Mechanisms Of Age-related Changes in Transcriptional Regulation in lympphocytes

赖氨酸特异性甲基转移酶2D(Kmt2d)催化组蛋白3赖氨酸4(H3K4me1)单甲基化，通过调节Foxp3基因表达，在调节性T细胞生成中发挥重要作用。在这里，我们报告了Kmt2d在初始CD8+T细胞存活中的作用。首先，我们发现与野生型(Kmt2dfl/flCD4cre-，WT)小鼠相比，Kmt2d(Kmt2dfl/flCD4cre+，KO)小鼠脾中CD8+T细胞，尤其是初始CD8+T细胞(CD62Lhi/CD44lo)的数量显著减少。其次，我们观察到在体外刺激下，初始CD8+T细胞的死亡率显著增加。第三，我们观察到在缺乏Kmt2d的情况下，增强剂中H3K4me1水平的降低降低了激活的NAVE CD8+T细胞中与凋亡相关的基因的表达。最后，我们证实了在Kmt2d KO小鼠感染李斯特氏菌后，体内抗原特异性的NAVE CD8+T细胞被激活诱导死亡。这些结果表明，Kmt2d通过调节CD8+T细胞凋亡增强子中H3K4me1的水平和免疫相关基因的表达来调节活化诱导的初治CD8+T细胞的存活。 CD8T细胞是抗感染和抗癌细胞免疫的重要细胞因子，由表型和功能决定的血液中CD8T细胞包括六个主要亚群(NAVE、SCM、中枢和效应记忆、EMRA和效应)。然而，目前尚不清楚这些亚群的异质性有多大，以及年龄对人类CD8T细胞亚群组成的影响。在这里，我们使用单细胞RNA测序(ScRNAseq)和流式细胞术多色免疫表型分析CD8T细胞的组成。通过对16例健康献血者分离的CD8 T细胞的scRNAseq分析，我们分析了80,402个细胞，共鉴定出10个CD8T细胞亚群，包括初代、中央和效应记忆细胞以及终末分化的效应记忆细胞(EMRA)。同时，我们用流式细胞术对165名捐献者的CD8T细胞进行了多色(16抗体组)免疫表型分析，将CD8T细胞分为10个亚群。根据mRNA(UMI计数)和蛋白质(MFI水平)表达的相关性，这两种方法确定的9个亚群是等价的。重要的是，这九个亚群的百分比及其随年龄的变化具有可比性。综上所述，我们的发现揭示了以前未识别的CD8T细胞亚群及其随年龄的变化。这些新发现的CD8T亚群的功能及其在衰老过程中的作用目前正在研究中。

Lysine specific methyltransferase 2D (Kmt2d) catalyzes the mono-methylation of histone 3 lysine 4 (H3K4me1) and plays a critical role in regulatory T cell generation via modulating Foxp3 gene expression. Here we report a role of Kmt2d in nave CD8+ T cell survival. First, we found that the number of CD8+ T cells, in particular nave CD8+ T cells (CD62Lhi/CD44lo), in spleen was greatly decreased in the absence of Kmt2d (Kmt2dfl/flCD4cre+, KO) compared to wild type (Kmt2dfl/flCD4cre-, WT) mice. Second, we observed significant increase in death of nave CD8+ T cells upon stimulation in vitro. Third, we observed reduced H3K4me1 level in enhancers reduced expressions of apoptosis-related genes in activated nave CD8+ T cells in the absence of Kmt2d. Finally, we confirmed the activation-induced death of antigen specific nave CD8+ T cells in vivo in Kmt2d KO mice upon challenge with Listeria monocytogen infection. These findings reveal that Kmt2d regulates activation induced nave CD8+ T cell survival via modulating H3K4me1 levels in enhancer of those apoptosis and immune-related gene expressions. CD8 T cells, the essential players in cell mediated immunity against infection and cancers, consist of six major subsets (nave, SCM, central and effector memory, EMRA, and effector) in blood defined by their phenotype and function. However, it is currently unknown how heterogeneous these subpopulations are, and what impact age has on the composition of CD8 T cell subpopulations in humans. Here, we used single cell RNA sequencing (scRNAseq) and multicolor immunophenotyping by flow cytometry to analyze the composition of CD8 T cells. From scRNAseq analysis of isolated CD8 T cells from 16 healthy donors, we analyzed 80,402 cells and identified a total of 10 subpopulations of CD8 T cells including nave, central and effector memory cells and terminally differentiated effector memory cells (EMRA). In parallel, our multi-color (16 antibody-panel) immunophenotyping analysis by flow cytometry of CD8 T cells from 165 donors categorized CD8 T cells into 10 subpopulations. Nine subpopulations identified by these two methods were equivalent based on the correlations of mRNA (UMI counts) and protein (MFI levels) expression. Importantly, the percentage of these nine subpopulations and their identified changes with age were comparable. Together, our findings reveal previously unidentified subpopulations of CD8 T cells and their changes with age. The functions of these newly identified CD8 T subpopulations and their role during aging are currently under investigation.