2E) To further confirm these findings, we purified CD4+

2E). To further confirm these findings, we purified CD4+

T cells from B6 BCG-vaccinated and unvaccinated DLNs at different time points postvaccination and measured cytokine mRNA induction in these cells. Consistent with data shown in Fig. 2D, IL-17 mRNA induction occurred in CD4+ T cells earlier than the induction of IFN-γ mRNA, which was detected on day 14 postvaccination (Fig. 2F). Together, our data show that BCG vaccination induces an early IL-23-dependent Th17-cell response that precedes the Th1-cell response, and is required for the induction of an effective BCG vaccine-induced MK-8669 mw Th1-cell response. Th17 cells are induced early in vivo following BCG vaccination and are important for subsequent generation of vaccine-induced Th1 cells at later time points (Figs. 1 and 2). Therefore, we then addressed whether the Th1- and Th17-cell polarizing cytokines namely IL-12 or IL-23 are induced in DCs in response to BCG exposure. We found that following BCG exposure, DCs produced both IL-23 and IL-12 cytokines (Fig. 3A and B). Interestingly, BCG also induced high levels of the anti-inflammatory cytokine, IL-10 in BCG-exposed DCs (Fig. 3C). IL-10 is an anti-inflammatory cytokine that inhibits IL-12 production and Th1-cell differentiation 26. Accordingly, AZD9291 datasheet IL-10 also inhibits IL-12 production in BCG-infected

DCs and the generation of IFN-γ-producing cells 27. Based on these data, we hypothesized that the absence of early Th1-cell responses in vivo following BCG vaccination was due to high BCG-induced IL-10 levels (Fig.

3C) and that IL-17 dependence to induce Th1-cell responses (Fig. 1) was a host strategy to overcome the IL-10-mediated inhibition. To address this hypothesis, we first treated BCG-stimulated DCs with IL-10-neutralizing antibody and measured IL-12 production in supernatants. GNA12 As expected 27, neutralization of BCG-induced IL-10 resulted in significantly increased production of IL-12 (Fig. 3D). We also determined the effect of IL-10 neutralization on Th1 cell generation by coculturing naïve OT-II TCR Tg T cells with BCG/OVA323–339-treated DCs in the presence of IL-10-neutralizing antibody. Consistent with our hypothesis, we report that T-cell-derived IFN-γ production was inhibited in the presence of BCG and neutralization of IL-10 reversed BCG-mediated inhibition of IFN-γ production in T-cell supernatants (Fig. 3E). These data suggest that despite induction of some IL-12 in BCG-exposed DCs, coincident induction of IL-10 inhibits Th1-cell responses. Importantly, Ag85B-specific Th1-cell responses detected in vivo were also increased in BCG-vaccinated il10−/− mice when compared with B6 BCG-vaccinated mice (Fig. 3F).

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