Project 4: APOB-specific CD4 and CD8 T cells exacerbate atherosclerosis

项目4摘要 在这个由4个项目和3个核心组成的PPG中，项目4专注于T细胞对载脂蛋白B(ApoB)的反应， 一种动脉粥样硬化抗原，存在于人类和小鼠体内。中心假设是自身免疫反应 最初是预防动脉粥样硬化的，但随着动脉粥样硬化的进展，它变得有利于动脉粥样硬化(开关)。 我们将在有和没有冠状动脉疾病的人类中解决这一问题(弗吉尼亚州心血管评估 [CAVA]和CT-CAVA队列)和新生成的APOE-/-FoxP3-ERT2-CRE-GFP ROSA26-fl-Stop-fl- RFP血统追踪小鼠。我们提出了三个具体目标。目标1是定义载脂蛋白B特异性的CD4和CD8T 冠心病组和对照组外周血单个核细胞。这包括T细胞转录本、TcRα和β序列、细胞 表面表型的抗体序列，并找到免疫优势的载脂蛋白B表位，他们的反应。这个 主要方法有单细胞(Sc)RNA-Seq、抗体(Ab)-Seq和T细胞受体(TCR)-Seq。要找到 免疫优势表位，对于基于细胞和免疫调节治疗的关键相关，我们将使用 大容量208个MHC结合的人载脂蛋白B多肽，随后由ELISPOT测试系统去卷积， 细胞内细胞因子染色(ICS)、细胞因子捕获试验(CCA)和抗原诱导标记试验(AIM)。 目标2是通过将新的小鼠模型与ApoB特异的T细胞相结合来测试ApoB特异性T细胞是如何发育的 四聚体。我们的初步数据支持这样的假设，即一些ApoB特异性的CD4T细胞已经存在于 非常年轻的老鼠。其中一些细胞是效应器T细胞(非树突状细胞)。小鼠体内的其他FoxP3+CD4+Tregs 随着动脉粥样硬化的进展，它们的转录本从抗动脉粥样硬化转换为促动脉粥样硬化。 从机制上讲，我们将测试Treg to exTreg Switch的代谢和表观遗传学假说。目标3是测试 心血管疾病患者载脂蛋白B特异性T细胞包括Tregs、exTregs和Tregs的假设 Non-Tregs，使用scRNA-Seq和去卷积(CiberSort)方法。当提议的工作完成后， 我们将知道人类载脂蛋白B中主要的MHC-II限制性表位和反应T细胞的极化 细胞经scRNA-Seq、Ab-Seq和TCR-Seq.我们将了解Treg表型转换的机制和 PBMC中Tregs、exTregs和其他载脂蛋白B特异性T细胞的比例(CAVA和CT-CAVA队列)和 动脉内膜切除术转录本(自行车队列)。

Project 4 Summary Project 4 in this PPG with 4 projects and 3 cores is focused on the T cell response to apolipoprotein B (ApoB), an atherosclerosis antigen, in humans and mice. The central hypothesis is that the autoimmune response is initially atheroprotective, but becomes pro-atherogenic (switches) as atherosclerosis progresses. We will address this in humans with and without coronary artery disease (Cardiovascular Assessment Virginia [CAVA] and CT-CAVA cohorts) and in newly generated Apoe-/- FoxP3-ERT2-Cre-GFP ROSA26-fl-STOP-fl- RFP lineage tracker mice. We propose 3 specific aims. Aim 1 is to define the ApoB-specific CD4 and CD8 T cells in PBMCs from CAD cases and controls. This includes T cell transcriptomes, TCRα and β sequences, cell surface phenotypes by Ab-Seq, and finding the immunodominant ApoB epitopes to which they respond. The main methods are single cell (sc) RNA-Seq, antibody (Ab)-Seq and T cell receptor (TCR)-Seq. To find the immunodominant epitopes, of key relevance for cell-based and immunomodulatory therapeutics, we will use a megapool of 208 MHC-binding human ApoB peptides, followed by systematic deconvolution tested by Elispot, intracellular cytokine staining (ICS), cytokine capture assays (CCA) and antigen-induced marker (AIM) assays. Aim 2 is to test how ApoB-specific T cells develop by combining the new mouse model with ApoB-specific tetramers. Our preliminary data support the hypothesis that some ApoB-specific CD4 T cells already exist in very young mice. Some of these cells are effector T cells (non-Tregs). Other FoxP3+ CD4+ Tregs in mice switch their transcriptomes from atheroprotective to pro-atherogenic with progression of atherosclerosis. Mechanistically, we will test the metabolic and epigenetic hypotheses of Treg to exTreg switch. Aim 3 is to test the hypothesis that ApoB-specific T cells in humans with cardiovascular disease include Tregs, exTregs and non-Tregs, using scRNA-Seq and deconvolution (CIBERSORT) methods. When the proposed work is done, we will know the dominant MHC-II-restricted epitopes in human ApoB and the polarization of responding T cells by scRNA-Seq, Ab-Seq and TCR-Seq. We will know the mechanism of Treg phenotype switching and the proportion of Tregs, exTregs and other ApoB-specific T cells in PBMCs (CAVA and CT-CAVA cohorts) and endarteriectomy transcriptomes (BIKE cohort).