Resistance to Leishmania major in mice is associated with the appearance of distinct T helper type 1 (Th1) and Th2 subsets. T cells from lymph nodes draining cutaneous lesions of resistant mice are primarily interferon gamma (IFN-gamma)-producing Th1 cells. In contrast, T cells from susceptible mice are principally Th2 cells that generate interleukin 4 (IL-4). Although existing evidence is supportive of a role for IFN-gamma in the generation of Th1 cells, additional factors may be required for a protective response to be maintained. A potential candidate is IL-12, a heterodimeric cytokine produced by monocytes and B cells that has multiple effects on T and natural killer cell function, including inducing IFN-gamma production. Using an experimental leishmanial model we have observed that daily intraperitoneal administration at the time of parasite challenge of either 0.33 micrograms IL-12 (a consecutive 5 d/wk for 5 wk) or 1.0 micrograms IL-12 per mouse (only a consecutive 5 d) caused a > 75% reduction in parasite burden at the site of infection, in highly susceptible BALB/c mice. Delay of treatment by 1 wk had less of a protective effect. Concomitant with these protective effects was an increase in IFN-gamma and a decrease in IL-4 production, as measured by enzyme-linked immunosorbent assay of supernatants generated from popliteal lymph node cells stimulated with leishmanial antigen in vitro. The reduction in parasite numbers induced by IL-12 therapy was still apparent at 10 wk postinfection. In addition, we observed that the administration of a rabbit anti-recombinant murine IL-12 polyclonal antibody (200 micrograms i.p. every other day for 25 d) at the time of infection to resistant C57Bl/6 mice exacerbated disease. These effects were accompanied by a shift in IFN-gamma production in vitro by antigen-stimulated lymph node cells indicative of a Th2-like response. These findings suggest that IL-12 has an important role in initiating a Th1 response and protective immunity.

In this study we characterized Th2 responses in the absence of IL-4. We show that ST2L, a stable Th2 marker, is expressed at similar levels in Leishmania major-infected IL-4-deficient (IL-4(-/-)) and wild-type BALB/c (IL-4(+/+)) mice. Th2 cytokines are secreted by in vivo differentiated lymphocytes in response to specific activation in the absence of IL-4. Although IL-13 is produced, its neutralization did not alter the outcome of infection. Thus, we demonstrate that Th2 differentiation as assessed by the expression of ST2L and the production of Th2 cytokines can occur in vivo in the absence of IL-4.

The contribution of the costimulatory molecules B7–1 and B7–2 to the in vivo differentiation of Th cells remains controversial. The infection of resistant and susceptible strains of mice with the parasite Leishmania major provides a well‐established model for studying in vivo differentiation of CD4+ T cells. We have infected B7–1/B7‐2‐deficient mice onthe BALB/c and 129 background with L. major and subsequently examined different parameters of infection and cytokine responses upon restimulation of lymph node cells in vitro. BALB/c B7–2‐deficient and B7–1/B7–2‐double deficient mice are resistant to L. major, whereas BALB/c B7–1‐deficient mice remain as susceptible as wild‐type BALB/c mice. Differential expression of B7–1 and B7–2 can explain the distinct roles observed for these B7 costimulators in L. major infection.

Previously we demonstrated that recombinant murine interleukin-12 (rmIL-12) administration can promote a primary Th1 response while suppressing the Th2 response in mice primed with 2,4,6-trinitrophenyl–keyhole limpet hemocyanin (TNP-KLH). The present studies examined the capacity of rmIL-12 to drive a Th1 response to TNP-KLH in the presence of an ongoing Th2-mediated disease. To establish a distinct Th2 response, we used a murine model of leishmaniasis. Susceptible BALB/c mice produce a strong Th2 response when infected with Leishmania major and develop progressive visceral disease. On day 26 postinfection, when leishmaniasis was well established, groups of mice were immunized with TNP-KLH in the presence or absence of exogenous rmIL-12. Even in the presence of overt infection, TNP-KLH-plus-rmIL-12-immunized mice were still capable of generating KLH-specific gamma interferon (IFN-γ) as well as corresponding TNP-specific immunoglobulin G2a (IgG2a) titers. In addition, the KLH-specific IL-4 was suppressed in infected mice immunized with rmIL-12. However, parasite-specific IL-4 and IgG1 production with a lack of parasite-specific IFN-γ secretion were maintained in all infected groups of mice including those immunized with rmIL-12. These data show that despite the ongoing infection-driven Th2 response, rmIL-12 was capable of generating an antigen-specific Th1 response to an independent immunogen. Moreover, rmIL-12 administered with TNP-KLH late in infection did not alter the parasite-specific cytokine or antibody responses.

Studies on murine candidiasis suggest that resistance to disease is linked to a Th1 response and production of IFN-γ, while failure to elicit protection is associated with a Th2 response and production of IL-4 and IL-10. Experimental infection of C57BL/6 mice, IL-12 treatment of these mice, or both infection and IL-12 treatment resulted in a characteristic Th1 cytokine mRNA profile as measured by quantitative competitive PCR. Specifically, little or no IL-4 transcripts were detected, while IFN-γ message was elevated, particularly with IL-12 treatment. Despite its role in driving increased IFN-γ expression and production, IL-12 treatment, paradoxically, promoted disease progression in our model. Therefore, we examined the effect of IFN-γ neutralization on IL-12-induced susceptibility to infection. None of the systemically infected mice receiving IL-12 alone survived, while IL-12- and anti-IFN-γ-treated mice had a 70% survival rate, similar to that after infection alone. These results suggested that IFN-γ induced by IL-12 treatment contributed to lethality. However, in separate studies, IFN-γ knockout mice were more susceptible to infection than their wild-type counterparts, suggesting that IFN-γ is required for resistance. Nonetheless, infected IFN-γ knockout mice treated with recombinant murine IL-12 exhibited enhanced resistance, suggesting that the toxicities observed with IL-12 are directly attributable to IFN-γ and that an optimal immune response to Candida infections necessitates a finely tuned balance of IFN-γ production. Thus, we propose that although IFN-γ can drive resistance, the overproduction of IFN-γ during candidiasis, mediated by IL-12 administration, leads to enhanced susceptibility.