Single cell RNA-seq reveals expansion of IGRP-reactive CD4+ T cells in recent onset type I diabetes (bulk RNA-seq of T-cell clone). Homo sapiens

Islet-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects are thought to be involved in disease pathogenesis, but full understanding of their role is complicated by their presence also in blood of in healthy subjects. To elucidate their role in T1D, we have combined flow cytometry and single cell RNA sequencing (RNA-seq) techniques to link prior antigen exposure, inferred from expanded TCR clonotypes, and functional capacities of islet antigen-reactive CD4+ memory T cells. We find that cells activated by pooled peptides from immunodominant islet antigens showed significantly higher clonotype sharing within recent onset T1D subjects than in healthy individuals, consistent with in vivo T cell expansion during disease progression. There was no clonotype sharing between donors, indicating a predominance of TCRs with distinct or “private” specificities. Expanded clonotypes could be stable, as one was detected at repeat visits by spanning more than a year by one subject. We identified distinct IGRP peptides as the targets of expanded TCR clonotypes from two T1D subjects, thereby implicating this molecule as a trigger for CD4+ T cell expansion during T1D. Transcriptome profiles of cells from T1D and healthy subjects differed, particularly in cells having the most highly expanded TCR clonotypes. As a group, cells with the most highly expanded TCR clonotypes showed Th2-like phenotypes, but at the single cell level there was phenotypic heterogeneity within and between donors. Our findings demonstrate unique specificities and phenotypes of individual islet-reactive CD4+ memory T cells that have expanded during disease progression. Overall design: This project contains RNA-seq files from four cell types: 1) T cell clone (N=149 single cell profiles); 2) T cell clone (N=9 bulk cell profiles); 3) CD8+ influenza-reactive T cells (N=45 single cell profiles); and 4) CD4+ pooled islet antigen-reactive T cells (N=246 single cell profiles).

从1型糖尿病（type 1 diabetes, T1D）受试者外周血中分离得到的胰岛反应性T细胞，被认为与疾病发病机制密切相关，但由于健康受试者外周血中同样存在此类细胞，因此全面解析其在疾病中的作用难度较大。为阐明此类细胞在T1D发病过程中的功能，本研究结合流式细胞术（flow cytometry）与单细胞RNA测序（single cell RNA sequencing, RNA-seq）技术，将基于扩增性T细胞受体（T cell receptor, TCR）克隆型推断得到的既往抗原暴露事件，与胰岛抗原反应性CD4+记忆T细胞的功能特性进行关联分析。
研究发现，由免疫优势胰岛抗原混合肽激活的细胞，在新发T1D受试者体内的克隆型共享比例显著高于健康个体，这与疾病进展过程中体内T细胞扩增的现象相符。不同供者间未检测到克隆型共享现象，提示具有独特或"私有"特异性的TCR占主导地位。扩增性克隆型可保持稳定，某一受试者在间隔一年以上的两次随访中均检测到了同一克隆型。我们从两名T1D受试者的扩增性TCR克隆型中，明确了其靶向的特异性IGRP肽段，由此表明该分子是T1D发病过程中CD4+ T细胞扩增的触发因素之一。
T1D受试者与健康个体的细胞转录组谱存在显著差异，在扩增程度最高的TCR克隆型对应的细胞中这一差异尤为突出。整体而言，扩增程度最高的TCR克隆型对应的细胞呈现出Th2样表型，但在单细胞水平上，供者内部及供者之间均存在表型异质性。本研究结果揭示了在疾病进展过程中扩增的胰岛反应性CD4+记忆T细胞所具有的独特特异性与表型特征。
整体实验设计：本项目包含四类细胞的RNA测序文件：1）T细胞克隆（共149个单细胞转录组数据）；2）T细胞克隆（共9个批量细胞转录组数据）；3）CD8+流感反应性T细胞（共45个单细胞转录组数据）；4）CD4+混合胰岛抗原反应性T细胞（共246个单细胞转录组数据）。