Role of ILT4/HLA-G interaction in Tr1 cell differentiation. (A-B) Induction of Tr1 cells requires ILT4/HLA-G interaction. Naive CD4⁺ T cells were stimulated with monocyte-derived DC-10 in the presence of anti-ILT4 [T(DC-10 anti-ILT4)] or control IgG [T(DC-10)] mAbs. As control, naive CD4⁺ T cells were stimulated with mDCs [T(mDC)]. After stimulation, T cells were collected and tested for their ability to proliferate in response to mDCs (A) and to suppress responses of autologous CD4⁺ T cells activated with mDCs (MLR) (B). [3H]-Thymidine was added after 2 days (A) and 4 days (B) of culture for an additional 16 hours. Results are representative of 4 independent experiments. Numbers represent the percentage of anergy of T(DC-10) or T(DC-10 anti-ILT4) cells compared with T(mDC) cells (A), and the percentage suppression of proliferation in MLR cocultured in the presence of T(DC-10) or T(DC-10 anti-ILT4) cells compared with MLR alone (B). (C-D) IL-10 induces up-regulation of HLA-G on naive T cells. (C) Naive CD4⁺ T cells were stimulated with monocyte-derived iDCs, DC-10, and mDCs for 48 hours, and T cells were analyzed by flow cytometry to determine levels of expression of HLA-G. Percentages of HLA-G-positive cells obtained by stimulating naive CD4⁺ T cells with iDCs, DC-10, and mDCs from each of the 10 donors tested are presented. Black lines represent the mean percentages of CD4⁺HLA-G⁺ T cells. (D) Naive CD4⁺ T cells were stimulated with monocyte-derived iDCs, DC-10, and mDCs for 48 hours in the presence of control IgG or anti-IL-10R mAbs. T cells were analyzed by flow cytometry to determine levels of expression of HLA-G. Percentages of CD4⁺HLA-G⁺ T cells are shown. Black lines represent the mean percentages of CD4⁺HLA-G⁺ T cells. (E-F) Naive CD4⁺ T cells were stimulated with monocyte-derived DC-10 in the presence of anti-HLA-G [T(DC-10 anti-HLA-G)] or control IgG [T(DC-10)] mAbs. As control, naive CD4⁺ T cells were stimulated with mDCs [T(mDC)]. After stimulation, T cells were collected and tested for their ability to proliferate in response to mDCs (E) and to suppress responses of autologous CD4⁺ T cells activated with mDCs (MLR) (F). [3H]-Thymidine was added after 2 days (E) and 4 days (F) of culture for an additional 16 hours. Results are representative of 3 independent experiments. Numbers represent the percentage of anergy of T(DC-10) or T(DC-10 anti-HLA-G) cells compared with T(mDC) cells (E), and the percentage suppression of proliferation in MLR cocultured in the presence of T(DC-10) or T(DC-10 anti-HLA-G) cells compared with MLR alone (F).(G-H) Naive CD4⁺ T cells were stimulated with monocyte-derived DC-10 in the presence of anti-ILT2 [T(DC-10 anti-ILT2)] or control IgG [T(DC-10)] mAbs. As control, naive CD4⁺ T cells were stimulated with mDCs [T(mDC)]. After stimulation, T cells were collected and tested for their ability to proliferate in response to mDCs (G) and to suppress responses of autologous CD4⁺ T cells activated with mDCs (MLR) (H). [3H]-Thymidine was added after 2 days (G) and 4 days (H) of culture for an additional 16 hours. Results are representative of 3 independent experiments. Numbers represent the percentage of anergy of T(DC-10) or T(DC-10 anti-ILT2) cells compared with T(mDC) cells (G), and the percentage suppression of proliferation in MLR cocultured in the presence of T(DC-10) or T(DC-10 anti-ILT2) cells compared with MLR alone (H).