Active suppression of antibody formation against FVIII by induction of regulatory T cells. (A) Adoptive transfer experiments. CD4⁻, CD4⁺CD25⁻, and CD4⁺CD25⁺ cells were purified via magnetic sorting from spleens and MLN of FVIII-fed mice (n = 3) at time-point 3 indicated in Figure 5A and pooled (with a final ratio of approximately 30% spleen and 70% MLN-derived CD4⁺ T cells). Cells (10⁶ per mouse) were adoptively transferred into naive BALB/c mice via tail vein injection. Control cells were from unchallenged naive mice of the same strain. Twenty-four hours later, all recipient mice (n = 5 per group) were challenged with 1 IU FVIII in adjuvant via subcutaneous injection. IgG titers against FVIII were determined 3 weeks later. All data are shown as averages ± SEM (*P < .05; **P < .01). (B) Frequencies of Treg subsets in FVIII fed and control BALB/c mice with hemophilia A. Cells derived from spleens, MLN, inguinal lymph nodes (ILN), and Peyer’s patches (PP) were isolated from mice that had either been fed with FVIII (HC+C2, “FVIII fed”) or WT plant material (“control”), followed by IV treatment with FVIII (“FVIII fed”). Stained cells were first gated for live CD4⁺ cells (positive CD4-eFluor 450 and negative viability dye eFluor 506 staining). The frequencies of CD4⁺CD25⁻ Latency Associated Peptide (LAP)⁺ cells, CD4⁺CD25⁺Foxp3⁺ cells, and type 1 regulatory T cells (CD4⁺LAG-3⁺CD49b⁺) were calculated using flow cytometric analysis. Data for individual animals as well as averages ± SEM are shown (n = 3-5/group). Unpaired 2-tailed Student t tests were used to calculate P values for all panels.