Changes in Treg frequencies in hemophilia B mice. Flow cytometric analyses of spleen, MLN, ILN, and PP cells of hemophilia B mice following oral delivery of FIX plant material and IV challenge with FIX. Control groups were fed with WT plant material (wt fed) or were fed with FIX material but not challenged with IV injections of FIX (FIX fed no injection). (A) CD4⁺CD25⁺FoxP3⁺ Tregs; (B,C) CD4⁺CD25⁻LAP⁺ Tregs; and (D) Tr1 cells (CD49b⁺LAG-3⁺CD4⁺). (C) Independent experiment comparing the CD4⁺CD25⁻LAP⁺ Treg frequencies of mice that were fed with FIX and intravenously challenged either with FIX or with and identical antigen dose of keyhole limpet hemocyanin (KLH). Data points are for individual mice. Averages ± SEM are also indicated (n = 3-6 per group). Representative flow cytometry plots are shown for spleen, MLNs, and PPs. (E,F) Quantitative RT-PCR analysis of IL-10 (E) and TGF-β (F) expression in different subsets of magnetically sorted cells from FIX-fed and control mice after a 48-hour lymphocyte culture established for individual animals (and comprising pooled MLN cells and splenocytes). Data indicate average cytokine expression levels (± SEM) for FIX-stimulated relative to unstimulated cultures (n = 6 mice/cell type, except for CD4⁺CD25⁺ cells, which was n = 3).