Induction of T-cell anergy and regulation in vitro with indirect donor allospecificity. CBK (H-2k + Kb) DCs were transduced with either EIAV-GFP (control), EIAV-CTLA4-KDEL or EIAV-IDO, followed by stimulation with LPS. The transduced (and untransduced) DCs were subsequently incubated in vitro with CBA/Ca-derived CD4+ T cells. After 10 days, T cells were harvested and rechallenged in vitro with (A) donor CBK DCs or (B) third-party B10.A (H-2k + Dd) DCs, and CD4+ T-cell proliferation was assessed by thymidine incorporation on days 3, 5 and 7. (C–D) Culture supernatant from the donor DC rechallenge assay was harvested for detection of the immunoregulatory cytokines (C) TGF-β and (D) IL-10 by ELISA. (E–F) In addition, after 10 days of the primary DC-CD4+ T-cell coculture, T cells were also harvested, irradiated and added (0–105 CD4+ T cells) to a primary MLR between freshly isolated CBA CD4+ T cells and (E) donor CBK DCs or (F) third-party B10.A DCs. T-cell proliferation was assessed by thymidine incorporation after 5 days. All results are shown as the mean ± SD of triplicate wells and are representative of three independent experiments performed. *p < 0.05, two-tailed t-test.