Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6269-6273, August 1989 Influence of Mycobacterium leprae and its soluble products on the cutaneous responsiveness of leprosy patients to antigen and recombinant interleukin 2 GILLA KAPLAN*t, ELIZABETH PEREIRA SAMPAIo*, GERALD P. WALSHt, ROCHEL A. BURKHARDT* TRANQUILINO T. FAJARDOf, LAARNI S. GUIDOt, ALICE DE MIRANDA MACHADO§, ROLAND V. CELLONAt, RODOLFO M. ABALOSt, EUZENIR NUNES SARNO§, AND ZANVIL A. COHN* *The Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021; 1The Leonard Wood Memorial Center for Leprosy Research, Eversley Childs Sanitarium, Cebu City, Philippines; and §Leprosy Division, Fundacao Oswaldo Cruz, Rio de Janeiro, Brazil Contributed by Zanvil A. Cohn, May 8, 1989

ABSTRACT Experiments were carried out in the skin of However, the presence of specific suppressor T cells in patients with leprosy to examine whether suppressor cell lepromatous patients has not been proven. The activity populations either exist in the skin of multibacillary leproma- expressed in culture systems that inhibits responses to lec- tous leprosy patients, can be activated with antigen, or are tins, bacillus Calmette-Guerin, and M. leprae is not specific induced to emigrate into a cutaneous site from the circulation. for lepromatous patients and is also seen with cells from For this purpose, purified protein derivative of tuberculin, a tuberculoid patients, normal individuals, and contacts (11). delayed-type antigen that generates a cell-mediated immune In some cases it was clear that the products ofM. leprae were response, was introduced into the skin alone or with nonviable themselves nonspecifically suppressive in these in vitro sys- Mycobacterium leprae bacilli. Areas of induration and the tems (11, 12). In addition, a role for monocyte/macrophage resulting numbers and phenotypes of emigratory cells were not suppressor products induced by M. leprae has been sug- influenced by M. leprae and its products. Further studies gested by many investigators (13-16). examined the ability of M. leprae and its soluble products to We have therefore carried out in vivo experiments within modify the cutaneous response to intradermal injection of the confines of leprosy lesions to explore the inhibitory recombinant interleukin 2 (IL-2), a lymphokine that mimics a properties of (i) preexisting cells of the infiltrate, (ii) partic- cell-mediated response. Neither the simultaneous injection of ulate M. leprae and its soluble products, and (iii) newly M. leprae and IL-2, nor the prior injection ofM. leprae followed emigrated blood cells in the presence or absence of exoge- in 2 days by IL-2, nor the prior aIministration of IL-2 followed nous antigens. We have quantitated suppression by virtue of in 4 days byM. leprae, into the same skin site, modified the zone the ability of these agents to modify the area of induration of induration generated by IL-2. In addition, the immunocyto- evoked by a reaction to the purified protein derivative of chemical and histopathological evaluation of biopsy specimens tuberculin (PPD) (17) or by the injection ofrecombinant IL-2, of skin sites showed no difference between sites injected with a lymphokine that mimics a cell-mediated immune reaction IL-2 and sites injected with IL-2 and M. leprae. We conclude (18). that suppressor T cells, if they exist, do not influence the gross or microscopic responsiveness of a cell-mediated skin reaction MATERIALS AND METHODS to antigen and IL-2. IL-2 did, however, enhance the respon- siveness of skin-test-positive tuberculoid patients and family Patient Population. Eighteen leprosy patients, 18-60 years contacts to M. leprae antigens by a synergistic effect on the zone old, 6 with polar lepromatous leprosy (LL) and 12 with of induration and local cell accumulation. borderline lepromatous leprosy (BL) (19), were selected for intradermal administration of PPD at the Leprosy Outpatient Patients with lepromatous leprosy fail to respond to Myco- Unit, Fundacao Oswaldo Cruz, Rio de Janeiro, Brazil. Thir- bacterium leprae antigens and do not generate a cell- ty-seven leprosy patients, 14-72 years old, 30 with LL, 5 with mediated immune reaction. This defect stems from the in- BL, and 2 with borderline tuberculoid leprosy (BT), as well ability of T cells to undergo blastogenesis in the presence of as 6 family contacts without any overt disease, were selected specific M. leprae epitopes and to secrete important lym- for the intradermal administration of recombinant IL-2 (Ce- phokines such as interferon y and interleukin 2 (IL-2) (1-4). tus) at the Clinical and Epidemiological Branches of the Although many authors have commented on the nature ofthis Leonard Wood Memorial Center for Leprosy Research, selective anergy, the underlying mechanism(s) is still unclear Cebu City, Philippines. Leprosy patients in reaction or on (5). Based upon in vitro experiments, the presence of T cells steroid treatment were excluded. Written consent was ob- that inhibit thymidine incorporation of lectin-stimulated pe- tained from all participants. ripheral blood T cells has been reported (6-8). Such inhibi- Patients had been treated for <1 month to 7 years and had tory cells were reported to be present in the blood ofpatients received multidrug with rifampin (600 mg/day) and with lepromatous leprosy and absent from patients with the dapsone (100 mg/day); about half of them had received tuberculoid form of the disease and from normal individuals clofazimine (50-100 mg/day and/or 300 mg/month) as well. not infected with M. leprae. More recently, the isolation of All chemotherapy was continued during the trial. Slit smears T-cell clones with inhibitor activity was reported by Modlin for bacterial index (B.I.) were performed within 6 months et al. (9, 10), who emphasized the possible suppressor role of such cells in leprosy. Abbreviations: B.I., bacterial index; BL, borderline lepromatous leprosy; BT, borderline tuberculoid leprosy; LL, polar lepromatous leprosy; IL-2, interleukin 2; PPD, purified protein derivative of The publication costs of this article were defrayed in part by page charge tuberculin. payment. This article must therefore be hereby marked "advertisement" tTo whom reprint requests should be addressed at: The Rockefeller in accordance with 18 U.S.C. §1734 solely to indicate this fact. University, 1230 York Avenue, New York, NY 10021.

6269 Downloaded by guest on September 26, 2021 6270 Immunology: Kaplan et al. Proc. Natl. Acad. Sci. USA 86 (1989) before IL-2 and PPD injection. The B.I. of patients tested ranged from 0 to 5+. E PPD Administration. Leprosy patients received "simulta- E neous" injections ofPPD and M. leprae or PPD and saline as z follows. Five units of PPD (Connaught Laboratories) in 0.1 0 ml was injected into two skin sites on the back. After 5 min, 0.1 ml of isotonic saline or A (3-4 x 107 M. leprae D a bacilli per ml; lot J 15-3, National Hansen's Disease Center, z Carville, LA) was injected into one of the sites injected L- previously with PPD. Erythema and induration were evalu- o ated at time intervals from 1 to 14 days and 4-mm punch biopsy samples were taken. Li IL-2 Administration. Lyophilized recombinant human IL-2 (3 x 106 units/mg of protein) was reconstituted in pyrogen- free sterile water, diluted in 5% dextrose to a concentration of 100 ,ug/ml, and used within 2 hr of reconstitution. Injec- CN 72OT B O-0 PPD tions of 100 ul (10 gg) were given into the skin of the back; E *-* PPD + M. Leproe patients and contacts received either 10 or 2 such injections. E Within a few minutes they received 100 p.l of either soluble z 540+ 0 M. leprae antigens (Rees antigen or leprosin; World Health 1= Organization) or lepromin A (3-4 x 107 M. leprae bacilli per 360+ ml, lot LV-9). This represented "simultaneous" injections. 0cx In other experiments either antigen or IL-2 was administered z first and was followed 1-4 days later by the complementary antigens or IL-2 into the same site. Appropriate control sites 0 180+ for both antigens and IL-2 alone were injected nearby. All injection volumes were 100 ,ul. el Local erythema and induration of the injected sites were 2 4 6 8 10 12 14 evaluated daily. Selected sites ofIL-2, antigen, and IL-2 plus antigen injection and matched control sites were biopsied (4-mm punch) 1-7 days after the first injection. 4T C o-o PPD A part of each biopsy specimen was fixed (I) * * PPD + M. Lerne Histopathology. -J -J in neutral buffered 10% formalin overnight, embedded in Lij paraffin, sectioned, and stained with hematoxylin and eosin C) 3 0 for histological diagnosis and acid-fast staining for enumer- OL F- ation of M. leprae. 0 + 2 Immunocytochemistry. Biopsy specimens were fixed in 0l°.EJ phosphate-buffered saline containing 3% paraformaldehyde, 0~~~~~~~~~~ 75 mM lysine, and 10 mM sodium metaperiodate (20) and + were processed as described (18). Frozen sections were I stained with mouse monoclonal as described (17) z00 to identify cell types and phenotypes. Monoclonal Antibodies. Mouse monoclonal antibodies 0 2 4 6 8 10 12 14 were used for the identification of specific cell types. Anti- Leu-4, anti-Leu-2a, and anti-Leu-3a (CD3, CD4, and CD8, DAYS AFTER PPD INJECTION anti-T cells) (21, 22) and anti-Leu-M5 (CD11c, anti-mono- FIG. 1. Effect of M. leprae administration on the cutaneous cyte/macrophage) (23) were obtained from Becton Dickin- response to PPD. (A and B) Induration induced by PPD in the son. OKT6 (24) (CD1, anti-Langerhans cells) was obtained presence (filled symbols) or absence (open symbols) of simultane- from Ortho Diagnostics. 9.3F10 (anti-major histo- ously injected M. leprae in three lepromatous patients (individual compatibility complex class II antigen) was produced in this values) (A) and 18 lepromatous patients (means ± SD) (B). No laboratory (25). significant differences were observed when M. leprae was injected with PPD. (C) T-cell subset ratios (CD4+/CD8+) observed in re- sponse to PPD injected in the presence (o) or absence (o) of M. RESULTS leprae; results are means for 2-4 individuals. When 4 individuals were tested (1-, 2-, and 7-day time points), results include SD. No The possible modulating influence of the lepromatous state differences in T-cell subsets were observed when M. leprae was and the administration ofM. leprae and its products was first injected together with PPD. examined in the presence ofa delayed-type antigen-mediated event. For this purpose injection of PPD alone or in the presence of nonviable M. leprae was carried out. As noted with our former studies (17, 27) in which the responsiveness previously, the response to PPD in sensitized human skin of PPD-sensitized lepromatous patients was virtually the includes an emigratory event (17), the generation of primary same as compared to normal controls and tuberculoid pa- and secondary cytokines (26), and complex responses of tients. endothelium, fibroblasts, Langerhans cells, and kerati- Emi rating Blood Cells Accumulating in Skin Sites INjected nocytes (27, 28). with PPD or PPD and M. leprae. Cellular infiltrates in re- Influence ofM. kprae Antigen on the Cutaneous Response to sponse to PPD contained predominantly T cells and mono- PPD. Fig. 1 shows a comparison between PPD plus M. leprae cytes that differentiated into epithelioid and multinucleated and PPD plus saline injection in 3 patients (A) and in all 18 giant cells. The simultaneous injection ofM. leprae and PPD patients (B). The rate of generation and the intensity of the did not evoke any difference in response. Neither the kinetics cell-mediated immune response induced by PPD were not of infiltration, nor the cellular composition of the reaction, influenced by the presence of M. leprae. This is consistent were modified (data not shown). However, the sites injected Downloaded by guest on September 26, 2021 Immunology: Kaplan et A Proc. Natl. Acad. Sci. USA 86 (1989) 6271 with M. leprae and PPD contained larger numbers of intact endogenous cellular infiltrate, number of bacilli, and expres- and fragmented bacteria as a result of the injected M. leprae. sion of cell-mediated immunity failed to modify responsive- The nature and phenotype of emigratory cells were eval- ness to this lymphokine. uated. Injection of PPD with or without M. leprae gave rise Effect of Simultaneous Injection ofM. kprae and its Soluble to the same increase in dermal cellularity, keratinocyte Products on Responsiveness to IL-2. Lepromatous patients HLA-DR antigen expression, and dermal CD1+ cells. The with a high B.I. (3+ or more), in various stages of treatment, majority of the emigratory T cells were of the CD4' pheno- were injected with 3-4 x 106 nonviable M. leprae bacilli. This type and the dermal CD4+/CD8' T-cell ratios were similar in quantity is routinely employed for evaluating skin reactivity the presence or absence of M. leprae (Fig. 1C). in the Mitsuda reaction. Within 15 min after the deposition of We conclude that endogenous and/or newly administered baccilli, 10 ug of IL-2 was introduced into the same area, exogenous M. leprae neither enhances nor suppresses the often via the same needle track. At the same time, but into panoply of responses generated by the PPD antigens. different sites, control injections of leprosy bacilli or IL-2 Responsiveness of Lepromatous and Tuberculoid Patients to alone were made. Zones of induration were measured over IL-2. The initial study examined the basal reactivity of the the next 7 days (Fig. 2B). Both the maximum response at 24 skin of multibacillary and paucibacillary patients to the hr and the rate of reduction in induration up to 7 days were injection of IL-2. Lepromatous patients contain within their not influenced by the presence of intact, extracellular M. dermis heavily parasitized macrophages and a sparse T-cell leprae. None of the lepromatous patients responded to the infiltrate in which cells of the CD8' "suppressor/cytotoxic" bacilli during the 7-day period. The experiment was repeated phenotype predominate. In contrast, BT patients exhibit a in a group oflepromatous patients with low B.I. (1.5+ or less) more exuberant, cell-mediated, granulomatous reaction with who had been treated for over 2 years. These experiments few bacilli and many T cells, largely of the CD4' "helper" employed intact M. leprae as well as the soluble M. leprae phenotype. The skin responses that resulted when IL-2 (10 antigens. Zones of induration were measured after simulta- ,ug) was introduced into these disparate milieus are shown in neous injection of bacilli or soluble antigens plus IL-2 or Fig. 2A. Within 5 hr and peaking at 24 hr the injected site injection of IL-2 or soluble antigens alone (Fig. 2C). Again, demonstrated mild erythema and firm induration with a peak responses and clearance were uninfluenced by the intact slightly raised surface. By 48 hr the zone lacked erythema and or soluble M. leprae antigens. consisted of a firm nodule that gradually receded in diameter We conclude that neither particulate nor soluble M. leprae over the next 4 days. The responsiveness of LL and BT antigens influence the intrinsic and emigratory cells of the patients to IL-2 was virtually identical, suggesting that the microenvironment and in turn the responsiveness to IL-2.

200- A B 0- 0 Lepromatous 150T *-e Tuberculoid

CN 100- E

zT 0 03 1 2 3 4 5 6

LL LL 205v% f D 0-0 IL-2 0-0 IL-2 + Sol. M. Leproe 0 o0-0 IL-2 + M. Leproe 1 _ i- A^A-A Sol. M. Leprae

1C 010+t ' I

c 0

0 0 1 0 1 2 3 4 5 6 7 DAYS AFTER IL-2 INJECTION FIG. 2. Effect of M. Ieprae on the dermal response to recombinant human IL-2. (A) Response to intradermal injection of IL-2 in 30 lepromatous (o) and 2 tuberculoid (e) patients. No difference in the area of induration was observed in the two types of leprosy patients. (B) Induration induced by IL-2 with (o) or without (e) simultaneous injection of M. Ieprae in 6 high-B.I. (3-5+) lepromatous patients. Injection of M. Ieprae alone (A) produced no induration. (C) Induration induced by IL-2 in the absence of M. leprae (e), with soluble M. leprae antigens (o), or with intact M. leprae (o) in 12 low-B.I. (0-1.5+) lepromatous patients. Injection of soluble antigens alone (A) produced no induration. (D) Effect of intact M. leprae on the response to IL-2 in 7 lepromatous patients. Bacilli were injected into one of the IL-2 sites at 4 days as indicated by arrow. No difference in response was observed when M. leprae was injected with or before IL-2. Results are expressed as mean area of induration in mm2 (A-D) + SD (A and B). Downloaded by guest on September 26, 2021 6272 Immunology: Kaplan et al. Proc. Natl. Acad. Sci. USA 86 (1989)

Response toM. lepraeAntigens in Cutaneous Sites Pren Jected A *-@ IL-2 with MI-2. In this experiment we wished to evaluate whether 0-0 M. Leprae 9. IL-2 prior injection ofIL-2 could modify the cutaneous responsive- A-A M. Loprae ness to M. leprae antigens. For this purpose 10 jxg ofIL-2 was injected intradermally and 4 days later M. leprae was injected into the same cutaneous site. A second M. leprae injection was given into a separate site, and the response at this site was compared to that at the IL-2-prepared M. leprae site (Fig. 2D). The prior injection of IL-2 did not lead to early cutaneous M. leprae responsiveness, nor did it modify the cutaneous re- sponsiveness to a second injection of M. leprae. This is in keeping with in vitro studies in which IL-2 failed to enhance thymidine incorporation into lymphocytes from lepromatous leprosy patients either after addition in vitro (4) or following O-EN% cutaneous administration (unpublished data). E Effect of M. leprae on the Response to IL-2 in Antigen- E Nonreactive Individuals. Nonviable M. leprae bacilli were z injected intradermally into lepromatous patients and allowed 0 to influence the preexisting cell populations and/or induce the accumulation ofcells emigrating from the peripheral blood. At 0 48 hr, 10 ,ug ofIL-2 was introduced into the same site. Control z IL sites on other areas of the back received either M. leprae or 0 IL-2 alone. The study was conducted with patients who did not give an immediate immune response to M. leprae (negative Fernandez reaction). Fig. 3A shows that the prior injection of M. leprae had no influence on the cutaneous response to a 2501 c 0--* IL-2 subsequent injection of IL-2. The nature of the immediate 0-X 0 -0 IL-2 + Sol. M. Leproe response and the regression of induration were essentially A--A Sol. M. Lepro. similar in the IL-2-injected site and in the M. Ieprae-primed 200 IL-2-injected site. We conclude that during the 48 hr after the introduction of 150+ M. leprae into unresponsive lepromatous patients' skin, cells A. with the capacity to inhibit the dermal response to IL-2 were 100{ not observed. Effect of M. keprae on the Response to IL-2 in Antigen- 501 A Reactive Individuals. To examine whether skin-test reactivity to M. leprae modulated IL-2 responsiveness, a similar ex- -3 1 2 3 4- 5 7 8i9 periment was carried out in skin of leprosy patients and family contacts who exhibited positive Fernandez and Mit- DAYS AFTER M. LEPRAE INJECTION suda reactions. M. leprae (3-4 x 106 bacteria) was injected into two sites on the back of family contacts and examined at FIG. 3. Effect of M. Ieprae on the dermal response to IL-2. (A) 24 and 48 hr. At this time IL-2 was injected directly into one Response to IL-2 administered into M. leprae-preinjected sites 2 of the M. leprae sites and at another, distant site. Zones of days after antigen (arrow) in 10 lepromatous leprosy patients. No induration were evaluated over the next 48 hr (Fig. 3B). Only change in response to IL-2 was observed in the presence of M. small, but definite, zones of induration occurred at the sites leprae. (B) Response to IL-2 administered into M. leprae-preinjected injected with M. leprae alone. Injection ofIL-2 alone gave the sites 2 days after antigen (arrow) in 3 M. leprae-responder family contacts. Injection of IL-2 into the M. Ieprae site gave a synergistic expected induration pattern noted previously. However, a response. (C) Response to IL-2 administered together with soluble significant enhancement in the extent of induration occurred M. Ieprae antigens in two BT responder patients. Response was when IL-2 was placed into a developing M. leprae reaction. additive when antigen and lymphokine were injected together. Re- The simultaneous injection of soluble M. leprae antigens sults are mean of area of induration in mm2 (A-C) ± SD (A). and IL-2 into the skin oftwo BT patients is depicted in Fig. 3C. IL-2 alone gave the expected induration pattern noted previ- the infiltration ofcells into the dermis in lepromatous patients ously. Soluble antigens gave a typical Fernandez response, (40-50%) but enhanced the extent of infiltration in the which persisted for up to 4 days and then subsided. A responder contacts to 70-80%o. significant enhancement in the extent of induration was ob- The predominance of CD8' T cells in the infiltrates of served at 68 hr when the two mediators were injected into the lepromatous patients (CD4+/CD8' ratio of 0.9) was reversed same site. in response to both IL-2 and IL-2 plus M. leprae. Forty-eight These findings are consistent with the ability of IL-2 to hours after injection, lepromatous patients showed CD4+/ enhance the cellular reactions occurring in response to an CD8' T-cell ratios of 3.1 ± 0.6 and 2.2 ± 0.5 in response to antigen-mediated event. Such experiments again failed to IL-2 and IL-2 plus M. leprae, respectively. The same ratios provide any evidence for the generation of an active sup- were observed in contacts. M. leprae-responsive sites in these pressor cell population under these conditions. individuals demonstrated a CD4+/CD8' T-cell ratio of 2.3. Immunocytochemical Analysis of the Effect of M. leprae on Keratinocyte HLA-DR and dermal accumulation of CD1M Numbers and Phenotype of the Emigratory Cells in Response Langerhans cells were induced similarly by IL-2 and by IL-2 to IL-2. The skin oflepromatous patients is characterized by plus M. leprae in lepromatous patients as well as family an inflammatory infiltrate involving 10-15% of the dermis. contacts. No infiltrate is observed in the skin of family contacts. After injection of IL-2, the percentage of the dermis infiltrated was DISCUSSION enhanced similarly in both groups, to 40-50% and 30-40%o, One of the difficulties in accepting a role for suppressor cells respectively. The addition of M. leprae to IL-2 did not affect as an explanation for the anergy ofLL patients is the disparity Downloaded by guest on September 26, 2021 Immunology: Kaplan et al. Proc. Natl. Acad. Sci. USA 86 (1989) 6273 between their expression in the host and in the test tube. Gallo and Jose Augusto da Costa Nery, Nadia Cristina Duppre, Those investigators who have considered the matter have Esther Borges, and Maria Fernanda Sardella Alvim from the Out- reported only partial inhibition of thymidine incorporation patient Clinic, Fundacao Oswaldo Cruz, Rio de Janeiro, for their help with high effector cell ratios. This fact, when added to (i) a during this study. We thank Judy Adams for her help with the graphic work and Myrna Garcia for typing the manuscript. This study was significant direct inhibitory role ofM. leprae macromolecules supported by National Institutes of Health Grant A122616 and by on monocyte-T-cell stimulation and (ii) a lack of specificity Cetus Corporation. This investigation also received financial support of the suppressor populations (11), does not make a compel- from United Nations Development Programme/World Bank/World ling case for T suppressor cells in the regulation ofthe disease Health Organization Special Programme for Research and Training process. in Tropical Diseases. We have attempted to define an experimental situation that utilizes the location of bacillary replication. It employs the 1. Godal, T., Mykelstad, B., Samuel, D. & Myrvag, B. (1971) Clin. cutaneous infiltrate as it exists in the lepromatous patient and Exp. Immunol. 9, 821-831. that 2. Haregewoin, A., Godal, T., Mustafa, A. S., Belehu, A. & Yemane- utilizes emigratory cell populations are evoked either by berhan, T. (1983) Nature (London) 303, 342-344. antigen or by lymphokine. A number of parameters at both 3. Nogueira, N., Kaplan, G., Levy, E., Sarno, E. N., Kushner, P., the gross and microscopic level serve to characterize the Granelli-Piperno, A., Vieira, L., Colmer-Gould, V., Levis, W., expression of cell-mediated immunity in the skin. Induration Steinman, R. M., Vip, Y. K. & Cohn, Z. A. (1983) J. Exp. Med. is a sensitive index of cell infiltration and has been used 158, 2165-70. effectively in both murine and human systems to characterize 4. Kaplan, G., Weinstein, D. E., Steinman, R. M., Levis, W. R., Elvers, U., Patarroyo, M. & Cohn, Z. A. (1985) J. Exp. Med. 162, cell-mediated reactions. It encompasses a variety of reac- 917-929. tions including chemoattraction, endothelial cell transmigra- 5. Hastings, R. C., Gillis, T. P., Krahenbuhl, J. L. & Frnazblau, S. C. tion, and the stimulation of the secretory repertoire of both (1988) Clin. Microbiol. Rev. 1, 330-348. macrophages and lymphocytes. Microscopically this is char- 6. Mehra, V., Mason, L. H., Fields, J. P. & Bloom, B. R. (1979) J. acterized by the presence of a predominant CD4' T-cell Immunol. 123, 1183-1188. infiltrate, expression of interferon y-induced protein (IP-10), 7. Mehra, V., Mason, L. H., Rothman, W., Reinherz, E., Schloss- man, S. F. & Bloom, B. R. (1980) J. Immunol. 125, 1183-1188. keratinocyte major histocompatibility complex class II de- 8. Mehra, V., Convit, J., Rubinstein, A. & Bloom, B. R. (1982) J. terminants, Langerhans cell differentiation, and the produc- Immunol. 129, 1946-1951. tion and function of cytotoxic effectors (17, 18, 26-30). 9. Modlin, R. L., Mehra, V., Wong, L., Fujimiya, Y., Chang, W. C., We use this wide variety of immunological events to Horwitz, D. A., Bloom, B. R., Rea, T. & Pattengale, P. K. (1986) evaluate the possible suppressor role of T cells. For the first J. Immunol. 137, 2831-2834. experiments we chose an event because it also 10. Modlin, R. L., Kato, H., Mehra, V., Nelson, E., Fan, X. D., Rea, antigen-driven T. H., Pattengale, P. K. & Bloom, B. R. (1986) Nature (London) incorporated a recognition phase and the clonal expansion of 322, 459-461. small T-cell populations. Subsequently we employed IL-2, a 11. Kaplan, G., Gandhi, R. R., Weinstein, D. W., Levis, W. R., Pa- major growth factor for T cells whose presence is required for tarroyo, M. E., Brennan, P. J. & Cohn, Z. A. (1987) J. Immunol. the expression of other lymphokines and cytokines. In both 138, 3028-3034. instances neither the cells involved in the lepromatous state 12. Prasad, H. K., Mishra, R. S. & Nath, I. (1987) J. Exp. Med. 165, 239-244. nor those (0) migrating into cutaneous sites, (ii) activated by 13. Sathish, M., Bhutani, L. K., Sharma, A. K. & Nath, I. (1983) M. leprae antigen, or (iii), stimulated by crossreacting myco- Infect. Immun. 42, 890-899. bacterial antigens (PPD) had a suppressive effect on the 14. Wadee, A. A., Sher, R. & Rabson, A. R. (1980) J. Immunol. 125, above parameters of cell-mediated immunity. We consider 1380-1386. this strong evidence that suppressor T cells play no major role 15. Salgame, P. R., Mahadevan, P. R. & Antia, N. H. (1983) Infect. in down-regulating the essential features ofcellular immunity Immun. 40, 1119-1126. 16. Sengupta, U., Sinha, S., Ramu, G., Lamb, J. & Ivanyi, J. (1987) in the skin of lepromatous patients. Clin. Exp. Immunol. 68, 58-64. It has recently come to our attention that the influence of 17. Kaplan, G., Laal, S., Sheftel, G., Nusrat, A., Nath, I., Mathur, M. leprae products on a tuberculin response had been N. K., Mishra, R. S. & Cohn, Z. A. (1988) J. Exp. Med. 168, investigated by Sengupta et al. (16). Those authors mixed 1811-1824. PPD and soluble molecules ofM. leprae prior to injection and 18. Kaplan, G., Kiessling, R., Teklemariam, S., Hancock, G., Sheftel, reported a decided reduction in induration. Such data conflict G., Job, C. K., Converse, P., Ottenhoff, T. H. M., Becx-Belumink, M., Dietz, M. & Cohn, Z. A. (1989) J. Exp. Med. 169, 893-907. with those reported here, and we suspect that mixing ofthese 19. Ridley, D. S. & Jopling, W. B. (1966) Int. J. Lepr. 34, 255-273. disparate molecules led to a reduction in tuberculin and 20. McLean, I. W. & Nakane, P. K. (1974) J. Histochem. Cytochem. leprosin activity. 22, 1077-1083. An understanding of the T-cell unresponsiveness of lepro- 21. Engleman, E. G., Warnke, R., Fox, R. I. & Levy, R. (1981) Proc. matous patients to M. leprae antigens is essential for a Natl. Acad. Sci. USA 78, 1791-1795. 22. Evans, R. L., Wall, D. W., Platsoucas, C. D., Siegal, E. P., Fikrig, rational approach to any form of immunotherapy. Such S. M., Testa, C. M. & Good, R. A. (1981) Proc. Natl. Acad. Sci. mechanisms must consider findings in which LL/BL patients USA 78, 544-548. exhibit either a complete absence of in vivo T-cell reactivity 23. Schwarting, R., Stein, H. & Wang, C. Y. (1985) Blood 65, 974-983. or a spectrum ofhyporesponsiveness (4). The present data do 24. Fithian, E., King, P., Goldstein, G., Rubenfield, M., Fenoglio, C. not support a role for suppressor T-cell populations in & Edelson, R. (1981) Proc. Natl. Acad. Sci. USA 78, 2541-2544. explaining this anergy and point toward various defects in 25. Van Voorhis, W., Steinman, R. M., Hair, L. S., Luban, J., Witmer, M. D., Koide, S. & Cohn, Z. A. (1983) J. Exp. Med. 158, 126-145. T-cell activation caused by ablation in the neonatal or post- 26. Kaplan, G., Luster, A. J. & Cohn, Z. A. (1987) J. Exp. Med. 166, natal period or defective antigen presentation. Numerous 1098-1108. publications dealing with this subject in murine systems have 27. Kaplan, G., Witmer, M. D., Nath, I., Steinman, R. M., Laal, S., implicated the thymus in the process. Exploration of the Prasad, H. K., Sarno, E. N., Elvers, U. & Cohn, Z. A. (1986) Proc. mechanisms of antigen presentation, T-cell receptor selec- Natl. Acad. Sci. USA 83, 3469-3473. and extent of T-cell deletions in with 28. Kaplan, G., Nusrat, A., Witmer, M. D., Nath, I. & Cohn, Z. A. tivity, patients lepro- (1987) J. Exp. Med. 165, 763-776. matous leprosy promises to yield important information. 29. Kaplan, G., Nusrat, A., Sarno, E. N., Job, C. K., McElrath, J., Porto, J. A., Nathan, C. F. & Cohn, Z. A. (1987) Am. J. Pathol. We thank Paulina S. Munalem, Cecilia T. Alberta, Maria Teresa 128, 345. N. Lee, and Guillerma L. Lim from the Leonard Wood Memorial 30. Hancock, G., Cohn, Z. A. & Kaplan, G. (1989) J. Exp. Med. 169, Center for Leprosy Research, Cebu, and Drs. Maria Eugenia Noviski 909-919. Downloaded by guest on September 26, 2021