122 C L I N I C A L I M M U N O L O G Y Newsletter Vol. 19, No. 10/11, 1999

Forward (Continued from pg. 121)

has been translational: to take original laboratory observations from in vitro and animal models into the clinic. The ultimate goal of these investigations is to exploit the unique properties of DC to create effective anti-tumor and anti-viral immuno- therapeutic modalities.

This issue of CIN provides an over- view of laboratory and clinical aspects of DC immunobiology as envisioned by the Pittsburgh group. Barratt-Boyes, Tanaka, Hirao and Lotze, and Donnenberg and Meyer discuss DC maturation and traf- ticking. Barrat-Boyes approaches this sub- ject from the standpoint of DC ontogeny, reviewing the data from animal models that relate DC maturational status to DC trafficking patterns. Donnenberg and Meyer present a step-by-step tutorial in rare event detection by flow cytometry as applied to the detection of DC that have migrated from injection sites in the skin to draining lymph nodes. Tanaka et al. approach the problem of DC trafficking

from a translational standpoint, reviewing the role of chemokines in DC recruitment and commenting on the prospects for ini- tiating anti-tumor responses by injecting engineered DC directly into tumors. Na~ak and Falo discuss the possibility of eliciting tumor specific immunotherapy by delivering antigen to DC, reviewing the critical pathways for antigen presenta- tion and the requirements for immuno- genicity. On this background, they discuss the relative merits of delivering tumor antigen to DC in different formulations, including peptide pulsing, apoptotic bod- ies, and genetic immunization by trans- fection. On the opposite side of the coin, Shurin examines the effects that tumor cells exert on DC growth and function. He argues that the escape of malignant cells from immune surveillance may result from defective DC differentiation and he reviews the mechanisms by which this may occur. Elder brings DC therapy from theory to practice, discussing DC preparation for use in clinical trials and the regulatory issues pertaining to the experimental use of DC immunotherapy.

The editors of CIN would like to thank these investigators for compiling this informative series of articles.

R e f e r e n c e s

1. Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature 392:245-252, 1998.

2. lnaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, Muramatsu S, Ste inman RM: Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulat- ing factor. J Exp Med 176:1693-1702, 1992.

3. Canx C, Dezutter-Dambuyant C, Schmitt D, Banchereau J: GM-CSF and TNF-alpha cooper- ate in the generation of dendritic Langerhans cells. Nature 360:258-261, 1992.

4. Sallusto F, Lanzavecchia A: Efftcient presenta- tion of soluble antigen by cultured human dendritic cells is maintained by granulocyte/ macrophage colony-stimulating factor plus Intedeukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179:1109-1118, 1994.

5. Romani N, Gmner S, Brang D, Kampgen E, Lenz A, Trockenbacher B, Konwalinka G, Fritsch PO, Steinman RM, Schuler G: Prolifer- ating dendritic cell progenitors in human blood. J Exp Med 180:83-93, 1994.

Do Dendr i t i c Cells Require Matura t ion in Vitro for Effect ive Traf f i ck ing in Vivo! (Continued from pg. 121)

inflammatory proteins (MIP) lo~ and 113, and monocyte chemotactic proteins 1 - 4. 24 Human DC derived from CD34 ÷ cells also express CCR6, the ligand for MIP- 3et. 5 Interaction with such inflammatory chemokines is thought to promote accu- mulation of antigen-acquiring immature DC at sites of tissue injury. Following exposure to a maturation signal, such as bacterial lipopolysaccharide, CD40L, or TNF-o~, DC switch receptor expression to CCR7 and CXCR4 and migrate in response to constitutive chemokines 6Ckine, MIP-38,

and stromal cell-derived factors -1 a and 113. 2 - 7 6 C k i n e and MIP-313 are expressed in lymphatics and lymphoid tissues and are likely to induce migration of antigen- stimulated and maturing DC to lymph nodes. 6s Indeed, mice that lack expres- sion of 6Ckine have a paucity of DC in lymph nodes, and administration of an antibody to this chemokine in mice blocks the migration of mature DC to lymph nodes. 7,8

These patterns of chemokine respon- siveness bear directly on the type of DC to be used in DC-based vaccination proto- cols. The data would suggest that antigen- loaded DC must receive a maturation signal in vitro prior to administration to patients, as only mature DC express the

appropriate receptors for migration to peripheral lymph nodes. However, results of several in vivo studies present a less clear picture of DC migration with respect to maturation state. In the murine system, splenic and bone marrow-derived DC appear to migrate from subcutaneous tis- sues to draining lymph nodes with and without prior antigenic stimulation or delivery of a maturation signal. 9~2 In human studies, a proportion of antigen- loaded DC given intradermally as a vac- cine to cancer patients homed to lymph nodes in the absence of any ex vivo matu- ration stimulus. 13 In our own studies, chimpanzee blood-derived DC adminis- tered without in vitro maturation still migrated from a subcutaneous site of

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injection to T-cell rich areas of draining lymph nodes) 4 Overall, these data sug- gest that induced maturation of DC prior to subcutaneous or intradermal adminis- tration is not a requirement for efficient migration of DC to lymph nodes. It is possible that the microenvironment pre- sent at the site of injection results in spontaneous maturation of immature DC, resulting in a switch of chemokine recep- tor expression and subsequent responsive- ness to constitutive chemokines that induce homing to lymph nodes.

We have critically addressed this prob- lem in a well-defined preclinical model using the rhesus macaque monkey, in which monocyte-derived DC closely resemble DC generated in the same man- ner from human blood (Barratt-Boyes, Zimmer, Harshyne et al., manuscript sub- mitted). Consistent with the human DC literature, immature monkey DC express low levels of CCR5 and do not express CCR7. Conversely, CD40L matured DC are negative for expression of CCR5 but express CCR7 at high levels. Importantly, immature monkey DC migrate in response to CCR5 ligand MIPlct during in vitro chemotaxis assays but not at all to CCR7 ligands 6Ckine and MIP-313. Upon matu- ration, chemotactic responses are com- pletely switched to MIP-313 and 6Ckine. Based on these chemokine receptor and chemotaxis assays, we predicted that mature but not immature monkey DC would migrate from a dermal injection site to draining lymph nodes. To test this directly in vivo, we labeled immature and mature DC with a lipophilic membrane dye called DiD TM (1, l'-dioctadecyl-3,3,3"3'- tetramethylindodi-carbocyanine perchlo- rate; excitation/emission spectra = 644 nm/663 nm). In separate experiments, DiD- labeled immature and mature DC were injected intradermaUy into the inguinal region of donor monkeys and the draining inguinal lymph nodes excised 36 hr later and subjected to rare event flow cyto- metric analysis (see article by Albert D. Donnenberg in this issue). Surprisingly, DiD + cells were identified in the draining lymph node of animals receiving both immature and mature DC at similar fre- quencies, representing approximately 1% of total lymph node DC cells. The mean fluorescence intensity of CD86 expres- sion on immature DC that localized to

lymph nodes was statistically significantly higher than that of immature DC prior to injection, suggestive of in vivo maturation. Moreover, injection of immature DC resulted only in a minor localized acute inflammatory response at the dermal site of injection, with no residual DC present at 36 hr. In marked contrast, a severe acute inflammatory infiltrate was present at the site of injection of animals that received injections of mature DC, and a large number of fluorescently-labeled, mature DC were detected in the dermis at 36 hr in these animals (Barratt-Boyes, Zimmer, Harshyne et al., manuscript submitted).

Our findings are consistent with data from the mouse using purified splenic myeloid (CD8¢c) DC, which are analo- gous to human monocyte-derived DC. These cells traffic to lymph nodes follow- ing subcutaneous injection in the absence of any in vitro maturation stimulus, repre- senting about 1% of total lymph node DC. 15 A recent report in the murine system indi- cates that immature bone marrow-derived DC migrate poorly to lymph nodes fol- lowing subcutaneous injection, relative to migration of GM-CSF transfected imma- ture DC. 16 Interestingly, GM-CSF trans- fection did not induce maturation of DC in this system, based on phenotypic and functional analyses. When the migration of immature and CD40L matured DC was compared in the same system, injected DC that homed to lymph nodes constituted from 1 to 2% of lymph node DC 48 hr after subcutaneous injection, regardless of maturation state. 12 Fossum has reported in earlier studies that purified lymph DC injected into footpads of mice mostly were retained at the site of injection. 17 Lymph DC represent a maturing DC population and hence these findings may be consis- tent with our results. More recently, anti- gen-stimulated blood-derived human DC used therapeutically in human cancer patients were only partially cleared from intradermal and subcutaneous injection sites) 3 Labeur et al. t2 also found that the majority of mouse DC remained at the site of subcutaneous injection, regardless of the in vitro maturation state prior to administration. Interestingly, in our chim- panzee studies, injected DC were not pre- sent in the subcutaneous tissue 48 hr after injection, indicating that migration from

skin in this system was very efficient) 4 While interpretation of these conflicting results is difficult, it remains possible that there is a fundamental difference between the capacity of immature and mature DC to be cleared from a skin injection site. Mature DC produce large quantities of pro-inflammatory cytokines and likely induced the severe inflammatory response observed in the dermis in our recent stud- ies. Therefore, while a mild inflammatory response may induce maturation of imma- ture DC and promote migration, it is pos- sible that a more severe inflammatory response impairs migration of mature DC out of skin. Whether retention of mature DC in inflamed skin contributes to the immune response to antigen needs to be addressed.

The field of DC-based immunotherapy is rapidly developing and progressing towards clinical application. However, it is clear that the behavior of DC follow- ing administration is not yet completely understood, and further studies are required to determine the optimal cell type to be used in DC-based vaccination protocols.

References

1. Banchereau J, Steinman RM: Dendritic cells and the control of immunity. Nature 392:245-252, 1998.

2. Lin CL, Suri RM, Rahdon RAet al.: Dendritic cell chemotaxis and transendothelial migration are induced by distinct chemokines and are regulated on maturation. Eur J Immunol 28:4114-4122, 1998.

3. Sozzani S, Allavena P, D'Amico Get al.: Differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking properties. J Immunol 161:1083-1086, 1998.

4. Sallusto F, Schaedi P, Loetscher Pet al.: Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur J Immunol 28:2760-2769, 1998.

5. Dieu MC, Vanbervliet B, Vicari Aet al.: Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in differ- ent anatomic sites. J Exp Med 188:373-386, 1998.

6. Ngo VN. Tang HL, Cyster JG: Epstein-Barr virus-induced molecule 1 ligand chemokine is expressed by dendritic cells in lymphoid tissues and strongly attracts naive T-cells and activated B cells. J Exp Med 188:181-191, 1998.

7. Saeki H, Moore AM, Brown MJ et al.: Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph

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124 C L I N I C A L I M M U N O L O G Y Newsletter Vol. 19, No. 10/11, 1999

nodes. J Immunol 162:2472-2475, 1999.

8. Gunn MD, Kyuwa S, Tam C et al.: Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization (see comments). J Exp Med 189:451-460, 1999.

9. Kupiec-Weglinski JW, Austyn JM, Morris PJ: Migration patterns of dendritic cells in the mouse. Traffic from the blood, and T-cell-dependent and -independent entry to lymphoid tissues. J Exp Med 167:632-645, 1988.

10. Ingulli E, Mondino A, Khoruts A et al.: In vivo detection of dendritic cell antigen presentation to CD4(+) T-cells. J Exp Med 185:2133-2141, 1997.

11. Morikawa Y, Furotani M, Matsuura Net al.: The

role of antigen-presenting cells in the regulation of delayed-type hypersensitivity. II. Epidermal Langerhans' cells and peritoneal exudate macro- phages. Cell Immunol 152:200-210, 1993.

12. Labeur MS, Roters B, Pers Be t al.: Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell mat- uration stage. J Immunol 162:168-175, 1999.

13. Morse MA, Coleman RE, Akabani Get al.: Migration of human dendritic cells after injec- tion in patients with metastatic malignancies. Cancer Res 59:56-58, 1999.

14. Barratt-Boyes SM, Watkins SC, Finn OJ: In vivo migration of dendritic cells differentiated in vitro: a chimpanzee model. J Immunol 158:4543-4547, 1997.

15. Smith AL, Fazekas de St. Groth B: Antigen- pulsed CD8ot ÷ dendritic cells generate an immune response after subcutaneous injection without homing to the draining lymph node. J Exp Med 189:593-598, 1999.

16. Curiel-Lewandrowski C, Mahnke K, Labeur M et al.: Transfection of immature murine bone marrow-derived dendritic cells with the granulocyte-macrophage colony-stimulating factor gene potently enhances their in vivo anti- gen-presenting capacity. J Immunol 163:174- 183, 1999.

17. Fossum S: Lymph-borne dendritic leucocytes do not recirculate, but enter the lymph node paracortex to become interdigitating cells. Scand J Immunol 27:97-105, 1988.

Principles of Rare Event Analysis by Flow Cytometry: Detection of Injected Dendrit ic Cells in Draining Lymphatic Tissue Albert D. Donnenberg and E. Michael Meyer Interim Director, Blood and Marrow Transplant Program, University of Pittsburgh Cancer Institute, 200 Lothrup St. West, 1051 BST, Pittsburgh, PA 15213. e-mail: donnenbergad@msx.upmc.edu

Supported by grants PO1 A143664 and RO1 AI41408

Finding a Needle in a Haystack

Rare event analysis by flow cytometry is the art of finding a needle in a haystack, proving that it really is a needle, and then making several measurements to deter- mine exactly what kind of a needle it is. The key elements in rare event detection are the frequency of the event of interest and the signal to noise ratio. This article will discuss practical aspects of dealing with both in the context of the detection of dendritic cells in the draining lymph node 36 hr after they have been injected in the skin.

Sample Flow Rate

The event frequency is a property of the sample and not something that we can mampulate, other than to decide how many total cells must be processed. Obviously the lower the frequency of the events of interest, the more events it will be neces- sary to acquire. The rate of sample acqui- sition can be manipulated in two ways: by concentrating the sample and by increas- ing the sample flow rate. Although it is tempting to shorten acquisition time by

increasing the flow rate, this has its limi- tations. Most analytical cytometers have low, medium, and high flow rate settings. Cell sorters have sheath fluid pressure and sample differential pressure settings. In both types of instruments increasing the sample pressure relative to the sheath pressure increases the sample flow rate. This results in a wider sample stream (core stream) within the sheath fluid stream. Unfortunately, this increases the oppor- tunity for two cells to pass through the detection system at the same time (coinci- dence) and also increases the coefficient of variation (CV) of all of the measured parameters. Both of these eventualities are undesirable. One can determine the maximum acceptable flow rate for a given sample concentration using a mix- ture of green and red fluorescent beads. At increasing flow rates, monitor the fre- quency of doublets (red and green beads which are seen by the cytometer as a sin- gle event) by looking for double positive (green and red) events. Monitor the CV of fluorescence of the single positive events as well. This simple experiment can help you determine a speed limit for event acquisition. At higher flow rates doublets can be gated out of the analysis by the use of doublet discrimination (comparison of

signal pulse height and width or area). An alternate approach that can greatly increase throughput is fluorescence threshold trig- gering, in which events below a certain threshold of fluorescence are not seen by the cytometer and therefore not collected as events. This method requires that the rare event be very brightly labeled and that the denominator (number of cells processed) be inferred from the sample flow rate.

Signal to Noise

Successful detection of rare events depends on maximizing the signal-to-noise ratio. Noise, in the sense used here, comes from many sources including nonspecific bind- ing of a fluorochrome of interest, cellular autofluorescence, disruptions in sample flow caused by cell clumps, and other electrical or mechanical problems. There are several tricks for maximizing the dif- ference between signal and noise, some of which will be illustrated in the exam- ple below. One very important aspect is to characterize the total noise using an appropriate negative control. Sometimes, as is the case with the example to be presented here, it is possible to devise a control sample that is identical to the

I I I I 0197-1859/99 (see frontmatter) © 1999 Elsevier Science Inc.