consumption123.

33

3) DC-regs may induce T cell anergy by ligating T cell inhibitory receptors including

ICOS196 or PD-1/PD-2195. Ligation of the ICOS receptor on T cells also leads to IL-10

production, a well-recognized immunosuppressive cytokine196.

4) DC-regs may induce T cell death 90.

To establish the mechanism of immuno-suppression in our system, the activation status of T cells

in allogeneic MLR was assessed first by measuring expression levels of CD25 and CD44.

BALB/c T cells incubated with C57BL/6 DCs showed high expression of both markers at day 3

(Figure 14). By contrast, co-culture with C57BL/6 DC-regs blocked CD25 and CD44

expression. Similarly, no T cell activation was detected in cultures of T cells incubated with DC-

regs alone. These findings indicated that DC-regs suppress T cell activation.

It was next assessed whether DC-regs induced T cell apoptosis. OT-II cells were recovered from

cultures and stained with propidium iodide (PI). In necrotic or late apoptotic cells, in which cell

membrane integrity is compromised, PI can permeate into the cell and intercalate between DNA

bases; this results in increased fluorescence (20-30 fold). PI is effectively excluded from viable

cells. In the absence of stimulation, most T cells stained positive for PI (Figure 15). Similarly, T

cells incubated with DC-regs alone stained positive for PI indicating that DC-regs do not provide

sufficient T cell survival signals, which may be related to their low MHCII expression (Figure 5)

and, therefore, the inability to ligate the TCR complex on OT-II cells. Few dead cells were

detected when T cells were incubated with both DCs and DC-regs as DCs provide TCR

stimulation vital for T cell survival. These results indicate that DC-regs in co-cultures with cDC

do not actively induce T cell death or prevent survival signals from reaching T cells.

It was hypothesized that CD4+ CD25+ FoxP3+ Tregs might be induced by DC-regs. To test this,

OT-II cells were incubated with DC-OVA +/- DC-reg-OVA or DC-reg. After 7 days, OT-II cells

were recovered, stained for CD4, CD25 and FoxP3, and analyzed by flow cytometry. We did not

detect CD4+ CD25+ FoxP3+ Tregs present under any of the culture conditions (Figure 16).

Collectively, these results suggested that suppression of T cell proliferation and activation by

DC-regs does not involve FoxP3+ CD25+ Tregs, or regulation of T cell survival or death. Instead,

it seemed likely that DC-regs produce factors that regulate T cell activation and cell cycle

progression.

34

Trans-well experiments were conducted to determine whether suppression mediated by DC-regs

was cell contact dependent. OT-II cells (0.5×106) and DC-OVA cells (1×105) were placed in the

bottom chamber of the trans-well and DC-reg-OVA (1×105) were placed in the top chamber. The

chambers were separated by a membrane whose 8 µm pore size excluded DC-regs from the

bottom chamber. DC-reg suppression was reduced by about half in the transwells, suggesting

that both soluble and membrane bound mediators were involved (Figure 17). Passage of cell

processes through membrane pores was possible but unlikely, as DC-regs did not have dendritic

processes (Figure 3B).

Qualitative RT-PCR was used next to determine whether DC-regs express previously recognized

immunosuppressive molecules including IL-10 148, IDO 127, PD-L1 195, ICOS-L 196, iNOS 126,

arginase1 126 and FasL 197. Erythrocyte-depleted spleen cells were used as positive control for all

assessed molecules. FasL expression was not observed in DC-regs (Figure 18). This was in

agreement with the previous finding that DC-regs did not induce T cell death (Figure 15). IDO

expression was not detected in DC-regs, indicating that tryptophan starvation is likely not

involved in DC-reg mediated T cell suppression127. ICOS-L, PD-L1 and IL-10 were

constitutively expressed by DC-regs, whereas arginase1 and iNOS were induced upon LPS

stimulation (Figure 18).

The activity of ARGI and iNOS enzymes in DC-regs was determined with biochemical assays.

ARG1 activity was determined by incubating cell lysate with L-arginine and measuring urea

production. iNOS activity was determined by measuring nitrite production in the supernatant,

which is a by-product of iNOS activity. Consistent with the RT-PCR data, DC-regs displayed

high levels of both enzymes after overnight stimulation with LPS (Figure 19). By contrast,

freshly isolated spleen DCs did not display any enzymatic activity with or without LPS

stimulation.

Expression of PD-L1 was also assessed by flow cytometry (Figure 20). Both spleen DCs and

DC-regs showed high levels of PD-L1 expression whether or not they were incubated with LPS.

The high PD-L1 expression level in both cell populations suggests that PD-L1 is not involved in

DC-reg-mediated suppression.

35

IL-10 production was measured by ELISA (Figure 21). High levels of IL-10 were detected in

DC-regs pulsed with LPS, but not by DC. In contrast to RT-PCR data, IL-10 was undetectable

in DC-regs without LPS stimulation. This may indicate storage of intracellular IL-10 and its

release upon stimulation.

Collectively, these results demonstrate that suppression of T cell proliferation and activation is

most likely mediated by iNOS, arginase, and/or IL-10. PD-L1 and ICOS-L that were expressed

by DC-regs were also observed in DCs, which makes them unlikely candidates for DC-reg-

specific immuno-suppressive function.

To clarify the functional relevance of PD-L1, IL-10, ARG1 and iNOS, transgenic CD8+ OT-I T

cells were incubated with OVA-peptide pulsed spleen DCs (DC-OVA) in the presence or

absence of DC-regs and corresponding blocking reagents. Anti-IL-10R and anti-PD-L1 were

used to block binding of IL-10 to its receptor and PD-1, respectively. Nω-hydroxy-nor-Arginine

(Nor-NOHA) and L-NG-monomethyl arginine citrate (L-NMMA) were used to block arginase1

and iNOS enzymatic activity, respectively. Similar to our studies of OT-II cells, OT-I T cells

incubated with DC-OVA proliferated vigorously whereas the addition of DC-regs blocked

proliferation (Figure 22). Addition of anti-IL-10R Ab, anti-PD-L1, and and nor-NOHA had little

effect on immunosuppression. However addition of iNOS inhibitor L-NMMA restored T cell

proliferation by 60-70%. Similar results were obtained using the OT-II cells (data not shown).

These results indicate that DC-regs inhibit T cell proliferation through an iNOS-dependent

mechanism.

3 Immunosuppressive activity of CD11clow MHCIIlow cells in vivo

It has been reported that in vitro generated regulatory DC can suppress T cell proliferation in

vivo 77, 99. Immuno-suppressive properties of DC-regs were, therefore, tested in an adoptive T

cell transfer model. B6.SJL mice expressing the CD45.1 congenic marker were i.v. injected with

CFSE-labelled CD45.2+ CD4+ OT-II cells. Twenty-four hours later, OVA-pulsed DCs with or

without OVA-pulsed DC-regs were injected i.p. Proliferation of spleen OT-II T cells was

assessed at 3 and 5 days later by flow cytometery (Figure 23). OT-II T cells from mice injected

with DC and DC-regs showed more proliferation than mice injected with DCs alone. They also

36

produced more IFN-γ after re-stimulation with OVA peptide in vitro (Figure 23). By contrast,

OT-II T cells recovered on day 5 from mice receving DC-OVA + DC-regs showed lower levels

of proliferation and IFN-γ production than mice that received DC-OVA alone.

The ability of DC-regs pulsed with OVA to stimulate OT-II T cell proliferation on day 3 in vivo

was unexpected. It was speculated that DC-regs pulsed with OVA might have resulted in

transfer of OVA to immunostimlatory recipient APCs. To investigate this possibility, DC-regs

that were not pulsed with OVA were used in the adoptive transfer (Figure 24). However, DC-

regs were still unable to suppresss OT-II proliferation or IFN-γ production induced by OVA-

pulsed DC.

Because of these unexpected results, it was decided to assess the ability of DC-regs to prime T

cells in vivo. Here, mice were injected with either DC-OVA or DC-reg-OVA and proliferation

was assessed 3 days later. T cell activation markers were also assessed. T cells from both

groups displayed the same levels of proliferation. Moreover, both groups expressed similar

levels of CD44 and CD69 (Figure 25) indicating that DC-regs are able to prime T cells in vivo.

In summary, these results demonstrate that under the experimental conditions employed, DC-

regs fail to suppress OT-II T cell proliferation and activation.

37

CHAPTER 5

Discussion

This study demonstrates that cDCs retain functional and phenotypic plasticity. This novel

finding challenges the prevalent view that cDCs are terminally differentiated. cDC co-cultured

on a supportive stromal monolayer lost cell surface expression of CD11c and MHCII and

differentiated into a cell population with potent immuno-suppressive properties, which were

termed DC-regs. DC-regs have lost the usual constellation of morphologic and functional

features that are used to define cDC including morphology, responsiveness to maturaton stimuli,

and the capacity to stimulte lymphocytes in vitro. DC-regs also acquired the capacity to produce

high levels of iNOS, IL-10, and arginase. It was further shown that iNOS contributes to their

immunsuppressive properties.

Failure of previous studies to demonstrate this novel pathway of cDC development is multi-

dimensional. First, the culture conditions used by other groups to generate cDC in vitro did not

recapitulate the natural pathway of cDC development. DCs or their precursors are commonly

cultured in liquid medium, which is drastically different from the in vivo developmental

conditions. The natural environment for DC differentiation first takes place in the bone marrow,

where progenitors and precursors are embedded in the bone marrow stroma. DC precursors then

migrate to lymphoid tissues to finish their differentiation in tight association with lymphoid

stroma. Therefore, the use of a stromal monolayer for in vitro modeling of DC development is

imperative. Zhang et al212 generated DCs in liquid culture and placed fully differentiated DCs on

a stromal monolayer for further developmental studies. This approach may not be optimal

because unnatural DC development in liquid system may have altered DC differentiation

program and skewed further development towards abnormal immuno-regulatory populations

such as diffDCs described in the study. Our laboratory was the first to study DC differentiation

by culturing an immediate DC precursor (pre-cDC) on a stromal monolayer. This system allows

for tracing the natural progression of DC differentiation in the ‘steady state-like’ environment.

Pre-cDC placed on stroma proliferated vigorously to give rise to a pure population of cDCs

within 3 days. This population resembled in vivo isolated DCs – it was moderately potent in

stimulating allogeneic T cells proliferation and the stimulatory capacity was dramatically

38

increased when the cells were matured with LPS or TNFα. Furthermore, these cDCs responded

well to GM-CSF and, similarly to the in vivo inflammatory state, expanded dramatically in

number. When cDCs were allowed to propagate in the absence of GM-CSF, however, they did

not undergo apoptosis. Instead, these cells were observed to lose their DC identity (presence of

dendrites as part of classical DC morphology, expression of CD11c and MHCII, T cell

stimulatory capacity, response to classical DC maturation stimuli) and become potent immuno-

regulatory cells, which suppressed allogeneic and Ag-specific T cell proliferation.

Secondly, cytokine cocktail used in previous studies may not be optimal for identification of DC-

reg population arising from DCs. GM-CSF is usually added to the liquid culture to mature and

expand DCs generated from bone marrow progenitors. Some studies generating regulatory DCs

in vitro have used low levels of GM-CSF and/or IL1077, 96, 97. In the present study, however, GM-

CSF was shown to block the pathway leading to further DC differentiation into DC-regs.

Interestingly, GM-CSF was not identified as a key factor playing a role in generation of

regulatory populations in other studies98. The dependence on GM-CSF to generate DC-regs may

be explored further in some therapeutic applications like cancer immuno-therapy, where further

transformation of DCs into DC-regs is undesirable.

The third drawback of previous studies is the use of crude bone marrow extracts to generate

DCs, which makes difficult the study of developmental relationships in the DC lineage. The use

of bone marrow progenitors to generate immuno-suppressive populations generated in some

studies puts their DC origin under question. Moreover, it becomes impossible to dissect the

developmental origin of these immune-suppressive populations. For example, Svensson et al98

placed lineage- c-kit+ progenitors on stroma and generated CD11clow CD45RB+ CD11b+ cells. It

is, however, without reason that Svensson calls these cells DCs as merely excluding lineage cells

does not prove that they are DCs. In our study we definitively demonstrate that DC-regs do

indeed belong to the DC lineage. A highly pure FACS sorted pre-cDCs were cultured on stroma.

These cells were previously demonstrated by our group to give rise to DCs62. In the present

study, pre-cDCs gave rise to cDCs by day 3 and gradual loss of MHCII was observed by day 6.

By day 9 all cells lost CD11c expression as well. These results were confirmed by clonal assay

where single pre-cDC clones were cultured and generation of CD11c+ MHCII+ DCs and

subsequently CD11clow MHCII low DC-regs was seen (data not shown).

39

In vivo detection of regulatory dendritic cell populations has proven to be particularly

challenging. Most studies attribute regulatory properties to immature DCs occurring in lymphoid

organs and the periphery85-90. For example, CD103+ DCs have been described as capable of

inducing peripheral tolerance towards Ag in the gut and splenic CD8+ DCs have been shown to

induce T cell tolerance in spleen90, 91, 95. In addition, immature DCs with reduced T cell priming

capacity have been described in tumors149, 153, 154. However, regulatory populations that lack

typical DC features (such as expression of CD11c and MHCII) described in most studies have

not been detected in vivo. Wakkach et al has recently described a population of CD11clow

CD45RB+ population that was isolated from IL-10 transgenic as well as C56Bl/6 and BALB/c

mice99. These cells were first generated in vitro and then detected in vivo. However, their DC

origin remains to be determined as they were generated from crude bone marrow extract and in

the presence of low levels of GM-CSF.

Detection of DC-regs described in this study is also difficult using previous approaches. Past

studies used adoptive transfer model to demonstrate developmental relationships between

precursors and their progeny. For example, Naik et al61 isolated potential DC precursors from

spleen based on their density and expression of CD11c, CD45RA, SIRPα and CD43 and injected

them back into uninfected non-irradiated mice. The occurrence of donor-derived spleen cDCs

was then quantified by flow cytometry. This approach could not be utilized for the current study

as DC-regs described here could not be detected in the steady state conditions. Moreover,

previous detection of DC-regs was difficult due to lack of specific markers that could be used to

identify this population in vivo. Expression of CD11b, F4/80 and SIRPα that was detected on

DC-reg surface is non-specific and has also been described in other populations such as DCs,

macrophages and monocytes. Therefore, the use of CD11c-Cre+/- Rosa26-EGFP transgenic mice

in this study proved to be particularly useful. The expression of GFP driven by CD11c promoter

allowed not only to detect CD11clow MHCII low DC-regs in tumors but also to trace their

developmental history from CD11c+ MHCII+ cDCs. Using this model, this population was

detected in tumors (data not shown).

Under steady state conditions, lymphoid and non-lymphoid tissues contain stable numbers of

DCs, which is achieved through dynamic interactions between influx of new recruits, emigration,

and death. Bone marrow-derived pre-cDC, the immediate precursor of cDC, migrate via blood

40

and enter peripheral lymphoid and non-lymphoid tissues, where they differentiate into cDC that

proliferate for several generations. Non-lymphoid tissue cDC emigrate continuously to

lymphoid tissues via lymphatics into draining lymph nodes. Like the resident populations of

lymphoid DCs, most migrant tissue cDCs die in lymphoid tissue and do not re-enter the blood

circulation. The estimated life span of lymphoid tissue cDC is 2-3 days198. It remains unclear

whether cDC terminate through apoptosis exclusively. In the study performed by Chen et al. 199,

DC apoptosis was inhibited by expressing p35 under CD11c promoter. However, an increase in

total DC number became evident only in in mice > 3 months of age. Of note, inhibition of DC

apoptosis did not affect DC proliferation or DC generation from precursors. Moreover, no

autoimmune Ab generation was observed up to 9 months of age. These results suggest the

existence of additional mechanisms of systemic DC removal and, thereby, maintenance of

peripheral tolerance. This study raises the possibility that DC-reg differentiation is an alternative

fate for cDC.

The notion of functional and phenotypic plasticity in cDC is not unique. Recent evidence points

to the essential role of microenvironment in shaping the immunophenotype and function of other

cells of the innate immune system. Tumor associated macrophages have been shown to retain

their functional plasticity within the tumor. Upon treatment with IL-12, these cells down-

regulated pro-tumorigenic factors IL-10, TGF-β and MCP-1, and upregulated anti-tumorigenic

factors TNFα, IL-15 and IL-18 204. Moreover, interaction of M2 macrophages with Th1 cells has

been shown to skew macrophage phenotype towards M1 205. Recent evidence points to plasticity

in neutrophils as well. It has been argued that granulocytic CD11b+ Ly6C- Ly6G+ MDSCs seen

in tumors arise from neutrophils that have been modified by tumor microenvironment219. Besides

having a constellation of cell surface markers indicative of neutrophils, granulocytic MDSC also

express functional molecules commonly seen in neutrophils. For example, these MDSCs produce

high amounts of L-arginine and iNOS as well as have high oxidative capacity via high

expression of NADPH oxidase.

Cell plasticity is also being increasingly recognized in the adaptive immune system. For

example, propagation of committed Th17 precursors in the presence of IL-23 but in the absence

of TGF-β resulted in progressive loss of IL17F secretion and appearance of Th1-like IFNγ-

producing cells. Moreover, stimulation of Th17 cells with IL-12 led to rapid loss of Th17-

41

associated transcription factors and emergence of a Th1 gene signature 200. Several studies have

also shown the ability of Tregs to reprogram into inflammatory Th17 type cells in tumor draining

lymph nodes in the absence of IDO and presence of IL-6 201. In mice, Tregs have been shown to

directly differentiate into Th17 in the presence of IL-6 and in the absence of exogenous TGFβ 202. In humans, Tregs are also able to reprogram into Th17 cells when stimulated by allogeneic

monocytes in the presence of IL-2 and IL-15 203.

Hematopoietic cell lineage plasticity urges us to reconsider the classical view of the immune

system in the context of health and disease. The possibility of manipulating the

microenvironment to alter the functionality of a desired cell population has been explored for

some time. For example, administration of cytokines and growth factors for cancer

immunotherapy has been used in clinical trails with relative success220. IFNα has been

demonstrated in a number of trials to be successful at inducing anti-cancer response and

prolonging the survival of chronic myelogenous leukemia (CML) patients221. This therapy has

been shown to increase a number of CML-specific CD8+ T cells. Moreover, administration of

IL-2 was shown to be effective in a subset of patients with renal cell carcinoma222. This has been

shown to enhance both NK and CD8+ T cell function as well as increase vascular permeability.

TNFα, IL-12, GM-CSF and other cytokines have also been tested in clinical trials220. These

cytokines were shown to increase tumor cell apoptosis; enhance T cell cytotoxicity and inhibit

tumor angiogenesis; as well as increase tumor Ag presentation for TNFα, IL-12 and GM-CSF

respectively220. Unfortunately, systemic administration of cytokines for cancer immunotherapy is

limited by severe toxicities associated with these treatments. Moreover, this approach needs to be

optimized to achieve full therapeutic benefit for human use.

DC-regs generated from cDC potently inhibited proliferation of allogeneic and antigen-specific

OT-II T cells in vitro. For allogeneic T cells, decreased proliferation was accompanied by

reduced expression of effector cytokines. By contrast, intracellular cytokine expression in OT-II

T cells was unaffected by DC-regs. This difference may be due to the sensitivity of clonal OTI-II

T cells to re-stimulation. This result was similar to observation made by Zhang et al where

diffDCs suppressed proliferation of OVA-specific transgenic DO11.10 CD4+ T cells but not

cytokine production212.

42

In this study, DC-regs express high levels of arginase1, iNOS and IL-10 activity after stimulation

with LPS. They also constitutively expressed PD-L1 and ICOS-L. Moreover, iNOS was

determined to be responsible, at least in part, for the immunosuppressive properties of DC-regs.

An immunosuppressive effect of iNOS was reported to be due to generation of reactive nitrogen

species, which suppress T cell proliferation via impairment of IL-2R signalling 114, 115, 116, 117. NO

produced by iNOS is short lived and acts locally in a paracrine manner. In this study, the

suppression was reduced by about half when DC-regs were separated by a semi-permeable

membrane. It is not surprising considering that NO acts in a paracrine manner and may have,

therefore, been degraded before it could reach T cells in the lower compartment of a transwell

system. Similar to this study, Lukacs-Kornek et al223 recently reported inhibition of T cell

proliferation by stromal cells via iNOS-dependent mechanism. In this report T cell proliferation

was restored when a transwell membrane was inserted between T cells and stromal cells.

Moreover, nitrite production was decreased to baseline. Both these and our results indicate that

direct contact or close cell proximity between T cells and regulatory cells may be necessary for

iNOS induction and subsequent immunosuppression.

In addition to cell contact requirement, Lukacs-Kornek reports the involvement of IFNγ

produced by activated T cells in the induction of immunosuppression. We have tested induction

of potential immune-suppressive molecules by LPS. Although iNOS was produced in this

system, LPS is not the stimulus for iNOS induction in T cell proliferation assays. Here, T cells

were activated by allogeneic or OVA-pulsed DCs. This may have triggered IFNγ production,

which in turn could activate or induce iNOS. DC-regs were also observed to suppress

proliferation of effector memory T cells (data not shown), which secrete high amount of IFNγ.

The role of IFNγ in inhibition of T cell proliferation remains to be directly tested in our system.

Although DC-regs produced high levels of IL-10 upon stimulation with LPS, it was not directly

involved in inhibition of T cell proliferation. In this respect, DCregs in this study were similar to

diffDCs described by Zhang et al 212. DC-regs suppression of T cell proliferation was stronger

when they were not pulsed with OVA. This may be due to transfer of OVA Ag between DC-reg-

OVA and DCs or minor stimulation of TCR receptor on T cells through MHCII on DC-regs.

DC-regs suppressed cytokine release in T cells upon restimulation only in allogeneic but not in

the OT-II system. The disparity in DC-reg suppression between the allogeneic and clonal system

43

may be attributed to difference in sensitivity to Ag stimulation of both systems. In addition to

direct suppression of T cell proliferation, we evaluated the ability of DC-regs to induce CD4+

FoxP3+ Treg population. However, development of Tr1 cells in co-cultures was also possible.

The presence of this population was not assessed directly. Induction of other Treg subtypes (γδ

T cells, NKT cells, CD8+ Tregs) in co-cultures of OT-II cells with DC-regs is also unlikely as

highly purified CD4+ T cells were used in the initial cultures, which excludes other T cell

populations.

iNOS and ARG1 have been shown to be the major players in MDSC and TAM-mediated

immuno-suppression 166. These cells occur mostly in cancer and promote tumor progression 166.

In this study, DC-regs did not appear to be MDSCs, which comprise a heterogenous population

of Gr-1+ monocytes, neutrophils and primitive myeloid cells. DC-regs do not express Gr-1 and

are a homogeneous population derived from fully differentiated DC. High levels of iNOS and

ARG1 expression has also been previously described in macrophages 111. DC-regs resemble in

many respects alternatively activated macrophages and TAMs: they all express F4/80 (data not

shown) and high levels of CD11b, and have immuno-suppressive properties. A recent study

reported the existence of FoxP3+ macrophages in spleen, bone marrow, lymph nodes, thymus,

liver and other tissues of naïve mice; these cells inhibit T cell proliferation mainly via

prostaglandin E2 (PGE2) 209. Since blocking iNOS alone did not restore T cell proliferation

completely in our studies, it possible that other factors such as PGE2 expresson may be involved

in DCreg suppression. Future studies will address this possibility.

The immunosuppressive potency of DC-regs is similar to that reported in other CD11clow DC

populations. CD11clow diffDC generated by culturing mature DCs on embryonic spleen stroma

suppressed mature DC-induced DO11.10 T cell proliferation by ~70% but did not suppress

cytokine release 212, which was similar to our results in the OT-II system. Moreover, regulatory

capacity of our DC-regs cells in allogeneic system was also similar to those of

CD11clow CD45RB+ described by Svensson et al 98. These cells were generated on spleen stroma

from lin- c-kit+ progenitors. When compared to MDSC, DC-regs had similar potency with these

cells described in some studies 214 but were more than twice as potent as MDSC described in

other studies. It should be noted, however, that suppression of T cell response by MDSC was

44

assessed when T cells were stimulated non-specifically with anti-CD3 and anti-CD28 Abs126 or

with peptides without stimulator APCs present in culture 175, 215.

The role of DC-regs in immuno-suppression in vivo was also investigated in this study. The

results demonstrate that DC-regs do not suppress proliferation or effector function of adoptively

transferred T cells when injected i.p. Moreover, it was found that DC-regs pulsed with OVA are

able to prime naïve OT-II cells in vivo, induce up-regulation of T cell activation markers and

proliferation. The discrepancy between the in vivo and in vitro finding is difficult to explain. It is

possible that the read-out used in this study was not appropriate for detecting in vivo suppression.

Previous studies have shown that induction of T cell proliferation at day 3 does not always

correlate with the development of subsequent T cell tolerance. For example, mice injected with

a DEC-205:OVA conjugate, which delivers Ag to endogenous DC without causing DC

maturation, showed marked reduction in OT-II T cell numbers at day 12 as compared to mice

injected with DEC-205:OVA and anti-CD40 Ab, which causes DC maturaton, even though both

treatment regimens induced OT-II T cell proliferation initially 90. Similarly, another study found

marked T cell proliferation at day 2 in mice injected with a tolerogenic DEC-205:HEL

conjugate; however, T cell proliferation returned to baseline by day 7. By contrast, T cell

proliferation was sustained beyond 7 days in mice injected with immunostimulatory HEL in

CFA 87. It may, therefore, be worthwhile to evaluate T cell responses at later time point in our

model.

It is also possible that the inability of DC-regs to suppress in vivo responses represents their

failure to migrate to sites of DC-T cell interaction in lymphoid tissues. Since DC-regs resemble

macrophages, which are tissue-resident with limited migratory capacity 211, it seems reasonable

to speculate that i.p. injected DC-regs remained in the peritoneal cavity. In future studies, the

migratory capacity and chemokine receptor expression of DC-regs will be evaluated.

The role of the microenvironment in shaping cDC fate has become increasingly apparent. This

study suggests that the relative concentration of GM-CSF in tissues has critical role in

determining the fate of proliferating cDC. In the presence of high GM-CSF levels, the

proliferating progeny of cDC maintained a cDC phenotype, whereas in the absence of exogenous

GM-CSF they differentiated into DC-regs. Although DC-regs were generated in vitro in the

45

present study, recent evidence from our laboratory indicates that a similar cell population

develops in tumors. This study suggests that manipulating GM-CSF levels in tissues or its

downstream target maybe a useful strategy in the treatment of cancer and inflammatory diseases.

46

CHAPTER 6

Conclusion and Future Directions

1 Conclusion

In summary, these studies demonstrate two previously unknown attributes of cDC life cycle.

First, proliferating cDCs are not terminally differentiated but retain differentiation potential and

plasticity. When cDCs are co-cultured on a stromal monolayer in the absence of exognenous

GM-CSF, a novel immunosuppressive regulatory population of DC-regs is generated. These

cells suppress allogeneic T cell response and Ag-specific T cell proliferation in vitro through an

iNOS-dependent mechanism. Second, when cultured on stroma in the absence of GM-CSF, cDC

life does not finish with apoptosis. Instead, in the absence of GM-CSF, these cells are capable of

avoiding death by changing the course of their differentiation. The importance of this finding in

vivo is unclear. In the steady state and inflammation, where we have not been able to detect the

occurance of DC-regs, cDCs are likely to encounter endogenous or foreign Ag and, after a few

rounds of cell division, die by apoptosis in the lymphoid tissues. However, in the pathological

conditions such as cancer, natural cDC life cycle may be altered by microenvironment to

produce a new cell type with regulatory properties. These cells may be an important cause of

diseases where DC dysfunction is known to play a key role. The relevance of DC-reg occurance

in such pathologies remains to be investigated.

2 Future directions

The key role of GM-CSF in the generation of CD11clow MHCII low DC-regs from DCs was

demonstrated by adding the cytokine on days 0-12 of culture. We observed that DC-reg failed to

emerge when GM-CSF was added up to day 6 of culture. After that time point, addition of GM-

CSF did not prevent the development of DC-regs from DCs. However, GM-CSF may not be the

only factor involved in this transformation. Contrary to stromal co-culture, DCs cultured in

liquid medium in the presence of GM-CSF die within 2 days. Soluble or membrane bound

molecules produced by stromal cells, therefore, appear to be critical in the maintenance of DC

longevity and generation of DC-regs. Incubation of DCs with stromal supernatant can determine

47

if soluble factors are involved in differentiation of DCs into DC-regs. Some of the cytokines that

might be involved include: TGFβ, IL-10, IL-6, VEGF, hepatocyte growth factor (HGF), PGE2

and others. Alternatively, fixation of stromal cells will determine if cell contact is important in

the transformation212. In the study by Zhang et al it was reported that fibronectin plays a critical

role in generation of diffDCs from maDCs 212. Other factors like members of the fibroblast

growth factor family, laminin and apoptotic debris from stroma may also be important.

It would also be imperative to investigate changes in the genetic program that occurs during

transformation of cDCs into DC-regs. To do this, genome wide microarray analysis would be

done first to identify genetic signature of DC-regs. This would also help determine if DC-regs

resemble or belong to any of the presently known cell lineage (like macrophages) or if they

constitute a totally new cell type. It would then be possible to pinpoint specific genes involved in

the transformation and identify key transcription factors that act as signatures of DC-reg lineage.

Identification of these molecules would allow for the design of DC-reg-specific inhibitors or

inducers which could then be used therapeutically for the treatment of pathologies requiring DC

activation such as cancer or suppression such as transplantation. Epigenetic changes involved in

the transformation would also be interesting to look at.

DC-regs were generated from DCs in vitro in the studies presented. It will be important to

determine whether the same process occurs in vivo. Using the CD11c-Cre+ Rosa26-EGFP

transgenic mouse model, it has not been possible to detect DC-derived GFP+ CD11c- DC-regs in

the steady state. However, it appears that DC regs do develop in subcutaneous tumors derived

from B16 melanoma and lewis lung carcinoma (data not shown). The immunosuppressive

microenvironment within the tumor likely plays a pivotal role in the development of DCregs,

similar to the accumulation of MDSC. Once factors involved in the promotion of DC-regs are

identified in the in vitro system, it may be possible to inhibit DC reg develpment in vivo by

infusing blocking monoclonal Abs i.v. or into the tumor. So far, in vitro evidence points to the

importance of GM-CSF in the maintenance of DC phenotype. To determine whether increasing

the concentration of GM-CSF in the tumors could influence cDC differentiation, tumor cells

could be induced with a retroviral vector encoding GM-CSF. In fact, several Phase I clinical

trials investigating the role of GM-CSF in tumor microenvironment have done just that. In these

studies, surgically excised tumors (metastatic melanoma or non-small-cell lung carcinoma) were

48

transduced with replication defective GM-CSF expressing retrovirus and re-injected back into

patients224-226. Another study injected allogeneic GM-CSF-secreting HER2-expressing tumor cell

line for vaccination against breast tumor227. In this study, one out of six patients developed anti-

HER2 Abs when injected with vaccine alone and 7 out of 22 patients when injected with vaccine

in combination with chemotherapy. Vaccination sites showed substantial infiltration of DCs,

macrophages and granulocytes224-226. Moreover, metastatic lesions that were excised after but not

before vaccination showed considerable infiltration of CD4+ and CD8+ T lymphocytes and B

cells as well as necrosis, fibrosis and oedema. It was also demonstrated in a pre-clinical mouse

model that tumors engineered to secrete GM-CSF induce massive infiltration and activation of

DCs into tumor tissue, which then prime Ag-specific CD4+ and CD8+ DCs228.

The occurrence of CD11clow DC-regs in other immune conditions is also possible. The

resolution phase of acute inflammation is a likely situation given that this process is

characterized by massive leukocyte apoptosis which occurs in association with increased

frequency of various regulatory cells. Key molecules involved in inflammation resolution

include prostaglandin E2, lipotoxin, resolvins and protectins 208. Involvement of lipids in DC

dysfunction has been reported before 216. It would be interesting to determine whether DCregs

develop during the resolution phase of inflammation and whether the immunosuppressive

molecules linked to this process promote DCreg development. Chronic inflammatory processes

may also favour the development of DCregs and should be investigated in the future.

In experimental conditions used, DC-regs did not show any immunosuppression in vivo. There

are several explanations for the observed phenomenon. One possibility is that T cell proliferation

measured at 3 days has no relationship with the establishment of tolerance mediated by DC-regs.

T cell proliferation or other read-outs for tolerance could be measured at later time points (eg.

after day 10). Moreover, alternative routes of T cell priming may be utilized. It was

demonstrated in this study that DC-regs upregulate various regulatory molecules upon exposure

to a TLR agonist LPS. In our in vivo model, we did not inject LPS into the system and OT- II

cells were primed by injection of OVA-pulsed DCs i.p. We could try challenging the mice with

LPS and testing if DC-regs could suppress systemic inflammatory response. OT-II cells could

also be primed with OVA in Complete Freud’s Adjuvant (CFA) to induce systemic T cell

priming and inflammation. Because CFA contains bacterial components, it is likely to act as

49

TLR agonist, activating regulatory molecule production similarly to LPS. This approach has

been taken by Kusmartsev et al229 where MDSC were observed to suppress OT-I T cell

proliferation in response to OVA in Incomplete Freud’s Adjuvant (IFA) challenge. Moreover, to

avoid DC-reg migration issue, DC-regs could be injected localy into the footpad to increase

interaction between T cells and DC-regs, which would be immunized with OVA in CFA. Since

DC-regs express high levels of IL-10 and iNOS, it would also be interesting to test whether DC-

regs could use therapeutically to down-regulate acute inflammatory responses.

50

Figures

MHC II

Lin-

+/- GM

No GM-CSFGM-CSF

MH

C II

CD

11c

CD11c

Pre-cDC

Day 3

Day 9

A

CD11clow

MHCIIlow CD11c+ MHCII+B

-T

NFα

+ T

NFα

C

Day 3 Day 10Day 6

CD11c

MH

C II

Day 0

Figure 3. Differentiation and proliferation of pre-cDC on stroma. A) FACS purified CD11c+ MHCII - lin- (CD3, CD19, B220, CD49b) pre-cDC were placed on a primary fibroblast stromal monolayer in the presence or absence of GM-CSF (4ng/mL). Pre-cDC generated proliferating cDC at day 3 under both conditions. In the absence of GM-CSF, the cDC progeny at 10 days did not express CD11c and MHCII markers and turned into CD11clow MHCII low cell type. B) Time course of pre-cDC development into CD11clow MHCII low cell through CD11c+ MHCII+ DC intermediate. C) Morphology of CD11clow MHCII low and CD11c+ MHCII+ cells following exposure to TNFα. Cells were Giemsa stained and viewed under the light microscope (40× magnification).

51

CD11c MHCII GFP

Day 0

Day 3

Day 10

CD11c-Cre

ROSA-stopFlox-GFP

CD11c-Cre-ROSA-GFP

X

A

B

Figure 4. CD11clow MHCIIlow arise from CD11c+ MHC II+cDC. A) CD11c-Cre mice were crossed with ROSA-stopFlox-GFP mouse to create CD11c-Cre-ROSA-GFP mouse. B) Pre-cDC from Cre+/- mice (white histogram) and Cre-/- littermate controls (grey histogram) were placed on stroma in the absence of GM-CSF. The histograms show expression of CD11c, MHC II, and GFP by pre-cDC at day 0, and their progeny at day 3 and day 10.

52

CD11c CD11bMHCII CD103 CD4 CD8 CD172a

Spleen cDC

CD11clow

MHCIIlow DC

Figure 5. Immunophenotype of cDC-derived CD11clow MHCIIlow cells. Cells were stained with the indicated markers and analyzed by flow cytometry.

53

DC DC+LPS

CD40

CD80

CD86

MHCII

DC-reg DC-reg+LPS

CD11c

Figure 6. DC-regs fail to up-regulate co-stimulatory molecules in response to maturation stimuli. Spleen DCs or cultured DC-regs were stimulated with LPS (2µg/mL) for 18 hours. Cell surface expression of co-stimulatory molecules (CD40, CD80, CD86) were assessed by flow cytometry. Data is representative of 3 independent experiments.

54

Figure 7. CD11clow MHCIIlow cells are poor stimulators of allogeneic T cell lymphocytes. BALB/c responder T cells were pulsed with C57BL/6 DCs or DC-regs at various ratios. T cell proliferation was determined on day 3 by 3H-thymidine incorporation.

55

Figure 8. CD11clow MHCIIlow DC-derived cells have increased phagocytic capacity. A) CD11clow MHCII low DC-derived cells and CD11c+ MHCII+ DCs were pulsed with OVA-FITC at 4º C and 37º C and mean fluorescence intensity was measured by flow cytometry. B) Cells were incubated with fluorescent dextran beads and viewed under confocal microscope (40× magnification). Results are representative of 3 independent experiments.

4oc 37 oc4oc 37oc

OVA

CD11clow MHCIIlow DC CD11c+ MHCII + DC

CD11clow

MHCIIlow DC CD11c +

MHCII+ DC

A

B

MFI=30 MFI=15

56

Figure 9. CD11clow MHCIIlow suppress T cell proliferation in allogeneic mixed lymphocyte reaction. Balb/c responder T cells were pulsed with DCs +/- DC-regs at various ratios. T cell proliferation was determined on day 3 by 3H-thymidine incorporation.

3

0

2

4

6

8

10

12

14

4:1 2:1 1:1 0.5:1 0:4 0:2 0:1 noAPC

3 H t

hym

idin

e in

corp

ora

tio

n (

cpm

) x

103

3

DC -reg: DC ratio

57

0

5

10

15

20

25

IFNy

% I

FN

y +

ve c

ells

0

2

4

6

8

10

12

IL4

% I

L-4

+ve

cel

ls

0

5

10

15

20

25

30

IL2

% I

L-2

+ve

ce

lls

DC DC+DC-reg DC-reg

DC DC+DC-reg DC-reg

DC DC+DC-reg DC-reg

*

* *

Figure 10. DC-regs suppress effector function of allogeneic T cells in mixed lymphocyte cultures. Balb/c spleen cells stimulated with DC+/- DC-regs for 7 days were harvested, washed, and re-stimulatedwith anti-CD3 and anti-CD28 monoclonal antibodies. Intracellular expression of IFN-γ, IL-2 and IL-4 was measured by flow cytometry. * p<0.01 by Student`s t-test. Data are representative of >3 independent experiments.

58

Figure 11. DC-regs suppress OT-II T cell proliferation in response to OVA-pulsed DCs. 1×105 OT-II T cells were stimulated with 1×104 DC-OVA +/- 1×104 DC-reg. T cells were harvested on day 4 and cell number was assessed by flow cytometry. * p<0.01 Results are pooled from 3 independent experiments.

0 2 4 6 8 10 12

treatment

cell number (*105)

No APC DC -OVA

DC-OVA + DC-reg-OVADC-OVA + DC-reg

DC-reg-OVA

DC- reg

*

*

*

*

59

CFSE

DC-OVADC-reg-OVA

DC-reg

+--

++-

+-+

-+-

--+

* White = no APC

Figure 12. DC-regs suppress OT-II T cell proliferation. 1×105 CFSE-labelled OT-II T cells were stimulated with 1×104 DC-OVA+/- 1×104 DC-regs. Cells were harvested on day 3 and T cell proliferation (CFSE dilution) was assessed by flow cytometry. Results are representative of 3 independent experiments.

60

0

5

10

15

20

25

30

35

40

IL2

% I

L2

+ve

cel

ls

DC-OVA

DC-reg

DC-reg-OVA

+ + + - -

- + - + -

- - + - +

0

2

4

6

8

10

12

14

16

IFNy

% I

FN

y +

ve c

ells

Figure 13. DC-regs do not suppress OT-II T cell cytokine release. 1×105 OT-II T cells stimulated with 1×104 DC-OVA +/- 1×104 DC-regs for 7 days. Cells were then harvested, washed and re-stimulated with anti-CD3 and anti-CD28 Abs for 12 hours. Intracellular cytokine production was assessed by flow cytometry.

61

DC DC + DC-reg DC-reg

Day 1

Day 3

CD25

Day 1

Day 3

CD44

Figure 14. Expression of CD25 and CD44 by T cells in allogeneic mixed lymphocyte reaction in the presence or absence of DC-regs. 1×105 BALB/c T cells were isolated from spleen and stimulated with 1×104 C57BL/6 DC +/- 1×104 C57BL DC-regs for 3 days. Expression of CD25 and CD44 was the assessed by flow cytometry. White histograms indicate un-stimulated T cells. Results are representative of 3 independent experiments.

62

DC-ovaDC-reg-ova

DC-reg

+--

++-

+-+

-+-

--+

* White = no APC

PI

Figure 15. DC-regs do not induce T cell death in mixed lymphocyte reactions. 1×105 OT-II T cells were stimulated with 1×104 DC-OVA +/- 1×104 DC-regs for 3 days. Propidium iodide (PI) staining of dead cells was assessed by flow cytometry. White histograms indicate un-stimulated cells. Results are representative of 3 independent experiments.

63

CD25

CD

4

CD25

Fo

xP3

DC

DC + DC-reg

1

0

66

64

CFSE

Res

po

nd

erT

cel

l No

.

Figure 16. DC-regs do not induce Foxp3+ Tregs in mixed lymphocyte cultures. CFSE-labelled 1×105 BALB/c T cells were stimulated with 1×104 C57BL/6 DC +/- C57BL/6 1×104 DC-regs for 7 days, harvested, stained for CD4, CD25, and FoxP3, and analyzed by flow cytometry. Results are representative of 3 independent experiments.

64

0 2000 4000 6000 8000 10000 12000

T + DC-ova

T + DC-ova + DC-reg-ova

T + DC-ova // DC-reg-ova

Cell number

Figure 17. Mechanism of DC-reg-mediated immuno-suppression involves both soluble and contact-dependent factors. 1×106 OT-II cells along with 1×105 DC-OVA were placed in the bottom chamber. 1×105 DC-reg-OVA were placed in the top chamber. Proliferation was assessed three days later by flow cytometry. Results represent data pooled from 3 independent experiments.

65

iNOS

DC

-reg

DC

-reg

+ LP

SS

ple

enS

ple

en +

LP

S

arginase1

FasL

PD-L1

IDO

ICOS-L

DC

-reg

DC

-reg

+ L

PS

Sp

leen

Sp

leen

+ L

PS

IL-10

Figure 18. RT-PCR expression of candidate molecules responsible for observed in vitro immuno-suppression. DC-regs or freshly isolated spleen mononuclear cells were pulsed with 2 µg/mL LPS overnight prior to gene expression evaluation.

66

02468

10121416

DC

DC

+ L

PS

DC

-reg

DC

-reg

+ L

PS

arg

inas

e ac

tivity

(mU

/ug

)

010

2030

4050

DC

DC

+ L

PS

DC

-reg

DC

-reg

+ L

PS

nitr

ite c

on

c (u

M)

A B

* * ** * *

Figure 19. DC-regs express high levels of arginase1 and iNOS (reflected by nitrite production) activity when stimulated with LPS. 1×106 cells/mL were pulsed with 2 µg/mL LPS overnight. A) Cells were harvested, washed and lysed. Arginase1 activity was determined by incubating cell lysate with L-arginine for 60 min and measuring resulting urea production. B) Supernatants were harvested and nitrite levels were detected by Griess reagent. * p<0.01 Data was pooled from 3 independent experiments.

67

DC DC-reg

DC + LPS DC-reg + LPS

PD-L1

Figure 20. Both DCs and DC-regs express high levels of PD-L1. 1×106 DC or DC-regs were pulsed with 2 µg/mL LPS overnight. Cells were then harvested, washed, stained, and assessed by flow cytometry. White histograms indicate isotype control. Result is representative of 3 independent experiments.

68

0

500

1000

1500

2000

2500

DC DC + LPS DC-reg DC-reg +LPS

IL10

(p

g/m

L)

ND ND ND

Figure 21. LPS-pulsed DC-regs express high levels of IL-10. Freshly isolated spleen DCs or DC-regs were pulsed with 2 µg/mL LPS overnight. Supernatants were harvested and IL-10 levels were detected by ELISA.

69

3 H-t

hym

idin

e in

corp

ora

tion

(cp

m)

1x03

DC-OVA + DC-reg

DC-OVA

L-NIL

Isoty

pe

Figure 22. DC-regs suppress T cell proliferation through an iNOS-dependent mechanism. 1×105 OT-I T cells were stimulated with 1×104 OVA peptide-pulsed DC +/- 1×104 DC-reg in the presence of IL-10R blocking Ab or chemical inhibitors Nor-NOHA and L-NIL that block arginase1 and iNOS activity, respectively. Cells were cultured for 3 days and proliferation was assessed by 3H- thymidine incorporation. Data is representative of 3 independent experiments.

70

Figure 23. DC-regs do not suppress T cell proliferation and activation in vivo. A) In vivo experimental scheme. CD45.1+ B6.SJL mice were i.v. injected with 1×106 CD45.2+ OT-II cells. The next day, mice were immunized with 1×106 DC +/- 1×106 DC-regs i.p. Mice were sacrificed on days 3 and 5. Cell proliferation was assessed by CFSE dilution. Spleen and lymph node cells were re-stimulated with OVA in vitro and cytokine production was assessed by flow cytometry 12 hours later. B) OT-II cells were injected into a mouse as in the experimental scheme above and primed with DC +/- DC-regs. Spleen and lymph nodes were harvested on days 3 and 5. OT-II cell proliferation was assessed by CFSE dilution. C) Spleen and lymph nodes were harvested on days 3 and 5 and re-stimulated with OVA in vitro. IFN-γ production was evaluated by flow cytometry.

CD45.2+ OT-II cells iv

Day 0

DC +/- DC- regip

Day 1

Assess T cell proliferation

+Cytokine

production

Day 3, 5

CD45.1+

A No APC

DAY 5

DAY 3

CFSE

CD

45.2

+ C

D4+

O

TII

Cel

l No

.

DC- ova

DC - ova + DC -reg- ova

B

0

10

20

30

40

50

-OVA DC-ova DC-ova +DC-reg-ova

DC-reg-ova

% IF

Ny

+ve

cells

Day 3

Day 5

C

71

No APCDC-ova

+ DC

DAY 5

DAY 3

CFSE

CD

45.2

+ C

D4+

O

TII

Cel

l No

.

DC-ova + DC-reg

A

05

1015

2025

3035

4045

-OVA DC-ova + DC DC-ova + DC-reg

% IF

Ny

+ve

cells

Day 3

Day 5

B

Figure 24. DC-regs fail to suppress T cell proliferation and activation in vivo. A) OT-II cells were injected into mouse as in the experimental scheme above and primed with DC +/- DC-regs. Number of APCs was normalized in both groups with co-injection of DCs that were not pulsed with OVA. Spleen and lymph nodes were harvested on days 3 and 5. OT-II cell proliferation was assessed by CFSE dilution. B) Spleen and lymph nodes were harvested on days 3 and 5 and re-stimulated with OVA in vitro. IFN-γ production was evaluated by flow cytometry.

72

No APC DC-reg-ovaDC-ova

CFSE

CD44

CD69

Figure 25. DC-regs induce OT-II cell activation in vivo. OT-II cells were injected into mouse as in the experimental scheme above and primed with DC +/- DC-regs. Spleens were harvested on day 3. Expression of activation markers (CD44, CD69) on CD45.2+ OT-II cells were evaluated by flow cytometry.

73

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Characterization of a Novel Dendritic Cell Population · 2013-10-17 · 1 CHAPTER 1 Introduction Dendritic cells comprise a heterogeneous population of cells that are specialized