Proliferating alloantigen-specific CD4⁺ T cells are exclusively CD154⁺. PBMCs of donor A were stimulated with PBMCs of donor B at a 1:1 ratio, mismatched for HLA class II, in the presence of the α-CD40 monoclonal antibody and the combination of soluble anti-CD28 and anti-CD49d for 24 hours. Alloreactive CD154⁺ T cells were then isolated using automated magnetic cell sorting; a typical flow cytometric example of cells before separation (i) as well as the CD154-depleted (ii) and -enriched (iii) fraction is depicted in panel A. Both fractions as well as the unseparated cells were labeled with CFSE and subsequently restimulated in absence or presence of irradiated allogeneic PBMCs for 6 days. On day 6, proliferation of CD3⁺CD4⁺ (B top panel) and CD3⁺CD8⁺ (B bottom panel) T lymphocytes was analyzed using flow cytometry. The dark blue peak at the far right of the x-axis represents the CFSE-labeled T-cell population that has not divided. With every cell division the CFSE-intensity is halved, and the different colors identify cell populations, which have undergone one or more rounds of cell division. The numbers within the figures indicate the percentage of cells that has divided. Shown is a representative example of 3 separate experiments performed.