FVIII-selective immunosuppression by expanded 17195TCR-Tregs. (A) FVIII-specific immunosuppression by 17195TCR-Tregs. To measure FVIII-2191-2220–specific proliferation of T effectors as FVIII-specific responder cells, 3-week expanded 17195TCR-T effectors were labeled with cell proliferation dye (as in Figure 2A) and cocultured with 3-week expanded mock-transduced Tregs or 17195TCR-transduced Tregs in the presence of anti-CD3ε antibody or FVIII-2191-2220 for 4 days without IL-2. The ratio of responder:Treg:irradiated APCs was 1:2:1. The histogram shows dye dilution indicating proliferation of only the GFP⁺ cell population. Representative results are shown for 1 of 3 repeat experiments carried out with cells from different donors. (B) FVIII-selective immunosuppression by 17195TCR-Tregs. Mock-transduced and 17195TCR-transduced CD4 T cells were expanded for 3 weeks following the FVIII-2191-2220/ODN protocol as in Figure 4B. GFP⁺-transduced Tregs were enriched by FACS sorting. The experimental setup was similar to that shown in A but with different Treg:T effector ratios, and the cells were stimulated with rFVIII (0.5 μg/mL) instead of FVIII-2191-2220. Immunosuppression was evaluated using a ³H-thymidine incorporation assay. Dot plots indicate GFP expression in mock-transduced and 17195TCR-transduced Tregs (left) and the expression of Foxp3 and Helios in GFP⁺ cells (right). Results were analyzed using a 1-tailed t test (*P < .05, **P < .005). Representative data from 1 of 3 experiments are shown. (C) Suppression of FVIII-specific cytokine secretion by 17195TCR-Tregs. Four-week expanded mock-transduced Tregs or 17195TCR-Tregs were mixed with 17195TCR-T effectors (responders) at the indicated ratios with γ-irradiated DR1-PBMCs and rFVIII (0.2 μg/mL) and cultured for 36 hours without IL-2. Cytokines in culture media were measured using a human Th1, Th2, Th17 CBA kit (BD Bioscience). The quality of the mock-transduced and 17195TCR-transduced Tregs was evaluated by measuring their GFP expression (left) and the expression of Foxp3 and Helios in the GFP⁺ cells (right). Raw mean fluorescence intensity data from the CBA assays were converted to cytokine concentrations according to standard curves generated for each experiment. Data are presented as mean ± standard deviation. (D) IL-10 is suppressed in the presence of 17195TCR-Treg. Production of IL-10 was measured by CBA assay (results using cells from 2 different donors). The experimental protocol was identical that described for C.