DC-10 induce Tr1 cell with suppressive activity. (A) Naive CD4⁺ T cells were cultured with allogeneic DC-10 [T(DC-10)] or myDCs [T(myDC)] isolated from peripheral blood by FACS sorting according to the expression of CD11c, CD83, and CD14, at the ratio of 10:1. After 14 days, T cells were tested for their ability to suppress responses of autologous CD4⁺ T cells activated with monocytes (MLR). Naive CD4⁺ T cells were stimulated with monocytes alone (MLR) or in the presence of T(DC-10) or T(myDC) cell lines at a 1:1 ratio. As control, T(DC-10) or T(myDC) cell lines were stimulated with monocytes. [³H]-thymidine was added after 3 days of culture for an additional 16 hours. Results of one experiment representative of 3 independent experiments are shown. (B) Suppression of IFN-γ production by CD4⁺ T cells in response to monocytes was measured in culture supernatants after 4 days of culture. Results representative of 2 independent experiments are shown. (C) Naive CD4⁺ T cells were stimulated with allogeneic monocyte-derived iDCs [T(iDC)], DC-10 [T(DC-10)], or mDCs [T(mDC)]. After 14 days, T cells were tested for their ability to suppress responses of autologous CD4⁺ T cells activated with mDCs (MLR). Naive CD4⁺ T cells were stimulated with mDCs alone (MLR) or in the presence of T(iDC), T(DC-10), and T(mDC) cells at a 1:1 ratio. [³H]-Thymidine was added after 3 days of culture for an additional 16 hours. Results of one experiment representative of 8 independent experiments are shown. (D) Suppression of IFN-γ production by CD4⁺ T cells in response to mDCs was measured in culture supernatants after 4 days of culture. Results representative of 3 independent experiments are shown. (E) Role of IL-10 and TGF-β in suppression mediated by T(DC-10) cells. After activation with DC-10, T(DC-10) cells were tested for their ability to suppress the IFN-γ production of CD4⁺ T cells in response to allogeneic monocytes in the absence or presence of anti–IL-10R (30 μg/mL) and anti–TGF-β (50 μg/mL) mAbs. Suppression of IFN-γ production was measured in culture supernatants 4 days after culture. Results are representative of 3 independent experiments. (F) Autocrine IL-10 is required for the differentiation of T(DC-10) cells by DC-10. Naive CD4⁺ T cells were stimulated with allogeneic DC-10 in the presence of anti-IL10R (10 μg/mL) or control IgG (10 μg/mL) mAbs. After activation, T cells were collected and tested for their ability to suppress the response of autologous CD4⁺ T cells activated with mDCs (MLR). [³H]-Thymidine was added after 3 days of culture for an additional 16 hours. Results of one experiment representative of 3 independent experiments are shown. Numbers represent the percentage suppression of proliferation in MLR cocultured in the presence of T(DC-10) compared with MLR alone.