T-cell differentiation and activation. (A) Enumeration of memory and effector T cells. Representative profiles (left) and summary data (right). (B) Analysis of proliferation of either naive CD4⁺ T cells (right) separated by negative selection with magnetic beads or CD4⁺ T cells within PBMCs (left) by dilution of cell trace violet after stimulation with CD3 and CD28. Cells were harvested and analyzed by flow cytometry on day 4 (blue) and day 6 (red). Histograms were gated on CD3 and CD4. Unstimulated control cells are shown (gray). In the same experiment, naive CD4⁺ T cells were analyzed for activation based on induction of CD69. The percentage of CD69⁺ cells at each division was determined (day 6, upper panel; day 4, lower panel) and plotted (controls, open blue; I.1, black filled). CTV, cell trace violet; T_CM, central memory T cells; T_EM, effector memory T cells; T_EMRA, CD45RA⁺ effector memory T cells.