Abstract P2093: Immunodominant Human Leukocyte Antigen-ii-restricted Epitopes In Human Apolipoprotein B

Abstract

Introduction: CD4 + T cell responses to apolipoprotein B are well characterized in atherosclerotic mice and also detectable in humans. CD4 + T cells recognize peptides displayed on highly polymorphic Human Leukocyte Antigen-II molecules. Immunogenicity of individual APOB peptides is largely unknown in humans. Only one HLA-II-restricted epitope was validated using a DRB1*07:01-APOB 3036-3050 tetramer. We reasoned that the T cell epitope repertoire of APOB is broader and may contain dominant antigenic regions. Hypothesis: Human APOB harbors discrete immunodominant sequences that are targeted by autoreactive CD4 + T cells in majority of donors. These trigger atherosclerosis-related autoimmune responses in humans. Objective: To optimize a restimulation-based workflow to examine antigenicity of multiple candidate peptides in HLA-typed donors, to chart TCR specificities and phenotypes of APOB-reactive cells and to profile T helper cytokine responses to APOB epitopes. Methods and Results: We selected twenty APOB peptides (APOB 20 ) from an in silico screen and validated peptide binding to the most commonly occurring HLA-II alleles. Using stringent controls, we confirmed specificity of expansion-based protocols to detect CD4 + T cytokine responses to APOB 20 pool. Ex vivo assessment of activation-induced markers revealed statistically significant autoimmune response to APOB 20 , but not to a ubiquitously-expressed negative control protein, actin. CD4 + T responses to the APOB 20 pool were resolved to the level of individual peptides using an IFNγ enzyme-linked immune-absorbent spot (ELISpot) assay. This led to the discovery of six immunodominant epitopes (APOB 6 ) that triggered robust T cell responses in most donors. High-throughput sequencing identified unique APOB 6 -specific expanded TCR clonotypes. Responding CD4 + T cells were enriched in antigen-experienced memory phenotypes. APOB 6 induced secretion of both pro-inflammatory and regulatory cytokines. In matched clinical samples, APOB 6 stimulation detected heightened expression of activation and memory markers and augmented secretion of proinflammatory cytokines in patients with more severe coronary artery disease. Conclusions: Using >60 donors expressing diverse HLA-II alleles and distributed over a screening and a validation cohort of healthy subjects and a clinical cohort of CAD patients, we discovered six immunodominant APOB epitopes that allowed interrogation of dynamic antigen-specific CD4 + T responses associated with human atherosclerosis.