ApoB-specific CD4 T cells in mouse and human atherosclerosis

项目4说明 已知动脉粥样硬化是由调节性T细胞(Treg)控制的，但既不是抗原特异性也不是 这些细胞保护的位置和机制是已知的。项目4正在测试假设 调节性CD4T细胞表达和分泌IL-10以响应其同源抗原ApoB，主要核心是ApoB 低密度脂蛋白(LDL)的蛋白质。这是基于发现大量特定于载脂蛋白B的 小鼠和人类体内的CD4T细胞，使用小鼠和人的MHC-II四聚体和右旋糖体负载 小鼠和人的载脂蛋白B多肽。具体目标1是通过研究在小鼠身上验证这一假说 APOE-/-小鼠的动脉粥样硬化和ApoB特异的CD4T细胞谱系的自然历史 流式细胞术(FACS)、质量细胞术(CyTOF)和RNA-Seq(通过core E)。最后测试一下是否 ApoB特异的CD4T细胞中的IL-10是动脉粥样硬化保护所必需的，我们将采集ApoB特异的CD4T 来自APOE-/-(IL-10充足)或CD4CreIL10fl/flApoe-/-(IL-10缺乏)供者的细胞，并分别转移它们 转化为受体APOE-/-CD4-/-小鼠。我们假设IL-10充足的CD4T细胞将具有动脉粥样硬化保护作用 而缺乏IL-10的CD4T细胞则不会。具体目标2是将这些发现转化为人类，使用冷冻的 来自台地队列(核心D)和长波紫外线队列(核心C)的PBMC。初步数据显示，我们可以检测到 亚临床心血管疾病患者外周血中人载脂蛋白B特异性CD4T细胞的研究 (CVD)。我们建议测试他们表达(通过流式细胞仪)和分泌(通过Elispot)IL-10的能力，评估他们的 通过CyTOF进行表型分析，并通过RNA-Seq(通过核心E)确定其转录组。我们发现了30个 结合许多人类MHC-II(DR)等位基因的人载脂蛋白B肽，我们估计我们可以询问 &gt；85%的样品使用17种不同的四聚体和葡聚体。通过与CORE D合作，我们提议 载脂蛋白B特异性CD4T细胞的数量和表型与冠脉疾病定义的亚临床心血管疾病的相关性 钙(CAC)评分和CAC评分进展。项目4将与项目1在抗原方面进行合作 单核细胞和活体显微镜的介绍，项目2，关于低密度脂蛋白修饰和 研究B1细胞对低密度脂蛋白的反应的项目3。当建议的工作完成后，我们会明白 载脂蛋白B特异性CD4T细胞在IL-10调节动脉粥样硬化中的作用机械式鼠标工作 为检测ApoB特异性CD4T细胞在CVD患者中的相关性提供了依据。载脂蛋白B特异性CD4T 细胞在动脉粥样硬化中可能是有用的免疫生物标志物，研究结果可以指导未来的治疗 和预防性的努力。

Project 4 description Atherosclerosis is known to be controlled by regulatory T cells (Tregs), but neither the antigen specificity nor the location nor the mechanism by which these cells protect are known. Project 4 is testing the hypothesis that regulatory CD4 T cells express and secrete IL-10 in response to their cognate antigen, ApoB, the main core protein of low density lipoprotein (LDL). This is based on the discovery of a significant number of ApoB-specific CD4 T cells in mice and in humans, using mouse and human MHC-II tetramers and dextramers loaded with mouse and human ApoB peptides, respectively. Specific Aim 1 is to test this hypothesis in mice by studying atherosclerosis in Apoe-/- mice and following the natural history of the ApoB-specific CD4 T cell repertoire by flow cytometry (FACS), mass cytometry (CyTOF) and RNA-Seq (through core E). To conclusively test whether IL-10 from ApoB-specific CD4 T cells is required for atheroprotection, we will harvest ApoB-specific CD4 T cells from Apoe-/- (IL-10 sufficient) or CD4CreIl10fl/flApoe-/- (IL-10-deficient) donors and separately transfer them into recipient Apoe-/- Cd4-/- mice. We hypothesize that the IL-10 sufficient CD4 T cells will be atheroprotective and the IL-10 deficient CD4 T cells will not. Specific Aim 2 is to translate the findings to humans, using frozen PBMCs from the MESA cohort (core D) and the UVa cohort (core C). Preliminary data show that we can detect human ApoB-specific CD4 T cells in frozen PBMCs from subjects with subclinical cardiovascular disease (CVD). We propose to test their ability to express (by FACS) and secrete (by EliSpot) IL-10, assess their phenotype by CyTOF and define their transcriptome by RNA-Seq (through core E). We have discovered 30 human ApoB peptides that bind many human MHC-II (DR) alleles and we estimate that we can interrogate >85% of all samples using 17 different tetramers and dextramers. In collaboration with core D, we propose to correlate the number and phenotype of ApoB-specific CD4 T cells with subclinical CVD as defined by coronary calcium (CAC) scores and CAC score progression. Project 4 will collaborate with project 1 on antigen presentation by monocytes and intravital microscopy, project 2 on the importance of LDL modifications and project 3 on studying the B1 cell response to LDL. When the proposed work is completed, we will understand the role of ApoB-specific CD4 T cells in modulating atherosclerosis by IL-10. The mechanistic mouse work provides the basis for testing the relevance of ApoB-specific CD4 T cells in CVD patients. ApoB-specific CD4 T cells are likely useful immunological biomarkers in atherosclerosis and the results can guide future therapeutic and preventive efforts.