In Silico Analysis of Functional Overlap and Complementarity of HLA-DQ5 and DQ9 in Gliadin Presentation: Expanding the Immunopeptidomic Landscape of Celiac Disease.

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.