NO-mediated T-cell immunosuppression by regMΦ. (A) Dose-dependent effect of regMΦ on NO production. OTII cells were stimulated for 48 hours at the conditions indicated. Before the concentration of NO in the medium was determined (mean ± SEM of 3 wells from a representative of 2 independent experiments). (B) Addition of L-NAME restored T-cell proliferation. The total number of DAPI⁻CD45.1⁺CD4⁺Vα2⁺ OTII cells was determined after 3 days of coculture conditions indicated (open bars, without; closed bars, with 1mM L-NAME; mean ± SEM of 3 wells from a representative of 2 independent experiments). (C) Supernatant from OTII cells that were activated by OVA-maDCs can induce NOS2 specific transcripts in regMΦ. Supernatants were harvested from OTII cells activated for 3 days by OVA-maDCs. One-half of culture medium of 10⁵ regMΦ (MDPs cultured on nnLNSCs for 5 days was exchanged by the supernatant of OVA-maDCs activated OTII cells. After further 48 hours, total RNA was prepared and mRNA specific for NOS2 and HPRT mRNA determined by RT-PCR. Data are representative of at least 3 independent experiments. (D) regMΦ generated from NOS2^−/− immature DCs were inefficient in suppressing T-cell proliferation. BM cells from WT BL6 mice and NOS2^−/− mice (BL6 background) were cultured with GM-CSF to generated BMDCs. On day 7, immature DCs were harvested and put into coculture with nnLNSCs. After another 7 days, WT (open bars) or NOS2-deficient (closed bars) regMΦ were used in in vitro T-cell immunosuppression assay. The total number of DAPI⁻CD45.1⁺CD4⁺ Va2⁺OTII cells was determined after 3 days of cocultures at the conditions indicated (mean ± SEM of 3 wells from 1 representative of 2 independent experiments).