Single-cell transcriptional profiling identifies a spectrum of unconventional intraepithelial T lineage cells in human cord blood [bulk RNA-Seq]

Conventional CD4 and CD8 single positive T cell lineages constitute the main differentiation pathway in the thymus. In human thymus, a minor TCRaß differentiation pathway diverges from the early double positive stage, consisting of CD10+ PD-1+ cells. These cells are phenotypically and functionally similar to murine agonist-selected intraepithelial T lymphocyte precursors (IELps) which home to the small intestine. Here, the progeny of the human agonist-selected IEL lineage was identified in antigen-inexperienced cord blood (CB) with a polyclonal T cell receptor (TCR) repertoire exhibiting a bias towards early TCR alpha chain rearrangements and elevated autoreactive indices. Single-cell RNA sequencing allowed further delineation of this unconventional lineage in CB. Trajectory analysis, along with TCR repertoire analysis and transcriptomics, suggests a precursor-progeny relationship with the thymic IELps. The distinct, heterogeneous CB population can now be defined as CD3+ TCRaß+ CD4- CCR7- CD26-. Besides recent thymic emigrants, this population also consists of newly identified effector clusters and previously described populations: the suppressive NK receptor expressing CD8+ Treg population, the KIR/NKG2A+ EOMES+ virtual memory population and the CD8aa+ T cell populations. The population shows a discriminating stable Helios expression and is exclusively able to downregulate CD8ß expression, resulting in double negative T cells. The functional properties of this population suggest that the cells expand on inflammatory cues and exert cytotoxic and proinflammatory activity. Overall design: To identify additional marker genes in cord blood for unconventional intraepithelial T cells, 3 T cell populations were sorted from cord blood: I. TCR?d+, II. CD3+/low PD-1+ (unconventional intraepithelial T cells) and III. CD3+ PD-1- (conventional T cells). For each sort 3 replicates from 3 different donors wereused in a differential expression analysis with DeSeq2 to identify additional markers. The same populations were sorted from post natal thymus (also 3 replicates from 3 different donors per cell population) to use as a comparison.

常规CD4与CD8单阳性T细胞谱系是胸腺内主要的分化通路。在人类胸腺中，一条次要的T细胞受体αβ（TCRαβ）分化通路从早期双阳性阶段分支而来，该通路由CD10+ PD-1+细胞组成。此类细胞在表型与功能上，与定居于小肠的小鼠激动剂选择型上皮内T淋巴细胞前体（intraepithelial T lymphocyte precursors, IELps）相似。在此研究中，研究人员在抗原未接触过的脐带血（cord blood, CB）中鉴定出了人类激动剂选择型IEL谱系的子代细胞，这群细胞拥有多克隆T细胞受体（TCR）库，其特征为偏向于早期TCRα链重排，且自身反应性指数升高。单细胞RNA测序（single-cell RNA sequencing）可进一步解析脐带血中的这一非常规谱系。轨迹分析结合TCR库分析与转录组学结果表明，该谱系与胸腺IELps存在前体-子代关系。该独特且具有异质性的脐带血细胞群可被定义为CD3+ TCRαβ+ CD4- CCR7- CD26-。除新近胸腺迁出细胞（recent thymic emigrants, RTE）外，该细胞群还包含新鉴定出的效应细胞簇以及此前已报道的细胞群：表达抑制性NK受体的CD8+调节性T（Treg）细胞群、KIR/NKG2A+ EOMES+ 虚拟记忆细胞群，以及CD8αα+ T细胞群。该细胞群具有特征性的稳定Helios表达，且仅能下调CD8β的表达，最终形成双阴性T细胞。该细胞群的功能特性表明，其可在炎症信号刺激下增殖，并发挥细胞毒性与促炎活性。实验整体设计：为鉴定脐带血中非常规上皮内T细胞的额外标记基因，研究人员从脐带血中分选了3群T细胞：I. TCRγδ+细胞，II. CD3+/low PD-1+细胞（非常规上皮内T细胞），以及III. CD3+ PD-1-细胞（常规T细胞）。针对每一分选群，均使用来自3名不同供体的3份生物学重复样本，通过DESeq2进行差异表达分析以鉴定额外标记基因。研究人员同时从产后胸腺中分选了相同的细胞群（每类细胞群同样使用来自3名不同供体的3份生物学重复样本）作为对照。