regMΦ show T-cell immunosuppression and induce antigen-specific immune tolerance in vivo. (A-B) regMΦ are T-cell immunosuppressive in vivo. OTII cells (1 × 10⁷⁾ were intraperitoneally transferred into recipients together without or with OVA-maDCs, OVA-regMΦ or OVA-maDCs together with regMΦ. After 5 days the percentage (A) of DAPI⁻CD45.1⁺CD4⁺Vα2⁺ viable OTII in DAPI⁻CD4⁺ cells and the absolute number (B) of DAPI⁻CD45.1⁺CD4⁺Vα2⁺ viable OTII cells in mLNs and spleen were determined (dots represent individual mice analyzed in 1 experiment; similar results were obtained in a second experiment). (C) NOS2 deficient regMΦ are incapable in T-cell immunosuppression in vivo. OTII cells (1 × 10⁷⁾ were intraperitoneally transferred into recipients together with or without OVA-maDCs, together with WT or NOS2^−/− regMΦ. After 5 days the percentage of DAPI⁻CD45.1⁺CD4⁺Vα2⁺ OTII in DAPI⁻CD4⁺ cells was determined (dots represent individual mice analyzed in 2 independent experiments). (D-F) regMΦ induce antigen-specific immune tolerance in vivo. OVA-maDCs, OVA-regMΦ, or OVA-maDCs together with regMΦ were injected intravenously in to syngeneic C57BL/6 mice. Seven days later, mice were immunized subcutaneously with OVA or KLH in CFA. Serum IgG1 levels against OVA (E) or KLH (F) were measured by ELISA. Serum was diluted serially as indicated (mean ± SEM, 3 mice per group). Similar data were obtained in a second experiment.