Kinetics of CD25 and CD127 expression of CD154⁺CD4⁺ T cells and proliferation of CD154⁺CD4⁺ T cells obtained from HLA-identical cocultures. PBMCs of one donor were cocultured with CFSE-labeled PBMCs of a different donor in the presence of costimulatory antibodies directed against CD28/CD49d and a monoclonal antibody against CD40. CFSE-labeled autologous PBMCs were used as a negative control and CMV-stimulated PBMCs, as a positive control. The cell surface expression of CD154 is plotted against CD25 (Ai-ii) or CD127 (Aiii-iv) at 6 (Ai,iii) and 24 (Aii,iv) hours (left panels). In the right panels of panel A, the expression of these markers is displayed for the different combinations of mismatches (MMs) for HLA-DR and CMV stimulation within the CD154⁺ fraction at 24 hours. Median fluorescence intensities (MFI) for CD25 and CD127 within the CD154⁺CD3⁺CD4⁺ T lymphocytes were related to the number of MMs on HLA-DR at 6 and 24 hours (B) and compared with the MFIs of CD25 (Bi) and CD127 (Bii) of CD154⁺ CMV-specific T cells and CD154⁻CD3⁺CD4⁺ T cells. *Significantly different compared with 6 hours. # and ^ indicate significantly different compared with CD154⁺ population with 0, 1, or 2 MMs on HLA-DR at 6 and 24 hours, respectively. Next, CD154⁺CD4⁺ T lymphocytes were isolated from combinations of PBMCs of 2 HLA-identical donors incubated at a 1:1 ratio for 24 hours. The CD154-enriched (Ci-iv top panel) as well as the CD154-depleted (Cv-viii bottom panel) fraction were CFSE-labeled and subsequently restimulated for 6 days using either irradiated PBMCs of the donors at a 1:1 ratio, 200 U/mL recombinant human (RHu) IL-2, or both. The dot plots show forward scatter (FSC) and side scatter (SSC) of the cells at day 6. Region 1 (R1) contains the apoptotic cells and region 2 (R2) signifies the viable CD154⁺ and CD154⁻ T cells. The percentage of divided CD3⁺CD4⁺ T cells from R2, restimulated with irradiated PBMCs and RHu-IL-2, is calculated from the dilution of CFSE dye as a result of cell division (C histograms on right).