FVIII-specific and bystander immunosuppression by ANS8 CAR Tregs. Sorted human Treg and T-effector cells were transduced with Mock, ANS8 CAR, 17195 TCR, or Ob2F3 TCR, expanded, and rested. (A) Cell proliferation dye (CPD)-labeled 17195 T-effector cells were cocultured with different numbers of Mock or ANS8 CAR Tregs in the presence of FVIII protein (0.4 μg/mL) for 4 days. ANS8 CAR Tregs suppressed FVIII-specific effector T-cell proliferation. (B) CPD-labeled Ob2F3 T effectors were cocultured with different numbers of either ANS8 CAR or 17195 TCR Tregs in the presence of FVIII protein and pMBP for 4 days. ANS8 CAR and 19175 TCR Tregs suppressed MBP-specific effector T-cell proliferation but only when both pMBP and FVIII were present. This figure shows representative cell proliferation data of 17195 TCR (A) or Ob2F3 TCR (B) T-effector cells in the presence of the different Treg populations (left). Graphs show absolute counts of divided GFP⁺ T-effector cells after coculture with differently engineered Tregs (right, dotted line and closed circle, mock Tregs; closed square, ANS8 CAR8 Tregs; open square, 17195 TCR Tregs). Data indicate mean values for triplicates ± standard error of the mean (SEM). The experiment was repeated with separately transduced T cells from different donors with similar results.