Vascular macrophages and T cells in atherosclerosis

摘要 这项申请提出了旨在为免疫治疗提供坚实的机制基础的研究 以及预防和治疗动脉粥样硬化的疫苗接种。用来自人的MHC-II限制性肽表位接种 载脂蛋白B（ApoB）通过诱导ApoB特异性调节性CD 4 T细胞（TcB）来改善动脉粥样硬化。 我已经开发了试剂（四聚体和右旋糖酐），以检测和分离这些载脂蛋白B特异性T细胞， 在小鼠和人类的单细胞水平上。拟议的工作具有T细胞方面，目前由R 01支持 HL 121697（2014-2018）和血管巨噬细胞方面，目前由R 01 HL 115232（2012- 2018）支持 2022年）。为了获得更好的机制见解，我建议采用转移ApoB特异性TdR（如果必要的话 在Rag 2-/-小鼠中扩增）转移到受体小鼠中并测量动脉粥样硬化。我还会把载脂蛋白B肽 特异性抗体正式测试可能的抗体效应，并测试B细胞在两种B细胞缺陷型中的作用。 鼠标线。我将研究疫苗接种后体内血管巨噬细胞功能的变化。提高 疫苗的有效性，我建议测试疫苗配方类似于将用于临床，测试 在另外两种动脉粥样硬化小鼠模型（Apoe-/-，食物饮食，Ldlr-/-，高脂饮食）中的动脉粥样硬化疫苗 饮食），优化疫苗接种方案，并添加低剂量IL-2以稳定TcB。去发现 为了将Treg转换为效应T细胞，我将使用FoxP 3（Treg定义转录因子）谱系追踪小鼠。 TCR-Seq将检验这样的假设，即明显的转换是由少数群体的生长引起的。 促炎性ApoB特异性CD 4 T细胞。为了测试细胞外源因子，我将孵育ApoB特异性CD 4 T细胞与正常或动脉粥样硬化斑块，并通过DNA和组蛋白测量表观遗传变化 FoxP 3基因座周围的甲基化。为了准备将疫苗转化为人类，我建议更多 人的工作，包括对外周血单核细胞（PBMC）进行的42- “颜色”面板。获得单细胞转录组的条形码scRNA-Seq将更深入地定义细胞类型。 我建议将目前的临床数据集（所有妇女，大多数艾滋病毒阳性）扩大到性别和艾滋病毒阴性。当这个 这项工作完成后，我们将很好地了解动脉粥样硬化疫苗的工作原理。我们会一起 从心血管疾病患者采集的PBMC中ApoB特异性CD 4 T细胞表型的广泛人体数据 疾病病例和控制。

Abstract This application proposes research designed to provide solid mechanistic underpinnings for immunotherapy and vaccination to prevent and treat atherosclerosis. Vaccination with MHC-II restricted peptide epitopes from apolipoprotein B (ApoB) ameliorates atherosclerosis by inducing ApoB-specific regulatory CD4 T cells (Tregs). I have developed reagents (tetramers and dextramers) to detect and isolate these ApoB-specific Tregs at the single cell level in mice and humans. The proposed work has a T cell aspect, currently supported by R01 HL121697 (2014-2018), and a vascular macrophage aspect, currently supported by R01 HL115232 (2012- 2022). To gain better mechanistic insight, I propose to adoptively transfer ApoB-specific Tregs (if necessary expanded in Rag2-/- mice) into recipient mice and measure atherosclerosis. I will also transfer ApoB peptide- specific antibodies to formally test possible antibody effects, and test the role of B cells in two B cell-deficient mouse lines. I will study changes in functions of vascular macrophages in vivo after vaccination. To improve the vaccine efficacy, I propose to test vaccine formulations similar to what would be used clinically, test the atherosclerosis vaccines in two other mouse models of atherosclerosis (Apoe-/- on chow diet, Ldlr-/- on high fat diet), optimize the vaccination protocol, and add low-dose IL-2 to stabilize Tregs. To discover how and why Tregs switch to effector T cells, I will use FoxP3 (the Treg defining transcription factor) lineage tracker mice. TCR-Seq will test the hypothesis that the apparent switch is caused by an outgrowth of a minor population of pro-inflammatory ApoB-specifc CD4 T cells. To test cell-exogenous factors, I will incubate ApoB-specific CD4 T cells with explanted normal or atherosclerotic aortas, and measure epigenetic changes by DNA and histone methylation around the FoxP3 locus. To prepare for translating the vaccine into humans, I propose more human work, including mass cytometry (CyTOF) on peripheral blood mononuclear cells (PBMCs) with 42- “color” panels. Barcoded scRNA-Seq to obtain single cell transcriptomes will define the cell types more deeply. I propose to expand the current clinical data set (all women, most HIV+) to both genders and HIV-. When this work is completed, we will have a good understanding how atherosclerosis vaccination works. We will have extensive human data for the phenotype of ApoB-specific CD4 T cells in PBMCs collected from cardiovascular disease cases and controls.