Accessory cell function of cells isolated from Mycobacterium leprae-induced granulomas

CELLULAR

IMMUNOLOGY

Accessory

102,346-354 (1986)

Cell Function of Cells isolated from Mycobacterium leprae-Induced Granulomas

N. MONTREEWASUWAT, JILL CURTIS, AND J. L. TURK Department of Pathology, Royal College of Surgeons of England, 35-43 Lincoln’s Inn Fields, London WCZA 3PN. England Received March l&1986; accepted July 2,1986 The large cells from Mycobacterium leprae-induced granulomas in guinea pig lymph nodes were separated by Percoll discontinuous density gradient centrifugation and on a fluorescenceactivated cell sorter (FACS) using cross-reacting monoclonal antibody to human MHC Class II antigens. Large Percoll-separated cells (83% Class II antigen positive and 52% macrophage-specific antigen positive) and FASseparated cells are able to act as antigen-presenting cells for T-cell proliferation to PPD. In previous studies, macrophage antigen-positive cells consistently failed to act as accessorycells. This indicates that there is a population of accessorycells which are macrophage antigen negative and MHC Class II antigen positive present in these M. lepraeinduced granulomas. @ 1986 Academic Ress, Inc. INTRODUCTION

An experimental model for the study of Mycobacterium leprae-induced granulomas in the guinea pig has been established in this laboratory ( 1,2). The granulomas in draining postauricular lymph nodes produced 5 weeks after the intradermal injection of cobalt-irradiated ikf. leprue into the dorsum of the ear of the guinea pig mainly consist of macrophages containing ingested organisms. These cells are nonspecific esterasepositive, peroxidase and Fc and C3 surface receptor negative, and 70% are glassadherent. Mathew et al. (3) also showed that these cells are MHC Class II antigen positive and carry a macrophage-specific antigen defined by a monoclonal antibody raised in this laboratory. It is widely accepted that proliferation of T lymphocytes induced by antigen or mitogen requires the participation of accessorycells expressing MHC Class II antigen (4-6). Gupta et al. (7) separated macrophage antigen-positive cells from M. leprue granulomas on a fluorescence-activated cell sorter (FACS) and found that these cells were unable to act as accessory cells for both mitogen (Con A)- and antigen (PPD)induced T-cell proliferation. However, the unseparated population of cells from M. leprue-induced granulomas gave proliferative responses to both Con A and PPD (S. Verghese, unpublished data). Thus the existence of accessory cell heterogeneity and the possibility that a population of accessory cells might be excluded from the macrophage antigen-positive cell fraction cannot be ruled out. Percoll (Pharmacia) is a standard reagent for the separation of lymphocyte, monocyte, and subpopulations (S- 10). Percoll discontinuous density gradient centrifuga346 0008-8749186$3.00

Copyright Q 1986 by Academic Fwss, Inc. Au rights of reproduction in any form nserwd.

M. leprue GRANULOMAS;

ACCESSORY CELL FUNCTION

347

tion was used in the present study to separate the large cells from M. leprae-induced granulomas. Large cells with MHC Class II antigens separated on a FACS were also studied for comparison with Percoll-separated cells for their accessorycell function. MATERIALS AND METHODS Animals Outbred Hartley strain guinea pigs of either sex weighing 300-400 g from stock bred at the Royal College of Surgeons or purchased from David Hall (New Church, Staffs,U.K.) were used. They were fed on Labsure RGP diet (F. Dixon & Sons, Ware, Herts) liberally supplemented with cabbageand hay. Organisms Armadillo-derived cobalt-irradiated M, leprae organisms were kindly provided by Dr. R. J. W. Reesof the Clinical Research Centre, Harrow, London. Monoclonal Antibodies Mouse antihuman HLA-DR (RFDR, , Batch 1784) that cross-reactswith guinea pig MHC Class II antigens was provided by Dr. L. Poulter of the Royal Free Hospital, London. Specific anti-macrophage monoclonal antibody was produced in our laboratory as previously described (3). Mouse anti-guinea pig “Pan T” (CT5) monoclonal antibody was kindly provided by Dr. Tan and Dr. Scheper of the Free University Hospital, Amsterdam. This reagent also cross-reacts with a small proportion (lo15%) of macrophages. Rabbit anti-guinea pig immunoglobulin conjugated to horseradish peroxidase (P 141) was purchased from Dako, Ltd. (High Wycombe, Bucks). Antigen Purified protein derivative (PPD) was obtained from the Central Veterinary Research Laboratory, Weybridge, Surrey. It was dialysed against 100 vol of PBS for 24 hr at 4°C filter sterilized, aliquoted, and stored at -20°C until use. Cell Preparation 1. Preparation of lymph node cell suspensions. The postauricular lymph nodes from a guinea pig injected into the dorsum of each ear with lo9 cobalt-irradiated M. leprae organisms 5 weeks previously were removed aseptically. They were cut into small pieces and gently pressed through a stainless steel mesh into MEM (Eagle’s minimum essential medium; Wellcome Reagents, Ltd., England). The cervical and jugular lymph nodes were also collected from the same guinea pig and used as a source of responder T lymphocytes. The cells were washed three times in MEM and resuspended in RPM1 1640 (Flow Laboratories, U.K.) supplemented with 10%heatinactivated fetal calf serum (FCS; Seralab, U.K.), 4 mM L-glutamine, 2 mM sodium pyruvate, 100 IU/ml penicillin, and 100 pg/ml streptomycin (Flow Laboratories). 2. PuriJication of granuloma cells in a fluorescence-activated cell sorter (FAGS-l). Fifty million cells from both postauricular lymph nodes were incubated with 1 ml of supernatant ( 1:10 dilution) containing monoclonal antibody against MHC Class II antigens for 45 min at room temperature. The cells were washed once with RPM1

348

MONTREEWASUWAT,

CURTIS,

AND

TURK

1640 containing 5% FCS and were then incubated with 800 ~1of sheep F(ab’)Z antimouse Ig-FITC conjugate (Sigma, U.K.) at 1:50 dilution, for 30 min on ice. The cells were washed three times with chilled medium containing 5% FCS and finally resuspended at a concentration of 4 X 1O6viable cells/ml. The negative controls were incubated with the FITC conjugate only. Fluorescein-labelled large cells were separated on FACS-1 (Becton-Dickinson, Rutherford, N.J.) excluding the population of labelled cells in the negative control. They were treated with mitomycin C (40 llg/m1/106 cells) for 30 min at 37°C then washed five times with ice-cold MEM, and finally resuspended in culture medium at a concentration of 1 X lo6 viable cells/ml. 3. Purijication of granuloma cells on Percoll discontinuous density gradients (9). Percoll [density (d) = 1.128 g/ml; Pharmacia Fine Chemicals, Sweden] was first made isoosmotic by adding nine parts (v/v) of Percoll to one part of 1.5 M phosphatebuffered saline (PBS). This isoosmotic preparation has a density of 1.122 g/ml and for simplicity was referred to as “ 100%Percoll.” It was then diluted with 0.15 M PBS to 70% (d = 1.090), 60% (1.077), 50% (1.067), 40% (1.056), and 30% (1.043). Cells (less than 100 X 106)from postauricular lymph nodes were resuspended in 2 ml of 100% Percoll and transferred to a 15-ml conical graduated plastic tube. Successively less dense solutions (2 ml), starting with 70% Percoll and reducing by 10% steps to 30% Percoll, were carefully layered by gentle pipetting. Centrifugation was carried out at 450g for 10 min at room temperature. At equilibrium after centrifugation, five distinct bands could be observed at various interfaces. Fractions at the interfaces between 40 and 50% and 50 and 60% were collected with a Pasteur pipette from the top and washed twice in MEM, then treated with mitomycin C, and finally resuspended in RPM1 1640 with 10% FCS and antibiotics at a concentration of 1 X lo6 viable cells/ml. They were mainly large cells with the morphology of macrophages by May-Griinwald-Giemsa stain. In some experiments, Percoll-separated fractions of large cells were further depleted of T cells by rosetting with rabbit erythrocytes (11). E-rosette-positive (E+) and E-rosette-negative (E-) fractions were recovered by Ficoll-Paque centrifugation. The latter was devoid of T cells but the former still contained a few macrophages. Since Percoll- and FACS-separated cells are comparable in properties and there is a very low cell recovery from FACS separation we did not rosette the FACS-separated cells. In some experiments, mesenteric and all the other lymph nodes below the diaphragm were collected. A small portion was sent for histology. There was no granuloma seen. They were then separated on Percoll density gradients and treated as described above. 4. Peritoneal exudate macrophages. Peritoneal exudate cells (PEC) were harvested from guinea pigs (immunized 5 weeks previously with M. leprae) 4 days after intraperitoneal injection of 20 ml sterile paraffin oil. PEC were washed three times with MEM and finally resuspended in culture medium. Cells ( 100 X 106)were separated on Percoll density gradients and mitomycin C treated as described above. 5. Lymphocyte proliferation assay. T lymphocytes were purified from cervical and jugular lymph nodes by passing through nylon wool columns as described elsewhere ( 12). They were resuspended in culture medium at a concentration of 5 X 1O6viable cells/ml. One hundred microliters of mitomycin C-treated granuloma cells ( 106/ml) separated by FACS or Percoll, or Percoll-separated PEC, were cultured with 100 ~1 of autologous nylon wool column purified T-cell suspension in 96-well flat-bottom

M. Zeprue GRANULOMAS;

349

ACCESSORY CELL FUNCTION TABLE 1

Properties of Large Cells Separated on Percoll Density Gradients or on a FACS after Labelling with Antibody to MHC Class II Antigens Large cells from M leprae granulomas separated by Peritoneal exudate cells: Percoll separated (%) Adherence Nonspecific esterase Macrophage antigen MHC Class II antigens “Pan T” antigen (CT5)

97,2 88 Ik 5 100 92+4 13+2

Surface immunoglobulins

45 + 6

FACS (%) NT 44 a2 50 25 loo M4 7.7+ 1.8 T30 k 1.5 9 k2

Percoll (%b) 5924 46k2 52+3 83-+4 Mqi 6kO.4 T31 k2.3 7kO.l

Note. Each figure is the mean f standard error of three to six determinations. NT, not tested; Mb, macrophage morphology; T, lymphocyte morphology.

microtitre plates at 37°C in humidified 5% COz in air, in the absence or presence of PPD (25 pg/ml) for 66 hr. The cultures were pulsed for the last 24 hr with [3H]thymidine (5 Ci/mmol sp act; Amersham International, England) and harvested on a MASH II microharvester (Microbiological Association, Inc.) onto glass hlter paper strips (Whatman GF/A). [3H]Thymidine incorporation was determined in a liquid scintillation counter (Packard, U.K.). 6. Characterization of large cells separated on a FACS or Percoll gradients. Cytocentrifuge preparations of cells separatedon a FACS or Percoll gradients were studied by May-Griinwald-Giemsa stain and nonspecific esterasestain (13). Expression of macrophage specific antigen, “Pan T,” and surface immunoglobulins on MHC Class II-positive cells separated by FACS was determined by an indirect immunoperoxidase technique as described previously (3, 14) using sheepF(ab’)2 anti-mouse immunoglobulin conjugated to horseradish peroxidase (Sigma, U.K.) to prevent nonspecific binding of the conjugate via Fc receptors. Percoll-separated cells were identified for macrophage antigen, MHC Class II antigens, “Pan T,” and surface immunoglobulins by means of the indirect immunofluorescence or indirect immunoperoxidase techniques. Ability to adhere to glass or plastic was also tested in the cells separated by Percoll density gradient centrifugation. RESULTS The characteristics of the large cells from M. leprae granulomas separated on a FACS or on Percoll discontinuous density gradients are compared with those from a peritoneal exudate in Table 1. Large cells from the M. leprae granulomas separated by the two methods contained similar proportions of macrophages (approximately 50%) and surface immunoglobulin positive cells (less than 10%). Percoll-separated PEC were 100%macrophages but 13%of these cells stained with CT5 “Pan T” monoclonal antibody. The large M. leprae granuloma cells contained approximately 30%

350

MONTREEWASUWAT,

CURTIS, AND TURK

TABLE 2 Comparison of Accessory Cell Function of Peritoneal Exudate Macrophages and Large Percoll Separated Cells from M. leprae Granulomas in Supporting Antigen (PPD)-Induced Proliferation of Lymph Node T Lymphocytes” Percoll-separated cells Peritoneal exudate cells

M. Zepraegranuloma cells Guinea pig no.

Lb

1 2 3 4 5 6

126’ 159 93 56 178 222

L+PPD 113 129 101 52 207 283

L+A 147 306 176 190 184 757

L+A+PPD 1,350 2,787 535 747 456 3,672

L 123 143 149 56 178

L+PPD 137 206 150 52 207

L+A 326 3,393 1,620 494 344

L+A+PPD 3,459 6,366 6,042 826 2,819

’ Mitomycin C-treated peritoneal exudate macrophages or M. leprae granuloma cells (1 X 105)were added to 5 X 10’ autologous T cells/well. b L, lymphocytes; A, accessorycells; PPD, 25 &ml. ‘Mean cpm of four replicate cultures. The standard deviation was 5- 10%of the mean.

T cells when cells with the obvious morphology of macrophages were excluded from the CTS-positive population. There was a high proportion of surface immunoglobulin-positive cells in the Percoll-separated PEC which must be macrophages with surface cytophilic antibodies. Antigen-Presenting Function of Percoll-Separated and MHC Class II Antigen-Positive Cells from A4. leprae Granulomas Sensitized lymph node T lymphocytes depleted of adherent cells by passageover nylon wool columns minimally responded to PPD (Tables 2-5). Addition of 1 X 10’

TABLE 3 Comparison of Accessory Cell Function of Peritoneal Exudate Macrophages and MHC Class II AntigenPositive Cells from M. leprae Granulomas in Supporting PPD-Induced Proliferation of Lymph Node T Lymphocytes”

Guinea pig no.

FACS-separated cells with MHC Class II antigens from hf. leprae granulomas

Percoll-separated peritoneal exudate cells

Lb

L

115c 186 129 222 142 144 a-CSame as Table 2.

L+PPD 75 369 187 283 167 180

L+A 204 524 249 343 332 161

L+A+PPD 1,621 3,124 621 870 1,628 1,274

57 124 199 187 145 292

L+PPD 52 120 133 207 140 275

L+A 494 397 600 344 426 748

L+A+PPD 826 2,924 5,260 2,892 2,257 2,344

M. leprue GRANULOMAS;

351

ACCESSORY CELL FUNCTION TABLE 4

Comparison of Accessory Cells Function of Peritoneal Macrophages and the E-Rosette Negative (E-) and E-Rosette-Positive (E+) Fractions of Large Percoll Separated Cells from M. leprue Granulomas in Supporting Antigen (PPD)-Induced Proliferation of Lymph Node T Lymphocytes’ Percoll-separated cells M. leprae granuloma cells

Guinea pigno. 1 2 3 4

Lb L+PPD 43 63 48 57

46 83 40 68

L+ESW 707d IOS 42’

L+E-+PPD 65 4,373 645 368

L+E+

Peritoneal exudate cells L+E++PPD

119’ 1576 64% 48f

L

88 127 66 47

L+PPD

63 48

L+A

83 40

L+A+PPD

326 207

1,536 1,315

a Mitomycin C-treated peritoneal exudate macrophages or M. leprae granuloma cells (1 X 10’) were added to 5 X 10’ autologous T cells/well. ’ L, lymphocytes; A, accessory cells; E-, T-depleted population of large PercoIl separated M. leprae granuloma cells; E+, T-enriched fraction of large Percoll-separated M. leprae granulomacells. ’ 5 X IO’E- per well. d I X IO5E- per well. ‘3 X 104E- per well.‘5 X lO’E+ per well. 8 1 X lO’E+perwell.

autologous mitomycin C-treated peritoneal macrophages considerably enhanced the antigen induced T-cell proliferation. Similarly the addition of 1 X lo5 mitomycin C-treated Percoll-separated cells or MHC Class II antigen-positive cells from hf. leprae granulomas to autologous nylon wool column-purified T lymphocytes led to 2- to IO-fold increases in the lymphocyte proliferation responsesto PPD (Tables 2 and 3). Further purification of large Percoll-separated cells from AI. leprae granulomas by rosetting with rabbit erythrocytes yielded T-cell-depleted accessory cells which supported responsesto PPD in three out of four guinea pigs although the number of accessory cells was fewer than usually used due to a low cell recovery (Table 4). On

TABLE 5 Comparison of Accessory Cell Function of Peritoneal Exudate Macrophage and Large Percoh-Separated Cells from Nongranulomatous Lymph Nodes in Supporting PPD-Induced Proliferation of Lymph Node T Lymphocytes“ Percoh-separated celk Nongranulomatous lymph node ceils Guinea pig no.

Lb

I 2 3 4 5

94c 186 45 52 68

a< Same as Table 2.

LSPPD 159 276 44 42 61

L+A 99 563 885 134 90

L+A-tPPD 146 363 1,360 164 144

Peritoneal exudate cells L

L+PPD

L+A

L+A+PPD

171

162

445

4,329

21

47

428

1,823

51

96

203

5,267

352

MONTREEWASUWAT,

CURTIS,

AND

TURK

the other hand, the E-rosette-positive fractions (E+) did not act as accessorycells for responsesto PPD (Table 4). Mitomycin C-treated Percoll-separated cells from nongranulomatous lymph nodes, i.e., other than those draining the injection site in M. leprue-sensitized animals, were not able to act as accessorycells for PPD-induced T-lymphocyte proliferation (Table 5). DISCUSSION One of the major functions of accessorycells is antigen presentation to the relevant T cells. It has been known that accessory cells which express MHC Class II antigens play a major role in the activation of T lymphocytes by antigen or mitogen (4-6). Accessory cells were originally identified as macrophages ( 15, 16) but recently other cells of the mononuclear phagocyte system (e.g., Langerhans cells, Kupffer cells, interdigitating cells, dendritic cells, or microglia) or nonmacrophage cell types [e.g., endothelial cells, follicular dendritic cells, mast cells, activated B cells, some activated T cells, natural killer (NK) cells, or tumour cell lines] have been reported to express accessory cell function (6, 17, 18). All accessory cells, be they macrophages or not, share one common characteristic, the expression of MHC Class II molecules on their surface. It appears that any Class II antigen-bearing cell can function as an accessory cell, indicating the existence of accessorycell heterogeneity. The previous study from our laboratory on M. leprae granuloma cells showed that the unfractionated population of the granuloma cells gave responsesto both Con A and PPD. However, a purified population of macrophages from the M. leprue granulomas separated on a FACS (using monoclonal antibody to guinea pig macrophages which failed to block antigen presentation by peritoneal exudate macrophages) consistently failed to act asaccessorycells for both Con A- and PPD-induced T-cell prolif eration (7). In the present study, we have tried to identify the accessorycell population in M. leprae granulomas by using different approaches, i.e., by Percoll discontinuous density gradient centrifugation and by FACS separation using monoclonal antibody to Class II antigens. The results showed that more than 80% of the large cells from M. leprae granulomas which are separated on PercolI density gradients expressClass II antigens and these cells strongly enhance lymphocyte proliferative responses to PPD. Similarly Class II antigen-positive large granuloma cells separated on a FACS are efficient accessorycells for PPD-induced T-cell proliferation. This indicates that the expression of MHC Class II antigens is more important for antigen-presenting function than the expression of macrophage-specific antigen. There are a number of observations that interleukin 1 (IL-l) is also necessary for T-cell activation in conjunction with Class II expression (19, 20). M. leprue granuloma cells, particularly those in the Percoll-separated fractions, secrete small amounts of IL-1 (manuscript in preparation). Nongranulomatous lymph nodes with normal histology distant from the infection site in M. leprae-immunized animals failed to act as accessorycells for proliferation of sensitized lymph node T lymphocytes to PPD. Thus the M. leprue granulomas contain antigen-presenting cells that are not present in normal lymph nodes or the A4. leprue granulomas are enriched for antigen presenting cells which are present in low numbers in normal lymph nodes. B cells have recently been shown to function as antigen-presenting cells to antigenspecific MHC-restricted T-cells (2 1, 22, 23, 24). However, they are not as potent as

M. leprue GRANULOMAS;

ACCESSORY CELL FUNCTION

353

dendritic cells or macrophages. Frohman and Cowing (23) found that one adherent macrophage is functionally equivalent to four LPS-activated B cells and 1000 resting B cells. Katz et al. (25) and Bandeira et al. (26) reported that B cells lost their ability to present ovalbumin or minor histocompatibility antigens to specific T-cell clones when they were irradiated or treated with mitomycin C. Thus it is unlikely that the B cells in the fractions separated by Percoll or FACS can act as antigen-presenting cells for PPD-induced T-cell proliferation because they were mitomycin C treated and present in such low numbers. Several observations have recently shown that cloned human T cells are able to synthesize Class II molecules and act asantigen-presenting cells (27-29) under appropriate conditions. In the present study, the T-cell-depleted population from Percollseparated M. leprue granuloma cells acted as accessory cells for PPD responsesbut the T-cell-enriched population did not. Furthermore, Gerrard et al. (28) has reported that irradiated activated human T cells were potent stimulators in allogeneic and autologous mixed-leukocyte responsesbut failed to present soluble antigen to autologous T cells. It seems,therefore, unlikely that the accessorycell function of the granuloma cell fractions separated by Percoll or on a FACS with anti-Class II is associated with the sensitized T lymphocytes present in these populations. Dendritic cells have previously been reported to be very potent accessorycells (17, 18,30-32). They are weakly adherent, nonphagocytic, nonspecific esterase-negative and Fc receptor-negative cells which strongly express Class II molecules but do not carry macrophage-specific antigen (F4/80) in the mouse (30). They are enriched in the low density fraction on discontinuous bovine serum albumin gradients (3 1, 32). Our study shows that the accessorycell population in M. leprae granulomas are Class II antigen-bearing cells which are devoid of macrophage-specific antigen, non-T and non-B cells. They are separated in the low density fraction on Percoll gradients and share some criteria of dendritic cells. It has been found using a double labelling technique that approximately 10%of large Percoll-separated cells from M. leprue granulomas are macrophage-specific antigen negative but strongly expressClass II molecules and these cells show dendritic cell morphology by phase contrast microscopy (S. Verghese,personal communication). It is, therefore, possible to postulate that the accessory cells in the M. leprue granulomas are dendritic cells. It is not technically feasible at present to isolate this subpopulation of cells from M. leprue granulomas in the guinea pig. The fact that hf. leprae granulomas macrophages also lack Fc and C3 surface receptors (2) makes it more difficult to isolate a pure population of dendritic cells by the conventional methods described previously by others (30-32). ACKNOWLEDGMENTS We thank Grace Lam, Laura Davis, and Deborah Kerr of the Imperial Cancer Research Fund for carrying out the FACS sorting, and Penny Hurford for typing the manuscript. This work was supported by grants from the Royal Thai Civil Service Commission.

REFERENCES 1. Narayanan, R. B., Jones, P. B., and Turk, J. L., J. Path&. 134,253-265, 1981. 2. Narayanan, R. B., Jones, P. B., Curtis, J., and Turk, J. L., J. P&o/. 138,2 16-233, 1982. 3. Mathew, R. C., Katayama, I., Gupta, S. K., Curtis, J., and Turk. J. L., Infect. Immun. 39, 344-352, 1983. 4. Yamashita, U., and Shevach, E. M.,J. Immunul. 119,1584-1588,1977.

354

MONTREEWASUWAT,

CURTIS, AND TURK

5. Rosenstreich, D. L., and Mizel, S. B., Immunol. Rev. 40, 102-l 14, 1978. 6. Unanue, E. R., Annu. Rev. Immunol. 2,395-428,1984. 7. Gupta, S. K., Curtis, J., and Turk, J. L., Cell. Immunol. 91,425-433, 1985. 8. Ulman, A. J., and Flad, H. D., J. Immunol. Methods 30, l-10, 1979. 9. Tatsumi, M., and Imamura, Y., Immunology 52,25-30,1984. 10. Roth, A., Kaufmann, M. T., Cruchaud, A., and Dayer, J. M., Eur. J. Immunol. 15,960-963, 1985. 11. Stadecker, M. J., Bishop, G., and Wortis, H. H., J. Zmmunol. 111,1834-1837,1973. 12. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Zmmunol. 3,645-649,1973. 13. Horwitz, D. A., Alison, A. C., Ward, P., and Knight, N., Clin. Exp. Immunol. 30,289-297, 1977. 14. Antoniou, A. V., Parker, D., Turk, J. L., Tan, B. T. G., and Scheper, R. J., Cell. Immunol. 97,386396,1986. 15. Rosenthal,A.S.,andShevach,E.M., J. Exp. Med. 138,1194-1212,1973. 16. Erb, P., and Feldman, M., J. Exp. Med. 142,460-472, 1975. 17. Erb, P., Ramila, G., Studer, S., Loffler, H., Cecka, J. M., Conscience, J. F., and Feldman, M., ZmmunobioZogy168,141-153, 1984. 18. Lee, K. C., and Guidos, C., Immunobiology 168,172-18 1, 1984. 19. Kurt-Jones, E. A., Virgin, H. W., IV, and Unanue, E. R., J. Zmmunol. 135,3652-3654,1985. 20. Scala, G., and Oppenheim, J. J., Lymphokines 12,39-56,1985. 21. Ashwell, J. D., De France, A. L., Paul, W. E., and Schwartz, R. H., J. Exp. Med. 159,881-905, 1984. 22. Chu, E. T., Lareau, M., Rosenwasser,L. J., Dinarello, C. A., and Geha, R. S., J. Immunol. 134,16761681,1985. 23. Frohman, M., and Cowing, C., J. Immunol. 134,2269-2215,1985. 24. Kreiger, J. I., Grammer, S. F., Grey, H. M., and Chestnut, R. W., J. Zmmunol. 135,2937-2945, 1985. 25. Katz, M. E., Jones, B., and Janeway, C. A., Jr., In “Ir Genes: Past, Present and Future” (C. W. Pierce, S. E. Cullen, J. A. Kapp, B. D. Schwartz, and D. C. Shreffler, Eds.), pp. 467-472. The Humana Press,Clifton, N.J., 1983. 26. Bandeira, A., Pober, G., Pettersson, S., and Coutinho, A., J. Exp. Med. 157,3 12-343, 1983. 27. Brown, M. F., Cook, R. G., Van, M., and Rich, R. R., Human Immunol. l&219-228,1984. 28. Gerrard, T. L., Volkman, D. J., Jurgensen, C. H., Fauci, A. S., Cell Zmmunol. 95,65-74, 1985. 29. Triebel, F., de Roquefeuil, S., Blanc, C., Charron, D. J., and Debre, P., Human Immunol. 15, 302315,1986. 30. Steinman, R. M., andNussenzweig, M. C., Immunol. Rev. 53,127-147,198O. 31. Sunshine,G. H., Katz,D. R.,andFeldman,M.,J. Exp. Med. 152,1817-1822,198O. 32. Kaye, P. M., Chain, B. M., andFeldmann, M., J. Immunol. 134,1930-1934,1985.