Stable β2-Microglobulin-HLA Class I Association Reshapes the Antigenic Landscape and TCR Recognition of Cancer-Associated Epitopes.

β2-Microglobulin (β2m) is essential for the stability and surface expression of human leukocyte antigen class I (HLA-I) molecules. While β2m's structural role is well established, its influence on the antigenic landscape remains incompletely understood. Here, we investigated how constitutive β2m binding to HLA-I influences the antigenic landscape in the context of cancer-associated epitopes. Using two engineered cell lines expressing β2m fused to the HLA-I heavy chain, we analysed changes in immunopeptidomes and epitope presentation. Despite cell line-specific transcriptional responses, the fusion of β2m to HLA-I consistently reduced peptide editing and resulted in convergent remodelling of the antigen presentation pathway (APP). The fusion further altered peptide selection, favouring the display of lower-affinity peptides derived from endogenous proteins while impairing high-affinity peptide presentation on HLA-I molecules. These changes were not attributable to APP component abundance but rather reflected mechanistic alterations in peptide selection and impacted T cell receptor recognition of tumour epitopes. Our findings suggest that β2m-HLA-I fusion can distort the physiological antigenic repertoire and T cell recognition, with implications for epitope discovery, immunotherapy design, and studies of HLA-I-mediated immune surveillance.