Figure 3. Suppressive function of F8CAR-Tregs were examined in ³H-thymidine incorporation assay and CFSE proliferation assay.

(A) F8CAR-mFoxp3 transduced T cells (F8CAR-Tregs) were cultured at decreasing ratios with either CD4⁺ T effector cells isolated from FVIII-primed HemA BL6 inhibitor mice (nTresp) or F8CAR transduced T cells (F8CAR-Tresp) in the presence of APCs and 10 U/ml human FVIII protein. ³H-Thymidine was added to the plate after 72 hrs incubation, and the plate was cultured for another 18 hrs. Thymidine incorporation was measured in counts per minute with Betaplate scintillation counter. There is no statistical significance between the nTresp and F8CAR-Tresp groups. However, significant differences were observed between the suppressed cells (Tregs+Tresp) and Tresp only (p < 0.01) for both groups. (B) In the CFSE proliferation assay, F8CAR-Tresps were stained with CFSE dye and cultured with anti-CD3/CD28 Dynabeads, FVIII protein plus APCs, FVIII protein only, or APCs only to stimulate T cells proliferation. (C) The suppressive function of F8CAR-Tregs was measured by co-culturing with F8CAR-Tresps, which were stained with CFSE dye before plating, at decreasing ratios in the presence of APCs and 10 U/ml FVIII protein. F8CAR-Tresps cultured in the presence of APCs and 10 U/ml human FVIII protein were used as controls. Four days later, CFSE expression was measured on flow cytometry. Top panel: representative flow cytometry figures of the FVIII-specific suppressive assay. Bottom panel: Summary of suppressive function of F8CAR-Tregs at decreasing ratios.