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。MHC-II限制性多肽表位疫苗的研究 载脂蛋白B(ApoB)通过诱导ApoB特异性调节性CD4T细胞(Tregs)来改善动脉粥样硬化。 我已经开发了试剂(四聚体和葡聚体)来检测和分离这些载脂蛋白B特异的Tregs 小鼠和人类的单细胞水平。拟议的工作具有T细胞方面，目前由R01支持 HL121697(2014-2018)和血管巨噬细胞方面，目前由R01 HL115232(2012- 2022年)。为了获得更好的机械性洞察力，我建议采用转移载脂蛋白B特定的树(如果必要 在Rag2-/-小鼠中扩展)进入受体小鼠，并测量动脉粥样硬化。我还将转移载脂蛋白B肽- 正式测试可能的抗体效果，并测试B细胞在两个B细胞缺陷中的作用 老鼠线。我将研究接种后体内血管巨噬细胞功能的变化。为了提高 疫苗效力，我建议测试类似于临床使用的疫苗配方，测试 动脉粥样硬化疫苗在另外两种动脉粥样硬化小鼠模型中的应用(APOE-/-饮食，Ldlr-/-高脂肪 饮食)，优化疫苗接种方案，并添加小剂量IL-2以稳定Tregs。了解如何以及为什么 Tregs转到效应器T细胞，我将使用FoxP3(Treg定义转录因子)谱系追踪小鼠。 TCR-Seq将检验这样的假设，即表面上的变化是由 促炎性载脂蛋白B特异性的CD4T细胞。为了测试细胞外源因子，我将培养载脂蛋白B特异的CD4 T细胞与移植的正常或动脉粥样硬化的动脉，并通过DNA和组蛋白测量表观遗传学的变化 FoxP3基因座周围的甲基化。为了准备将疫苗移植到人类身上，我提出了更多 人类工作，包括外周血单个核细胞(PBMCs)的质粒术(CyTOF) “彩色”面板。条形码scRNA-Seq获得单细胞转录本将更深入地定义细胞类型。 我建议将目前的临床数据集(所有女性，大多数是HIV+)扩大到性别和HIV-。当这件事 工作完成后，我们将对动脉粥样硬化疫苗的工作原理有一个很好的了解。我们会有 从心血管收集的PBMCs中ApoB特异性CD4T细胞表型的大量人类数据 疾病病例和对照。

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.