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

在1型糖尿病（type 1 diabetes, T1D）患者外周血中检出的胰岛反应性T细胞（islet-reactive T cells），被认为与疾病发病机制相关，但此类细胞同样存在于健康个体外周血中，这使得完整阐明其功能角色变得更为复杂。为阐明此类细胞在T1D中的作用，本研究结合流式细胞术（flow cytometry）与单细胞RNA测序（RNA-seq）技术，关联了从扩增型T细胞受体（T cell receptor, TCR）克隆型中推断出的既往抗原暴露史，以及胰岛抗原反应性CD4+记忆T细胞的功能特性。研究发现，经免疫显性胰岛抗原混合肽段激活的细胞，在新发T1D患者体内的克隆型共享率显著高于健康个体，这与疾病进程中体内T细胞扩增的现象相符。不同供者间未检测到克隆型共享，提示具有独特或"private"特异性的TCR占据主导地位。扩增的克隆型可保持稳定，一名受试者在间隔一年以上的两次随访中均检出了同一克隆型。我们从两名T1D受试者的扩增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进程中CD4+ T细胞扩增的触发因素。T1D患者与健康个体的细胞转录组谱存在差异，在扩增程度最高的TCR克隆型对应细胞中这一差异尤为显著。整体而言，扩增程度最高的TCR克隆型对应细胞呈现出辅助性T细胞2型（Th2）样表型，但在单细胞层面，供者内部及供者间均存在表型异质性。本研究结果证实，在疾病进程中发生扩增的个体胰岛反应性CD4+记忆T细胞，具有独特的特异性与表型特征。整体实验设计：本项目包含四类细胞的RNA-seq测序数据文件：1）T细胞克隆（N=149个单细胞转录组图谱）；2）T细胞克隆（N=9个批量细胞转录组图谱）；3）CD8+流感反应性T细胞（N=45个单细胞转录组图谱）；4）CD4+混合胰岛抗原反应性T细胞（N=246个单细胞转录组图谱）。