DCs were cultured together with DX5+CD4+ or DX5−CD4+ supernatant in the presence of blocking antibodies against IL-4 or IL-10. Our results show that inhibition of IL-10 present in the DX5+CD4+ supernatant restored the

ability of DCs to produce IL-12. In contrast, neutralization of IL-4 did not result in the restoration of IL-12 production by DCs (Fig. 3). Together, these findings indicate that IL-10 but not IL-4 secreted by DX5+CD4+ T cells is responsible for the suppression of IL-12 production. The results presented above indicate that DX5+CD4+T cells can modulate the expression and secretion of various molecules involved in T-cell activation and skewing. To analyze whether DX5+CD4+ T-cell-modulated DCs display altered abilities to activate naïve T cells, we next investigated the impact of DC modulation by DX5+CD4+ T cells on the outcome of T-cell responses. To this selleckchem end, we incubated DCs with supernatants of DX5+CD4+ or DX5−CD4+ T-cell selleck chemical cultures. After extensive washing, the DCs exposed to supernatant from DX5+ (DX5+DCs) or DX5− (DX5−DCs) T-cell cultures

were co-cultured with OVA-specific CD4+ D0.11.10 T cells and OVA peptide. After 3 days, IFN-γ production by OVA-specific CD4+ T cells was analyzed by flow cytometry. Interestingly, OVA-specific CD4+ T cells primed with DX5+DCs produced less IFN-γ as compared with CD4+ T cells primed with either DX5−DCs or DCs exposed to medium only (medium DCs) (Fig. 4A and B and Supporting Information Fig. 3). These data indicate that DCs exposed to the action of DX5+CD4+ T cells are affected in their ability to prime CD4+ T cells for IFN-γ production. As DX5+CD4+ T cells produced factors that inhibited IL-12 production by DCs and as IL-12 is a prominent cytokine capable of inducing IFN-γ production, we next determined whether the reduced IL-12 production was responsible for the effects observed. To this end, we supplemented cultures of naïve OVA-specific T cells and OVA-peptide-loaded DX5+ DC with exogeneous IL-12. Addition

of IL-12 was sufficient to restore the potential CYTH4 of DX5+DC-primed CD4+ T cells to secrete IFN-γ (Fig. 4C and D and Supporting Information Fig. 3). As inhibition of IL-12 production was dependent on IL-10 present in the DX5+CD4+ T-cell supernatants, we next blocked IL-10 in the supernatant of DX5+CD4+ T-cell cultures upon addition to DCs. These DCs were subsequently used to prime OVA-specific D0.11.10 cells as described above. DCs exposed to anti-IL-10-treated DX5+ supernatant regained their capacity to prime CD4+ T cells for IFN-γ production, as OVA-specific CD4+ T cells were able to produce IFN-γ at levels comparable with (or higher than) that produced by T cells primed by DX5−DCs or medium DCs. Conversely, IFN-γ-production by responding CD4+ T cells was not restored after treatment of DX5+DCs with anti-IL-4 (Fig. 5A and B and Supporting Information Fig. 3).