Cutaneous leishmaniasis in anti-IgM-treated mice: enhanced resistance due to functional depletion of a B cell-dependent T cell involved in the suppressor pathway

The contribution of B cells and antibodies to either the resistance or susceptibility to cutaneous leishmaniasis has been investigated in mouse strains rendered B cell-deficient by treatment with anti-mouse IgM antisera from birth (mu-suppressed). These studies confirm that immunity to cutaneous disease in a normally resistant mouse strain (C3H/HeJ) is independent of antibody, but that B cells and/or antibodies are required for the evolution of suppressed DTH and the consequent disease susceptibility of BALB/c mice. Anti-IgM-treated BALB/c mice, which lacked detectable anti-leishmanial antibodies during the course of infection, displayed a sustained DTH response to leishmanial antigen and were able to control their cutaneous lesions. The enhanced resistance of mu-suppressed mice could be completely abrogated by transfer of suppressor T cells from infected control animals into mu-suppressed mice before their infection. Thus the suppressor T cells, which are generated during leishmanial infection in BALB/c mice, can effect suppression in the absence of antibody. Evidence that B cells or antibodies are required for the generation of suppressor T cells was demonstrated by using BALB/c mice in which suppressor T cells fail to be generated during infection as a result of prior sublethal irradiation. Splenic T cells from normal mice could overcome the resistance conferred by sublethal irradiation, whereas splenic T cells from mu-suppressed mice could not. Thus the enhanced resistance of mu-suppressed BALB/c mice appears to be a consequence of their lack of functional expression of a B cell-dependent T cell critical to the suppressor pathway.     

The chemotherapeutic effect of praziquantel against Schistosoma mansoni is dependent on host antibody response

To assess the role of host humoral immune responses in the mechanism of action of praziquantel (PZQ) against Schistosoma mansoni, the efficacy of the drug was compared in infected B cell-depleted (mu-suppressed) vs immunologically intact C3H/HeN mice. We found that PZQ was on the average only 20% as effective in eliminating adult schistosomes from mu-suppressed as compared with control animals. Indeed, in three of four experiments performed, the drug failed to significantly reduce adult worm burdens in the mu-suppressed mice. These results were not due to a delay in parasite death in the infected B cell-depleted animals, because adult worms recovered from these mice as late as 7 wk after chemotherapy were indistinguishable in number and appearance from those recovered from non-drug-treated animals. The efficacy of PZQ against schistosomes in mu-suppressed mice was completely restored by passive transfer of immune serum from donor mice infected for 6 wk and partially restored with IgG purified from the same sera. Moreover, IgG as well as IgM antibodies were detected by immunofluorescence on the surface of adult worms recovered from intact mice as early as 1 hr after administration of the drug in vivo. The tubercles of the male worms appeared to be a major site for antibody binding. These results formally demonstrate that the mechanism of action of PZQ, the most important anti-schistosomal compound in current use, involves a synergy between the drug and the humoral immune response of the host, and suggest that the relevant effector antibodies act directly against parasite antigens which become exposed on the surface of the worms as a consequence of interaction with the drug.     

Macrophages as effector cells of protective immunity in murine schistosomiasis. VI. T cell-dependent, lymphokine-mediated, activation of macrophages in response to Schistosoma mansoni antigens

Macrophages from Schistosoma mansoni-infected mice kill significant numbers of skin stage schistosomula and murine fibrosarcoma cells in vitro. In order to determine whether the macrophage tumoricidal and larvicidal activation observed in mice as a result of S. mansoni infection are mediated through T cell-dependent (lymphokine) or B cell-dependent (antibody or immune complex) mechanisms, the development of macrophage populations with cytotoxic activity against schistosome larvae or tumor cells was monitored in S. mansoni-infected nude or mu-suppressed mice. Whereas peritoneal cells from S. mansoni-infected congenitally athymic mice had no activity in either assay, cells from mu-suppressed S. mansoni-infected mice showed cytotoxic activity equivalent to that of cells from untreated S. mansoni-infected counterparts. Cells from mu-suppressed uninfected mice were not activated. The mu-suppressed animals had no detectable nonspecific IgM or specific antischistosome IgM, IgG, or IgE antibodies and showed a 90% reduction in numbers of splenic IgM+ cells upon fluorescence activated cell sorter analysis. These results indicate that antibody is not required for in vivo activation of macrophages during S. mansoni infection. Further experiments showed that lymphoid cells from S. mansoni infected mice respond in culture with various specific antigens (such as living or dead whole schistosomula or soluble adult worm antigens) by production of factors capable of activating macrophages from uninfected control mice to kill schistosomula or tumor cells in vitro. Macrophage-activating factors were produced by T cell-enriched, but not T cell-depleted or B cell-enriched, populations from spleens of schistosome-infected mice in response to schistosome antigen. Similar lymphokines may be responsible for the macrophage activation observed during chronic murine schistosomiasis. These observations emphasize the potential contribution of T cell-mediated immune mechanisms in resistance to S. mansoni infection.     

The role of tumor-specific Lyt-1+2- T cells in eradicating tumor cells in vivo. I. Lyt-1+2- T cells do not necessarily require recruitment of host's cytotoxic T cell precursors for implementation of in vivo immunity

The present study determines the Ly phenotype of T cells mediating tumor cell rejection in vivo and investigates some of cellular mechanisms involved in the in vivo protective immunity. C3H/HeN mice were immunized to syngeneic X5563 plasmacytoma by intradermal (i.d.) inoculation of viable X5563 tumor cells, followed by the surgical resection of the tumor. Spleen cells from these immune mice were fractionated by treatment with anti-Lyt antibodies plus complement, and each Lyt subpopulation was tested for the reconstituting potential of in vivo protective immunity in syngeneic T cell-depleted mice (B cell mice). When C3H/HeN B cell mice were adoptively transferred with Lyt-1-2+ T cells from the above tumor-immunized mice, these B cell mice exhibited an appreciable cytotoxic T lymphocyte (CTL) response to the X5563 tumor, whereas they failed to resist the i.d. challenge of X5563 tumor cells. In contrast, the adoptive transfer of Lyt-1+2- anti-X5563 immune T cells into B cell mice produced complete protection against the subsequent tumor cell challenge. Although no CTL or antibody response against X5563 tumors was detected in the above tumor-resistant B cell mice, these mice were able to retain Lyt-1+2- T cell-mediated delayed-type hypersensitivity (DTH) responses to the X5563 tumor. These results indicate that Lyt-1+2- T cells depleted of the Lyt-2+ T cell subpopulation containing CTL or CTL precursors are effective in in vivo protective immunity, and that these Lyt-1+2- T cells implement their in vivo anti-tumor activity without inducing CTL or antibody responses. The mechanism(s) by which Lyt-1+2- T cells function in vivo for the implementation of tumor-specific immunity is discussed in the context of DTH responses to the tumor-associated antigens and its related Lyt-1+2- T cell-mediated lymphokine production.     

Administration of monoclonal anti-IFN-gamma antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major

C3H/HeN mice that are naturally resistant to cutaneous disease and systemic infections with the protozoan parasite, Leishmania major, were treated at the time of infection, and weekly thereafter, with mouse anti-rat IFN-gamma mAb or an irrelevant antibody of similar isotype. Anti-IFN-gamma-treated mice developed cutaneous lesions; parasites spread to the regional lymph nodes and then metastasized to spleens and livers. The course of disease in these animals was similar to that of genetically susceptible BALB/c mice. Two exceptions in the pathology of L. major infections were noted between BALB/c and anti-IFN-gamma-treated C3H/HeN mice: 1) BALB/c mice died of systemic complications, whereas C3H/HeN mice did not, and 2) multinucleated giant cells were observed in lymph nodes and spleens of infected BALB/c mice, whereas these cells were not observed in infected C3H/HeN mice. Control mice, those treated with either saline or irrelevant antibody of the same isotype as the anti-IFN-gamma monoclonal, showed no evidence of cutaneous disease (development of footpad lesions) or systemic infection (by histopathology). Total abrogation of the natural resistance of C3H/HeN mice could be achieved by treatment with as little as 0.5 mg/mouse/wk of anti-IFN-gamma antibody, or by a single dose of 1 mg/mouse anti-IFN-gamma antibody administered at the time of parasite inoculation. If antibody treatment was delayed for as little as 1 wk after parasite inoculation, the infections in treated animals resembled that of untreated or control antibody-treated mice: no cutaneous lesions (by footpad swelling or viable counts of leishmania in footpad tissue) or systemic disease (by microscopic analysis of touch preparations of internal organs, and histopathology of same). The production of IFN-gamma during the initial interaction of the parasite and host cells appears to be a major component of genetic control of natural resistance to infection with L. major in C3H/HeN mice.     

Leishmania mexicana: Immunology and histopathology in C3H mice

C3H mice were infected subcutaneously with 10⁵ promastigotes of Leishmania mexicana and subsequent lesions were examined at 3, 5, and 8 months. All animals developed persistent nonulcerating nodules of variable size which did not metastasize. The nodules contained amastigotes with a mononuclear infiltrate of histiocytes, lymphocytes, and plasma cells, but without formation of tuberculoid-type granulomas. Neutrophils and eosinophils were also encountered in some cases. Specific antileishmanial antibodies and delayed-type hypersensitivity to leishmanial antigen were present at 3, 5, and 8 months postinfection. L. mexicana infection in C3H mice differs from classic self-healing cutaneous leishmaniasis by the pesistence of nonhealing, nonulcerating, nonmetastasizing lesions, despite evidence of cellular and humoral immunity.     

Delayed-type hypersensitivity response in mice to Pneumocystis carinii.

Resistance to Pneumocystis carinii infection appears to be mediated by T lymphocytes but the mechanism and subsets of T cells involved are poorly understood. We used the BALB/c mouse model to study the delayed-type hypersensitivity (DTH) response to rat P. carinii. Mice were sensitized to P. carinii for seven days and then challenged with P. carinii antigens in the right rear footpads and normal rat lung antigens in the left rear footpads. A typical DTH response was observed in the right footpads as evidenced by significant swelling and substantial mononuclear cell infiltration at 24-h post-challenge. The DTH response could be transferred to naive syngeneic mice by adoptively transferring spleen cells from P. carinii-sensitized mice. In addition, by using anti-thy-1, anti-mouse Ig, anti-L3T4 and anti-Lyt-2.2 monoclonal antibodies in in vitro cytolysis experiments, we were able to demonstrate that the DTH response was dependent upon T lymphocytes. The response appeared to require cooperation between both L3T4+ and Lyt 2+ subsets of T lymphocytes.     

Delayed-type hypersensitivity to hen egg-white lysozyme. I. The surface phenotypes of suppressor T cells induced by intravenous injection of lysozyme-modified spleen cells, and their molecular interactions

Suppressor T cells (Ts) induced by lysozyme-modified syngeneic lymphocytes were characterized. Hen egg-white lysozyme (HEL)-specific delayed-type hypersensitivity (DTH) was suppressed when HEL-induced Ts were transferred into naive mice. These HEL-induced Ts had surface markers of both Thy-1 antigen, and I-J gene products. The suppression of HEL-specific DTH was greatly increased, when these Ts had been enriched with HEL-coated petri dishes. Isolated anti-HEL antibodies from B10.BR or A/Sn mice were inoculated into rabbits to induce anti-cross-reactive idiotype (CRI) antibodies. The rabbit antisera were extensively absorbed with normal B10.BR or A/Sn immunoglobulins (Igs) and MOPC 104E ascites Igs to render them idiotype (Id) specific. Using these anti-CRI antibodies, we observed that these Ts possessed Id receptors on their cell surface. Results of both fluorescence techniques and cytotoxicity tests revealed that about 10% of the enriched T cells containing these Ts were Id positive. Moreover, these enriched T cells were substantially killed by anti-I-J antiserum plus complement. However, this killing was completely blocked by HEL antigen. These results suggest that both Id receptors and I-J gene products might be forming the same molecular complexes or might coexist in the vicinity of the molecule.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Both T and B cells shed infectious mouse mammary tumor virus.

Mouse mammary tumor virus (MMTV) infected both B and T tissue culture cells and primary B and T cells in vivo after milk-borne transmission of the virus. The infected tissue culture cells processed viral proteins, and both these and primary B and T cells shed virus when cultured in vitro. Moreover, the infected B and T tissue culture cells transmitted virus to uninfected mammary gland cells in vitro. The level of infection of these different cell types in vivo was dependent on the strain of mouse, with C3H/HeN mice showing greater B-cell infection and BALB/c mice greater T-cell infection after nursing on MMTV-infected C3H/HeN mothers. Although their B cells were less infected, BALB/c mice developed tumors more rapidly than C3H/HeN mice. These results indicate that both infected T and B cells are potential carriers of MMTV in vivo.     

Prophylactic immunization against experimental leishmaniasis. V. Mechanism of the anti-protective blocking effect induced by subcutaneous immunization against Leishmania major infection

The responsiveness of BALB/c mice to protective i.v. immunization with 150,000-rad irradiated or heat-killed Leishmania major promastigotes can be totally suppressed by prior subcutaneous (s.c.) injection of the same "vaccine." Induction of this effect is leishmania specific for although prevention of protection against L. major infection can be obtained with either homologous or Leishmania donovani promastigotes, it does not follow s.c. administration of an immunogenic Trypanosoma cruzi epimastigote preparation. Multiple s.c. injections of irradiated L. major promastigotes do not inhibit the subsequent antibody response of any major isotype to i.v. immunization, but rather induce some priming. The same s.c. injections induced delayed-type hypersensitivity (DTH) reactivity that could be transferred locally or systemically, although it was weaker than in mice with cured infections. Parallel cell-mediated immunity (CMI) responses were also reflected in vitro in specific lymphocyte transformation assays. Despite this evidence of a DTH/helper type of T cell response, transfer of 5 X 10(7) viable T cell-enriched spleen cells from 4 X s.c. immunized donors to normal recipients completely abrogated the protective response to i.v. immunization. Conversely, T cell-depleted (anti-Thy-1.2 + C treated) cells were without effect. The inhibitory T cells were defined by monoclonal antibody pretreatment as possessing an Lyt-1+2-,L3T4+ phenotype. T cells from s.c. immunized donors were also shown, by mixed transfer experiments, to counteract completely the protective effect of T cells from i.v. immunized donors in 550-rad irradiated recipients. They were as potent as suppressor T cells from donors with progressive disease both in this capacity and in abrogating the prophylactic effect of sublethal irradiation itself. The similarities and differences between suppressor and immune effector T cells induced by s.c. or i.v. immunization and those arising in response to leishmanial infection itself are discussed.     

T-T cell interaction in the induction of delayed-type hypersensitivity (DTH) responses: vaccinia virus-reactive helper T cell activity involved in enhanced in vivo induction of DTH responses and its application to augmentation of tumor-specific DTH responses

The role of antigen-specific helper T cells in augmenting the in vivo development of delayed-type hypersensitivity (DTH) responses was investigated. C3H/HeN mice were inoculated i.p. with vaccinia virus to generate virus-reactive helper T cell activity. These vaccinia virus-primed or unprimed mice were subsequently immunized subcutaneously (s.c.) with either trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self), vaccinia virus-infected spleen cells (virus-self), or cells modified with TNP subsequent to virus infection (virus-self-TNP). Seven days later, these mice were tested for anti-TNP DTH responses either by challenging them directly with TNP-self into footpads or by utilizing a local adoptive transfer system. The results demonstrated that vaccinia virus-primed mice failed to generate significant anti-TNP DTH responses when s.c. immunization was provided by either virus-self or TNP-self alone. In contrast, vaccinia virus-primed mice, but not unprimed mice, could generate augmented anti-TNP DTH responses when immunized with virus-self-TNP. Anti-vaccinia virus-reactive helper activity was successfully transferred into 600 R x-irradiated unprimed syngeneic mice by injecting i.v. spleen cells from virus-primed mice. These helper T cells were found to be antigen specific and were mediated by Thy-1+, Lyt-1+2- cells. DTH effector cells enhanced by helper T cells were also antigen specific and were of the Thy-1+, Lyt-1+2- phenotype. Furthermore, vaccinia virus-reactive helper T cell activity could be applied to augment the induction of tumor-specific DTH responses by immunization with vaccinia virus-infected syngeneic X5563 tumor cells. T-T cell interaction between Lyt-1+ helper T cells and Lyt-1+ DTH effector T cells is discussed in the light of the augmenting mechanism of in vivo anti-tumor-specific immune responses.     

Effect of treatment with cyclophosphamide in low doses upon the onset of delayed type hypersensitivity in mice chronically infected with Trypanosoma cruzi: involvement of heart interstitial dendritic cells

Acute infection with Trypanosoma cruzi results in intense myocarditis, which progresses to a chronic, asymptomatic indeterminate form. The evolution toward this chronic cardiac form occurs in approximately 30% of all cases of T. cruzi infection. Suppression of delayed type hypersensitivity (DTH) has been proposed as a potential explanation of the indeterminate form. We investigated the effect of cyclophosphamide (CYCL) treatment on the regulatory mechanism of DTH and the participation of heart interstitial dendritic cells (IDCs) in this process using BALB/c mice chronically infected with T. cruzi. One group was treated with CYCL (20 mg/kg body weight) for one month. A DTH skin test was performed by intradermal injection of T. cruzi antigen (3 mg/mL) in the hind-footpad and measured the skin thickness after 24 h, 48 h and 72 h. The skin test revealed increased thickness in antigen-injected footpads, which was more evident in the mice treated with CYCL than in those mice that did not receive treatment. The thickened regions were characterised by perivascular infiltrates and areas of necrosis. Intense lesions of the myocardium were present in three/16 cases and included large areas of necrosis. Morphometric evaluation of lymphocytes showed a predominance of TCD8 cells. Heart IDCs were immunolabelled with specific antibodies (CD11b and CD11c) and T. cruzi antigens were detected using a specific anti-T. cruzi antibody. Identification of T. cruzi antigens, sequestered in these cells using specific anti-T. cruzi antibodies was done, showing a significant increase in the number of these cells in treated mice. These results indicate that IDCs participate in the regulatory mechanisms of DTH response to T. cruzi infection.     

Involvement of the Th1 subset of CD4+ T cells in acquired immunity to mouse infection with Trypanosoma equiperdum.

Heat- or merthiolate-inactivated Trypanosoma equiperdum was administered to recipient mice that were subsequently challenged with viable inocula of the same stabilate. Only mice inoculated with merthiolate-killed parasites were completely protected from a challenge inoculum of 10(3) trypanosomes, an effect that was abolished by prior immunosuppression of mice. Immune sera from protected animals contained high levels of interferon (IFN)-gamma and specific IgG2a antibodies. Spleen cells from these mice produced high amounts of interleukin (IL)-2 and IFN-gamma in vitro in response to specific antigen or concanavalin A, whereas splenocytes from mice receiving heat-killed parasites produced high amounts of IL-6. In contrast, the production of tumor necrosis factor (TNF)-alpha and colony-stimulating activity (CSA) was not significantly different in mice receiving either killed parasite preparation. The protection in immunized mice was associated with the detection of strong delayed-type hypersensitivity (DTH) to T. equiperdum antigens, an effect that could be adoptively transferred onto naive recipients by specifically immune CD4+ lymphocytes. These results suggest that the development of protective immunity in mice to T. equiperdum by our immunization protocol may involve the activity of helper/DTH T cells, particularly those of the Th1 subset.     

Experimental autoimmune thyroiditis in mice chronically treated from birth with anti-IgM antibodies

The role of T lymphocytes in the pathogenesis of experimental autoimmune thyroiditis in mice is well established while the role of B lymphocytes is unclear. Mice with thyroid lesions have thyroglobulin antibodies whereas these antibodies can occur in mice immunized with Tg that do not develop thyroid lesions. To determine whether thyroglobulin antibodies are necessary for the development of the thyroid infiltrates with mononuclear cells, which are characteristic for experimental autoimmune thyroiditis, AKR mice chronically treated from birth with goat anti-mouse IgM antibodies were immunized with mouse thyroglobulin in Freund's complete adjuvant when they were 7 weeks old. Control mice, similarly immunized, were chronically injected from birth with normal goat gamma-globulin. Three weeks after immunization, all mice were sacrificed, thyroglobulin antibodies in the serum were measured by hemagglutination assay and enzyme-linked immunosorbent assay, and thyroid pathology was assessed. The serum concentration of IgG and IgM, the percentage of B and T lymphocytes in the spleen (flow cytometry), and the in vitro proliferative response of spleen lymphocytes to stimulation by PHA, LPS, and Tg were also measured. All mice treated with anti-IgM antibodies did not have detectable thyroglobulin antibodies but 63% of these mice and 88% of control mice (all of which had thyroglobulin antibodies) had thyroid lesions. Mice treated with anti-IgM antibodies that did not have thyroid lesions had a more pronounced depression of B lymphocytes than similarly treated mice that had thyroid lesions. These experiments suggest that thyroglobulin antibodies are not necessary for the development of thyroid infiltrates with mononuclear cells. B lymphocytes could still participate in the production of experimental autoimmune thyroiditis by presenting thyroglobulin to helper T lymphocytes.     

Suppression of polyclonal antibody production in Trypanosoma cruzi-infected mice by treatment with anti-L3T4 antibodies

Acute Trypanosoma cruzi infection of mice results in a very marked polyclonal activation of B and T lymphocytes, accompanied by high numbers of Ig-secreting PFC and lectin-dependent effector CTL. Treatment of mice with monoclonal anti-L3T4 antibodies from the time of infection (days 0, 4, and 8) completely suppresses the polyclonal PFC response and CTL generation. Treatment of nude mice with antibody does not alter the lipopolysaccharide-induced polyclonal PFC response, and it only modulates the isotypic profile of the PFC response to T. cruzi infection, without reducing its magnitude. Furthermore, antibody-treated, T. cruzi-infected euthymic mice do not develop the typical B cell blastogenic response, but show high numbers of activated Lyt-2+ lymphoblasts in the spleen. These results indicate that effector cell generation in T. cruzi-infected mice is predominantly helper T cell-dependent.     

Susceptibility of inbred mice to Leishmania tropica infection: correlation of susceptibility with in vitro defective macrophage microbicidal activities

Eleven mouse strains were inoculated in footpads with amastigotes of Leishmania tropica and observed for 12 weeks. Liver and spleen impression smears from infected mice were examined for the presence of intracellular parasites. Four strains (BALB/cJ, C57L/J, NZW/N, and P/J) failed to heal the subcutaneous lesion and showed evidence of systemic infection; the remaining seven strains (A/J, C3H/HeJ, C3H/HeN, C3HeB/FeJ, C57BL/6J, C57BL/10J, and C57BL/10ScN) were each resistant to infection and resolved their lesions by Week 10. Macrophages from the four susceptible strains could not be activated to kill L. tropica amastigotes by treatment with soluble lymphocyte products in vitro. In contrast, macrophages from all seven resistant strains responded to lymphokine treatment and eliminated 80-90% of intracellular parasites. These results suggest that in vitro macrophage microbicidal activities predict the course of systemic leishmanial disease.     

Suppression of the delayed-type hypersensitivity response to hen egg white lysozyme (HEL) by HEL peptides in a genetically high-responder mouse strain: evidence for requirement of the loop structure for induction of suppressor T cells

The delayed-type hypersensitivity (DTH) response of C3H/HeN mice to hen egg white lysozyme (HEL) can be blocked by a single iv injection of a solution of HEL in buffered saline 7 days before sensitization of animals with HEL in complete Freund's adjuvant (CFA). The minimal structure of HEL required for the suppression was examined by determining the abilities of various HEL-derivative peptides to inhibit HEL-DTH. Treatment of normal mice with Ploop I X II, sequence 57-107 (Cys64-Cys80, Cys76-Cys94), or P17 (sequences 1-27 and 123-129 linked by Cys6-Cys127) 7 days before immunization with HEL resulted in marked suppression of the DTH response. This inhibition of DTH involved generation of suppressor T cells (Ts). The results suggested that two suppressor pathways are involved. These data, together with another recent finding (1) that an entirely different portion of HEL is a suppressor determinant (SD) in A/J mice, indicate that different epitopes act as SDs in different strains of mice. Of the loop region peptides tested, Plc (intact loop I joined to a linear peptide, residues 84-97) was found to be the minimum structure capable of suppressing the HEL-DTH response; loop I or II alone did not cause suppression. Activation of Ts cells by the loop peptide depended on its conformational structure; completely reduced and carboxymethylated Ploop I X II did not cause suppression.     

Immunologic regulation of experimental cutaneous leishmaniasis. V. Characterization of effector and specific suppressor T cells

Resistant CBA mice infected with Leishmania tropica promastigotes develop concomitant and convalescent immunity against reinfection. This can be adoptively transferred by splenic and lymph node T cells with a threshold dosage of 1 to 2.5 x 10(7). The effector cells are of Thy-1+, Lyt-1+2- phenotype. The same immune cell population also adoptively transfers specific DTH to L. tropica, which is restricted by the major histocompatibility complex. On the other hand, highly susceptible BALB/c mice infected with L. tropica develop antigen-specific suppressor T (Ts) cells (previously shown to inhibit the induction and expression of DTH), which are capable, on transference, of reversing the healing of lesions induced by prior sublethal irradiation of BALB/c mice. As few as 10(6) of these T cells are effective in abrogating the potent prophylactic effect of 550 rad. The Ts cells are of Thy-1+, Lyt-1+2-, and I-J- phenotype. Sublethally irradiated and infected BALB/c mice produce antibody responses quantitatively and isotypically similar during the critical first 40 days after infection whether or not they are injected with 10(7) Ts cells (nonhealing vs healing). Thus, impairment of DTH and curative immune responses in BALB/c mice cannot be attributed to a helper function of these Thy-1+, Lyt-1+2- cells for the formation of suppressive antibody.     

Vaccination against cutaneous leishmaniasis in a murine model. II. Immunologic properties of protective and nonprotective subfractions of soluble promastigote extract

We have previously demonstrated that BALB/c mice can be protected against a fatal infection with Leishmania major by i.p. immunization with a soluble leishmanial antigen (SLA) preparation in conjunction with the adjuvant, Corynebacterium parvum (CP). In this study, SLA was separated into nine distinct fractions by anion exchange liquid chromatography, and the fractions were analyzed for their ability to stimulate T cells obtained from immunized mice, to be recognized by vaccine-induced antibodies, and to induce protective immunity. While all but one of the fractions were recognized by antibodies from SLA + CP immunized mice, only two fractions (fractions 1 and 9) stimulated lymphocytes to produce macrophage-activating factor and elicited significant delayed-type hypersensitivity in vivo. When mice were immunized with the fractions, only fraction 9 stimulated significant immunity (76% protection in seven experiments). Proteins (accounting for 1.3% of the total in SLA) appear to be responsible for the protection elicited with fraction 9, since protease treatment of this fraction destroyed its immunogenicity. Thus, a partially purified protective protein antigen fraction has been obtained and protection with this fraction correlated with cell-mediated immune responses. However, these results also demonstrate that the ability of leishmanial antigens to be recognized by T cells and produce macrophage-activating factor does not in itself predict whether such molecules will induce immunity, suggesting that protective leishmanial antigens may have additional unique properties.