Cytokine production by FOXP3-deficient Th17 and Th1 cell lines. Th1 and Th17 cells transduced with siFOXP3 or control siLuc and purified based on ΔLNGFR expression were restimulated with anti-CD3/anti-CD28–coated beads (1 bead: 32 cells) in the presence of IL-2. (A) On day 8 after stimulation, cells were washed and replated at 1 × 10⁶ cells/mL, and supernatants were collected 48 hours later for analysis by cytometric bead array. Each dot represents the fold change in cytokine production by siFOXP3-transduced T cells relative to control siLuc-transduced T cells for one donor. (B-C) On days 0, 8, and 11 of the expansion, Th1 and Th17 cells were restimulated with PMA and ionomycin and stained intracellularly for IFN-γ and IL-17A. Analysis was conducted on ΔLNGFR⁺ cells. (B) Representative plots of IL-17A and IFN-γ expression 11 days after activation. (C) The average fold changes in the percentage of IFN-γ⁺IL-17⁻ Th17 cells (n = 4), IFN-γ⁺ of IL-17⁺ Th17 cells (n = 4), and IFN-γ⁺ Th1 cells (n = 3) in siFOXP3 relative to control siLuc over the course of the experiment. At day 8 after activation, the fold change in the percentage of IFN-γ⁺IL-17⁻ Th17 cells is significant (P = .0436) and at day 11 after activation, the fold change in the percentage of IFN-γ⁺ of IL-17⁺ is significant (P = .0033).