VIP-differentiated DCs generate human regulatory T cells in vitro. Purified naive CD4 T cells exposed to allogeneic DC_controls (T_{reg controls}) or DC_VIPs (T_{reg VIPs}) were evaluated for suppressive/regulatory activity. (A) T_{reg VIPs} (□) or T_{reg controls} (▪) were cocultured at different ratios with syngeneic T_H1 cells in the presence of allogeneic mature DCs (mDCs). Proliferation of T cells and IL-2 production was determined. T_{reg VIPs}, alone or with allogeneic mDCs, did not proliferate. (B) T_H1 cells and T_{reg VIPs} were generated by priming of CD4 T cells from donor C with allogeneic DC_controls or DC_VIPs from donor A (T_H1^anti-A/T_{reg VIPs}^anti-A) or donor B (T_H1^anti-B/T_{reg VIPs}^anti-B) and were cocultured in the presence of mDCs from donor A or donor B, or both, as indicated. Proliferation of responder T cells was determined. Each result is the mean ± SD of 3 experiments performed in duplicate. (C) Polarized T_H1 cells were cocultured with T_{reg VIPs} and allogeneic mDCs in the presence or absence of blocking anti-IL-10, anti-TGFβ, anti-CTLA-4, or IL-2 antibody. Additionally, T_H1 + mDCs were separated from T_{reg VIPs}+mDCs in a Transwell system. The proliferative response of T_H1 cells was determined. (D) Phenotype of T_{reg VIPs} and T_{reg controls}. Expression of surface markers (CD25, CD69, CD70, CD154), intracellular IL-10 and CTLA-4, and cytosolic and membrane-bound TGFβ were determined by flow cytometry. Numbers represent the percentage of positive cells in each quadrant. Foxp3 and neuropilin 1 (Nrp1) mRNA expression in sorted CD4⁺, CD4⁺CD25⁺, and CD4⁺CD25^- T_{reg controls} (▪) or T_{reg VIPs} (□) was determined by real-time RT-PCR. Results are representative of 5 independent experiments.