c-maf Expression Correlates with IL-10 Production in Th1, Th2, and Th17 Cells
To investigate further the downstream factors involved in regulating IL-10 production, we differentiated DO11.10 CD4+ Tcells with increasing doses of OVA, in the presence or absence of IL-12, and quantified the expression of cytokines and transcription factors by real-time RT-PCR. Low-dose antigen resulted in transcription of Il4 and this was abrogated by both high antigen doses and IL-12 (Figure6A). A low amount of transcription of Ifngamma was induced by IL-12 when cells were differentiated with low antigen dose, but this effect of IL-12 was markedly upregulated with increasing doses of antigen (Figure6A). A low amount of Il10 transcription was observed at low doses of antigen accompanying Il4 expression (Th2 cell response), and this was abrogated by increased doses of antigen as was Il4 expression (Figure6A). At low doses of antigen, IL-12 had little effect to increase IL-10 mRNA expression (Figure6A) in keeping with the protein data (Figure1A). However, IL-12 induced a high amount of Il10 transcription as well as Ifngamma expression with increased antigen doses (Figure6A), again in keeping with the protein data (Figure1A). 
CD4+ Tcells differentiated with increasing doses of antigen did not express high amounts of Tbx-21 (T-bet) mRNA, unless they were cocultured with IL-12 (Figure6B). In contrast, high amounts of GATA-3 mRNA expression were only observed under Th2 cell differentiation conditions (low-dose antigen) (Figure6B), and this expression was markedly downregulated by both increasing antigen dose and coculture in IL-12 (Figure6B). Differentiation of Tcells under low antigen dose led to expression of c-maf, in keeping with the Th2 cell profile (Ho etal., 1996), which was almost completely abrogated by increasing doses of antigen (Figure6B). Interestingly, IL-12 sustained the high expression of c-Maf mRNA even at the highest antigen dose (Figure6B). Moreover, IL-12 maintenance of c-maf expression required STAT4 activation (data not shown). 
In Th17 cells that expressed IL-17a as well as IL-10 mRNA (Figure6C), T-bet and GATA-3 mRNA were undetectable (data not shown), whereas that of ROR-gammat was high (Figure6C) (Ivanov etal., 2007). Th17 cells also expressed high amounts of c-maf (Figure6C), confirming a recent report (Bauquet etal., 2009). c-Maf is therefore expressed in all IL-10-expressing Tcell populations tested (Figures 6B and 6C) and may not be just a Th2 cell-specific transcription factor as originally thought (Ho etal., 1996). We showed also that like Il10 expression, c-maf expression was inhibited in Th1 and Th17 cells in the presence of the MEK1 and MEK2 inhibitor (PD184352), whereas T-bet and RORgammat expression was hardly affected (Figure6D). 
