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.