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  • The molecular mechanisms regulating the differentiation of T


    The molecular mechanisms regulating the differentiation of Th1 versus Tfh paf receptor from IL-12-stimulated CD4+ T cells remain largely uncharacterized in humans. This topic has been extensively studied in mice (Weinmann, 2014), because IL-12 stimulation promotes mouse naive CD4+ T cells to express both Tfh and Th1 molecules (Eto et al., 2011, Nakayamada et al., 2011). These studies largely agree with the inhibitory role of T-bet in Tfh cell differentiation. T-bet-deficient CD4+ T cells expressed higher levels of CXCR5, IL-21, and Bcl-6 in response to IL-12 stimulation in vitro, and T-bet-deficient mice showed enhanced Tfh and GC response upon infection with Toxoplasma gondii (Nakayamada et al., 2011). T-bet was found to bind the promoter regions of the murine Bcl6 gene and was suggested to directly suppress Bcl-6 transcription (Nakayamada et al., 2011). T-bet was also shown to inhibit the function of Bcl-6 by masking the DNA-binding domain through the formation of a complex with Bcl-6 (Oestreich et al., 2012). In contrast, Bcl-6 represses the expression of T-bet (Nurieva et al., 2009, Yu et al., 2009). Thus, whether a CD4+ T cell differentiates into a Th1 or a Tfh cell seems largely regulated by the balance between T-bet and Bcl-6 in mice (Weinmann, 2014). Whether this regulatory mechanism also operates in humans remains to be established, because the dominant signal pathways promoting the expression of Tfh molecules seem different between the two species (Ueno et al., 2015). For example, although IL-12 is the most potent cytokine to induce human naive CD4+ T cells to express IL-21 (Ma et al., 2009, Schmitt et al., 2009, Schmitt et al., 2013), IL-6 and IL-21 are more potent than IL-12 in mouse naive CD4+ T cells (Suto et al., 2008). Furthermore, whereas IL-12 stimulation induces CD4+ T cells stably expressing IL-21 in humans (Ma et al., 2009, Schmitt et al., 2009, Schmitt et al., 2013), IL-12-stimulated mouse CD (mCD)4+ T cells express IL-21 only transiently (Nakayamada et al., 2011, Suto et al., 2008). In addition, although the stimulation with type I IFNs promotes the expression of Th1 molecules such as T-bet and IFN-γ in both mice and human CD4+ T cells, the expression of Tfh molecule is totally different between the two species. Type I IFNs promote the expression Bcl-6, CXCR5, and PD-1, but not IL-21, in mCD4+ T cells (Nakayamada et al., 2014). In contrast, type I IFNs suppress the expression of Bcl-6, CXCR5, and ICOS but promote the expression of Blimp-1 and IL-21 in human CD4+ T cells (Schmitt et al., 2014, Wong et al., 2010). In this study, we aimed to determine the molecular mechanisms regulating the differentiation of Th1 versus Tfh cells in human CD4+ T cells. We show that T-bet does not strongly inhibit the Tfh cell differentiation program per se in humans but inhibits the helper functions of IL-12-stimulated CD4+ T cells. Our study suggests that IRF4 serves as an important factor to tip the balance of differentiation of IL-12-stimulated CD4+ T cells toward Tfh cells and away from Th1 cells in humans.
    Discussion Human CXCR5+ CD4+ T cells generated by IL-12 stimulation expressed both Tfh molecules (CD40L, ICOS, IL-21, Batf, and Bcl-6) and Th1 molecules (IFN-γ and T-bet). Therefore, as demonstrated in mice (Nakayamada et al., 2011, Pepper et al., 2011), these CXCR5+ CD4+ T cells appear to be endowed with the ability to differentiate into both Tfh and Th1 cells. Our current study shows mechanisms that control their differentiation and functions. Mouse studies suggested that the balance between T-bet and Bcl-6 largely regulates the differentiation of CD4+ T cells into either the Th1 cell lineage or the Tfh cell lineage (Weinmann, 2014). However, our study suggests that T-bet does not strongly inhibit Tfh cell differentiation programs per se in humans. While T-bet bound to human BCL6 and PRDM1 genes (also shown by sequencing data) (Figure S7), T-bet inhibition or overexpression did not strongly affect the expression of Bcl-6 or Blimp-1 proteins in IL-12-stimulated CD4+ T cells. Furthermore, T-bet expression by human CD4+ T cells in response to stimulation via CD3 and CD28 and by IL-12 stimulation seems transient; therefore, the time window within which T-bet inhibits Bcl-6 function by masking its DNA-binding domain (Oestreich et al., 2012) seems narrow. Rather, we found that T-bet overexpression promoted CXCR5 expression by human CD4+ T cells. T-bet also downregulated the expression of CCR7 and EBI2 by human CD4+ T cells. Therefore, our results suggest that T-bet might contribute to some extent to human Tfh cell differentiation at early stages by promoting the acquisition of the phenotype required for migration toward B cell follicles and for retention within follicles.