In Silico Analysis of Functional Overlap and Complementarity of <scp>HLA</scp> ‐ <scp>DQ5</scp> and <scp>DQ9</scp> in Gliadin Presentation: Expanding the Immunopeptidomic Landscape of Celiac Disease

ABSTRACT Celiac disease (CD) is classically associated with the HLA‐DQ2.5 and HLA‐DQ8 heterodimers; however, a clinically relevant subset of patients fulfilling diagnostic criteria lacks these canonical risk HLA‐DQ molecules, indicating the presence of alternative antigen‐presentation pathways. Among potential contributors, HLA‐DQ5 and HLA‐DQ9 remain poorly characterised at the immunopeptidomic level. In this study, we applied an integrated in silico framework to systematically investigate the gliadin‐derived peptide repertoire presented by canonical and non‐canonical HLA‐DQ molecules. Peptides derived from α‐, γ‐ and ω‐gliadin families were generated following simulated tissue transglutaminase–mediated deamidation and multi‐enzyme gastrointestinal digestion. Binding affinities to HLA‐DQ2.5, DQ8, DQ5 and DQ9 were predicted using NetMHCIIpan 4.3 and integrated with peptide stability and sequence abundance into a composite Biological Plausibility score. The analysis identified γ‐gliadin–derived peptides as the dominant immunogenic drivers across all investigated HLA‐DQ molecules, owing to their enhanced digestive stability and high allelic promiscuity. Hierarchical clustering of binding profiles revealed a repertoire‐dependent functional overlap between HLA‐DQ9 and the canonical DQ8 molecule, particularly for γ‐gliadin motifs. In contrast, HLA‐DQ5 displayed functional complementarity, selectively presenting a distinct subset of gliadin peptides that were poorly recognised by classical risk HLA‐DQ molecules. Collectively, these findings provide a mechanistic framework for CD pathogenesis in individuals lacking HLA‐DQ2.5 and HLA‐DQ8, demonstrating that non‐canonical HLA‐DQ molecules can sustain pathogenic CD4 + T‐cell responses through convergent and complementary antigen‐presentation pathways. As a predictive in silico framework, this work refines the genetic paradigm of CD by emphasising functional organisation of the gluten immunopeptidome over simple allele presence and provides a mechanistic rationale for future experimental validation of non‐canonical HLA‐DQ–restricted gliadin presentation.