Immunochemical Studies of a Purified Glycoprotein Fraction

INFECTION AND IMMUNITY, JUlY 1988, p. 1766-1770 0019-9567/88/071766-05$02.00/0 Copyright C 1988, American Society for Microbiology

Vol. 56, No. 7

Novel 17-Kilodalton Leishmania Antigen Revealed by Immunochemical Studies of a Purified Glycoprotein Fraction Recognized by Murine T Lymphocytes MAURICIO MARTINS RODRIGUES,'* MARCIA TOSTA XAVIER,2 LUCIA MENDONQA-PREVIATO,2 AND MARCELLO ANDRE BARCINSKI3 Discipline of Parasitology, Escola Paulista de Medicina, Sao Paulo, SP 04023,1 and Institute of Microbiology2 and Carlos Chagas Filho Institute of Biophysics,3 Federal University of Rio de Janeiro, RJ 21941, Brazil Received 7 January 1988/Accepted 18 April 1988

Recently, a glycoprotein fraction, designated gplO/20, purified from Leishmania mexicana amazonensis was shown to induce a cellular immune response mediated by murine L3T4+ T lymphocytes. This fact led us to pursue further the characterization of this fraction. The present study demonstrated that gplO/20 is a degradation product of a 17-kilodalton antigen present in promastigotes and amastigotes of L. mexicana amazonensis. This antigen was easily detected in promastigotes of L. mexicana mexicana, L. donovani, L. chagasi, L. major, and L. tropica. However, culture forms of L. braziliensis complex expressed either low amounts of the 17-kilodalton antigen or an antigenically unrelated antigen. The recognition of gplO/20 by several serum samples of patients with kala-azar was also shown.

Leishmaniasis is a granulomatous disease caused by an obligatory intracellular protozoan of the genus Leishmania. In the murine model of cutaneous leishmaniasis, L3T4+ T lymphocytes have been associated with the resistance (11, 14) or susceptibility (11, 23, 24) observed among different inbred mouse strains. The importance of immunity to a surface glycoprotein (gp63) and to a lipophosphoglycan purified from leishmanias has also been described (7, 13, 21). The studies of glycoproteins recognized by L3T4+ T lymphocytes are therefore important in understanding hostparasite interplay. Recently, a purified glycoprotein fraction from Leishmania mexicana amazonensis, designated gplO/20, was shown to induce a cellular immune response mediated by L3T4+ T lymphocytes (20). It was also observed that the induction of specific immunity to this fraction significantly accelerates primary lesion progression in infected BALB/c mice (19). In this paper, we report that gplO/20 is a product of a 17-kilodalton (kDa) antigen present in the promastigote and amastigote parasite differentiation forms. This antigen is widely distributed among different species of Leishmania. The recognition of gplO/20 by several serum samples of patients with kala-azar is also reported.

Laboratories, Detroit, Mich.) supplemented with hemin (0.01 g/liter) and fetal calf serum (10%, vol/vol). Promastigotes of all other species were cultivated in biphasic Novy, MacNeal, and Nicolle (NNN) rabbit blood agar slopes with a fluid phase of brain heart infusion medium (Difco). After 3 to 5 days in culture, the cells were collected and washed at least three times in phosphate-buffered saline (PBS) before use.

Amastigotes of L. mexicana amazonensis and L. mexicana mexicana were obtained from infected hamsters or BALB/c mice 30 to 60 days after inoculation with 106 amastigotes. The lesion was aseptically dissected, washed in sterile PBS-fetal calf serum (10%, vol/vol) and ground in a glass homogenizer. The suspension was then centrifuged at 200 x g to remove large debris. The amastigotes from the supernatant were washed at least twice in PBS-fetal calf serum and shaken for 4 h to remove macrophage phagolysosomal membranes. Purification of gplO/20 fraction. Frozen cells were rapidly thawed and extracted as described previously (12). The aqueous extract was fractionated by gel filtration chromatography on a Bio-Gel P-10 column (120 by 2 cm; Bio-Rad Laboratories, Richmond, Calif.). The material collected in the void volume was designated gplO/20. Antigen amount is given in terms of dry mass. Immunization. BALB/c mice were immunized in the hind footpads with 100 ,ug of gplO/20 emulsified in complete Freund adjuvant (Difco), as described elsewhere (20). Hyperimmune serum samples were obtained from mice boosted two to three times with 100 ,ug of gplO/20 in incomplete Freund adjuvant (Difco) at 2 and 3 weeks after the first immunization. T-cell proliferation assay. The culture medium was RPMI 1640 medium (Flow Laboratories, Inc., McLean, Va.) supplemented with penicillin (0.014%, wt/vol; Sigma Chemical Co., St. Louis, Mo.), streptomycin (0.01%, wt/vol; Sigma), 2-mercaptoethanol (5 x 10-5 M; Sigma), L-glutamine (2 mM), and inactivated normal human serum (5%, vol/vol). At 2 weeks after immunization, the draining lymph nodes were collected, and a single cell suspension of whole lymph node

MATERIALS AND METHODS Parasites. The leishmanias used in the present study were the following: L. mexicana amazonensis H21 and MHOM/ BR/73/M2269, L. mexicana mexicana MNYC/BR62/M379, L. braziliensis braziliensis MHOM/BR75/M2903, L. braziliensis guyanensis M4147, L. chagasi M6041 (kindly provided by J. Shaw, Wellcome Parasitology Unit, Instituto Evandro Chagas, Belem, Brazil), L. donovani 1S (provided by D. Dwyer, National Institutes of Health, Bethesda, Md.), L. major MHOM/IL/67/JERICHO II, and L. tropica MHOM/SU/74/K27 (kindly donated by G. Grimaldi, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil). Promastigotes of L. mexicana amazonensis and L. donovani were grown in liver infusion tryptose medium (Difco *

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cells was prepared. These cells were cultivated at a density of 4 x 105 cells per well in 96-well round-bottom microdilution plates (Linbro Inc., Hamden, Conn.) in a final volume of 0.22 ml for 72 h at 37°C in a humid environment containing 5% CO2. At 18 to 20 h before harvest, each well received 1 ,uCi of tritiated thymidine (specific activity, 6.7 Ci/mmol; New England Nuclear Corp., Boston, Mass.). Cultures were harvested into fiber glass filters with the aid of a semiautomatic harvesting device. The amount of tritiated thymidine incorporated was measured in a liquid scintillation counter. Results are expressed in mean counts per minute of triplicate cultures. Enzyme-linked immunosorbent assay. Briefly, 96-well flexible plates were coated overnight with 1 jig of gplO/20 per well in carbonate buffer (0.1 M [pH 9.6]). The plates were saturated with PBS buffer containing Tween 20 (0.1%, vol/ vol) and gelatin (0.5%, wt/vol). After incubation with the antibodies diluted in this buffer for 1 h at 37°C, the plates were washed three times with PBS-Tween 20 before rabbit anti-mouse immunoglobulin G (IgG) or anti-human IgG with horseradish peroxidase (0.001%, vol/vol; Dako Co., Santa Barbara, Calif.) was added. The substrate solution consisted of citric acid (5 mg/ml), sodium bicarbonate (7.3 mg/ml), H202 (0.3 ,ul of a 30% solution per ml), and o-phenyldiaminobenzidine (0.4 mg/ml). The reaction was interrupted with 2 N sulfuric acid, and the optical density at 470 nm was measured. Immunoblotting. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed as described by Laemmli (10). Proteins with molecular sizes ranging from 94 to 14.4 kDa (Pharmacia, Uppsala, Sweden) were used as molecular weight markers. Immunoblotting was carried out essentially as described by Towbin et al. (25). Briefly, samples dissolved in boiling SDS-polyacrylamide gel electrophoresis sample buffer containing 4% SDS and 1% 2mercaptoethanol were electrophoresed in 12 to 15% polyacrylamide slabs of gel. After electrophoresis, proteins were transferred to nitrocellulose sheets overnight at 200 mA. The transferred polypeptides and molecular weight markers were visualized by staining the sheets with Ponceau-S (0.1%, wt/ vol) in acetic acid (10%, vol/vol). The nitrocellulose sheets were then soaked in blot buffer (0.15 M NaCl, 1 mM EDTA, 0.04% Tween 20, 30 mM Tris hydrochloride [pH 7.5], 0.5% gelatin) for 45 min. After incubation with antiserum to gplO/ 20 or with preimmune serum (1% diluted in blot buffer) for 1 h at room temperature, the sheets were subjected to three washes of 15 min each in blot buffer. Bound IgG was visualized after incubation for 1 h with rabbit anti-mouse IgG or anti-human IgG with horseradish peroxidase (Dako) diluted to 0.05% (vol/vol) in blot buffer, followed by three 15-min washes in Tris-buffered saline (pH 7.5) and reaction with diaminobenzidine (0.2 mg/ml) and H202 (5 ,ul of a 30% solution per 30 ml of TBS). Affinity purification of gplO/20-specific antibodies. Preparative SDS-polyacrylamide gel electrophoresis was performed with 1.5 mg of the gplO/20 fraction, and the gel was subsequently blotted as described above. The two major bands, visualized with Ponceau-S and detected with the antiserum to gplO/20, were excised from the nitrocellulose sheet and used to adsorb the specific antibodies from a pool of mouse hyperimmune sera (10%, vol/vol; diluted in blot buffer). After an extensive wash, the antibodies were eluted by incubation with 2 ml of glycine (0.1 M [pH 2.8]) for 5 min at room temperature. The pH of the eluted material was raised to 7.5 with 30 ,u of 2 M Tris (pH 11). Metabolic labeling and immunoprecipitation. Metabolic

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FIG. 1. Cellular and humoral immune responses to gplO/20. BALB/c mice (n = 3) were immunized with 100 jig of the purified gplO/20 fraction 2 weeks before the assays. (A) In vitro lymph node cell proliferation response to gplO/20 as detected by tritiated thymidine incorporated after 72 h in culture. (B) ELISA titers of serum of immunized mice ( ) and of the preimmune serum ( -). KCPM, Counts per minute (103); O.D., optical density.

labeling of L. mexicana amazonensis promastigotes was carried out by incubation for 4 h at 25°C of 108 culture forms in 1 ml of methionine-free Eagle minimum essential medium (Flow) supplemented with L-glutamine to which 100 ,iCi of L-[35S]methionine (1,200 Ci/mmol; Amersham International, Little Chalfont, United Kingdom) was added. Then the cells were washed five times in PBS, and the pellet was solubilized in lysis buffer. Lysis buffer consisted of PBS supplemented with Nonidet P-40 (0.5%, vol/vol), aprotinin (2 trypsin-inhibiting units/ml), antipapain and leupeptin (each at 25 ,ug/ml), and 1 mM phenylmethylsulfonyl fluoride. The extract was centrifuged at 12,000 x g for 10 min, and the pellet was discarded. Immunoprecipitation of labeled parasite extract was performed by using 10% formaldehydetreated Staphylococcus aureus Cowan I to bind the immunocomplex. The extracts were incubated overnight with anti-gplO/20 polyclonal serum or preimmune serum (3%, vol/ vol) at 4°C. The precipitates were washed at least five times in NET-bovine serum albumin (0.5% Nonidet P-40, 1 mM EDTA, 0.15 M NaCl, 50 mM Tris, 2 mg of bovine serum albumin per ml). The pellets were then boiled with sample buffer for 3 min and centrifuged, and the supernatant was applied to a slab of gel. RESULTS gplO/20 is related to a 17-kDa molecule and contains a minor 56-kDa contaminant. In immunized BALB/c mice, gplO/20 induced a primary IgG response, as measured by ELISA and a potent lymph node cell proliferative response in vitro (Fig. 1; 20). Immunoblot analysis with BALB/c mouse hyperimmune serum (titer greater than 1:105) showed the specific staining of two bands in L. mexicana amazonensis promastigotes which migrated, under reducing conditions, to 17- and 56-kDa positions (Fig. 2, lanes C and D). The same pattern was observed in the immunoprecipitation L-[355]methionine-labeled promastigotes (Fig. 2, lane f). The relationship between the gplO/20 fraction and the two stained bands (17 and 56 kDa) from whole-promastigote

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FIG. 2. Immunoblot (A) and immunoprecipitation (B) with hyperimmune anti-gplO/20 serum. The samples loaded on the SDSpolyacrylamide gel for immunoblot were 20 ,ug of gplO/20 (lanes A and C) and 107 promastigotes of L. mexicana amazonensis (lanes B and D). Samples were reacted with preimmune mouse serum (lanes A and B) or with hyperimmune mouse serum (lanes C and D). Metabolically labeled promastigote extract (lane E) was immunoprecipitated with hyperimmune mouse antiserum to gplO/20 (lane F) or with preimmune mouse serum (lane G). The positions of molecular size markers are indicated. KD, Kilodaltons.

samples was investigated by purifying the antibodies specific to each band of the gplO/20 fraction. Antibodies purified with

either individual band on the nitrocellulose recognized the 17-kDa band in promastigote lysates. They also bound to both bands of the purified gplO/20 fraction (Fig. 3). This evidence indicates that both bands are products of the same 17-kDa protein and are probably generated by degradation during the purification procedure. The 56-kDa protein stained in the immunoblot was not related to the 17-kDa antigen but was, rather, a minor contaminant of the purified gplO/20 fraction. This fact became even clearer when large amounts of the purified material were used in the immunoblot with the immune serum; the 56-kDa antigen was occasionally weakly stained (data not shown). The 17-kDa antigen is a family of antigenically related proteins distributed in several Leishmania species. To investigate whether the 17-kDa antigen is stage specific or species

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FIG. 3. Identification of gplO/20 antigenically related polypeptide in promastigotes of L. mexicana amazonensis by using affinitypurified antibodies. Lanes A, B, C and D were loaded with 2 x 106 promastigotes. Lanes E and F were loaded with 10 ,ug of gplO/20 fraction. Lanes A, C, and E were stained with affinity-purified antibodies to the 11-kDa band. Lanes B, D, and F were stained with affinity-purified antibodies to the 17-kDa band. The positions of molecular size markers are indicated. KD, Kilodaltons.

FIG. 4. Identification of 17-kDa antigen of various Leishmania species by using affinity-purified antibody to gplO/20. Lanes: A, L. donovani; B, L. chagasi; C, L. mexicana mexicana; D, L. mexicana ainazonensis; E, L. mexicana amazonensis amastigotes; F, L. major; G, L. tropica; H, L. braziliensis guyanensis. All lanes were loaded with 2 x 106 culture forms, except for lane E which was loaded with 7 x 106 cells. The positions of molecular size markers are indicated. KD, Kilodaltons.

specific, we performed an immunoblot analysis of several Leishmania samples. First, we observed that the expression of the 17-kDa antigen is not developmentally regulated. In Fig. 4, the presence of the antigen in L. mexicana amazonensis promastigote and amastigote forms is demonstrated (Fig. 4, lanes D and E). The 17-kDa antigen was also present in promastigotes of L. mexicana mexicana (Fig. 4, lane c). The expression of this molecule did not change in culture forms harvested at different points of the growth curve (data not shown). A slightly different pattern was observed when promastigotes of different species of Leishmania were compared. We observed two bands around 17 kDa for culture forms of L. tropica and one band of 17 kDa for those of L. major (Fig. 4, lanes G and F). For both leishmanias of the kala-azar complex (L. donovani and L. chagasi), we observed only a single band just above 17 kDa (Fig. 4, lanes A and B). However, for L. braziliensis guyanensis, the 17-kDa antigen was only weakly stained (Fig. 4, lane H), and often, it was visualized near the detection limit of the method. The same result was observed with L. braziliensis braziliensis (data not shown). This difference could represent variations in the amount of antigen expressed or in the number of crossreactive epitopes or both. Recognition of gplO/20 by serum samples of patients with kala-azar in enzyme-linked immunosorbent assay. Since the existence of cross-reactive epitopes between gplO/20 and a 17-kDa molecule of leishmanias of the kala-azar complex was demonstrated, we examined whether serum samples from patients with kala-azar will recognize this fraction. Enzyme-linked immunosorbent assay titrations of 11 serum samples from patients from Bahia, Brazil, were performed. Of 11 samples tested, 6 were significantly different from a serum pool from healthy normal donors (n = 8). Figure 5A shows the titration curves of "responders" and that of a serum pool from normal controls. Immunoblot analysis demonstrated that responders reacted with gplO/20, whereas the normal controls failed to recognize this fraction (Fig. 5B). DISCUSSION The glycoprotein fraction designated gplO/20 from L. mexicana amazonensis has been recently purified, and its ability to induce a cellular immune response was studied.

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FIG. 5. Recognition of ELISA of gplO/20 by serum samples of patients with kala-azar. (A) Titration curves of responders (sera from patients with kala-azar) ( ) and of serum pool from healthy normal donors (0 0). (B) Immunoblot with human sera (diluted to 1:25). Lane A, Pool of responders (sera from patients with kala-azar); lane B, serum pool from normal donors from an area where kala-azar is endemic. The position of the 14-kDa (KD) marker is indicated.

This fraction has an exquisite capacity to stimulate a cellular immune response mediated by the L3T4+ T lymphocytes of infected and immunized mice (20). Unexpectedly, however, results of subsequent studies showed that the immunity to this fraction enhances the size of the lesion induced by L. mexicana amazonensis in susceptible BALB/c mice (19). The indication of a possible participation of gplO/20 in the immunopathological process of experimental American cutaneous leishmaniasis led us to pursue further the characterization of gplO/20. The two bands present in the gplO/20 fraction are related to a 17-kDa antigen (Fig. 3), a finding which raises some questions concerning the origin of the lower-molecular-mass band. This antigen is purified without protease inhibitors, and since the proteolytic enzymes of L. mexicana amazonensis are extremely active (5), some proteolytic degradation is expected. However, other types of chemical degradation should be considered. The 17-kDa antigen is a non-stage-specific antigen widely distributed among Leishmania species, and it seems to comprise a family of antigenically related polypeptides. Several pieces of evidence indicate that this molecule is internally located in both forms of L. mexicana amazonensis. Infections in which an impairment of immunological effector mechanisms is observed apparently correlate with leishmanias which express easily detectable levels of the 17-kDa antigen. Suppression of the cellular immune response has been described in patients with New-World diffuse cutaneous leishmaniasis, which is mainly caused by subspecies of the L. mexicana complex (15, 17, 18), and in mice infected with L. major (4, 6, 22). Patients with kala-azar and experimental animals infected with L. donovani and L. chagasi show profound impairment of T-lymphocyte func-

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tion during the acute phase of the disease (1, 3, 8, 16). In contrast, the cutaneous and mucocutaneous leishmaniasis induced by parasites of the L. braziliensis complex, in which the parasites express low amounts of the 17-kDa antigen or an antigenically unrelated antigen or both, do not give any indication of an immunosuppressive mechanism (2, 9). On the basis of previous data showing that immunization of BALB/c mice with gplO/20 prior to infection accelerates disease progression (19), the possibility exists that the 17kDa molecule is involved in the generation of immunosuppressive mechanisms. The recognition of this molecule by sera from patients with kala-azar indicates that the antigenicity of this molecule is not solely restricted to the murine model. The reasons why only 6 of 11 sera from patients with kala-azar recognized the 17-kDa antigen will be addressed further to determine whether recognition is correlated with patient clinical data. The characterization performed in the present work will allow the purification of the native 17-kDa antigen from individual Leishmania species and subsequent detailed studies in humans and in mice. The possible biological role of this molecule and the specific cellular immune response in visceral and cutaneous leishmaniasis induced by L. tropica and L. major remain poorly understood but can now be addressed. ACKNOWLEDGMENTS We thank Renato Motara, Nobuko Yoshida, and Rosana Puccia for helpful discussions and careful review of the manuscript. We also thank M. L. Cardoso de Almeida for helpful discussions. This work was supported by grants from the Brazilian National Research Council, Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, and the UNDP/WORLD BANK/WHO Special Programme for Research and Training in Tropical Disease.

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LITERATURE CITED Carvalho, E. M., R. Badaro, S. Reed, T. Jones, and W. D. Johnson. 1985. Absence of gamma interferon and interleukin 2 during active visceral leishmaniasis. J. Clin. Invest. 76:20662069. Carvalho, E. M., W. D. Johnson, E. Barreto, P. D. Mandger, J. L. Costa, S. Reed, and H. Rocha. 1985. Cell mediated immunity in American cutaneous and mucosal leishmaniasis. J. Immunol. 135:4144 4148. Carvalho, E. M., R. S. Teixeira, and W. D. Johnson, Jr. 1981. Cell-mediated immunity in American visceral leishmaniasis: reversible immunosuppression during acute infection. Infect. Immun. 33:498-502. Cillari, E., F. Y. Liew, and R. Lelchuk. 1986. Suppression of interleukin-2 production by macrophages in genetically susceptible mice infected with Leishmania major. Infect. Immun. 54:386-394. Coombs, G. H. 1982. Proteinases of Leishmania mexicana and other flagellate protozoa. Parasitology 84:149-155. Farrel, J. P., and C. E. Kirkpatrick. 1987. Experimental cutaneous leishmaniasis. II. A possible role for prostaglandins in exacerbation of disease in Leishmania major-infected BALB/c mice. J. Immunol. 138:902-907. Handman, E., and G. F. Mitchell. 1985. Immunization with Leishmania receptor for macrophage protects mice against cutaneous leishmaniasis. Proc. Natl. Acad. Sci. USA 82:59105914. Ho, M. D., E. Koech, D. W. Iha, and A. D. Brycenson. 1983. Immunosuppression in Kenya visceral leishmaniasis. Clin. Exp. Immunol. 51:207-214. Jones, T. C., W. D. Johnson, A. C. Barreto, E. Lago, R. Badaro, B. Cerf, S. G. Reed, E. M. Netto, M. S. Tada, F. Franca, K. Wiek, L. Golightly, E. Fikrig, J. M. Costa, C. C. Cuba, and P.

1770

RODRIGUES ET AL.

Marscen. 1987. Epidemiology of American cutaneous leishmaniasis due to Leishmania braziliensis braziliensis. J. Infect. Dis. 156:73-83. 10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 221:680-685. 11. Liew, F. Y., C. Hale, and J. G. Howard. 1982. Immunological regulation of experimental cutaneous leishmaniasis. V. Characterization of effector and specific suppressor T cell. J. Immunol.

128:1917-1922. 12. Mendonca-Previato, L., P. A. J. Gorin, A. F. Braga, J. Scharfstein, and J. 0. Previato. 1983. Chemical structure and antigenic complexes from epimastigotes of Trypanosoma cruzi. Biochemistry 22:4380-4387. 13. Mitchell, G. F., and E. Handman. 1986. The glycoconjugate derived from a Leishmania major receptor for macrophages is a suppressogenic, disease-promoting antigen in murine cutaneous leishmaniasis. Parasite Immunol. 8:255-262. 14. Mitchell, G. T. 1985. Murine cutaneous leishmaniasis: resistance in reconstituted nude mice and several F 1 hybrids infected with Leishmania major. J. Immunogenet. 10:395-409. 15. Murray, H. M., B. Rubin, S. Carriero, and A. M. Acosta. 1984. Reversible defect in antigen-induced lymphokine and gamma interferon in cutaneous leishmaniasis. J. Immunol. 133:22502254. 16. Murray, H. W., H. Masur, and J. Keithly. 1982. Cell mediated immune response in experimental visceral leishmaniasis. I. Correlation between resistance to Leishmania donovani and lymphokine-generating capacity. J. Immunol. 129:344-350. 17. Petersen, E. A., F. A. Neva, A. Barral, R. Correz-Coronas, H. Bogaert-Diaz, D. Martinez, and F. E. Ward. 1984. Monocyte suppression of antigen-specific lymphocyte responses in diffuse cutaneous leishmaniasis patients from Dominican Republic. J. Immunol. 132:2603-2606.

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18. Petersen, E. A., F. A. Neva, C. N. Oster, and H. B. Diaz. 1982. Specific inhibition of lymphocyte-proliferation responses by adherent suppressor cells in diffuse cutaneous leishmaniasis. N. Engl. J. Med. 306:387-392. 19. Rodrigues, M. M., L. Mendonsa-Previato, R. Charlab, and M. A. Barcinski. 1987. The cellular immune response to a purified antigen from Leishmania mexicana subsp. amazonensis enhances the size of the leishmanial lesion on susceptible mice. Infect. Immun. 55:3142-3148. 20. Rodrigues, M. M., M. T. Xavier, L. M. Previato, and M. A. Barcinski. 1986. Characterization of cellular immune response to chemically defined glycoconjugates from Leishmania mexicana subsp. amazonensis. Infect. Immun. 51:80-86. 21. Russell, D. G., and J. Alexander. 1988. Effective immunization against cutaneous leishmaniasis with defined membrane antigens reconstituted into liposomes. J. Immunol. 140:1274-1279. 22. Scott, P. A., and J. P. Farrell. 1981. Experimental cutaneous leishmaniasis. I. Non-specific immunodepression in BALB/c mice infected with Leishmania tropica. J. Immunol. 127:23952400. 23. Titus, R. G., R. Ceredig, J. C. Cerottini, and J. A. Louis. 1985. Therapeutic effect of anti-L3T4 monoclonal antibody GK 1.5 on cutaneous leishmaniasis in genetically susceptible BALB/c mice. J. Immunol. 135:2108-2114. 24. Titus, R. G., G. C. Lima, H. D. Engers, and J. A. Louis. 1984. Exacerbation of murine cutaneous leishmaniasis by adoptive transfer of parasite specific helper T cell population capable of mediating L. major specific delayed type hypersensitivity. J. Immunol. 133:1594-1600. 25. Towbin, H., T. Staehlin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4359.