- Email: [email protected]
Subsets of specific T cells and experimental cutaneous leishmaniasis
IMMUNITY TO LEISHMANIA
755
[30] SCOTT, P.A. & FARRELL, J.P., Experimental cutaneous leishmaniasis. - 1. Non-specific immunosuppression in BALB/c mice infected with Leishmania tropica. J.Immunol., 1981,127,2395-2400. [31] REINER, N.E. & FINKE, J.H., Interleukin 2 deficiency in murine Leishmania donovani and its relationship to depressed spleen cell responses to phytohemagglutinin. J. Immunol., 1983, 131, 1487-1491. [32] OLLARI, E., LIEw, F.Y. & LELCHUK, R., Suppression of interleukin-2 production by macrophages in susceptible BALB/c mice infected with Leishmania major. Inject. Immun., 1986, 54, 386-394. [33] MIRKOVICH, A.M., GALELLI, A., ALLISON, A.C. & MODABBER, F.Z., Increased myelopoiesis during Leishmania major infection in mice: generation of « safe target », a possible way to evade the effector immune mechanism. Clin. expo Immunol., 1986,64,1-7. [34] LELCHUK, R., GRAVELEY, R. & LIEW, F.Y., Susceptibility to murine cutaneous leishmaniasis correlates with the capacity to generate interleukin-3 in response to leishmania antigen in vitro. Cell. Immunol., 1987 (in press). [35] GERGENS, R.L. & MARR, J.J., Growth of L. donovani amastigotes in a continuous macrophage-like cell culture. J. Protozool., 1979, 26, 453-462. [36] HOOVER, D.L. & NACY, C., Macrophage activation to kill L. tropica: defective intracellular killing of amastigotes by macrophages elicited with sterile inflammatory agents. J. Immunol., 1984, 132, 1487-1493.
SUBSETS OF SPECIFIC T CELLS AND EXPERIMENTAL CUTANEOUS LEISHMANIASIS J.A. Louis, T. Pedrazzini, R.G. Titus, I. Muller, J.P. Farrell, V. Kindler, P. Vassalli, G. Marchal and G. Milon
WHO Immunology Research and Training Centre, Institute oj Biochemistry, University oj Lausanne, Epalinges, Switzerland; Department oj Pathology, University oj Geneva, Switzerland and the Cellular Immunophysiology Unit, Institut Pasteur, Paris The murine model of infection with
Leishmania major represents an impor-
tant system for the analysis of the cellular parameters which entail susceptibility or resistance to infection with L. major. Indeed, depending on the genetic background of the mice, the entire spectrum of the clinical manifestations observed in human leishmaniasis can be seen. After s.c. infection with L. major, resistant mice (e.g. CBA) develop locally small lesions which resolve spontaneously, whereas in susceptible mice (e.g. BALB/c), these lesions are much more severe, do not heal and visceralisation occurs [1, 2].
Several studies have shown that the expression of disease in experimentally induced murine leishmaniasis depends upon the activity of macrophages the host cells in which L. major replicates [3, 1]. Furthermore, the virulence of Leishmania could be related to surface molecules involved in its binding to and survival in mononuclear phagocytes [4]. A considerable body of evidence from various laboratories also strongly indicates that specific T-cell responses generated during infection playa crucial role both in the resolution and progression of cutaneous leishmaniasis (reviewed in [5, 6, 7]). Rather than reviewing
756
20th FORUM IN IMMUNOLOGY
the literature on this last issue, the manuscript will summarize the work performed in our laboratories pertaining to the assessment of the possible role of T-cell responses in favouring susceptibility or resistance to infection with L. major in mice. Importance of L3T4 + T cells: in vivo analysis. In an attempt to evaluate the role of L3T4 + T cells in susceptibility or resistance to infection with L. major, we have studied the course of infection in mice in which the pool of L3T4 + cells had been reduced by administration of anti-L3T4 monoclonal antibodies. It was observed that virtual elimination (> 95 %) of L3T4 + T cells by intensive treatment with anti-L3T4 mAb resulted in the development of severe uncontrolled lesions in both susceptible and resistant mice [8]. These results clearly show that L3T4 + cells play a major role in the resolution of L. majorinduced lesions. Interestingly, although the elimination of only 60-70 % of L3T4 + cells from lymphoid tissues still led to an exacerbation of lesions in genetically resistant CBA mice, this treatment completely reversed the exquisite susceptibility of BALBIc mice to infection with L. major [9, 10]. These results strongly indicate that L3T4 + T cells can also be detrimental to the host. In a second approach, the number and phenotype of parasite-specific T cells triggered in lymphoid tissues as a result of infection were compared in susceptible and resistant mice. T cells were scored by determining their ability to locally transfer a delayed-type hypersensitivity reaction to viable L. major promastigotes and their frequencies determined by classical limiting dilution analysis. Results have shown that the frequency of specific T cells was significantly higher (up to 50 times) in the lymph nodes draining the lesions of susceptible mice than in resistant mice, as assessed various times after infection. Furthermore, in susceptible BALB/c mice, 90 % of these specific T cells (i.e., able to mediate DTH) expressed the L3T4 + cell surface phenotype [11].
Therefore, using a DTH assay to detect
L. major-specificT cells, it appears that
the immune response of susceptible mice to infection with L. major is characterized by the generation of high numbers of specific L3T4 + T cells. Although it is realized that our DTH assay might not detect all specific T cells, these results together with observations showing that reduction of L3T4 + cells in vivo by treatment with anti-L3T4 mAb renders genetically susceptible mice resistant to infection with L. major led us to speculate that the protective role of specific L3T4 + T cells may depend upon the actual number of such cells that are generated during infection [10, 11]. Alternatively, it is possible that the L3T4 + response is qualitatively different between susceptible and resistant mice. According to this hypothesis, two types of specific L3T4 + T cells are generated during infection with L. major: one cell type having mainly a protective effect (preferentially induced in resistant mice), another type favouring the development of lesions (preferentially induced in susceptible mice). The outcome of infection would then be dependent upon the balance between these functional subsets [6]. Indirect evidence which could suggest the triggering in lymphoid tissues of two functionally distinct parasitespecific L3T4 + T-cell populations has recently been obtained. We have observed that the cutaneous lesions of BALBIc mice treated with a single injection of anti-L3T4 mAb (GK 1.5) 10 days after initiation of infection resolve completely around 60 days after infection. At that time, the absolute number of L3T4 + T cells in the lymph nodes of these mice was found to be similar to that seen in normal mice. Compared to control mice similarly infected but not treated with mAb GK 1.5, the L3T4 + lymph node cells of mice treated with a unique dose of anti-L3T4 mAb provided similar helper activity, as tested in a secondary antiDNP antibody response in vitro using DNP-L. major as the immunogen. In sharp contrast, the frequency of L3T4 + T cells able to locally transfer the DTH reaction was drastically redu-
IMMUNITY TO LEISHMANIA ced in lymph nodes of these mice (T. Pedrazzini, G. Milon, G. Marchal and J. Louis, in preparation). Importance of Lyt-2 + T cells: in vivo analysis. Depletion of Lyt-2 + cells in vivo by administration of anti-Lyt-2 mAb before and during infection with L. major resulted in the exacerbation of lesions in both susceptible and resistant mice [8]. However, it is important to emphasize that resistant mice severely depleted of Lyt-2 + T cells were still capable of healing their lesions. Furthermore, compared to susceptible mice, higher numbers of specific Lyt-2 + T cells able to mediate DTH reactions were present in lymph nodes draining the lesions of resistant mice just before the onset of resolution of lesions. These results suggest that specific Lyt-2 + cells, although not being the main protective cells, also contribute to the immunological control of experimental leishmaniasis. L. major-specificL3T4 + T cells that exacerbate cutaneous leishmaniasis.
Because of the centrol role of T cells in resistance and susceptibility to infection with L. major, efforts were made in our laboratory to derive homogeneous populations and clones of parasite-specific T cells in vitro, to characterize these cells phenotypically and functionally and to study their effect on the course of cutaneous l~ishmaniasis. Following a protocol described in detail elsewhere, L3T4 + T-cell populations and clones specific for L. major antigens were derived and functionally characterized [12, 13]. Recent studies have revealed that, after activation by L. major antigen in vitro, these L3T4 + T cells release, in addition to macrophage-activating factors (MAF), considerable amounts of haemopoietic stimulating factors, namely multilineage colony-stimulating factor (interleukin 3 or IL3) and granulocyte macrophage colonystimulating factors (GM-CSF) ([10] and
757
unpublished observations). Interestingly, all L3T4 + T-cell lines and clones tested so far were shown, after adoptive transfer i. v. in syngeneic recipients, to exacerbate the course of cutaneous leishmaniasis [14]. This effect was neither the consequence of the production of specific antibodies by the host as a result of adoptive transfer of «helper» T cells, nor the result of the induction of suppressor cells by the host [10]. On the basis of a study showing that significant numbers of the i. v. injected L3T4 + T cells localized rapidly into the lesion site, we proposed that these cells exacerbate the development of lesions by continuously recruiting, to the site of infection, blood-derived phagocytes, the host cells required for the growth of Leishmania [9, 11]. This hypothesis has now been experimentally substantiated [10]. Furthermore, since, after activation, these L3T4 + T cells release molecules capable of modulating the proliferation and the differentiation of haemopoietic progenitor cells, it is possible that these factors increase the pool of circulating phagocytes recruitable to the lesions, thus favouring the multiplication of the parasites. This contention is supported by recent observations which have shown that treatment of susceptible BALB/c mice with recombinant IL-3 resulted in the development of more severe lesions (V. Kindler, P. Vassali and J. Louis, in preparation). Although the exacerbation of cutaneous leishmaniasis by these L3T4 + T cells can already be explained by their capacity to release molecules capable of (a) increasing the pool of circulating phagocytes and (b) recruiting these phagocytes to the site of lesions, recent observations indicate that, in addition, the incubation of some of these haemopoietic colony-stimulating molecules (IL-3 and GM-CSF) with cultures of macrophages infected with L. major promotes the growth of intracellular L. major in vitro (V. Kindler, J. Louis and P. Vassalli, in preparation). Inasmuch as, in titration experiments, these L3T4 + T cells either exacerbated the development of lesions or had no effect on the course of
758
20th FORUM IN IMMUNOLOGY
disease, it is likely that they represent a functionally homogenous population of L3T4 + T cells capable of promoting the growth of parasites in vivo. So far, we have not been able to derive and maintain in vitro homogenous populations of specific L3T4 + T cells having a protective activity against cutaneous leishmaniasis. This failure could indicate that «protective» and «dele-
terious» L3T4 + T cells have different requirements for growth, at least in vitro. It is clear that studies aimed at delineating either the specificity, the functional activities or the parameters of activation of these two types of parasite-specific L3T4 + T cells will be greatly facilitated by the possibility of maintaining them in vitro.
References. [1] BEHIN, R., MAUEL, J. & SORDAT, B., Leishmania tropica: pathogenicity and in vitro macrophage function in strains of inbred mice. Exp. Parasito/., 1979, 48, 81-91. [2] HANDMAN, E., CEREDIG, R. & MITCHELL, G.F., Murine cutaneous leishmaniasis: disease patterns in intact and nude mice of various genotypes and examination of some differences between normal and infected macrophages. Aust. J. expo Bio/. med. Sci., 1979, 57, 9-18. [3] NACY, C.A., FORTIER, A.H., PAPPAS, M.G. & HENRY, R.R., Susceptibility of inbred mice to Leishmania tropica infection: correlation of susceptibility with in vitro defective macrophage microbicidal activities. Cell. immuno/., 1983, 77, 298-307. [4] HANDMAN, E., SCHNUR, L.F., SPITHILL, T.W. & MITCHELL, G.F., Passive transfer of leishmania lipopolysaccharide confers parasite survival in macrophages. J. Immuno/., 1986, 137, 3608-3613. [5] LIEw, F.Y., In «Mechanisms of host resistance to infectious agents, tumors and allografts» (R.M. Steinman and R.J. North) (p. 305). Rockefeller University Press, New York, 1986. [6] MITCHELL, G.F., Host-protective immunity and its suppression in a parasitic disease: murine cutaneous leishmaniasis. Immuno/. Today, 1984, 5, 224-226. [7] MITCHELL, G.F. & HANDMAN, E., T lymphocytes recognize Leishmania glycoconjugates. Parasito/. Today, 1985, 1, 61-64. [8] TITUS, R.G., MILON, G., MARCHAL, G., VASSALLI, P., CEROTTINI, J.C. & LOUIS, J.A., Involvement of specific Lyt-2 + T cells in the immunological control of experimentally induced murine cutaneous leishmaniasis. Europ. J. Immuno/., 1987 (in press). [9] TITUS, R.G., CEREDIG, R., CEROTTINI, J.e. & LOUIS, J.A., Therapeutic effect of anti-L3T4 monoclonal antibody GK 1.5 on cutaneous leishmaniasis in genetically-susceptible BALB/c mice. J. Immuno/., 1985, 135, 2108-2114. [10] LOUIS, J.A., MENDONl;A, S., TITUS, R.G., CEROTTINI, J.C., CERNY, A., ZINKERNAGEL, R., MILON, G. & MARCHAL, G., The role of specific T-cel1 subpopulations in murine cutaneous leishmaniasis, in «Progress in immunology VI» (B. Cinader and R.G. Miller) (pp. 762-769). Academic Press, New York, London, 1986. [11] MILON, G., TITUS, R.G., CEROTTINI, J.C., MARCHAL, G. & LOUIS, J.A., Higher frequency of Leishmania major-specific L3T4 + T cells in susceptible BALB/c as compared with resistant CBA mice. J. Immuno/., 1986, 136, 1467-1471. [12] LOUIS, J.A., LIMA, G., PESTEL, J., TITUS, R. & ENGERS, H.D., Murine T-cell responses to protozoan and metazoan parasites: functional analysis of T-cell lines and clones specific for Leishmania tropica and Schistosoma mansoni. Contemp. topics Immunobio/., 1984, 12, 201-224. [l3] LOUIS, J.A., ZUBLER, R.H., COUTINHO, S.G., LIMA, G., BEHIN, R., MAUEL, J. & ENGERS, H.D., The in vitro generation and functional analysis of murine T-cell populations and clones specific for a protozoan parasite, Leishmania tropica. Immuno/. Rev., 1982, 61, 215-243. [14] TITUS, R.G., LIMA, G.e., ENGERS, H.D. & LOUIS, J .A., Exacerbation of murine cutaneous leishmaniasis by adoptive transfer of parasite-specific helper T-cel1 populations capable of mediating Leishmania major-specific delayed-type hypersensitivity. J. Immuno/., 1984, 133, 1594-1600. This work has been supported by grants from the Swiss National Science Foundation, the UNDPlWorid Bank/WHO Special Programme on Tropical Diseases, the CNRS and the Institut Pasteur. The excellent technical assistance of K. Hug and A. Porret is gratefully acknowledged.
755
[30] SCOTT, P.A. & FARRELL, J.P., Experimental cutaneous leishmaniasis. - 1. Non-specific immunosuppression in BALB/c mice infected with Leishmania tropica. J.Immunol., 1981,127,2395-2400. [31] REINER, N.E. & FINKE, J.H., Interleukin 2 deficiency in murine Leishmania donovani and its relationship to depressed spleen cell responses to phytohemagglutinin. J. Immunol., 1983, 131, 1487-1491. [32] OLLARI, E., LIEw, F.Y. & LELCHUK, R., Suppression of interleukin-2 production by macrophages in susceptible BALB/c mice infected with Leishmania major. Inject. Immun., 1986, 54, 386-394. [33] MIRKOVICH, A.M., GALELLI, A., ALLISON, A.C. & MODABBER, F.Z., Increased myelopoiesis during Leishmania major infection in mice: generation of « safe target », a possible way to evade the effector immune mechanism. Clin. expo Immunol., 1986,64,1-7. [34] LELCHUK, R., GRAVELEY, R. & LIEW, F.Y., Susceptibility to murine cutaneous leishmaniasis correlates with the capacity to generate interleukin-3 in response to leishmania antigen in vitro. Cell. Immunol., 1987 (in press). [35] GERGENS, R.L. & MARR, J.J., Growth of L. donovani amastigotes in a continuous macrophage-like cell culture. J. Protozool., 1979, 26, 453-462. [36] HOOVER, D.L. & NACY, C., Macrophage activation to kill L. tropica: defective intracellular killing of amastigotes by macrophages elicited with sterile inflammatory agents. J. Immunol., 1984, 132, 1487-1493.
SUBSETS OF SPECIFIC T CELLS AND EXPERIMENTAL CUTANEOUS LEISHMANIASIS J.A. Louis, T. Pedrazzini, R.G. Titus, I. Muller, J.P. Farrell, V. Kindler, P. Vassalli, G. Marchal and G. Milon
WHO Immunology Research and Training Centre, Institute oj Biochemistry, University oj Lausanne, Epalinges, Switzerland; Department oj Pathology, University oj Geneva, Switzerland and the Cellular Immunophysiology Unit, Institut Pasteur, Paris The murine model of infection with
Leishmania major represents an impor-
tant system for the analysis of the cellular parameters which entail susceptibility or resistance to infection with L. major. Indeed, depending on the genetic background of the mice, the entire spectrum of the clinical manifestations observed in human leishmaniasis can be seen. After s.c. infection with L. major, resistant mice (e.g. CBA) develop locally small lesions which resolve spontaneously, whereas in susceptible mice (e.g. BALB/c), these lesions are much more severe, do not heal and visceralisation occurs [1, 2].
Several studies have shown that the expression of disease in experimentally induced murine leishmaniasis depends upon the activity of macrophages the host cells in which L. major replicates [3, 1]. Furthermore, the virulence of Leishmania could be related to surface molecules involved in its binding to and survival in mononuclear phagocytes [4]. A considerable body of evidence from various laboratories also strongly indicates that specific T-cell responses generated during infection playa crucial role both in the resolution and progression of cutaneous leishmaniasis (reviewed in [5, 6, 7]). Rather than reviewing
756
20th FORUM IN IMMUNOLOGY
the literature on this last issue, the manuscript will summarize the work performed in our laboratories pertaining to the assessment of the possible role of T-cell responses in favouring susceptibility or resistance to infection with L. major in mice. Importance of L3T4 + T cells: in vivo analysis. In an attempt to evaluate the role of L3T4 + T cells in susceptibility or resistance to infection with L. major, we have studied the course of infection in mice in which the pool of L3T4 + cells had been reduced by administration of anti-L3T4 monoclonal antibodies. It was observed that virtual elimination (> 95 %) of L3T4 + T cells by intensive treatment with anti-L3T4 mAb resulted in the development of severe uncontrolled lesions in both susceptible and resistant mice [8]. These results clearly show that L3T4 + cells play a major role in the resolution of L. majorinduced lesions. Interestingly, although the elimination of only 60-70 % of L3T4 + cells from lymphoid tissues still led to an exacerbation of lesions in genetically resistant CBA mice, this treatment completely reversed the exquisite susceptibility of BALBIc mice to infection with L. major [9, 10]. These results strongly indicate that L3T4 + T cells can also be detrimental to the host. In a second approach, the number and phenotype of parasite-specific T cells triggered in lymphoid tissues as a result of infection were compared in susceptible and resistant mice. T cells were scored by determining their ability to locally transfer a delayed-type hypersensitivity reaction to viable L. major promastigotes and their frequencies determined by classical limiting dilution analysis. Results have shown that the frequency of specific T cells was significantly higher (up to 50 times) in the lymph nodes draining the lesions of susceptible mice than in resistant mice, as assessed various times after infection. Furthermore, in susceptible BALB/c mice, 90 % of these specific T cells (i.e., able to mediate DTH) expressed the L3T4 + cell surface phenotype [11].
Therefore, using a DTH assay to detect
L. major-specificT cells, it appears that
the immune response of susceptible mice to infection with L. major is characterized by the generation of high numbers of specific L3T4 + T cells. Although it is realized that our DTH assay might not detect all specific T cells, these results together with observations showing that reduction of L3T4 + cells in vivo by treatment with anti-L3T4 mAb renders genetically susceptible mice resistant to infection with L. major led us to speculate that the protective role of specific L3T4 + T cells may depend upon the actual number of such cells that are generated during infection [10, 11]. Alternatively, it is possible that the L3T4 + response is qualitatively different between susceptible and resistant mice. According to this hypothesis, two types of specific L3T4 + T cells are generated during infection with L. major: one cell type having mainly a protective effect (preferentially induced in resistant mice), another type favouring the development of lesions (preferentially induced in susceptible mice). The outcome of infection would then be dependent upon the balance between these functional subsets [6]. Indirect evidence which could suggest the triggering in lymphoid tissues of two functionally distinct parasitespecific L3T4 + T-cell populations has recently been obtained. We have observed that the cutaneous lesions of BALBIc mice treated with a single injection of anti-L3T4 mAb (GK 1.5) 10 days after initiation of infection resolve completely around 60 days after infection. At that time, the absolute number of L3T4 + T cells in the lymph nodes of these mice was found to be similar to that seen in normal mice. Compared to control mice similarly infected but not treated with mAb GK 1.5, the L3T4 + lymph node cells of mice treated with a unique dose of anti-L3T4 mAb provided similar helper activity, as tested in a secondary antiDNP antibody response in vitro using DNP-L. major as the immunogen. In sharp contrast, the frequency of L3T4 + T cells able to locally transfer the DTH reaction was drastically redu-
IMMUNITY TO LEISHMANIA ced in lymph nodes of these mice (T. Pedrazzini, G. Milon, G. Marchal and J. Louis, in preparation). Importance of Lyt-2 + T cells: in vivo analysis. Depletion of Lyt-2 + cells in vivo by administration of anti-Lyt-2 mAb before and during infection with L. major resulted in the exacerbation of lesions in both susceptible and resistant mice [8]. However, it is important to emphasize that resistant mice severely depleted of Lyt-2 + T cells were still capable of healing their lesions. Furthermore, compared to susceptible mice, higher numbers of specific Lyt-2 + T cells able to mediate DTH reactions were present in lymph nodes draining the lesions of resistant mice just before the onset of resolution of lesions. These results suggest that specific Lyt-2 + cells, although not being the main protective cells, also contribute to the immunological control of experimental leishmaniasis. L. major-specificL3T4 + T cells that exacerbate cutaneous leishmaniasis.
Because of the centrol role of T cells in resistance and susceptibility to infection with L. major, efforts were made in our laboratory to derive homogeneous populations and clones of parasite-specific T cells in vitro, to characterize these cells phenotypically and functionally and to study their effect on the course of cutaneous l~ishmaniasis. Following a protocol described in detail elsewhere, L3T4 + T-cell populations and clones specific for L. major antigens were derived and functionally characterized [12, 13]. Recent studies have revealed that, after activation by L. major antigen in vitro, these L3T4 + T cells release, in addition to macrophage-activating factors (MAF), considerable amounts of haemopoietic stimulating factors, namely multilineage colony-stimulating factor (interleukin 3 or IL3) and granulocyte macrophage colonystimulating factors (GM-CSF) ([10] and
757
unpublished observations). Interestingly, all L3T4 + T-cell lines and clones tested so far were shown, after adoptive transfer i. v. in syngeneic recipients, to exacerbate the course of cutaneous leishmaniasis [14]. This effect was neither the consequence of the production of specific antibodies by the host as a result of adoptive transfer of «helper» T cells, nor the result of the induction of suppressor cells by the host [10]. On the basis of a study showing that significant numbers of the i. v. injected L3T4 + T cells localized rapidly into the lesion site, we proposed that these cells exacerbate the development of lesions by continuously recruiting, to the site of infection, blood-derived phagocytes, the host cells required for the growth of Leishmania [9, 11]. This hypothesis has now been experimentally substantiated [10]. Furthermore, since, after activation, these L3T4 + T cells release molecules capable of modulating the proliferation and the differentiation of haemopoietic progenitor cells, it is possible that these factors increase the pool of circulating phagocytes recruitable to the lesions, thus favouring the multiplication of the parasites. This contention is supported by recent observations which have shown that treatment of susceptible BALB/c mice with recombinant IL-3 resulted in the development of more severe lesions (V. Kindler, P. Vassali and J. Louis, in preparation). Although the exacerbation of cutaneous leishmaniasis by these L3T4 + T cells can already be explained by their capacity to release molecules capable of (a) increasing the pool of circulating phagocytes and (b) recruiting these phagocytes to the site of lesions, recent observations indicate that, in addition, the incubation of some of these haemopoietic colony-stimulating molecules (IL-3 and GM-CSF) with cultures of macrophages infected with L. major promotes the growth of intracellular L. major in vitro (V. Kindler, J. Louis and P. Vassalli, in preparation). Inasmuch as, in titration experiments, these L3T4 + T cells either exacerbated the development of lesions or had no effect on the course of
758
20th FORUM IN IMMUNOLOGY
disease, it is likely that they represent a functionally homogenous population of L3T4 + T cells capable of promoting the growth of parasites in vivo. So far, we have not been able to derive and maintain in vitro homogenous populations of specific L3T4 + T cells having a protective activity against cutaneous leishmaniasis. This failure could indicate that «protective» and «dele-
terious» L3T4 + T cells have different requirements for growth, at least in vitro. It is clear that studies aimed at delineating either the specificity, the functional activities or the parameters of activation of these two types of parasite-specific L3T4 + T cells will be greatly facilitated by the possibility of maintaining them in vitro.
References. [1] BEHIN, R., MAUEL, J. & SORDAT, B., Leishmania tropica: pathogenicity and in vitro macrophage function in strains of inbred mice. Exp. Parasito/., 1979, 48, 81-91. [2] HANDMAN, E., CEREDIG, R. & MITCHELL, G.F., Murine cutaneous leishmaniasis: disease patterns in intact and nude mice of various genotypes and examination of some differences between normal and infected macrophages. Aust. J. expo Bio/. med. Sci., 1979, 57, 9-18. [3] NACY, C.A., FORTIER, A.H., PAPPAS, M.G. & HENRY, R.R., Susceptibility of inbred mice to Leishmania tropica infection: correlation of susceptibility with in vitro defective macrophage microbicidal activities. Cell. immuno/., 1983, 77, 298-307. [4] HANDMAN, E., SCHNUR, L.F., SPITHILL, T.W. & MITCHELL, G.F., Passive transfer of leishmania lipopolysaccharide confers parasite survival in macrophages. J. Immuno/., 1986, 137, 3608-3613. [5] LIEw, F.Y., In «Mechanisms of host resistance to infectious agents, tumors and allografts» (R.M. Steinman and R.J. North) (p. 305). Rockefeller University Press, New York, 1986. [6] MITCHELL, G.F., Host-protective immunity and its suppression in a parasitic disease: murine cutaneous leishmaniasis. Immuno/. Today, 1984, 5, 224-226. [7] MITCHELL, G.F. & HANDMAN, E., T lymphocytes recognize Leishmania glycoconjugates. Parasito/. Today, 1985, 1, 61-64. [8] TITUS, R.G., MILON, G., MARCHAL, G., VASSALLI, P., CEROTTINI, J.C. & LOUIS, J.A., Involvement of specific Lyt-2 + T cells in the immunological control of experimentally induced murine cutaneous leishmaniasis. Europ. J. Immuno/., 1987 (in press). [9] TITUS, R.G., CEREDIG, R., CEROTTINI, J.e. & LOUIS, J.A., Therapeutic effect of anti-L3T4 monoclonal antibody GK 1.5 on cutaneous leishmaniasis in genetically-susceptible BALB/c mice. J. Immuno/., 1985, 135, 2108-2114. [10] LOUIS, J.A., MENDONl;A, S., TITUS, R.G., CEROTTINI, J.C., CERNY, A., ZINKERNAGEL, R., MILON, G. & MARCHAL, G., The role of specific T-cel1 subpopulations in murine cutaneous leishmaniasis, in «Progress in immunology VI» (B. Cinader and R.G. Miller) (pp. 762-769). Academic Press, New York, London, 1986. [11] MILON, G., TITUS, R.G., CEROTTINI, J.C., MARCHAL, G. & LOUIS, J.A., Higher frequency of Leishmania major-specific L3T4 + T cells in susceptible BALB/c as compared with resistant CBA mice. J. Immuno/., 1986, 136, 1467-1471. [12] LOUIS, J.A., LIMA, G., PESTEL, J., TITUS, R. & ENGERS, H.D., Murine T-cell responses to protozoan and metazoan parasites: functional analysis of T-cell lines and clones specific for Leishmania tropica and Schistosoma mansoni. Contemp. topics Immunobio/., 1984, 12, 201-224. [l3] LOUIS, J.A., ZUBLER, R.H., COUTINHO, S.G., LIMA, G., BEHIN, R., MAUEL, J. & ENGERS, H.D., The in vitro generation and functional analysis of murine T-cell populations and clones specific for a protozoan parasite, Leishmania tropica. Immuno/. Rev., 1982, 61, 215-243. [14] TITUS, R.G., LIMA, G.e., ENGERS, H.D. & LOUIS, J .A., Exacerbation of murine cutaneous leishmaniasis by adoptive transfer of parasite-specific helper T-cel1 populations capable of mediating Leishmania major-specific delayed-type hypersensitivity. J. Immuno/., 1984, 133, 1594-1600. This work has been supported by grants from the Swiss National Science Foundation, the UNDPlWorid Bank/WHO Special Programme on Tropical Diseases, the CNRS and the Institut Pasteur. The excellent technical assistance of K. Hug and A. Porret is gratefully acknowledged.