In contrast T bet downregulated the expression of Tfh
In contrast, T-bet downregulated the expression of Tfh functional molecules, CD40L, ICOS, and IL-21; accordingly, T-bet overexpression abrogated the helper function of IL-12-stimulated CD4+ T cells. Thus, T-bet seems more involved in the downregulation of the helper functions than in the differentiation of Tfh cells in humans. This hypothesis is supported by the fact that among blood cTfh cells in humans, the subset expressing T-bet (which expresses the chemokine receptor CXCR3) is the least efficient at providing help to HQNO (Locci et al., 2013, Morita et al., 2011). Even when CXCR3+ cTfh cells provide help to B cells, for example, after split-virus seasonal influenza vaccination, their helper activity seems limited to memory B cells and does not help naive B cells, partly because of limited expression of IL-21 (Bentebibel et al., 2013). Overall, these observations suggest that T-bet expressed by human Tfh lineage cells might act as a rheostat, regulating their helper capacities. Our study also showed that IRF4 plays an important role in the regulation of the differentiation of IL-12-stimulated CD4+ T cells. IRF4 was rapidly induced in response to CD3 and CD28 stimulation but was not largely affected by IL-12 stimulation. Despite a short-lived effect, IRF4 siRNA transfection significantly decreased the expression of Tfh molecules, including CXCR5, IL-21, ICOS, and Bcl-6, by IL-12-stimulated CD4+ T cells. In contrast, IRF4 siRNA transfection increased their IFN-γ expression. Furthermore, IRF4 siRNA transfection decreased the helper capacity of IL-12-stimulated CD4+ T cells. These observations suggest that IRF4 tips the balance of IL-12-stimulated CD4+ T cell differentiation toward the Tfh cell and away from the Th1 cell. While both T-bet and IRF4 are rapidly induced in response to CD3 and CD28 stimulation (Figure 1D), enhanced IFN-γ expression by IRF4 inhibition was not due to increased T-bet expression. T-bet overexpression by HDV transfection also did not affect IRF4 expression (data not shown). Therefore, the expression of T-bet and IRF4 might be independently regulated in human CD4+ T cells, and these two molecules may not directly affect each other. Instead, we found that IRF4 inhibition induced a marked induction of Eomes, an IFN-γ-promoting TF typically expressed by activated CD8+ T cells. Previous studies show that CD3 stimulation is sufficient to induce robust expression of Eomes in CD8+ T cells but not CD4+ T cells (Pearce et al., 2003). Consistently, we did not observe substantial Eomes expression by human naive CD4+ T cells stimulated with anti-CD3 and CD28 even in the presence of IL-12. Eomes expression became evident only when IRF4 was inhibited in IL-12-stimulated CD4+ T cells. This is consistent with a previous observation in mCD8+ T cells that IRF4 downregulates Eomes expression (Nayar et al., 2012). Furthermore, Eomes+ CD4+ T cells induced by IRF4 inhibition highly expressed the Th1 molecules IFN-γ and T-bet but poorly expressed the Tfh molecules CXCR5, IL-21, and Bcl-6. These observations suggest that IRF4 plays an important role in inhibiting Th1 cell differentiation by suppressing Eomes. Given that the expression of IRF4 by CD4+ T cells is in proportion to the strength of signals via T cell receptors (Man et al., 2013), strong IRF4 expression might be one of the mechanisms by which high-affinity T cells preferentially differentiate into the Tfh lineage (Fazilleau et al., 2009, Tubo and Jenkins, 2014). In conclusion, our study shows that the differentiation and the function of developing human Tfh cells are regulated by multiple TFs. While T-bet regulates the helper function of developing Tfh cells, IRF4 tips the balance of Th1 versus Tfh cell differentiation toward the Tfh lineage. Our study provides novel insights into vaccine designs and therapeutic approaches for human autoimmune diseases. We surmise that vaccines that preferentially induce T-bet− Tfh cells will be more effective than those that preferentially induce T-bet+ Tfh cells for efficient Ab responses. In autoimmune diseases, correction of the cTfh subset balance from T-bet− dominant to T-bet+ dominant might be beneficial for treatment by decreasing the number of functional Tfh cells and thus limiting the generation of autoantibodies.