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

项目4：APOB特异性CD4和CD8 T细胞加剧动脉粥样硬化

• 批准号: 10334097
• 负责人: Klaus F. Ley
• 金额: $ 4.3万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-06-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10334097
• 关键词: Address; Alleles; Amino Acids; Antibodies; Antigens; Apolipoprotein E; Apolipoproteins B; Atherosclerosis; Autoantibodies; Autoantigens; Autoimmune; Autoimmune Responses; B-Lymphocytes; Binding; Biological Assay; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cardiovascular Diseases; Cardiovascular system; Cell Communication; Cell surface; Cells; Coronary Arteriosclerosis; Data; Data Set; Disease; Epigenetic Process; Epitopes; Eragrostis; FOXP3 gene; General Population; Genomics; Heat-Shock Response; Helper-Inducer T-Lymphocyte; Human; Immune; Immune response; Immunodominant Epitopes; Immunoglobulin Class Switching; Immunomodulators; Inflammatory; Injections; Length; Metabolic; Methods; Mus; Oligonucleotides; Peptides; Peripheral Blood Mononuclear Cell; Phenotype; Regulatory T-Lymphocyte; Resolution; Stains; Surface; T cell response; T-Cell Receptor; T-Lymphocyte; Tamoxifen; Techniques; Testing; Ursidae Family; Virginia; Work; atheroprotective; base; case control; cohort; cytokine; effector T cell; high dimensionality; macrophage; monocyte; mouse model; peptide B; response; single-cell RNA sequencing; transcriptome

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).

项目4摘要 该PPG的项目4具有4个项目，3个核心集中在T细胞对载脂蛋白B（APOB）的响应上 人类和小鼠的动脉粥样硬化抗原。中心假设是自身免疫反应 最初是动脉保护性的，但随着动脉粥样硬化的发展而变成促动脉粥样硬化（开关）。 我们将在患有有或没有冠状动脉疾病的人类中解决这个问题（心血管评估弗吉尼亚 [cava]和ct-cava队列）以及新生成的apoe - / - foxp3-ert2-cre-gfp rosa26-fl-stop-fl- RFP谱系跟踪器小鼠。我们提出了3个具体目标。 AIM 1是定义APOB特异性CD4和CD8 T 来自CAD病例和对照的PBMC中的细胞。这包括T细胞转录组，TCRα和β序列，细胞 AB-Seq的表面表型，并找到其反应的免疫主导APOB表位。这 主要方法是单细胞（SC）RNA-SEQ，抗体（AB）-Seq和T细胞受体（TCR）-Seq。找到 免疫主导表位，与基于细胞和免疫调节疗法的关键相关性，我们将使用 208 MHC结合的人ApoB肽的大型巨型植物，然后是由Elispot测试的系统反向卷积， 细胞内细胞因子染色（ICS），细胞因子捕获测定法（CCA）和抗原诱导的标记（AIM）测定。 AIM 2是通过将新鼠标模型与APOB特异性组合结合的APOB特异性T细胞如何发展 四聚体。我们的初步数据支持以下假设：某些APOB特异性CD4 T细胞已经存在于 非常年轻的老鼠。这些细胞中的一些是效应T细胞（非三重细胞）。小鼠中的其他FOXP3+ CD4+ Tregs 随着动脉粥样硬化的进展，将其转录组从动脉粥样硬化为促动源性。 从机械上讲，我们将测试Treg的代谢和表观遗传假设以推开开关。目标3是测试 假设心血管疾病的人类中APOB特异性的T细胞包括Tregs，Extreg和 非treg，使用scrna-seq和deonvolution（cibersort）方法。拟议的工作完成后， 我们将知道人类APOB中的主要MHC-II限制性表位和响应的极化 细胞由scrna-seq，ab-seq和tcr-seq。我们将知道Treg表型切换的机制和 PBMC（CAVA和CT-CAVA队列）和其他APOB特异性T细胞的比例（CAVA和CT-CAVA队列）和 内部切除术转录组（自行车队列）。