FOXP3-null CD4⁺CD25⁻ Tconv cell clones proliferate to a greater extent and produce more IFN-γ and IL-2 than WT Tconv cell clones. Tconv cell clones from a subject who is hemizygous for a null mutation in FOXP3 were labeled with CFSE and stimulated with different ratios of anti-CD3/anti-CD28–coated beads. After 4 days, Tconv clones were stained with anti-CD4 and anti-FOXP3 Abs (236A/E7) and read on a FACSCanto. (A) Activation-induced FOXP3 expression in WT but not FOXP3-null Tconv cell clones. Two representative WT and 2 FOXP3-null Tconv clones are shown for clones that were stimulated with 1 anti-CD3/anti-CD28–coated bead per 32 cells for 4 days. (B) One representative experiment; numbers in each plot represent the division index. (C) Average division index of multiple WT (n = 7) and FOXP3-null (n = 11) Tconv clones. (D) Division indices (DI) of the FOXP3⁺ and FOXP3⁻ populations are given within each plot of 2 representative WT Tconv clones stimulated with 1 anti-CD3/anti-CD28–coated bead per 32 cells for 4 days. (E) Average IFN-γ and IL-2 production by WT (n = 7) and FOXP3-null (n = 11) Tconv clones (5 × 10⁵ cells/mL). Supernatants from cultures were collected 20 hours after T-cell activation and analyzed by ELISA. (F) Percent suppression of IFN-γ production in Tconv clone cocultures. FOXP3-null clones were labeled with CFSE and cocultured at the indicated ratios with WT (black) or FOXP3-null (gray) Tconv clones labeled with CPD. Cocultures were activated with anti-CD3/anti-CD28–coated beads (1 bead: 32 cells). After 4 days, cocultures were restimulated with PMA and ionomycin and stained for IFN-γ. Shown is the average percent suppression against 3 different FOXP3-null responders by each WT or null clone. The average FOXP3 expression on day 4 for the WT clones is also shown (n = 3). Error bars represent SEM. *P < .05.