scRNA-seq and scTCR-seq of autoreactive CD8 T cells from NOD mice

Type 1 diabetes (T1D) is an autoimmune disorder defined by CD8 T cell-mediated destruction of pancreatic ß cells. Our previous work has shown that diabetogenic CD8 T cells in the islets of the non-obese diabetic (NOD) mouse model of T1D are phenotypically heterogenous, but CD8 T cell clonal heterogeneity in this model remains relatively unexplored. Here, we use paired single-cell RNA sequencing (scRNA-seq) and single-cell T cell receptor sequencing (scTCR-seq) to characterize autoreactive CD8 T cells from the islets and spleens of NOD mice. Clonal analysis of scTCR-seq data demonstrates that CD8 T cells targeting the immunodominant ß cell epitope IGRP206-214 exhibit highly restricted TCR gene usage, with over 70% of IGRP206-214-reactive cells utilizing the same TCR V alpha, J alpha, and V beta genes. Despite this, we observe only 5% overlap of IGRP206-214-reactive CD8 T cell clones between two groups of 10 NOD mice, demonstrating the immense TCR heterogeneity generated by junctional diversity during V(D)J recombination. scRNA-seq identifies two new clusters of autoreactive CD8 T cells in the islets and six clusters of diabetogenic CD8 T cells in the spleen, including multiple memory-like clusters and a population of exhausted cells. Strong clonal overlap between IGRP206-214-reactive CD8 T cells in the islets and spleen suggests that these cells may exit the islets and enter the peripheral circulation. Finally, we identify correlations between TCR J beta gene usage, which is less restricted than that of other TCR genes, and CD8 T cell clonal expansion as well as effector fate. Collectively, our work demonstrates that IGRP206-214-specific CD8 T cells are phenotypically heterogeneous but clonally similar, raising the possibility of selectively targeting either conserved or divergent TCR structures of these cells for therapeutic benefit. Overall design: scRNA-seq and scTCR-seq of IGRP+ and IGRP- CD8 T cells from islets and IGRP+ CD8 T cells from spleens of NOD mice

1型糖尿病（Type 1 diabetes, T1D）是一种以CD8 T细胞介导的胰腺β细胞破坏为特征的自身免疫性疾病。我们此前的研究表明，在1型糖尿病非肥胖糖尿病（non-obese diabetic, NOD）小鼠模型的胰岛中，致糖尿病性CD8 T细胞具有表型异质性，但该模型中CD8 T细胞的克隆异质性仍相对未被充分探索。本研究采用配对的单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）与单细胞T细胞受体测序（single-cell T cell receptor sequencing, scTCR-seq）技术，对NOD小鼠胰岛及脾脏中的自身反应性CD8 T细胞进行表征。对scTCR-seq数据的克隆分析显示，靶向免疫显性β细胞表位IGRP206-214的CD8 T细胞的TCR基因使用具有高度限制性，超过70%的IGRP206-214反应性细胞使用相同的TCR Vα、Jα和Vβ基因。尽管如此，我们在两组各10只NOD小鼠中仅观察到5%的IGRP206-214反应性CD8 T细胞克隆重叠，这体现了V(D)J重组过程中连接区多样性所产生的巨大TCR异质性。scRNA-seq分析在胰岛中鉴定出两类新的自身反应性CD8 T细胞亚群，在脾脏中鉴定出六类致糖尿病性CD8 T细胞亚群，其中包含多个记忆样亚群以及一群耗竭性细胞。胰岛与脾脏中的IGRP206-214反应性CD8 T细胞之间存在显著的克隆重叠，这提示这些细胞可能离开胰岛并进入外周循环。最后，我们发现TCR Jβ基因的使用（其限制性弱于其他TCR基因）与CD8 T细胞克隆扩增及效应细胞命运之间存在相关性。综上，本研究表明IGRP206-214特异性CD8 T细胞具有表型异质性但克隆相似性，这为通过靶向这些细胞的保守或差异化TCR结构以实现治疗收益提供了可能性。整体实验设计：对NOD小鼠胰岛来源的IGRP阳性与IGRP阴性CD8 T细胞，以及脾脏来源的IGRP阳性CD8 T细胞进行scRNA-seq与scTCR-seq测序。