(A) DC.Foxp3- or control DC-induced autologous or allogeneic naïve (CD45ROneg) or memory (CD45RAneg) CD4+ T cells were analyzed for Foxp3 and CD25 co-expression by flow cytometry on day 21 of culture. (B) Day 21 expanded CD4+ T cells, gated on the CD25+ sub-population of CD4+ T cells, were analyzed for expression of the markers Foxp3, GITR, CTLA-4 and neuropilin-1 (NRP-1) by flow cytometry. CD4+CD25+ T cells were MACS-isolated from day 21 DC-T cell cultures and added to naïve autologous or allogeneic CD8+ T cells in the presence of anti-CD3/CD28 Dynabeads™ (Invitrogen) and rhIL-2 (for CFSE proliferation assays-only). Four days after the initiation of these cultures, we assessed DC-T cell cluster size (based on visual inspection using light microscopy; panel C) and T cell proliferation (based on CFSE dilution; panel D). On day 8 of culture, T cells were analyzed for intracellular IFN-γ by flow cytometry (E). For each panel, similar data were obtained in 3 independent experiments. Transduction efficiency for DC.Foxp3: 67% in panel A, 46% in panel B, 63% in panels C-E.