HLA-Restricted Autoantigen Presentation in Hashimoto’s Thyroiditis: An In Silico Immunopeptidomics Approach

Introduction: Hashimoto's thyroiditis (HT) is the most prevalent endocrine autoimmune disorder and leading cause of hypothyroidism in iodine-sufficient populations. Robust HLA class II associations implicate CD4⁺ T-cell antigen presentation as central to pathogenesis, yet specific peptide–HLA interactions driving thyroid autoimmunity remain poorly characterized. Objective: To quantitatively characterize HLA class II-restricted presentation of major HT-associated thyroid autoantigens using integrated in silico immunopeptidomics. Methods: Canonical thyroperoxidase, thyroglobulin, and thyrotropin receptor sequences were retrieved from curated databases and analyzed against 10 HT-associated HLA-DR, -DQ, and -DP alleles. NetMHCIIpan predicted peptide–HLA binding affinity, complemented by IEDB antigen processing and immunogenicity algorithms. Epitope candidates were prioritized by binding strength, allelic promiscuity, processing likelihood, and population coverage. Sliding-window fragmentation of the three autoantigen sequences generated 4,423 overlapping 15-mers, which were subsequently filtered using binding percentile thresholds, promiscuity (≥4 alleles), and predicted processing efficiency. Results: Of 4,423 peptides, 312 (7.1%) were strong binders (%Rank ≤2.0) and 891 (20.1%) weak binders (%Rank ≤10.0). After promiscuity filtering, 87 peptides (2.0%) remained; 52 (59.8%) were validated by combined binding/processing criteria. High-confidence epitopes comprised 24 thyroglobulin- (46.2%), 19 thyroperoxidase- (36.5%), and 9 thyrotropin receptor-derived peptides (17.3%). Binding affinities differed significantly across autoantigens (Kruskal–Wallis H=14.73, p=0.0006). The HLA panel achieved estimated population coverage of 72.4% in Europeans, 68.1% in East Asians, and 58.3% in sub-Saharan Africans. Conclusion: Quantitative in silico immunopeptidomics identified a restricted, numerically defined set of high-affinity, HLA class II-restricted thyroid epitopes in HT, refining thyroid autoimmunity's immunogenetic architecture and providing a robust, testable foundation for epitope-focused experimental and translational research.