Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6269-6273, August 1989 Immunology 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 therapy 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 lepromin 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 antibodies 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.