Examination of gene expression in different populations of the embryonic thymus at E15.5 and E19.5 by bulk RNA-seq

ILC2 contribute to immune homeostasis, protective immunity and tissue repair. However, an understanding of the microenvironmental factors and transcriptional circuits that support development of these innate lymphocytes within lymphoid organs, alongside their adaptive T and B cell relatives, is only starting to emerge. Here we demonstrate that functional ILC2 arise in the embryonic thymus, from shared T cell precursors, and precede the emergence of CD4+CD8+ (double-positive) T cells. Strikingly, RORa expression repressed T cell development, whilst promoting ILC2 in the thymus. Thymic ILC2 go on to contribute to the innate type-2 response at mucosal tissues with preferential colonisation of the intestinal lamina propria. From RNAseq, ATACseq and ChIPseq data we propose a revised transcriptional circuit to explain the enigmatic co-development of T cells, ILC2 and NK cells from common progenitors in the thymus. When Notch signalling is present, Bcl11b dampens Nfil3/Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORa overrides the Nfil3/Id2 repression, allowing Id2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORa expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus. Overall design: Examination of gene expression in different populations of the embryonic thymus at E15.5 and E19.5

2型固有淋巴细胞（ILC2）参与免疫稳态、保护性免疫与组织修复过程。然而，针对这些固有淋巴细胞在淋巴器官中与其适应性T、B细胞同源群体共同发育的微环境因素及转录调控回路的认知，目前才刚刚起步。
本研究证实，功能性ILC2源自共同的T细胞前体，于胚胎胸腺中产生，且早于CD4+CD8+双阳性（double-positive）T细胞的出现。
值得注意的是，视黄酸相关孤儿受体α（RORα）可抑制T细胞发育，同时促进胸腺内ILC2的生成。
胸腺来源的ILC2可参与黏膜组织的2型固有免疫应答，并优先定植于小肠固有层。
基于RNA测序（RNA-seq）、转座酶可及性测序（ATAC-seq）及染色质免疫沉淀测序（ChIP-seq）的数据，我们提出了一套修正后的转录调控回路，用以解释胸腺中T细胞、ILC2与自然杀伤细胞从共同前体细胞发生神秘共同发育的机制。
当存在Notch信号通路时，B细胞淋巴瘤/白血病11b蛋白（Bcl11b）会抑制核因子白细胞介素3调控因子（Nfil3）与抑制素2（Id2）的表达，使得E蛋白家族转录因子介导的T细胞定向定型得以实现。
但与此同时，RORα的表达可逆转Nfil3/Id2的抑制效应，使Id2能够抑制E蛋白家族转录因子，进而促进ILC2的分化。
综上，我们证实RORα的表达是胚胎胸腺中T细胞与ILC2谱系分支节点的关键检查点。
实验整体设计：检测E15.5与E19.5时期胚胎胸腺不同细胞群体的基因表达水平。