Characterization of CD8⁺CD28⁻ T cells generated from the primed human naive CD8⁺ T cells with allogeneic regulatory DCs.

(A) Human allogeneic naive CD8⁺ T cells (5 × 10⁶) were cultured with allogeneic normal or regulatory DCs (5 × 10⁵) for 5 days, and CD8⁺ T cells isolated from the coculture were assayed for phenotype (left panel) and cytokine profile (right panel) by FACS. The results are representative of 5 experiments with similar results; data are represented by a dot plot. (B) CD8⁺CD28⁺ T cells or CD8⁺CD28⁻ T cells were isolated from the coculture of human naive CD8⁺ T cells (5 × 10⁶) with allogeneic normal mDCs or regulatory iDCs (5 × 10⁶) for 5 days. Ag-primed CD4⁺ T cells, CD8⁺CD28⁺ T cells, or CD8⁺CD28⁻ T cells (10⁵) were cultured with or without allogeneic normal mDCs (10⁴). In another experiment, Ag-primed CD4⁺ T cells (10⁵) were cultured with allogeneic normal mDCs (10⁴) in the presence or absence of different numbers of CD8⁺CD28⁺ T cells and CD8⁺CD28⁻ T cells in 96-well plates, and the proliferative response was measured on day 5. For Transwell experiments, CD8⁺CD28⁺ T cells or CD8⁺CD28⁻ T cells (10⁶) plus allogeneic normal mDCs (10⁵) were either added directly to the coculture of Ag-primed CD4⁺ T cells (10⁶) with allogeneic normal mDCs (10⁵) or were separated in 24-well plates. Following depletion of DCs, T cells (10⁵) were transferred to 96-well plates to measure the proliferative response on day 5. Data were expressed as mean values ± SD of 5 individual experiments. *P < .01 compared with Ag-primed CD4⁺ T cells plus allogeneic normal mDCs by Student paired t test. Error bars express SD of mean values.