Mechanisms Of Age-related Changes in Transcriptional Regulation in lympphocytes

淋巴细胞转录调节与年龄相关的变化机制

• 批准号: 10252561
• 负责人: Nan-ping Peter Weng
• 金额: $ 29.57万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10252561
• 关键词: ATAC-seq; Age; Aging; Antibodies; Antigens; Apoptosis; Blood; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell Survival; Cell physiology; Cells; Cellular Immunity; Cessation of life; ChIP-seq; Chromatin; Color; DNA Methylation; Enhancers; FOXP3 gene; Flow Cytometry; Gene Expression; Gene Expression Profiling; Generations; Genes; Genetic Transcription; Goals; Human; Immune; Immunophenotyping; In Vitro; Infection; Investigation; Knockout Mice; Listeria; Lysine; Malignant Neoplasms; Measures; Memory; Messenger RNA; Methods; Methyltransferase; Mus; Phenotype; Play; Population; Proteins; Regulatory T-Lymphocyte; Reporting; Role; Spleen; T memory cell; T-Lymphocyte; Tissue-Specific Gene Expression; Transcriptional Regulation; age related; base; comparative; differential expression; genomic locus; histone methylation; in vivo; interest; single-cell RNA sequencing; transcriptome; transcriptome sequencing

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.

赖氨酸特异性甲基转移酶2D（Kmt 2d）催化组蛋白3赖氨酸4（H3 K4 me 1）的单甲基化，并通过调节Foxp 3基因表达在调节性T细胞生成中起关键作用。在这里，我们报告了Kmt 2d在幼稚CD 8 + T细胞存活中的作用。首先，我们发现，与野生型（Kmt 2dfl/flCD 4cre-，WT）小鼠相比，在不存在Kmt 2d（Kmt 2dfl/flCD 4cre+，KO）的情况下，脾脏中的CD 8 + T细胞，特别是原始CD 8 + T细胞（CD 62 Lhi/CD 44 lo）的数量大大减少。第二，我们观察到在体外刺激时幼稚CD 8 + T细胞的死亡显著增加。第三，我们观察到增强子中H3 K4 me 1水平的降低降低了在不存在Kmt 2d的情况下活化的原始CD 8 + T细胞中的凋亡相关基因的表达。最后，我们证实了在用单核细胞增生李斯特菌感染攻击后，Kmt 2d KO小鼠体内抗原特异性幼稚CD 8 + T细胞的激活诱导的死亡。这些发现表明，Kmt 2d通过调节H3 K4 me 1水平来调节活化诱导的原始CD 8 + T细胞存活，从而增强这些凋亡和免疫相关基因的表达。 CD 8 T细胞是细胞介导的抗感染和癌症免疫中的重要参与者，由血液中的六个主要亚群（原始、SCM、中枢和效应记忆、EMRA和效应子）组成，由其表型和功能定义。然而，目前尚不清楚这些亚群的异质性如何，以及年龄对人类CD 8 T细胞亚群组成的影响。在这里，我们使用单细胞RNA测序（scRNAseq）和流式细胞术分析CD 8 T细胞的组成。通过对来自16名健康供体的分离的CD 8 T细胞的scRNAseq分析，我们分析了80，402个细胞，并鉴定了总共10个CD 8 T细胞亚群，包括原始、中央和效应记忆细胞以及终末分化效应记忆细胞（EMRA）。同时，我们通过流式细胞术对来自165名供体的CD 8 T细胞进行多色（16个抗体组）免疫表型分析，将CD 8 T细胞分为10个亚群。基于mRNA（UMI计数）和蛋白质（MFI水平）表达的相关性，通过这两种方法鉴定的9个亚群是等同的。重要的是，这九个亚群的百分比及其随年龄的变化是相当的。总之，我们的研究结果揭示了以前未识别的CD 8 T细胞亚群及其随年龄的变化。这些新发现的CD 8 T亚群的功能及其在衰老过程中的作用目前正在研究中。