Expression of FOXP3, FOXP3⁺Hel-FL, and FOXP3⁺Hel-Δ3B reduces cell expansion and survival. (A) Cell counts of eTregs growing in IL-2–supplemented media over 9 days. n = 4 for each group from 4 different donors. *P ≤ .05 compared with empty vector control based on a 1-tailed Mann-Whitney U test for each time point. (B) Numbers of live (Zombie Green and Annexin V negative) eTregs after stimulation for 2, 4, and 6 days with anti-CD3 and anti-CD28 plate-bound antibody. Cells were plated as follows: 2 × 10⁵ empty vector control cells (n = 5), 2 × 10⁵ FOXP3 eTregs (n = 5), 2 × 10⁵ FOXP3⁺HEL-FL eTregs (n = 6), or 2 × 10⁵ FOXP3⁺Hel-Δ3B eTregs (n = 6). T cells from 4 to 6 different donors were used. *P ≤ .05 compared with empty vector control based on a 1-tailed Mann-Whitney U test. (C) Representative contour plots of activation-induced cell death in eTregs or empty vector control cells after stimulation for 2, 4, and 6 days.