Mature DCs are required for the generation of CD44^hiCD8⁺ and CD44^loCD8⁺ T cells ex vivo. (A) CFSE-labeled C3H.SW CD8⁺ T cells were cultured in the presence of B6 BM-derived DCs (T cell/DC ratio = 3:1), B6 peritoneum macrophages, and cytokines including IL-2 (5 ng/mL), IL-7 (3 ng/mL), IL-15 (3 ng/mL), IL-2 plus IL-7 and IL-2 plus IL-15. Surviving T cells were numerated at day 6 after culture. All values represent means ± SD of triplicate wells. (B) CFSE-labeled whole C3H.SW CD8⁺ T cells and separated C3H.SW CD44^loCD8⁺ T cells were cultured in the presence of B6 DCs, B6 DCs plus IL-2 plus IL-15, or IL-2 plus IL-15 for 5 days. Cells were collected and stained with rat anti-CD8 and anti-CD44 antibodies or control rat IgG for flow cytometry analysis. (C) C3H.SW CD8⁺ naive T cells were cultured in the presence of B6 DCs, with or without IL-2 plus IL-15 addition, for 5 days. Cells were harvested, stained with rat anti-CD8 Ab and other indicated Abs, and analyzed by flow cytometry. Dot plots shown are gated CD8⁺ T cells. Results are representative of at least 5 independent experiments. Numbers in quadrants represent percentages of total cells in each cell fraction. (D) Unstimulated CD8⁺ T cells and B6 DC-induced CD44^hiCD8⁺ and CD44^loCD8⁺ T cells were stained with anti-CD8 Ab, then permeabilized, stained with the indicated Abs, and analyzed by flow cytometry. Histograms shown are gated on CD8⁺ T cells.