Blimp-1 and c-Maf regulate Il10 and negatively regulate common and unique proinflammatory gene networks in IL-12 plus IL-27-driven T helper-1 cells [ATAC-Seq]. Blimp-1 and c-Maf regulate Il10 and negatively regulate common and unique proinflammatory gene networks in IL-12 plus IL-27-driven T helper-1 cells [ATAC-Seq]

CD4+ Th1 cells producing IFN-g are required to eradicate intracellular pathogens, however if uncontrolled these cells can mediate immunopathology. The cytokine IL-10 is produced by multiple immune cells including Th1 cells during infection and regulates the immune response to minimise collateral host damage. In this study we aimed to elucidate the transcriptional network of genes controlling the expression of Il10 and proinflammatory cytokines, including Ifng in Th1 cells. We show that the transcription factors Blimp-1 and c-Maf each have unique and common effects on cytokine gene regulation and not only co-operate to induce Il10 gene expression in IL-12 plus IL-27 differentiated Th1 cells, but additionally directly negatively regulate key proinflammatory cytokines including Ifng, thus providing mechanisms for reinforcement of regulated Th1 cell responses. Overall design: We have applied computational analysis of gene regulation derived from temporal profiling of gene expression clusters obtained from bulk RNA sequencing (RNA-Seq) of flow cytometry sorted CD4+ Th1 effector cells differentiated in vitro with IL-12 and IL-27 to produce Ifng and Il10, compared to control dirven-CD4+ T cells. Data was integrated with analysis of active genomic regions from these Th1 cells using ATAC-seq, integrated with literature derived-ChIP-seq data and the RNA-seq data, to elucidate the transcriptional network of genes controlling expression of Il10 and pro-inflammatory effector genes in Th1 cells. The co-dominant role for the transcription factors, Prdm1 (encoding Blimp-1) and Maf (encoding c-Maf), in cytokine gene regulation in Th1 cells, was confirmed using T cells obtained from mice with T-cell specific deletion of these transcription factors.

分泌干扰素-γ（IFN-γ）的CD4+ Th1细胞是清除胞内病原体的必需细胞群，但若调控失控，这类细胞可介导免疫病理损伤。感染过程中，包括Th1细胞在内的多种免疫细胞均可分泌白细胞介素-10（IL-10），该细胞因子可调控免疫应答以最大程度减轻宿主的附带组织损伤。本研究旨在解析调控Th1细胞中IL-10及促炎细胞因子（包括IFN-γ）表达的基因转录网络。

研究结果显示，转录因子Blimp-1与c-Maf各自对细胞因子基因调控具有独特及共通的作用；二者不仅可协同诱导经IL-12联合IL-27体外分化的Th1细胞中IL-10基因的表达，还可直接负向调控包括IFN-γ在内的关键促炎细胞因子，由此为强化受调控的Th1细胞应答提供了分子机制。

总体实验设计：本研究对体外经IL-12与IL-27诱导分化、可分泌IFN-γ及IL-10的CD4+ Th1效应细胞进行流式分选，随后开展批量RNA测序（RNA-Seq），通过对获得的基因表达簇进行时序分析，实施基因调控的计算分析，并以定向驱动的CD4+ T细胞作为对照。此外，本研究将该转录组数据与通过转座酶可及性测序（ATAC-seq）获得的上述Th1细胞开放染色质区域分析数据相结合，并整合已发表的染色质免疫沉淀测序（ChIP-seq）数据与上述RNA-seq数据，以此解析调控Th1细胞中IL-10及促炎效应基因表达的转录调控网络。最终，通过获取携带T细胞特异性敲除上述转录因子（即编码Blimp-1的Prdm1与编码c-Maf的Maf）的小鼠的T细胞，验证了这两种转录因子在Th1细胞细胞因子基因调控中的协同主导作用。