Cytokine profiles of CD154⁺ and CD154⁻ CD3⁺CD4⁺ T lymphocytes after polyclonal stimulation. A representative flow cytometric example of the cytokine profile of CD154⁺ and CD154⁻ CD3⁺CD4⁺ T lymphocytes after a 6-hour stimulation with either costimulation (cos only) alone or combined with PMA and ionomycin (P/I) is shown in panel A. For this purpose, alloantigen-specific CD154⁺ T cells are isolated upon a 24-hour coculture of PBMCs of 2 different donors using magnetic cell separation. These fractions were stimulated with costimulation alone or a polyclonal stimulus. Cytokine-producing cells are enumerated using flow cytometry staining for IL-2 in combination with either IFN-γ (top panel), IL-17 (second panel), or IL-10 (third panel). In addition, IFN-γ was also combined with IL-17 (bottom panel). Averages (+ SEM) of 10 independent experiments of total cytokine-producing CD3⁺CD4⁺ T lymphocytes compared between the CD154⁺ (closed bars) and CD154⁻ (open bars) fraction are depicted in panel B. Next, we analyzed the total percentages of cytokine-producing cells for each CD4⁺CD154⁺ T-cell subset. White bars represent Tnaive; gray bars, Tcm; and the black bars, Tem cytokine-producing cells (C). These were subsequently dissected to display single-positive and double-positive cytokine expression profiles of these CD3⁺CD4⁺ CD154⁺ T lymphocytes within the different T-cell subsets being naive (Di), Tcm (Dii), and Tem (Diii) using CCR7 and CD45RO monoclonal antibodies (D). *Significantly different (P < .05).