ApoB-specific CD4 T cells in mouse and human atherosclerosis

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Project 4 description Atherosclerosis is known to be controlled by regulatory T cells (Tregs), but neither the antigen specificity nor the location nor the mechanism by which these cells protect are known. Project 4 is testing the hypothesis that regulatory CD4 T cells express and secrete IL-10 in response to their cognate antigen, ApoB, the main core protein of low density lipoprotein (LDL). This is based on the discovery of a significant number of ApoB-specific CD4 T cells in mice and in humans, using mouse and human MHC-II tetramers and dextramers loaded with mouse and human ApoB peptides, respectively. Specific Aim 1 is to test this hypothesis in mice by studying atherosclerosis in Apoe-/- mice and following the natural history of the ApoB-specific CD4 T cell repertoire by flow cytometry (FACS), mass cytometry (CyTOF) and RNA-Seq (through core E). To conclusively test whether IL-10 from ApoB-specific CD4 T cells is required for atheroprotection, we will harvest ApoB-specific CD4 T cells from Apoe-/- (IL-10 sufficient) or CD4CreIl10fl/flApoe-/- (IL-10-deficient) donors and separately transfer them into recipient Apoe-/- Cd4-/- mice. We hypothesize that the IL-10 sufficient CD4 T cells will be atheroprotective and the IL-10 deficient CD4 T cells will not. Specific Aim 2 is to translate the findings to humans, using frozen PBMCs from the MESA cohort (core D) and the UVa cohort (core C). Preliminary data show that we can detect human ApoB-specific CD4 T cells in frozen PBMCs from subjects with subclinical cardiovascular disease (CVD). We propose to test their ability to express (by FACS) and secrete (by EliSpot) IL-10, assess their phenotype by CyTOF and define their transcriptome by RNA-Seq (through core E). We have discovered 30 human ApoB peptides that bind many human MHC-II (DR) alleles and we estimate that we can interrogate >85% of all samples using 17 different tetramers and dextramers. In collaboration with core D, we propose to correlate the number and phenotype of ApoB-specific CD4 T cells with subclinical CVD as defined by coronary calcium (CAC) scores and CAC score progression. Project 4 will collaborate with project 1 on antigen presentation by monocytes and intravital microscopy, project 2 on the importance of LDL modifications and project 3 on studying the B1 cell response to LDL. When the proposed work is completed, we will understand the role of ApoB-specific CD4 T cells in modulating atherosclerosis by IL-10. The mechanistic mouse work provides the basis for testing the relevance of ApoB-specific CD4 T cells in CVD patients. ApoB-specific CD4 T cells are likely useful immunological biomarkers in atherosclerosis and the results can guide future therapeutic and preventive efforts.