Transcription factor RUNX3 mediates plasticity of ThGM cells toward Th1 phenotype.

GM-CSF-producing T helper (Th) cells play a crucial role in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Recent studies have identified a distinct population of GM-CSF-producing Th cells, named ThGM cells, that also express cytokines TNF, IL-2, and IL-3, but lack expression of master transcription factors (TF) and signature cytokines of commonly recognized Th cell lineages. ThGM cells are highly encephalitogenic in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Similar to Th17 cells, in response to IL-12, ThGM cells upregulate expression of T-bet and IFN-g and switch their phenotype to Th1. Here we show that in addition to T-bet, TF RUNX3 also contributes to the Th1 switch of ThGM cells. T-bet-deficient ThGM cells in the CNS of mice with EAE had low expression of RUNX3, and knockdown of RUNX3 expression in ThGM cells abrogated the Th1-inducing effect of IL-12. Comparison of ThGM and Th1 cell transcriptomes showed that ThGM cells expressed a set of TFs known to inhibit the development of other Th lineages. Lack of expression of lineage-specific cytokines and TFs by ThGM cells, together with expression of TFs that inhibit the development of other Th lineages, suggests that ThGM cells are a non-polarized subset of Th cells with lineage characteristics. Human RNA-sequencing: Human ThGM, Th1, and GM-CSF+Th1 cells were isolated from peripheral blood of healthy donors using a combination of fllow cytometry cell sorting and cytokine sceretion assay. RNA from human ThGM, Th1, and GM-CSF+Th1 cells were isolated and their transcriptome were analyzed by RNA sequencing. MouseRNA sequencing: Mouse naive CD4+ T cells were polarized to ThGM and Th1 cells and RNA from several time points were isolated and analyzed using RNA sequencing.

分泌粒细胞-巨噬细胞集落刺激因子（GM-CSF）的辅助性T（Th）细胞在多发性硬化（MS）等自身免疫性疾病的发病机制中发挥关键作用。近期研究已鉴定出一类独特的分泌GM-CSF的Th细胞亚群，命名为ThGM细胞，该亚群可表达肿瘤坏死因子（TNF）、白细胞介素-2（IL-2）与白细胞介素-3（IL-3）等细胞因子，但不表达常见公认Th细胞谱系的核心转录因子（TF）与特征性细胞因子。ThGM细胞在多发性硬化的小鼠模型——实验性自身免疫性脑脊髓炎（EAE）中具有极强的致脑炎性。与Th17细胞类似，在白细胞介素-12（IL-12）刺激下，ThGM细胞会上调T-bet与干扰素-γ（IFN-γ）的表达，并将表型转换为Th1细胞。本研究证实，除T-bet外，转录因子RUNX3也参与调控ThGM细胞向Th1细胞的表型转换。在EAE模型小鼠的中枢神经系统（CNS）中，T-bet缺陷的ThGM细胞的RUNX3表达水平较低；在ThGM细胞中敲低RUNX3的表达，可消除IL-12诱导的Th1表型转换效应。对ThGM细胞与Th1细胞的转录组进行比较分析后发现，ThGM细胞表达一组可抑制其他Th细胞谱系发育的转录因子。ThGM细胞既不表达谱系特异性细胞因子与转录因子，同时又表达抑制其他Th细胞谱系发育的转录因子，这表明ThGM细胞是一类具有谱系特征的非极化辅助性T细胞亚群。人类转录组测序：本研究通过流式细胞术分选与细胞因子分泌检测联用的方法，从健康志愿者的外周血中分离得到人类ThGM细胞、Th1细胞与GM-CSF阳性Th1细胞；提取上述细胞的总RNA，通过RNA测序对其转录组进行分析。小鼠转录组测序：将小鼠初始CD4+ T细胞极化为ThGM细胞与Th1细胞，于多个时间点提取总RNA，并通过RNA测序进行转录组分析。