Research Projects

Rationale: Atherosclerosis, the most common pathology underlying cardiovascular disease, is associated with chronic inflammation (Roy et.al., Nature Reviews Immunology, 2022). My postdoctoral work has shown that chronic inflammatory conditions in atherosclerosis trigger loss of regulatory T cell function (Freuchet and Roy et.al., Nature Immunology, 2023) and aberrant autoreactivity that correlate with the severity of cardiovascular disease (Roy et.al., Circulation Research, 2022; Roy and Suthahar et.al., Frontiers in Immunology, 2024; Roy et.al., Nature Cardiovascular Research, 2025). Studies on autoantigen-specific adaptive immune cells in the context of atherosclerosis have largely focused on B cells, followed by CD4+T cells. However, recent single cell sequencing of transcriptomes and T cell receptor (TCR) profiling have shown that clonally expanded effector and memory CD8+T cell subsets are abundant in mouse and human plaques. This suggests that CD8+T cells respond to endogenous triggers at atherosclerotic tissues, but the antigenic identities and phenotypic diversity of autoreactive CD8+T cells in atherosclerosis are unknown. Hypothesis: Preclinical studies and clinical observations have established that the most relevant antigenic target in atherosclerosis is apolipoprotein B (ApoB), the core protein that forms the backbone of all proatherogenic lipid-carrier molecules (LDL, VLDL). Based on my preliminary studies and literature survey, I put forward an overarching hypothesis that atherosclerosis is associated with augmented inflammatory and cytotoxic activity of CD8+T cells against apolipoprotein B (ApoB). Objectives: I propose to identify the top antigenic MHC Class-I-restricted epitopes in mouse ApoB protein (Aim1), characterize the phenotypes of in vivo activated ApoB-specific CD8+T cells (Aim2), and examine the dynamics of activated CD8+T cells in the periphery (blood and secondary lymphoid organs) and at atherosclerotic tissues (whole aorta and aortic root sections) in high fat diet-fed mouse models (Aim3). Experiments: In my newly established lab, I propose to use an integrated pipeline that involves in-silico tools, immunization models, peptide-induced stimulation assays, low-input RNA sequencing, functional, metabolic and biochemical assays to chart MHC Class I epitopes in ApoB and dissect the phenotypic diversity of activated CD8+T cells in the periphery and aortic tissue in high-fat diet fed mouse models. We will first screen hundreds of putative epitope candidates using computational tools. The antigenicity of the top candidates will be individually assessed in vitro with stimulation assays and in vivo with immunized mice. ApoB-specific CD8+T cells against the dominant epitopes will be isolated and subjected to in-depth transcriptomic analyses. Differentially expressed molecular and phenotypic markers (cytokines, chemokines, lytic effectors, receptors, and signaling pathways) will be validated using high-dimensional flow cytometry, qPCR, and western blotting. Metabolic status of ApoB-specific CD8+T cells will be examined using new FACS-based techniques like Met-Flow and SCENITH. The functional capacity will be measured in degranulation and cytotoxic assays. To explore the dynamics of ApoB-specific CD8+T cells in high fat diet-fed mice, activated immune cells in the blood, spleen, lymph nodes, heart and aortic blood vessels will be assessed using multiparametric flow cytometry, microscopy, and histology. Significance: Completion of this study will shed light on the autoimmune component of CD8+ T cell responses in atherosclerosis and generate detailed information about the perturbed programs in atherogenic T cells. This will allow – a) tracking the antigen-specific CD8+T cells specifically related to cardiovascular disease; b) understanding the regulatory network underlying their activation dynamics; c) targeting them for therapy.