In vitro suppression of anti-FVIII antibody production by engineered FVIII-specific human Tregs. Pooled splenocytes from 2 immunized DR1/E16-FVIII-KO mice were used as B-cell responders. In a T25 flask, 1 × 107 splenocytes were cocultured with 17195TCR-transduced or mock-transduced Tregs at different ratios in the absence/presence of 1 µg/mL rFVIII. After 6 days in culture, FVIII-specific ASCs were detected using an ELISPOT assay as described in “Methods.” (A) FVIII-specific ASCs detected in splenocyte cultures that contained no TCR- or mock-transduced Tregs. (B) FVIII-specific ASCs detected in splenocytes cocultured with various ratios of 17195TCR-transduced or mock-transduced Tregs. Coculture with 17195TCR-Tregs profoundly inhibited anti-FVIII ASC formation, even with responder to suppressor ratios as low as 1:0.0625 (1/16). The histograms in C and D summarize the ELISPOT data in A and B, respectively. Data are presented as mean ± standard error of the mean. (E) The quality of the Tregs used in these experiments was evaluated by measuring their GFP expression (left) and the expression of Foxp3 and Helios in the GFP+ cells (right).
