Zfp362 potentiates inflammatory T cell responses independent of Th17 cell differentiation

Th17 cells play an important role for mucosal barrier integrity and pathogen clearance, but also have been implicated in inflammatory and autoimmune disorders. Recently, we had described the DNA methylation profile of Th17 cells, and identified Zinc finger protein (Zfp)362 among the genes being uniquely demethylated in Th17 cells (Yang et al., DOI: 10.1093/nar/gkv014 ). Here, we prepared nuclei from CD4+ T cells deficient in Zfp362 (CD4creZfp362flox/flox mice) or CD4+ T cells capable to express Zfp362 (Zfp362flox/flox mice), subjected them to single-nucleus RNA-sequencing (snRNA-seq) and analyzed the influence of Zfp362 on the transcriptome. Cells from colon and small intestine were prepared from the tissue of seven Zfp362flox/flox mice or CD4creZfp362flox/flox mice and CD8a-CD11c-CD45R-F4/80-CD4+CD44+CD62L- effector T cells isolated by Fluorescence-activated cell sorting (FACS). Nuclei from sorted cells were subjected to a droplet-based library preparation technique (10x Genomics). The resulting libraries were sequenced on a Novaseq 6000 (Illumina).Th17细胞（Th17 cells）在维持黏膜屏障完整性及清除病原体方面发挥关键作用，同时也参与炎症性疾病与自身免疫性疾病的病理过程。此前本团队已完成Th17细胞的DNA甲基化谱分析，并在Th17细胞特异性去甲基化的基因中鉴定出锌指蛋白（Zinc finger protein, Zfp）362（Yang等，DOI: 10.1093/nar/gkv014）。本研究中，我们分别从Zfp362缺陷型CD4+ T细胞（CD4creZfp362flox/flox小鼠）以及可正常表达Zfp362的CD4+ T细胞（Zfp362flox/flox小鼠）中提取细胞核，对其进行单细胞核RNA测序（single-nucleus RNA-sequencing, snRNA-seq），并分析Zfp362对细胞转录组的调控作用。我们从7只Zfp362flox/flox小鼠或CD4creZfp362flox/flox小鼠的结肠与小肠组织中分离样本，并通过荧光激活细胞分选（Fluorescence-activated cell sorting, FACS）纯化得到CD8a⁻CD11c⁻CD45R⁻F4/80⁻CD4⁺CD44⁺CD62L⁻效应T细胞。将分选获得的细胞的细胞核采用液滴式建库技术（10x Genomics）进行文库制备，最终构建的测序文库在NovaSeq 6000（Illumina）平台上完成测序。