In vitro suppression of antibody production by FVIII-specific CAR Tregs. Whole splenocytes isolated from E16×DR1 mice with high antibody titers (measured by ELISA) were cultured in the presence of FVIII for 6 days and antibody-secreting cells were enumerated by ELISPOT assay. Bars indicate mean ± SEM. (A) FVIII-specific antibody-secreting cells were detected in splenocyte cultures only in the presence of FVIII. (B) Coculture with either ANS8 CAR or 17195 TCR Tregs (but not Mock Tregs) inhibited formation of FVIII-specific antibody-secreting cells. The experiment was repeated with separately transduced Tregs from different donors with similar results.