Alloantigen-specific cytokine-producing CD3⁺CD4⁺ T cells. A typical flow cytometric example of cytokine-producing cells is shown in panel A. For this purpose, labeled PBMCs were stimulated with unlabeled PBMCs of a different donor at a 1:1 ratio for 6 hours in the presence of soluble costimulation. As a negative control, these PBMCs were stimulated with unlabeled autologous PBMCs, whereas CMV and the combination of PMA and ionomycin were used as positive controls, the first serving as another antigen-specific stimulus and the latter as polyclonal stimulus. Percentages are of CD3⁺CD4⁺ T lymphocytes, whereas in brackets the percentage of cytokine-positive cells within CD154⁺CD4⁺ cells is given. (B) A summary of the results of 11 independent experiments, each using 2 different donors, where cytokine-producing cells are identified as CD3⁺CD4⁺ or subsequently using intracellular staining as positive for CD154, IL-2, IFN-γ, IL-10, or IL-17. (C) We determined the fraction of cytokine-positive cells within the CD154⁺ fraction.