The identity of human tissue-emigrant CD8+ T cells (scRNAseq, scTCRseq, bulk RNA-seq)

Lymphocyte migration is essential for adaptive immune surveillance. However, ourcurrent understanding of this process is rudimentary, because most human studieshave been restricted to immunological analyses of blood and various tissues. Toaddress this, we used an integrated approach to characterize tissue-emigrant immunecells in thoracic duct lymph (TDL). The most prevalent immune cells in human andnon-human primate efferent lymph were T cells. Cytolytic CD8+ T cell subsets witheffector-like epigenetic and transcriptional signatures were clonotypically skewed andselectively confined to the intravascular circulation, whereas non-cytolytic CD8+ T cellsubsets with stem-like epigenetic and transcriptional signatures predominated intissues and TDL. Moreover, these anatomically distinct gene expression profiles wererecapitulated within individual antigen-specific clonotypes, suggesting paralleldifferentiation programs independent of the expressed antigen receptor. Our collectivedataset provides a framework of the migratory immune system and defines the natureof tissue-emigrant CD8+ T cells that recirculate via TDL. Overall design: scRNAseq and scTCRseq of 1) HIV-specific or CMV-specific CD8+ T-cells and 2) memory CD8+ T-cells between TDL and blood bulk RNA-seq of FACS sorted CD8+ Tcell memory subsets from human peripheral blood (samples re-used from GSE148234).

淋巴细胞迁移对于适应性免疫监视至关重要。然而，目前学界对这一过程的认知仍较为粗浅，因为绝大多数人体相关研究仅局限于血液与多种组织的免疫学分析。为解决这一研究局限，本研究采用整合分析策略，对胸导管淋巴（thoracic duct lymph, TDL）中的组织迁出免疫细胞进行了系统表征。人与非人灵长类动物输出淋巴中最主要的免疫细胞为T细胞。具有效应样表观遗传与转录特征的细胞毒性CD8+ T细胞亚群呈现克隆型偏倚，且选择性局限于血管内循环；而具有干细胞样表观遗传与转录特征的非细胞毒性CD8+ T细胞亚群，则主要分布于组织与胸导管淋巴中。此外，这些具有解剖学特异性的基因表达谱在单个抗原特异性克隆型中也得以重现，这提示存在不依赖于所表达抗原受体的平行分化程序。本研究的整合数据集为迁移免疫系统构建了研究框架，并明确了经由胸导管淋巴再循环的组织迁出型CD8+ T细胞的本质属性。实验整体设计：1. 对胸导管淋巴与血液中的HIV特异性或巨细胞病毒（cytomegalovirus, CMV）特异性CD8+ T细胞、记忆性CD8+ T细胞进行单细胞RNA测序（single-cell RNA sequencing, scRNAseq）与单细胞T细胞受体测序（single-cell T cell receptor sequencing, scTCRseq）；2. 对来自人外周血、经荧光激活细胞分选（fluorescence-activated cell sorting, FACS）纯化的CD8+ T细胞记忆亚群进行批量RNA测序（样本复用自GSE148234数据集）。