Cytokine-induced IFN-γ⁺ Th cells lack 4-1BB expression. (A) Expression of activation markers on live IFN-γ⁺ memory Th cells detected with the cytokine secretion assay after stimulation with the cytokine cocktail or αCD3 + αCD28 for 36 hours. Filled histograms represent stimulated; open histograms, unstimulated control samples. (B) PBMCs were stimulated with CMV lysate or with the cytokine cocktail. 4-1BB expression was analyzed on live IFN-γ⁺ Th cells at indicated time points by the cytokine secretion assay. (C) CMVpp65-specific Th1 cells were restimulated with CMVpp65 or IL-12 + IL-18 + IL-15 for 14 hours. Numbers indicate relative frequencies of 4-1BB⁺ and 4-1BB⁻ cells among the IFN-γ⁺ Th cell population; numbers in parentheses indicate frequencies among total Th cells. (D) Memory Th cells were stimulated for 36 hours with the cytokine cocktail, followed by FACS sorting of activated cells according to IFN-γ secretion. After 2 cycles of αCD3 + αCD28 restimulation, cells were activated as indicated. Numbers indicate relative frequencies of 4-1BB⁺ and 4-1BB⁻ cells among the IFN-γ⁺ Th cell population; numbers in parentheses indicate frequencies among total Th cells. One of 3 independent experiments is shown.