DC_VIP generate human CD8 T_reg. (A) Human naive CD8⁺ T cells (5 × 10⁶) were cultured with allogeneic DC_controls or DC_VIPs (5 × 10⁵ cells). After 5 days, CD8 T cells were isolated from the cocultures and assayed for phenotype (left panel) and cytokine profile (right panel) by flow cytometry. Human naive CD8⁺ T cells were used as controls. Numbers represent the percentage of positive cells in each quadrant. Cytokine levels in cultured supernatants (determined by ELISA) were consistent with the intracellular staining (not shown). Results are representative of 4 experiments with similar findings. (B) Total CD8⁺, CD8⁺CD28⁺, or CD8⁺CD28^- T cells were isolated from the coculture of human naive CD8⁺ T cells (5 × 10⁶) with allogeneic DC_controls or DC_VIPs (5 × 10⁵ cells) for 5 days and were cultured with human allogeneic mDCs (10⁴). Proliferation of CD8 T cells was determined. CD8 T cells without allogeneic mDCs did not proliferate. Each result is the mean ± SD of 3 experiments performed in duplicate. (C) Total CD8⁺, CD8⁺CD28⁺, or CD8⁺CD28^- were isolated from the coculture of human naive CD8⁺ T cells (5 × 10⁶) with allogeneic DC_controls or DC_VIPs (5 × 10⁵ cells) for 5 days and were cocultured at different ratios with human syngeneic T_H1 cells (10⁵) in the presence of allogeneic mDCs (10⁴). (D) Human T_H1 cells were cultured with allogeneic mDCs (10⁴) and CD8⁺ T cells (CD8_VIP, 2 × 10⁴) isolated from cocultures with DC_VIPs, in the presence or absence of blocking anti-IL-10, anti-TGFβ, anti-CTLA-4, or IL-2. Isotype IgG was used as control. Additionally, T_H1 + mDCs were separated from CD8_VIP + mDCs in a Transwell system. The proliferative response of T_H1 cells and the production of IFNγ were determined after 4 days of culture. mDCs alone did not proliferate. Each result is the mean ± SD of 3 experiments performed in duplicate. (E) CTLA-4 expression of naive CD8 T cells or CD8 T cells isolated from cocultures of human naive CD8 T cells and allogeneic DC_controls or DC_VIPs was determined by flow cytometry. Dashed lines correspond to isotype IgG controls. Results are representative of 4 independent experiments.