DataSheet_2_BHLHE40 Regulates IL-10 and IFN-γ Production in T Cells but Does Not Interfere With Human Type 1 Regulatory T Cell Differentiation.pdf

Type 1 regulatory T (Tr1) cells are subset of peripherally induced antigen-specific regulatory T cells. IL-10 signaling has been shown to be indispensable for polarization and function of Tr1 cells. However, the transcriptional machinery underlying human Tr1 cell differentiation and function is not yet elucidated. To this end, we performed RNA sequencing on ex vivo human CD49b+LAG3+ Tr1 cells. We identified the transcription factor, BHLHE40, to be highly expressed in Tr1 cells. Even though Tr1 cells characteristically produce high levels of IL-10, we found that BHLHE40 represses IL-10 and increases IFN-γ secretion in naïve CD4+ T cells. Through CRISPR/Cas9-mediated knockout, we determined that IL10 significantly increased in the sgBHLHE40-edited cells and BHLHE40 is dispensable for naïve CD4+ T cells to differentiate into Tr1 cells in vitro. Interestingly, BHLHE40 overexpression induces the surface expression of CD49b and LAG3, co-expressed surface molecules attributed to Tr1 cells, but promotes IFN-γ production. Our findings uncover a novel mechanism whereby BHLHE40 acts as a regulator of IL-10 and IFN-γ in human CD4+ T cells.

I型调节性T细胞（Tr1细胞）为外周诱导的抗原特异性调节性T细胞的一个亚群。研究表明，IL-10信号通路对于Tr1细胞的极化和功能至关重要。然而，关于人类Tr1细胞分化和功能背后的转录机制尚无定论。为此，我们对体外培养的人CD49b+LAG3+Tr1细胞进行了RNA测序。我们发现转录因子BHLHE40在Tr1细胞中高度表达。尽管Tr1细胞特征性地产生高水平的IL-10，但我们的研究发现，BHLHE40抑制IL-10并增加初始CD4+T细胞的IFN-γ分泌。通过CRISPR/Cas9介导的敲除实验，我们确定IL10在sgBHLHE40编辑细胞中显著增加，且BHLHE40对于初始CD4+T细胞在体外分化为Tr1细胞是可替代的。有趣的是，BHLHE40过表达诱导CD49b和LAG3的表面表达，这两者是归因于Tr1细胞的共表达表面分子，但同时也促进了IFN-γ的产生。我们的研究揭示了BHLHE40作为人类CD4+T细胞中IL-10和IFN-γ调节因子的一种新型作用机制。