Single cell and spatial transcriptomics analysis of kidney double negative T lymphocytes in normal and ischemic mouse kidneys [spatial transcriptomics]

T cells are important in the pathogenesis of acute kidney injury (AKI), and TCR+CD4-CD8- (double negative-DN) are T cells that have a regulatory role. However, little is understood about these cells in comparison to traditional CD4+ and CD8+ cells. To elucidate the molecular and spatial dynamics of DN T cells during AKI, we performed single-cell RNA sequencing (scRNA-seq) on sorted murine DN, CD4+, and CD8+ cells, combined with spatial transcriptomic profiling of normal and post-AKI mouse kidneys. scRNA-seq revealed distinct transcriptional profiles for DN, CD4+, and CD8+ T cells of mouse kidneys, with enrichment of Kcnq5, Klrb1c, Fcer1g, and Klre1 expression in DN T cells compared to CD4+ and CD8+ T cells in normal kidney tissue. We validated the expression of these genes in mouse and human kidney DN, CD4+ and CD8+ T cells using RT-PCR and in the NIH kidney precision medicine project (KPMP). Spatial transcriptomics in normal and ischemic mouse kidney tissue showed a localized cluster of T cells in the outer medulla expressing DN T cell genes including Fcer1g. These results provide a template for future studies in DN T as well as CD4+ and CD8+ cells in the normal and diseased kidney. Normal kidneys and kidneys treated with ischemic reperfusion injury were analyzed using spatial transcriptomics.

T细胞在急性肾损伤（AKI）的发病机制中具有重要作用，而TCR+CD4-CD8-（双阴性-DN，double negative-DN）T细胞是一类具备调节功能的T细胞亚群。然而，相较于传统CD4+与CD8+ T细胞，学界对这类细胞的了解仍相对有限。为阐明急性肾损伤发生过程中DN T细胞的分子与空间动态变化特征，本研究对分选得到的小鼠DN、CD4+及CD8+ T细胞开展了单细胞RNA测序（scRNA-seq），并结合正常小鼠肾脏与AKI后小鼠肾脏的空间转录组分析。单细胞RNA测序结果显示，小鼠肾脏中的DN、CD4+及CD8+ T细胞具有各自独特的转录谱；与正常肾脏组织内的CD4+和CD8+ T细胞相比，DN T细胞中Kcnq5、Klrb1c、Fcer1g及Klre1的表达显著富集。本研究通过逆转录聚合酶链反应（RT-PCR）验证了上述基因在小鼠及人类肾脏DN、CD4+与CD8+ T细胞中的表达，并在美国国立卫生研究院（National Institutes of Health, NIH）肾脏精准医学项目（kidney precision medicine project, KPMP）的数据集内完成了验证。对正常小鼠肾脏及缺血性小鼠肾脏组织的空间转录组分析显示，肾髓质外层存在一处局部T细胞簇，其表达包括Fcer1g在内的DN T细胞特征基因。本研究结果为后续针对正常与病变肾脏中DN T细胞，以及CD4+、CD8+ T细胞的相关研究提供了参考框架。本研究采用空间转录组技术分析了正常肾脏及经缺血再灌注损伤（ischemic reperfusion injury）处理的肾脏组织。