γδTCR-dependent and independent differentiation of innate lymphoid cells

摘要 先天性淋巴样细胞（ILC）存在于组织中，并作为各种环境线索的哨兵， 营养素、神经肽、微生物和病原体。它们是具有不同功能的异质种群 和发展起源。我们以前曾提出，除了骨髓，胸腺可以 基于肺ILC 2含有重排的T细胞的发现， 受体基因，可能是由于发育中的胸腺细胞进入ILC命运的分歧。一致性，T细胞 来自胸腺的前体可以在体外分化为ILC 2。检测到单细胞RNA测序研究 血液中ILC前体的异质群体，其在无胸腺裸鼠中大大减少， 表明胸腺也参与了这些细胞在细胞质中具有CD 3蛋白，但在细胞膜上没有。 表面，从而使细胞内CD 3（icCD 3）成为胸腺来源的ILC的标志物。事实上，icCD 3 + ILC亚群 在肺和小肠中检测到。有趣的是，icCD 3 + ILC 1的产生和对细胞的增殖作用， ILC 3依赖于Tcrd，而icCD 3 + ILC 2不依赖于Tcrd。这促使我们建议国际法委员会 胸腺中的分化涉及Tcrd依赖性和非依赖性机制。这样做的目的 应用是研究胸腺源性ILC是如何产生的，并研究这些细胞的功能。 有三个目标。Aim 1将检验具有非生产性TCR基因的发育中的T细胞 重排有助于多能ILC前体，并且未成熟γδ T细胞产生ILC 1-或ILC 3-。 在下调其TCR后偏向前体。我们将采用体外培养等方法 分化培养和TCR基因重排事件的分析。Aim 2将表征icCD 3 + ILC 通过检测ILC 1和ILC 2与T细胞的关系的年龄依赖性分化， 肠道微生物群的发展和变化，并通过比较icCD 3 + ILC 1的1型反应与那些 传统ILC 1和NK细胞以及γδ T细胞。AIM 3将研究类型2和类型3之间的相互作用。 3免疫应答，并比较icCD 3 + ILC 2与其icCD 3-对应物的行为。综合起来看， 这些研究可能会在发育免疫学的概念上取得进展，并阐明 胸腺来源的ILC对年龄相关的免疫差异的贡献。

ABSTRACT Innate lymphoid cells (ILCs) reside in tissues and act as sentinels to a variety of environmental cues from nutrients, neuropeptides, microbiota to pathogens. They are heterogeneous populations with diverse functions and developmental origins. We have previously suggested that in addition to the bone marrow, the thymus can contribute to ILC pools in peripheral tissues based on findings that lung ILC2s contain rearranged T cell receptor genes, likely due to the divergence of developing thymocytes into the ILC fate. Consistently, T cell precursors from the thymus can differentiate into ILC2s in vitro. Single cell RNA sequencing studies detected a heterogeneous population of ILC precursors in the blood, which is greatly diminished in athymic nude mice, suggesting the involvement of the thymus. These cells possess CD3 proteins in the cytoplasm but not on the surface, thus making intracellular CD3 (icCD3) a marker for thymus-derived ILCs. Indeed, icCD3+ ILC subsets have been detected in the lung and small intestine. Interestingly, the production of icCD3+ ILC1s and to a lesser extent, ILC3s depends on Tcrd but that of icCD3+ ILC2s does not. This prompted us to propose that ILC differentiation in the thymus involves Tcrd-dependent and independent mechanisms. The purpose of this application is to study how thymus-derived ILCs are generated and to investigate the functions of these cells. There are three aims. Aim1 will test the hypothesis that developing T cells with non-productive TCR gene rearrangement contributes to multipotent ILC precursors and immature γδ T cells gives rise to ILC1- or ILC3- biased precursors after down-regulation of their TCRs. We will employ approaches such as in vitro differentiation cultures and analyses of TCR gene rearrangement events. Aim2 will characterize icCD3+ ILCs in the small intestine by examining the age-dependent differentiation of ILC1s and ILC2 in relationship to T cell development and changes in gut microbiota, and by comparing the type 1 responses of icCD3+ ILC1s to those of conventional ILC1s and NK cells as well as γδ T cells. Aim3 will investigate the interplay of type 2 and type 3 immune responses and compare the behaviors of icCD3+ ILC2s to their icCD3- counterparts. Taken together, these studies may make conceptual advancement in developmental immunology and shed light on the contribution of thymus-derived ILCs to age-related differences in immunity.