CAR-mediated stimulation of FVIII-specific CAR Tregs. Sorted human Tregs were transduced with ANS8 CAR or Mock, expanded in the presence of IL-2, rested, and then stimulated with FVIII. Gating strategy for GFP⁺ and GFP^– cells is shown on the left. Intracellular staining for LAP and GARP in GFP⁺ cells was increased in the presence of FVIII but not in the presence of OVA. Foxp3 expression is increased in the ANS8 CAR GFP⁺ population in the presence of FVIII. Data are representative of 3 independent experiments.