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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: J Immunol. 2018 Jul 18;201(5):1434–1441. doi: 10.4049/jimmunol.1701800

Figure 4. Mechanism of suppression of FVIII-specific B cells by FVIII-BAR hTregs in vivo.

Figure 4

(A) Outline of the protocol. At the end of the in vivo experiment described in Figure 3 panel A, the mice were sacrificed and splenocytes were pooled from the FVIII-BAR hTreg or control OVA-BAR hTregs recipients. B and T cells, respectively, were purified from the pooled splenocytes by magnetic-activated cell sorting and co-cultured (B:T cell ratio = 2:1) in different combinations as indicated for 6 days in the presence of 0.1 μg/ml human rFVIII, followed by FVIII-specific B cell ELISPOT assay. (B) FVIII-specific ASC detected by the B cell ELISPOT assay. No anti-FVIII spot formation could be detected when the B cells were from the tolerant donors that received FVIII-BAR hTregs. (C) Summarized data from results shown in panel B. The data summarized from quadruplicate repeats (n = 4) were expressed as mean ± SEM. **p < 0.01, as compared to all other cell combinations except for condition #4 (the B and T cells were from the control mice and tolerant mice, respectively) or #1 (Both B and T cells were from the control mice group). The data was analyzed by student’s t test. N.D., none detected.