IL-10 signaling is required for the maintenances of Foxp3 expression. (A) IL-10 GFP and Foxp3 red fluorescent protein (RFP) dual-reporter mice (Tiger-Foxp3- internal ribosomal entry site-mRFP (Tiger-FIR) mice on C57BL/6 background) were crossed with B6 wild-type (Wt) or transgenic (Tg) mice, and these mice were then also immunized with type II collagen (CII) according to the same protocol that we used for collagen-induced arthritis. Upon gating on CD4+ T cells, cells from the lymph nodes of unimmunized Wt and Tg mice were analyzed for RFP and GFP expression (left). Cells from draining lymph nodes of Wt and Tg mice on day 28 after immunization with CII and Complete Freund's Adjuvant (CFA) were also analyzed (right). The results of one representative experiment are shown. (B) The percentages of CD4+GFP+ cells among CD4+RFP+ cells in Wt and Tg mice described in (A) are shown (three mice in each group). Tregs, regulatory T cells. (C) Splenocytes from B6 Wt and Tg mice (five mice in each group) were stimulated with phorbol 12-myristate 13-acetate and ionomycin for 4 hours. The expression levels of Foxp3 and transforming growth factor β (TGF-β) were measured by intracellular staining. A representative example is shown for one of three repeated experiments. (D) CTLA4 was analyzed by intracellular staining in CD3+CD4+Foxp3+ and CD3+CD4+Foxp3- cells from the lymph nodes of unimmunized and immunized C57BL/6 Wt and Tg mice (day 28 after immunization with CII and CFA). The results shown are from one of three repeated experiments. (E) B6 Wt and Tg Foxp3 RFP mice used in (A) were immunized with CII, with CFA or left untreated, and the percentage of RFP+ cells among gated CD4+ cells in Wt and Tg mice are shown (three mice in each group). (F) RFP+CD4+ T cells were sorted from B6 Thy1.1 Wt and Tg mice, transferred to B6 Thy1.2 Wt recipient mice and immunized with CII and CFA. One week later the level of Foxp3 expression in Thy1.1+ cells from the host mice was analyzed on the basis of the percentage of RFP+ cells (three mice in each group; P < 0.05). (G) The results of one representative experiment of the three repeated experiments described in (F) are shown.