Culture of human T cells under various conditions: effects on cell cycle, expansion, division, transduction efficiency, and proliferative responses to recall and allogeneic antigens. Purified hT cells were cultured in the presence of CD3 + IL-2 (▪); CD3/CD28 + IL-2 (▦); and CD3/CD28 (□). Except in panel C, results are expressed as mean ± SEM of 3 (A, D, E, F) to 5 separate experiments (B) carried out with hT cells obtained from 3 to 5 different donors, respectively. (A) Cell cycle was analyzed at different time points of culture. Data represent percent of cells into S + G₂/M phase. (B) Viable cells were counted at different time points of the cultures. Results represent total number of viable cells. (C) Cell division of cultured hT cells initially stained with CFSE at day 0. The solid line represents CFSE fluorescence intensities in gated CD4⁺ and CD8⁺ hT cells analyzed by flow cytometry at day 6 of culture. The gray line represents the modelization of cell division using the FlowJo software, which gives the percentage of cells that have divided since day 0. One representative experiment of 3 is shown. (D) Retroviral transduction of hT cells after 2 to 6 days of culture. The transduction efficiency was determined by expression of CD3⁺CD90⁺ 2 days after infection. Top panel: dot plot flow cytometry analysis of transduced hT cells at day 4; bottom panel, percentage of transduced T cells (CD3⁺CD90⁺ cells), values from noninfected T cells being subtracted from the indicated percentages. (E-F) After being cultured for 2, 4, 6, and 15 days, hT cells were plated in triplicates at a 10/1 T-cell/APC ratio in the presence of either irradiated autologous DCs loaded with tuberculin/candidin antigens (E) or irradiated EBV B cells used as allogeneic APCs (F). Cell proliferation was measured by ³H-Tdr incorporation 4 days later. Data were normalized comparatively to values from noncultured CD3-purified hT cells (10⁵ cpm), considered as 100% of the response, as represented by the dashed horizontal lines.