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Recent evidence suggests that T cells and their associated cytokines critically influence outcome in mice experimentally infected with Borrelia burgdorferi (Bb), the causative agent of human Lyme disease. In vivo T cell subset and cytokine depletion studies suggest that CD4+ T cell-derived IL-4 plays a critical role in control of spirochete growth in vivo, whereas CD8+ T cell-derived IFN-gamma appears to promote disease, particularly in susceptible mouse strains. To further investigate the immunologic basis of protection and the role of IL-4, we have examined the effects of early rIL-4 treatment on outcome in susceptible mice infected with Bb. In this study, we show that administration of rIL-4 to susceptible C3H mice during the first week of infection with Bb leads to early control of their infections, as evidenced by significant reductions in joint swelling at wk 5, 6, and 7 postinfection, and in the numbers of spirochetes recovered from their joints and skin at wk 7 when compared with sham-treated mice. Increased resistance in rIL-4-treated mice was accompanied by significant reductions in their in vitro splenic Bb-specific IFN-gamma responses and in serum levels of specific IgG2a and IgG3 Abs and significant increases in specific IgG1 Abs. We also show that the inherent susceptibility of Ab-deficient, C57BL/6-IgM knockout (B6-MKO) mice to Rh infection is intermediate relative to C57BL/6 severe combined immunodeficient (B6-SCID) mice (susceptible) or normal C57BL/6 mice (resistant), confirming the importance of both Ab-dependent and Ab-independent, T cell-dependent immune mechanisms in control of Bb infections. The additional finding that early treatment with rIL-4 significantly reduced the severity of Bb infections in B6-MKO mice indicates that IL-4 may augment anti-spirochetal immunity via an Ab-independent mechanism.

Because T cells appear to modulate the severity of murine Borrelia burgdorferi infections, we decided to examine the possible involvement of T cell-associated cytokines in disease outcome. Comparison of in vitro B. burgdorferi Ag-induced cytokine production in disease-susceptible and -resistant strains revealed striking differences; spleen cells from susceptible C3H mice produced significantly higher levels of IL-2 and IFN-gamma and lower levels of IL-4 than spleen cells from resistant BALB/c mice. Lymph node responses were even more divergent, with C3H mice producing high levels of IFN-gamma, and BALB/c mice producing little or none. This apparent Th1/Th2 cytokine imbalance was also reflected in vivo, since serum from C3H had significantly higher levels of B. burgdorferi-specific IgG2a Ab and lower levels of IgG1 Ab than serum from BALB/c mice. In vivo studies confirmed the importance of IL-4 in early control of spirochete growth, since treatment of either strain with neutralizing anti-IL-4 mAb led to increased joint swelling and higher spirochete burdens in joints compared with those in control mAb-treated mice. In contrast, IFN-gamma may hinder early control of spirochete growth in susceptible C3H mice, since treatment of mice with neutralizing anti-IFN-gamma mAb reduced both joint swelling and joint spirochete burdens compared with those in control mAb-treated mice. These studies indicate opposing roles for IL-4 and IFN-gamma in the modulation of spirochete growth and disease development in B. burgdorferi-infected mice and suggest that differential cytokine production early in infection may contribute to strain-related differences in susceptibility.

The possible involvement of specific T cells in resolution of infections with Borrelia burgdorferi (Bb), the causative agent of human Lyme disease, has not been adequately studied. To investigate the potential role of T cell subsets in resistance, we have depleted mice of CD4+ and CD8+ T cell subsets in vivo by the administration of specific mAbs and have examined outcomes after infection with Bb. Our results indicate that CD4+ T cells are required for immunologic control of spirochete levels, because their depletion in both susceptible C3H/HeN and resistant BALB/c mice increased the severity of arthritis and the numbers of spirochetes found in joints and skin, as compared with Bb-infected mice treated with a control mAb. In contrast, the CD8+ T cell compartment, particularly in susceptible C3H/HeN mice, appears to promote the disease process, possibly by interfering with the generation of protective immunity, as abrogation of this subset in vivo led to a reduction in both arthritis and in spirochete levels found in joints and skin when compared with Bb-infected control mice. Our inability to establish a correlation between resistance and Bb-specific IgG Ab levels in these mice raises the possibility that Ab-independent mechanisms are important in protection. These findings suggest that the final outcome in Bb-infected hosts may be the net effect of antagonistic influences exerted by CD4+ and CD8+ T cell subsets.

Recent in vivo depletion studies in mice demonstrated that CD8+ T cells play a critical role in acute resistance to experimental Trypanosoma cruzi infections. As part of efforts to characterize these protective CD8+ T cell effector populations, we report here that splenic lymphocytes from mice chronically infected with T. cruzi can be induced to express high levels of cytolytic activity after stimulation in vitro with irradiated T. cruzi-infected macrophages. Cytolytic activity can either be detected using a nonspecific lectin-dependent 51Cr-release assay or using 51Cr-labeled T. cruzi-infected target cells. Fresh splenocytes from chronically infected mice stimulated with T. cruzi-infected macrophages exhibit relatively "promiscuous" killing activity inasmuch as significant lysis of both T. cruzi-infected and uninfected syngeneic and allogeneic cells is detected. However, subsequent rounds of in vitro stimulation lead to the expression of lytic activity that is T. cruzi Ag-specific and MHC-restricted. Several short term in vitro maintained cytolytic T cell lines were shown to have mixed phenotypes by FACS analysis; approximately 50% to 75% of the cells in these populations were CD4-, CD8+, whereas 20% to 40% were CD4-, CD8-. Experiments in which effector cells were positively selected by adherence to anti-CD8 mAb-treated plates confirmed that CD8+ T cell could exhibit Ag-specific cytolytic activity against T. cruzi-infected target cells. Efforts are under way to clone these CTL to test their in vivo function and to determine their Ag specificity.

Murine T cell lines responsive to the protozoan parasite Trypanosoma cruzi were generated in vitro by stimulating hyperimmune C57BL/6 lymphoid cells with trypomastigote stage antigen. A spleen-derived line designated ST1 and eight clones derived from ST1 were characterized. All lines bear the surface phenotype Thy-1.2+, Ly-1.2+, 2.2- and respond to T. cruzi antigen only in the presence of antigen-presenting cells matched at the I-A subregion of the H2 locus. Clonal specificity analyses indicated that these T. cruzi-selected T cells are species specific and recognize antigenic determinants that are expressed predominantly in the trypomastigote stage. On the basis of their distinct patterns of response to a panel of different T. cruzi strains, clones recognizing strain-specific, shared, or common determinants were identified. Functional studies indicated that ST1 and some but not all of the clones are capable of expressing antigen-specific T helper function in vitro and in vivo. In addition, co-incubation of T. cruzi-specific T cells with cultured T. cruzi-infected syngeneic macrophages led to the dose-dependent destruction of intracellular parasites. Most notably, ST1 and several of the cloned T. cruzi-specific T cell lines were able to passively protect syngeneic recipients from lethal T. cruzi challenge infection. Efforts to identify the parasite antigens recognized by these T cell lines, particularly the protective clones, are currently in progress.