UC San Diego

Background

CD (cluster of differentiation) 4⁺ T-cell responses to APOB (apolipoprotein B) are well characterized in atherosclerotic mice and detectable in humans. CD4⁺ T cells recognize antigenic peptides displayed on highly polymorphic HLA (human leukocyte antigen)-II. Immunogenicity of individual APOB peptides is largely unknown in humans. Only 1 HLA-II-restricted epitope was validated using the DRB1*07:01-APOB_3036-3050 tetramer. We hypothesized that human APOB may contain discrete immunodominant CD4⁺ T-cell epitopes that trigger atherosclerosis-related autoimmune responses in donors with diverse HLA alleles.

Methods

We selected 20 APOB-derived peptides (APOB₂₀) from an in silico screen and experimentally validated binding to the most commonly occurring human HLA-II alleles. We optimized a restimulation-based workflow to evaluate antigenicity of multiple candidate peptides in HLA-typed donors. This included activation-induced marker assay, intracellular cytokine staining, IFNγ (interferon gamma) enzyme-linked immunospot and cytometric bead array. High-throughput sequencing revealed TCR (T-cell receptor) clonalities of APOB-reactive CD4⁺ T cells.

Results

Using stringent positive, negative, and crossover stimulation controls, we confirmed specificity of expansion-based protocols to detect CD4⁺ T cytokine responses to the APOB₂₀ pool. Ex vivo assessment of AIM⁺CD4⁺ T cells revealed a statistically significant autoimmune response to APOB₂₀ but not to a ubiquitously expressed negative control protein, actin. Resolution of CD4⁺ T responses to the level of individual peptides using IFNγ enzyme-linked immunospot led to the discovery of 6 immunodominant epitopes (APOB₆) that triggered robust CD4⁺ T activation in most donors. APOB₆-specific responding CD4⁺ T cells were enriched in unique expanded TCR clonotypes and preferentially expressed memory markers. Cytometric bead array analysis detected APOB₆-induced secretion of both proinflammatory and regulatory cytokines. In clinical samples from patients with angiographically verified coronary artery disease, APOB₆ stimulation induced higher activation and memory phenotypes and augmented secretion of proinflammatory cytokines TNF (tumor necrosis factor) and IFNγ, compared with patients with low coronary artery disease.

Conclusions

Using 3 cohorts, each with ≈20 donors, we discovered and validated 6 immunodominant, HLA-II-restricted APOB epitopes. The immune response to these APOB epitopes correlated with coronary artery disease severity.