Characterization of differential acquisition of CD80 by memory CD4 T cells. (A) Memory CD4 T cells were generated as described in “Materials and methods,” under “Generation of CD4 T cells in culture and acquisition of CD80.” Naive and memory CD4 T cells were stained for the markers CD80, CD28, and CTLA-4 (solid line) or respective isotype control (dashed line) and FACS analyzed. Graphs are representative of 4 experiments and the percentage represents the average of positive cells with standard deviation (SD). (B) Naive or memory CD4 T cells (1 × 10⁶) were cultured with 1 × 10⁵ (high-CD80–expressing fibroblasts) APCs in the presence of 0.001 μg/mL PCC for the indicated time and analyzed for CD80 acquisition. Data are representative of multiple repeats. (C) Memory CD4 T cells (1 × 10⁶) were cultured with 1 × 10⁵ (high-CD80–expressing fibroblasts) APCs in the presence of 0.001 μg/mL PCC. Following incubation for 24 hours, cells were separated from APCs and cultured in the absence of APCs and PCC. FACS analysis was performed for CD4 and CD80, IE^k, and CTLA-4 markers at indicated time points. Data are representative of 3 repeats. (D) Memory CD4 T cells were treated with either 20 μg/mL of anti-CD28 (bottom panel) or isotype control (top panel) antibody and then cultured with APCs and PCC (0.001 μg/mL) for 24 hours. Data are representative of 2 repeats. (E) Memory CD4 T cells (1 × 10⁶) were cultured with either APCs expressing very low levels of CD80 (DCEK, ⋄) or APCs expressing high levels of CD80 (P13.9, ▪) with varying concentrations of PCC. Experiments were repeated 3 times and values are averages with SD. *Dead-cell FACS profile in the memory CD4 T-cell population.