Kinetic change in disappearance of CD80 and IE^k, continued proliferation of CD4⁺CD80^acq T cells, and acquired CD80-dependent proliferative response. (A) Naive CD4⁺ T cells were incubated with PCC-pulsed P13.9 APCs for 20 hours to acquire MHC class II-IEk and CD80. CD4⁺CD80^{acq hi} T cells were cultured in the absence of APCs and PCC peptide at various time points, and acquired IEk and CD80 on these T cells were assessed by using multicolor FACS analysis. (B) CFSE-stained naive CD4⁺ T cells were stimulated with PCC-pulsed P13.9 APCs for 20 hours. The cells were further cultured in the presence or absence of APCs for an additional 24 hours. Proliferative response of the cells in the presence or absence of APCs was examined by the FACS analysis. (C) Naive CD4⁺ T cells were cocultured with either PCC-pulsed APCs expressing low levels of CD80^low (DCEK) or PCC-pulsed APCs expressing high levels of CD80^hi (P13.9) for 20 hours. After the separation from DCEK and P13.9 APCs, both CD4⁺CD80^{acq low} and CD4⁺CD80^{acq hi} T cells were incubated in the absence or presence of an additional 1 μg/mL PCC without APCs for 6 (▪) or 24 hours (). Proliferation of CD4⁺CD80^{acq low} and CD4⁺CD80^{acq hi} T cells (3 × 10⁵ cells per well, 96-well plate) was examined using ³H uptake assay. Data represent mean ± SE of 11 different experiments. Percentages are the percent positive cells in the respective quadrant.