T-Cell Responsiveness of American Cutaneous Leishmaniasis Patients to PurifiedLeishmania pifanoiAmastigote Antigens andLeishmania braziliensisPromastigote Antigens: Immunologic Patterns Associated with Cure

EXPERIMENTAL PARASITOLOGY ARTICLE NO. 0100

84, 144–155 (1996)

T-Cell Responsiveness of American Cutaneous Leishmaniasis Patients to Purified Leishmania pifanoi Amastigote Antigens and Leishmania braziliensis Promastigote Antigens: Immunologic Patterns Associated with Cure SERGIO G. COUTINHO,* MARCIA P. OLIVEIRA,* ALDA M. DA-CRUZ,* PAULA M. DE LUCA,* SERGIO C. F. MENDONC¸A,* ALVARO L. BERTHO,* LYNN SOONG,† AND DIANE MCMAHON-PRATT† *Laboratory of Cellular and Humoral Immunology, Department of Protozoology, Oswaldo Cruz Institute-FIOCRUZ, Ave. Brasil, 4365, Rio de Janeiro 21045-900 RJ, Brasil; and †Department of Epidemiology and Public Health Yale University School of Medicine, New Haven, Connecticut 06520-8034 U.S.A.

COUTINHO, S. G., OLIVEIRA, M. P., DA-CRUZ, A. M., DE LUCA, P. M., MENDONC¸A, S. C. F., BERA. L., SOONG, L., AND MCMAHON-PRATT, D. 1996. T-cell responsiveness of American cutaneous leishmaniasis patients to purified Leishmania pifanoi amastigote antigens and Leishmania braziliensis promastigote antigens: Immunologic patterns associated with cure. Experimental Parasitology 84, 144–155. Patients suffering from American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) or by three proteins (A-2/P-2, P-4, and P-8) derived from Leishmania pifanoi amastigotes were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-g), interleukin 2 (IL-2) and interleukin 4 (IL-4) produced were also determined. Results show two different patterns of Lb-induced T cell responses: (a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-g, IL-2, and IL-4) during the active disease, (b) similar proportions of responding CD4+ and CD8+ cells and type 1 cytokine production (presence of IFN-g and IL-2 and very low IL-4) at the end of therapy (healed lesions). Thus, this last pattern is probably associated with a beneficial T cell response. The A-2/P-2 amastigote cysteine proteinase provided only marginal (s.i. ≅ 2.5) T cell stimulation in 25% of patients studied; in contrast, the L. pifanoi P-4 and P-8 amastigote antigens induced significant stimulation (s.i. ≅ 5) in approximately 50% of the patients. In comparison to Lb-stimulated cultures, lower proliferative responses of T lymphocytes to P-4 or P-8 were observed. However, the P-4- or P-8-stimulated cultures had similar percentages of reactive CD4+ and CD8+ cells, as well as type 1 cytokines (presence of IFN-g and IL-2, and low levels or absence of IL-4) in the supernatants both before and at the end of therapy. The consistent induction of apparently beneficial T cell responses by the P-4 and P-8 amastigote glycoproteins points to the possibility that these molecules be considered as candidates for future defined vaccines against leishmaniasis. © 1996 Academic Press, Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Human American cutaneous leishmaniasis; Leishmania braziliensis; Leishmania pifanoi; CD4+/CD8+ T cells; P-4, P-8, and A-2/P-2 antigens; IFN-g, gamma interferon; IL, interleukin; TNF, tumor necrosis factor; TGF-b, transforming growth factor beta; PBMC, peripheral blood mononuclear cells; LPR, lymphocyte proliferative response; DTH, delayedtype hypersensitivity.

THO,

INTRODUCTION Leishmania are protozoan parasites that infect man and other animals. Their life cycle is characterized by a flagellated promastigote form that proliferates in the gut of the Phlebotominae insect vector and by an amastigote form that parasitizes vertebrate macrophages. Trans144 0014-4894/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

mission to man and other vertebrate hosts occurs when the promastigote forms are injected into the dermis during the bite of the sand fly vector. In the dermis, recently inoculated promastigote forms will be internalized by phagocytic cells and undergo transformation into amastigote forms inside the parasitophorus vacuole. The amastigotes maintain the parasit-

CYTOKINES AND T CELL PHENOTYPES IN CUTANEOUS LEISHMANIASIS

ism in the vertebrate hosts by replicating in the parasitophorus vacuole and eventually leading to the destruction of the host cells. In South America several species of Leishmania have been found to infect man, causing a range of clinical symptoms (Grimaldi and Tesh 1993). Certain Leishmania species (e.g., L. amazonensis) are able to produce diffuse cutaneous leishmaniasis, a severe disease associated with impairment of the T-cell-mediated immune responses to leishmanial antigens. The species L. braziliensis is the most frequent and widely distributed Leishmania parasite in Brazil, occurring throughout the country, except North of the Amazon river. The disease is characterized by one or more skin ulcers (cutaneous leishmaniasis) or, less frequently, secondary metastatic mucosal lesions (mucosal leishmaniasis). L. pifanoi appears to be a geographic variant of the L. mexicana complex that occurs mainly in Central America and Venezuela (Grimaldi and Tesh 1993; Convit and Pinardi 1974). The main clinical features produced by L. pifanoi are skin lesions at the site of parasite inoculation by the Phlebotominae insect vector, which occasionally disseminate. The cutaneous lesions produced by L. braziliensis or L. pifanoi are usually susceptible to the antimonial therapy, and healing occurs at the end of the therapy. In the mouse model, there is evidence that the mechanisms for cure of or resistance to L. major infection are associated with macrophages activation and production of nitric oxide, leading to the destruction of intracellular parasites. Type 1 cytokines like IFN-g and TNF-a and -b when produced by the Th1 subset of CD4+ lymphocytes play a pivotal role in this process of macrophage activation and parasite destruction (Titus et al. 1984, 1989; Liew et al. 1990; Heinzel et al. 1991; Scott 1991; Coffman et al. 1991; Liew and O’Donnell 1993). On the other hand, the mechanisms for aggravation of the disease in mice are related to the effects of type 2 cytokines, such as IL-4, IL-10, and TGF-b, which are primarily produced by the Th2 subset of CD4+ lymphocytes as well as other cell types. These cytokines are able to inhibit the differen-

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tiation of Th1 CD4+ lymphocytes and the production of IFN-g and TNF (Liew et al. 1989; Sadick et al. 1990; Ding et al. 1990; Heinzel et al. 1991; and Barral et al. 1993). A similar reciprocal effect between IFN-g and IL-4 on human L. donovani parasitized macrophages has also been observed (Zwingenberger et al. 1990). CD8+ T lymphocytes also appear to be important in the immunologic mechanisms responsible for cure of murine leishmaniasis caused by L. major, L. amazonensis, and L. donovani (Titus et al. 1987; Hill et al. 1989; Chan 1993; Rachamim and Jaffe 1993). Activated antigen specific CD8+ T cells have been shown to produce IFN-g and can have a cytolytic effect (CTL) on parasitized macrophages (Muller et al. 1993, 1994; Conceic¸a˜o-Silva et al. 1994). It has been found that mice resistant to L. major infection have higher percentages of reactive CD8+ lymphocytes when compared with susceptible mice (Milon et al. 1986). Further, the in vivo administration of anti-CD8 antibodies exacerbated the lesions produced by L. major in naive mice (Titus et al. 1987) as well as increased L. donovani parasite burdens in protectively immunized mice (Rachamim and Jaffe 1993). In humans, we have shown that cure of cutaneous leishmaniasis caused by L. braziliensis is associated with higher percentages of Leishmania reactive CD8+ T cells and higher production of IFN-g than that seen in the same patients during the active disease (Da-Cruz et al. 1994). Now we extended these results with the characterization of an apparently beneficial T cell response pattern to L. braziliensis antigens (Lb). The observation that protective immunity against Leishmania infection can be acquired in susceptible mice (Howard et al. 1982; Scott et al. 1987), as well as in humans (Gunders 1987), indicated the possibility for the development of a vaccine against leishmaniasis. Vaccination trials in human populations have been done in Brazil utilizing crude preparations of promastigote antigens (Mayrink et al. 1979, 1985). According to Antunes et al. (1986) a potentially protective response with that preparation oc-

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curred in 50% of the vaccinated population. Nevertheless, new investigations on better defined antigens as potential candidates for a vaccine are a priority. In this connection, studies of protection against cutaneous leishmaniasis in susceptible mice after immunization with promastigote-derived molecules such as lipophosphoglycan, gp63, defined gp63 epitopes, and GP46/M-2 have been done (Handman and Mitchell 1985; Champsi and McMahon-Pratt 1988; Russell and Alexander 1988; Yang et al. 1990; Jardim et al. 1990; Yang et al. 1991; McMahon-Pratt et al. 1993). However, molecules derived from the amastigote stage should provide relevant candidates for a future vaccine, as it is this intracellular stage that produces disease in the vertebrate host. As recent evidence (Soong et al. 1995) indicated that two purified L. pifanoi amastigote proteins (P-4 and P-8) induced significant protection against infection by L. pifanoi or L. amazonensis in susceptible BALB/c and CBA/J mice, we decided to investigate the T cell response pattern to those proteins, and to the A-2/P-2 protein, in humans with active cutaneous leishmaniasis and also in cured patients. Data presented here indicate that P-4 and P-8 responder T cells have predominant phenotypes and cytokine production profiles that apparently characterize a beneficial T cellmediated immune response. MATERIALS, METHODS,

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SUBJECTS

Subjects. Thirty-two patients (17 male and 15 female) with localized cutaneous leishmaniasis (LCL) were studied. All of them had acquired the disease in endemic areas of L. braziliensis infection in Rio de Janeiro, Brazil. The patients were diagnosed according to clinical, epidemiological, and immunological parameters (DTH to leishmanin, indirect immunofluorescent test) or by demonstration of the parasite in lesion biopsy samples by culture using NNN medium overlaid with RPMI 1640 (Sigma Chemical Co., St. Louis, MO) containing 10% heat-inactivated fetal calf serum (Microbiologica, Rio de Janeiro, Brazil). The ages of the patients ranged from 14 to 70 years (mean ± SEM 4 31 ± 13.9). All patients were treated with antimony (Glucantime, Rhodia) in three 10-day courses of daily intramuscular injections of N-methylglucamine at doses of 15 mg of Sb/kg of body weight. A group of eight healthy adult individuals were used as control. Leishmanin skin test (DTH). A volume of 0.1 ml of Leishmania promastigote antigens (leishmanin, kindly provided

by Dr. W. Mayrink, Federal University of Minas Gerais, Brazil) containing 40 mg N/ml was injected intradermally. After 48 hr, endurations with a diameter >5 mm were considered to be positive. Indirect immunofluorescence antibody test for leishmaniasis. Promastigote forms of the MHOM/BR/76/JOF strain of L. amazonensis were used as antigen. Positive antibody binding was revealed with fluorescein-labeled anti-human IgG conjugate (Sigma, USA). Patient sera were diluted in phosphate-buffered saline (PBS); reactivity was examined using serial twofold dilutions starting from 1:45. Titers ù45 were considered positive. Antigens used for in vitro T cell stimulation. Lb (crude L. braziliensis promastigote antigens) were prepared from L. braziliensis (MHOM/BR/75/2903) promastigotes, cultured in NNN medium supplemented with RPMI 1640 medium (Sigma) and 10% heat-inactivated fetal calf serum (Microbiologica). Stationary phase promastigotes were washed three times by centrifugation, adjusted to a concentration of 108 promastigotes/ml in PBS, and disrupted by 10 repeated cycles of freezing and thawing, followed by ultrasonication. The promastigote lysates were kept at −20°C until use. P-2/A-2, P-4, and P-8 amastigote antigens were purified from axenically cultured L. pifanoi (MHOM/VE/60/Ltrod) amastigotes, as previously reported (Soong et al. 1995). Briefly, cultured amastigotes of L. pifanoi (Pan et al. 1993) were washed, resuspended in a Tris–HCl lysis buffer containing proteinase inhibitors and disrupted by nitrogen cavitation. After differential centrifugation, the membrane fraction was collected and solubilized with detergent (Soong et al. 1995) for the purification of P-4 and P-8. The cytoplasmic fraction was employed for the purification of P-2/A-2. The amastigote antigens were purified from immunoaffinity columns employing, respectively, either A-2, P-4, or P-8 monoclonal antibody (Soong et al. 1995; Pan and McMahon-Pratt 1988). Peripheral blood mononuclear cells (PBMC) cultures. For the LPR assays, PBMC were separated by centrifugation over a gradient of Ficoll–Hypaque (Histopaque 1077, Sigma). Mononuclear cells were resuspended in RPMI 1640 (Sigma) supplemented with 10% heat-inactivated human AB Rh+ serum, 10 mM Hepes, 1.5 mM L-glutamine, 0.04 mM 2-mercaptoethanol, and antibiotics (200 IU/ml penicillin and 200 mg/ml streptomycin) and were adjusted to 3 × 106/ml. The cells (3 × 105/well) were distributed in triplicate in 96-well round-bottomed microtiter plates (Nunc A/S, Roskilde, Denmark). The cultures were incubated for 5 days at 37°C in a humidified atmosphere of 5% CO2 in air, in a final volume of 200 ml/well, in the presence of 20 mg/ml of concanavalin A (Pharmacia, Uppsala, Sweden) or four different leishmanial antigens separately: Lb, in an amount corresponding to 106 disrupted L. braziliensis promastigotes/ml, or A-2/P-2, P-4, or P-8 antigen, derived from L. pifanoi amastigotes, in a quantity of 5 mg/ml. Sixteen hours before harvesting, 1 Ci of [3H]thymidine (Amersham International, Amersham, UK), with a specific activity of 5 Ci/ mmol, was added to all wells, including negative controls

CYTOKINES AND T CELL PHENOTYPES IN CUTANEOUS LEISHMANIASIS without antigen or mitogen. Cells were harvested on fiber filters (Titertek, Flow Laboratories, Rockville, MD) using a cell harvester (Titertek Cell Harvester, Flow Laboratories). Radioactivity uptake was measured in a scintillation beta counter (1600 CA, Packard Instrumental Company, Downers Grove, IL, USA). Results were expressed as the stimulation index (s.i.): mean cpm in wells containing antigen divided by background (mean cpm in nonstimulated wells). Indices equal to or higher than 2.5 were considered positive. The mean background count was 523 ± 96 cpm. Phenotypic analysis of the antigen-responding T cells. In order to investigate the phenotypes of leishmanial antigenreactive T cells stimulated in vitro, PBMC (3 × 106/well) were cultured in 24-well flat-bottomed plates (Nunc) in a final volume of 2 ml/well under the conditions described above. After 5 days in culture, the cells were harvested and washed and then centrifuged over a discontinuous Percoll (Sigma) gradient. The blast T cells, which were specifically stimulated by the antigen (Louis et al. 1982) were separated, washed twice, and resuspended in a fixative solution containing 0.1% formalin and 0.05% sodium azide in PBS. The fixed cell concentration was then adjusted to 106 cells/200 ml and incubated for 0.5 hr at 4°C in the presence of 5 ml of monoclonal antibody specific for either the CD4 (T4-FITC, Coulter Diagnosis, FL, USA) or CD8 (T8-RD1, Coulter Diagnosis) human T lymphocyte subset determinant. After incubation, the blast cells were washed three times prior to analysis by flow cytometry (EPICS 751, Coulter). The supernatant of each culture was collected on Day 3 for determination of IL-2 and IL-4 concentrations and on Day 5 for determination of IFN-g. Supernatants were stored at −70°C until use. Cytokine assays. The levels of IFN-g, IL-2, and IL-4 were assayed in the supernatants from PBMC cultures in 24-well plates using the following ELISA kits, respectively: Intertest, Intertest 2×, and Intertest 4 (Genzyme, Cambridge, MA, USA). The experimental data were compared with standard curves obtained with IFN-g, IL-2, and IL-4 standards provided in the kits. Statistical analysis. The Mann–Whitney U test was used for statistical analysis.

RESULTS Clinical and diagnostic findings. All patients had active leishmanial skin ulcers at the beginning of the study. Twenty-one patients had a single lesion and 11 patients had multiple (two to six) lesions. The mean ± SEM period of illness was 3.7 ± 0.6 months. Parasites were isolated in cultures in 10 of 11 cases tested. A positive DTH (leishmanin) test was found in 90% of cases; positive indirect-immunofluorescence antibody titers were found for 61.2% of the patients. All patients were considered clini-

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cally cured at the end of the antimonial therapy because they had healed lesions. Lymphoproliferative responses (LPR) of peripheral blood mononuclear cells (PBMC) induced by Lb, P-4, P-8, and A-2/P-2 antigens. Twenty-five patients were tested before therapy (BT) and 17 at the end of the therapy (ET). The LPR induced by Lb in PBMC cultures were positive (s.i. > 2.5) in all patients tested before therapy as well as in all patients tested at the end of the therapy, when the lesions were already healed. The mean ± SEM stimulation indices BT 4 21.2 ± 4.2, and ET 4 18.6 ± 5.2 were not significantly different. The proliferative response to concanavalin A, before and at the end of therapy, were similar and positive in all patients (BT 4 36.7 ± 7.8; ET 4 34.1 ± 6.2). In preliminary experiments employing the A-2/P-2 amastigote cysteine proteinase antigen, only a low level proliferative response (s.i. ≅ 2.5) was observed in 25% (5/25) of the patients examined. As a consequence, further analyses of the T cell responses to the cysteine proteinase antigen were not undertaken. No significant difference was observed between the mean s.i. induced by P-4 and P-8 in the patients tested before therapy (P-4 4 4.5 ± 0.8; P-8 4 5 ± 1.1) and at the end of the therapy (P-4 4 4.6 ± 1.6; P-8 4 5.0 ± 1.3). However, in both occasions (BT and ET), P-4 and P-8 induced responses significantly lower than those induced by Lb (P < 0.01; Fig. 1). Moreover, whereas 100% of LPR induced by Lb were positive (s.i. > 2.5) before therapy and at the end of the therapy, the LPR induced by P-4 and P-8 were positive in only 13 cases (52%) before therapy and in, respectively, 8 cases (47%) and 9 cases (52.9%) at the end of the therapy. The LPR induced by Lb, P-4, and P-8 in PBMC cultures of eight uninfected individuals were all negative (s.i. ranged between 0.9 and 1.3), whereas the LPR induced by concanavalin A were all positive (39.7 ± 6.9) and similar to those of the leishmaniasis patients. Phenotypic analysis of PBMC after stimulation in vitro with Lb, P-4, and P-8 antigens. Preliminary results showed that the best P-4 and P-8 concentrations for T cell stimulation in vitro

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FIG. 1. Lymphoproliferative responses of peripheral blood mononuclear cells in cultures stimulated with crude Leishmania braziliensis promastigote antigens (Lb) or the antigen preparations P-4 and P-8, both derived from amastigote forms of Leishmania pifanoi. Data represent the mean stimulation index (s.i.) ± SEM observed in cultures performed before therapy ( ) or at the end of the therapy (j), when the lesions were healed.

ranged between 1 and 5 mg/ml, whereas the best Lb concentration has been determined as 106 promastigotes/ml. With regard to the Lb-stimulated T cells, 12 patients were studied before therapy and 10 patients at the end of the therapy. The mean percentages of responder CD4+ cells were significantly higher before therapy (38.5 ± 3.7) than at the end of therapy (25.4 ± 4.7) (P 4 0.03). On the other hand, the mean percentages of responder CD8+ cells in the same cultures was lower before therapy (16.3 ± 2.1) than at end of therapy (23.7 ± 3.6) although this difference was not statistically significant (Fig. 2). The percentage of CD4+ responder cells was more than twice the percentage of responding CD8+ cells (CD4+/CD8+ 4 2.3) during active disease, whereas the percentages of responding CD4+ and CD8+ cells observed in cured patients were similar (CD4+/CD8+ 4 1.1). Thus the Lbresponsive cell populations from cured patients showed decreased percentage of CD4+ cells and a tendency toward an increased percentage of CD8+ cells when compared with those obtained during active disease, before therapy (Fig. 2). The phenotypes of the P-4- or P-8-respond-

ing T cells were characterized only in cultures with positive LPR (s.i. > 2.5). The phenotype of the P-4-stimulated cells were determined in nine patients before therapy (BT) and in six patients at the end of the therapy (ET). Figure 2 shows that the percentages of responding CD4+ and CD8+ T cells at both time points were very similar (CD4+: BT 4 25 ± 3.9, ET 4 24.3 ± 5.9; CD8+: BT 4 17.9 ± 4.3, ET 4 21 ± 3.9) as were the CD4+/CD8+ ratios (respectively, BT 4 1.4 and ET 4 1.1). Similar results were also obtained with the P-8-stimulated cells in eight patients studied before therapy and in three patients studied at the end of therapy (Fig. 2). The percentages of responding T cell subpopulations were, respectively, CD4+, BT 4 24.5 ± 3.6, ET 4 22.9 ± 4.3; and CD8+, BT 4 17.8 ± 2.4, ET 4 18 ± 3.9. The CD4+/CD8+ ratios were, respectively, BT 4 1.3 and ET 4 1.2, similar to those observed in cultures stimulated with P-4. Comparing the cell phenotypes from cultures stimulated with the three antigen preparations before therapy, it was observed that the mean percentage of responding CD4+ T cells was sig-

FIG. 2. Phenotypic analysis of the responder T cells in peripheral blood mononuclear cell cultures stimulated with crude Leishmania braziliensis promastigote antigens (Lb) or the antigen preparations P-4 and P-8, both derived from amastigote forms of Leishmania pifanoi. Data represent the mean percentages ± SEM of antigen-responding CD4+ and CD8+ cells in cultures performed before therapy ( ) or at the end of the therapy (j).

CYTOKINES AND T CELL PHENOTYPES IN CUTANEOUS LEISHMANIASIS

nificantly higher (P 4 0.05) in the Lb-stimulated cultures (38.5 ± 3.5) than in the cultures stimulated with P-4 (25 ± 3.9) or P-8 (24.5 ± 3.6), whereas no significant difference was observed in the percentages of CD8+ responder cells among cultures stimulated with each different antigen (Fig. 2). Thus, during active disease P-4 and P-8 apparently stimulated CD8+ T cells at a level comparable to Lb but induced weaker stimulation of CD4+ T cells. At the end of the therapy (healed lesions), similar percentages of CD4+ and CD8+ responder cells were observed in the cultures stimulated with Lb, P-4, or P-8 (Fig. 2). Cytokine production in PBMC cultures stimulated with Lb, P-4, and P-8 antigens. IFNg, IL-2, and IL-4 were determined in culture supernatants of PBMC stimulated with each antigen. Figure 3 shows the results related to the type 1 lymphokines, IFN-g, and IL-2. With regard to Lb-stimulated cultures, the production of IFN-g was determined in 21 patients before therapy and in 12 patients at the end of therapy. The results showed a tendency, although not significant, of increased levels of IFN-g at the

FIG. 3. Type 1 lymphokines (IFN-g and IL-2) production in peripheral blood mononuclear cell cultures stimulated with crude Leishmania braziliensis antigen (Lb) or the antigen molecules P-4 and P-8, both derived from amastigote forms of Leishmania pifanoi. Data represent the mean determinations of IFN-g and IL-2 in supernatants from cultures performed before therapy (BT) and at the end of the therapy (ET), when the lesions were healed.

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end of therapy (3005 ± 900 pg/ml) when compared with the levels found before therapy (2255 ± 563 pg/ml). In the case of the P-4- or P-8-stimulated cultures, all supernatants available were tested, including those from cultures with very low proliferative responses (s.i. < 2.5), because activation of T cells may result in proliferation, in cytokine production or in both phenomena. The production of IFN-g was determined in 14 or 13 patients, respectively, before therapy and at the end of therapy. The response of 12 cured patients in the case of P-4 and that of 8 patients in the case of P-8 were studied. The results followed a similar tendency as observed in Lb stimulated cultures, leading to increased production of IFN-g at the end of therapy (P-4 4 1651 ± 662 pg/ml and P-8 4 2214 ± 900 pg/ml) in comparison with the results before therapy (P-4 4 1089 ± 426 pg/ml and P-8 4 659 ± 399 pg/ml). No significant differences were observed in the level of IFN-g production induced by either Lb, P-4, or P-8 leishmanial antigens. The mean production of IL-2 was studied in 9 patients before therapy and in 11 patients at the end of the therapy. The results observed in the supernatants from cultures stimulated with Lb (BT 4 208 ± 90 pg/ml; ET 4 370 ± 104 pg/ml), with P-4 (BT 4 160 ± 92 pg/ml; ET 4 360 ± 152 pg/ml), or with P-8 (207 ± 140 pg/ml; ET 4 43 ± 37 pg/ml) did not show significant differences between the levels found before and at the end of the therapy (Fig. 3). Figure 4 shows the results related to the type 2 lymphokine IL-4, determined in cultures stimulated with each antigen (Lb, P-4, and P-8). The mean production of IL-4 in cultures stimulated with Lb was determined in 11 patients. A decrease, although weakly significant, at the end of therapy (17 ± 10 pg/ml) in comparison with the mean determination in cultures before therapy (300 ± 173 pg/ml) (P 4 0.06) was observed. In contrast, the mean production of IL-4 in the supernatant of cultures stimulated with P-4 and P-8 showed extremely low levels or even not detectable IL-4 before therapy (P-4 4 13.3 ± 13 pg/ml and P-8 4 0 pg/ml) as well as at the

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FIG. 4. Type 2 lymphokine (IL-4) production in peripheral blood mononuclear cell cultures stimulated with crude Leishmania braziliensis antigen (Lb) or the antigen molecules P-4 and P-8, both derived from amastigote forms of Leishmania pifanoi. Data represent the mean determinations in supernatants from cultures performed before therapy (BT) and at the end of the therapy (ET), when the lesions were healed.

end of the therapy (P-4 and P-8 4 0 pg/ml). These data refer to 9 patients studied BT and 10 patients studied ET with both antigens. The differences observed between the production of IL-4 in cultures stimulated with Lb and in cultures stimulated with P-4 or P-8 before therapy were significant (P < 0.01). At the end of therapy, the levels of IL-4 induced by the three antigens Lb, P-4, or P-8 were comparable and always negligible (Fig. 4). DISCUSSION The mouse model has been extremely useful for studying the T cell-mediated immune responses to Leishmania infection and the mechanisms for cure or aggravation of the disease. Two polar clinical–immunological features have been described depending on the susceptibility or resistance of the mouse strain to L. major infections (Heinzel et al. 1991). The severe disease produced in the Leishmania susceptible BALB/c mouse strain presents many similarities with the severe human diffuse cutaneous leishmaniasis. In both cases, a depressed

T cell-mediated immune response is observed, with negative skin test to leishmanial antigens (DTH), production of type 2 cytokines by CD4+ Th2 cells, and inhibition of type 1 cytokine production. Inhibition of macrophage activation occurs, leading to enormous proliferation of amastigote forms in the parasitophorous vacuole macrophages (Carvalho et al. 1985; Caste´s et al. 1984, 1988). The inapparent/subclinical disease or the tendency for self-healing small lesions observed in the Leishmania-resistant mouse strains (e.g., C57BL/6) are similar to the human cases of natural resistance (partial or complete) to dermotropic Leishmania species. In such cases a CD4+ Th1 cell mediated immune response occurs, with positive DTH and production of type 1 cytokines (Carvalho et al. 1995), leading to activation of macrophages, destruction of parasites, and consequent tendency for self-healing lesion. In addition, evidence from the mouse model studies suggests that CD8+ T cells producing IFN-g are involved in the control of murine leishmaniasis caused by L. major (Titus et al. 1987; Hill et al. 1989; Muller et al. 1993, 1994; Conceic¸a˜o-Silva et al. 1994) and L. donovani (Rachamim and Jaffe 1993; Murray et al. 1995). However, a suitable mouse model for the chronic American cutaneous and mucocutaneous human diseases produced by L. braziliensis does not exist. In these patients, the lesions can remain active for months or years with presence of potent T cell-mediated immune responses with positive or even exacerbated DTH and scarceness of parasites in the lesions (Caste´s et al. 1984; Carvalho et al. 1985; Coutinho et al. 1987). In this context, the advancement of knowledge of the immunopathogenesis of the chronic American tegumentary leishmaniasis has come mainly from the study of human patients. The present study demonstrates two patterns of in vitro Lb induced T cell-mediated immune responses: (a) During the active disease the majority of the Lb-responding cells belong to the CD4+ phenotype and produce both type 1 and type 2 cytokines (IFN-g, IL-2, and IL-4). In these cases of chronic active lesions, the immune response was neither able to destroy the

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parasites leading to healing lesions (type 1/Th1 response) nor permissive to parasite proliferation, as observed in the severe diffuse cutaneous disease (type 2/Th2 response). Type 1 and type 2 cytokine-related mRNA have been detected in the lesions (Caceres-Dittmar et al. 1993; Pirmez et al. 1993; Tapia et al. 1993), in agreement with our results, during the active disease. (b) At the end of the antimonial therapy when the lesions were healed, similar percentages of CD4+ and CD8+ T cells or even predominance of CD8+ cells (Da-Cruz et al. 1994; Coutinho et al. 1996) are observed as well as an apparently beneficial type 1 cytokine profile with production of IFN-g and IL-2, but not IL-4. Consequently upon cure, decreased levels of Lb reactive CD4+ cells and an increased proportion of reactive CD8+ cells were observed. In addition, a change in cytokine production from a mixed type 1–type 2 to type 1 response occurred. A similar increased production of IFN-g, IL-2, and TNF has also been noted in patients cured of Old World cutaneous leishmaniasis (Frankenburg et al. 1993). In a previous paper (Da-Cruz et al. 1994), we observed a similar pattern/change in the proportions of antigen responsive CD4+ and CD8+ T cells in patients upon cure. In that study it was determined, using T-cell responses of leishmaniasis patients to Toxoplasma gondi as a control, that the changes in the profile of the CD4+ and CD8+ Lb-reactive T cells before and after cure were not a direct effect of antimonial treatment. Thus, it is tempting to postulate that the immunologic mechanism for healing of chronic American cutaneous leishmaniasis is associated with a switch from predominant Lb-reactive CD4+ (Th1 and Th2) responses to balanced CD4+/CD8+ responses, or predominant CD8+ (Da-Cruz et al. 1994; Coutinho et al. 1996) response, with production of type 1 cytokines (IFN-g and IL-2). Interestingly, T cells from human volunteers vaccinated against American cutaneous leishmaniasis with a preparation made of killed promastigotes of five dermotropic Leishmania strains also showed similar responses to Lb antigen to those observed in cured patients, i.e., the presence of IFN-g and

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predominance of responding CD8 + T cells (Mendonc¸a et al. 1995). The observed decrease in the proportion of Lb-reactive CD4+ cells, associated with production of IFN-g but not IL-4 in cured patients, may also point to a better modulation of the T cell-mediated immune responses, after destruction of parasites by the antimony therapy and decrease of the parasite load. We can speculate that differences in parasite antigens found in active versus healing lesions would be relevant in the mechanisms for differential T cell activation; in addition, a reduction of the parasite load by the antimonial therapy may alter the T cell responses to Lb antigen. Relevant to this point, it has been shown (Bretscher et al. 1992) that BALB/c susceptible mice infected with a low level of live L. major promastigotes became resistant to challenge with a high parasite inoculum. Further, investigations have demonstrated (Scott 1994) that the reduction of the parasite load by antimonial treatment facilitated the development of an IL-12 driven type 1 T cell response. Since we have defined a similar immunological pattern associated with cured patients and with vaccinated individuals after active immunization, we decided to investigate the in vitro T cell responsiveness induced by the amastigote A-2/P-2 cysteine proteinase and membrane associated proteins P-4 and P-8 in PBMC cultures from patients with active lesions and with healed lesions at the end of the therapy. Soong et al. (1995) have demonstrated that immunization with P-4 or P-8 antigens, purified from axenically cultured amastigotes of L. pifanoi, together with C. parvum as an adjuvant induced substantial levels of immunoprotection in BALB/c and CBA/J mice against infection with either infective L. pifanoi or L. amazonensis promastigotes. In contrast, the A-2/P-2 cysteine proteinase provided minimal protection, evident only at the lowest challenge dose of L. pifanoi parasites. Protectively vaccinated mice exhibited proliferative responses to parasite antigens and production of IFN-g. In these studies of the human responses to the P-4 or P-8 purified amastigote antigens, the

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stimulation indices observed were significantly lower than those induced by Lb; however, comparable levels of the cytokines, IFN-g, and IL-2 were found. Similar stimulation indices were observed both before and at the end of the therapy for the amastigote antigens. In contrast to the consistent proliferative response to Lb antigen, only 50% of the patients responded to either the P-4 or the P-8 molecules. These data should, however, be expected because: (a) P-4 and P-8 are derived from L. pifanoi and the patients were infected with L. braziliensis. Nevertheless, cross-reactivity was detected between both species; the definition of the cross-species reactive epitopes should be of interest for future vaccine studies. (b) P-4 and P-8 are single molecules with consequently fewer epitopes than Lb antigen, which is whole parasite lysate. The present results show that while 50% of the patients examined had significant proliferative responses (s.i. ≅ 5) to the P-4 and P-8 molecules, only 25% of the patients exhibited a borderline level of responsiveness (s.i. ≅ 2.5) to the A-2/ P-2 cysteine proteinase. Assuming that the L. pifanoi P-2/A-2, P-4, and P-8 molecules are equivalently phylogenetically divergent from their L. braziliensis homologs, this result appears of interest. Further, the level of the response found appears to correlate with the level of protection observed in mouse model studies (Soong et al. 1995). Interestingly, studies by Wolfram et al. (1995) indicated that a comparable cysteine proteinase of L. mexicana fails to be presented by murine macrophages infected with live parasites but that antigen presentation does occur upon parasite destruction. The mechanism responsible for the lack of antigen presentation of the cysteine proteinase by live organisms, however, is at present unclear. However, a limitation of antigen presentation would undoubtedly impact on the level of protection induced by the A-2/P-2 cysteine proteinase in the mouse model in vivo and the consequent level of proliferative response observed in the current study of infected human patients. With regard to the phenotype of the proliferating cells, P-4 and P-8 induced significantly lower proportions of CD4+ cells than Lb during

the active disease, whereas the three antigens induced similar percentages of CD4+ and CD8+ T cells at the end of therapy. A well balanced CD4+/CD8+ T cell response induced by Lb was found only in cured patients (present results). However, P-4 and P-8 induced this apparently beneficial pattern (CD4+/CD8+ ratios near 1) not only in cured patients but even during the active disease. In terms of cytokine production, the supernatants of P-4- and P-8-stimulated cultures consistently showed a typical type 1 profile (presence of IL-2 and IFN-g and near absence of IL-4). Moreover, the amount of IFN-g produced at the end of therapy had a tendency to be higher than those obtained during active disease. Thus the apparently beneficial type 1 cytokine pattern found in Lb-stimulated cultures in cured patients was observed in cultures stimulated with P-4 and P-8 in both during active disease and upon cure. Considering that the majority of cured leishmaniasis patients acquires resistance to further infections, the pattern of their T cell mediated immune responses should be associated with an immune-protective effect. Hence an antigenic candidate for a future vaccine should elicit in vitro T cell responses similar to those observed in cured patients. This could be considered an important parameter for selection of antigen candidates for a vaccine. Following this rationale, P-4 and P-8 should certainly be considered for future studies of immunoprotection in primates and potentially for vaccination trials in human populations. ACKNOWLEDGMENTS We are grateful to S. L. Mello and M. A. Santiago for clinical and laboratorial assistance. This work was supported by grants from the European Economic Community, Brazilian National Council for Scientific and Technologic Development (CNPq), and a grant from the National Institutes of Health (U.S.A.) AI-27811.

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Received 16 May 1996; accepted with revision 9 August 1996