Figure 3. Tregs produced by FVIII specific expansion protocol showed increased FVIII-specific suppressive activity.

F8S Tregs were selectively expanded using APCs and FVIII protein. The proliferation and suppressive effect of F8S-SE Tregs were compared to that of Naive-NSE Tregs. (A) Timeline of the F8S Treg expansion protocol. To increase the F8S Treg populations in the polyclonal Tregs, F8S Tregs were isolated from spleens of HemA mice with anti-FVIII IgG and first cultured for 3 days with FVIII protein and APCs. They were then transferred to a new plate and stimulated with anti-CD3/CD28 beads. Anti-Crry and 2000U/mL rmIL-2 were included in all cultures. (B) Fold expansion of Tregs undergoing FVIII-specific expansion and Tregs undergoing non-specific expansion. (C) F8S-SE Tregs or Naïve-NSE Tregs were cultured with Teff at a 1:2 ratio. Suppression of Teff proliferation was measured using the [³H]-Thymidine incorporation assay. Data is presented as averages from repeated experiments with error bars indicating standard deviation. *p < 0.05. The experiments have been repeated at least once without significant variance between experiments.