donovani challenge Neither the humoral polyclonal antibody response nor the cell-mediated DTH response could entirely explain the observed disease progression in LAg + adjuvant immunized mice following challenge with L. donovani. We therefore asked whether LAg specific recall cytokine responses could provide use with a further mechanistic insight. To do so, we cultured splenocytes from experimental
cohorts 10 days post-immunization, and 4 months after L. donovani challenge infection. Splenocytes from mice vaccinated with alum + LAg secreted significantly KPT-8602 ic50 higher levels of IL-12 in comparison to free adjuvant-immunized controls (Figure 4A, p < 0.05). In addition, IFN-γ measured in splenocyte cultures was also significantly higher compared to both PBS and free adjuvant-immunized controls (Figure 4C, p < 0.05). We performed blocking experiments with anti-CD4 and anti-CD8 monoclonal antibodies to assess the relative contributions of CD4+ and CD8+ T cells to this cytokine production, revealing that IFN-γ secretion in
alum + LAg immunized mice was produced mainly from CD8+ T cells, whereas CD4+ T-cell blocking had only a negligible effect. In contrast, the levels of IL-4 produced by CD4+ T cells was significantly higher not only in comparison to controls (Figure 4E, p < 0.001), but also to other remaining INK1197 mouse groups (p < 0.05). A low IFN-γ:IL-4 ratio (0.8) was observed in the alum + LAg Tryptophan synthase vaccinated group and furthermore significant IL-10 production was not observed, remaining comparable to both PBS and free adjuvant-immunized controls (Figure 4G). Figure 4 Cytokine response in vaccinated mice following immunization and L. donovani challenge infection. Ten days post-vaccination and 4 months after L. donovani challenge infection splenocytes were restimulated in vitro with LAg (10 μg/mL) in media alone or in the presence of anti-CD4 or anti-CD8 monoclonal antibody
(1 μg/106 cells). After 72 h supernatants were collected and assayed for IL-12 ((A, B), IFN-γ (C, D), IL-4 (E, F) and IL-10 (G, H)) by ELISA. Each sample was Sepantronium nmr examined in duplicate. The results are shown as the mean ± SE for five individual mice per group, representative of two independent experiments with similar results. * p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to PBS as well as free adjuvant immunized groups as assessed by one-way ANOVA and Tukey’s multiple comparison test. In contrast, splenocytes from saponin + LAg immunized mice produced significantly higher levels of IL-12 and IFN-γ in comparison controls (Figure 4A, C; p < 0.001). Notably, elevated levels of IL-4 and IL-10 were also produced by splenocytes of the saponin + LAg group (p < 0.001 compared to controls). Production of both IL-4 and IL-10 was substantially inhibited by addition of anti-CD4 blocking antibody to cultures, indicating that both of these cytokines were likely produced by the CD4+ T cell subset (Figure 4E, G).