Imaging Mass Cytometry‐Based Immune Profiling of Human Peyer's Patches in Patients with Crohn's Disease

Lymphoid follicles in the human gut are critical immune hubs, yet their role in Crohn's disease pathogenesis remains poorly understood. Here, we apply multiplexed imaging mass cytometry to spatially profile Peyer's patches and lymphoid follicles in biopsies from healthy controls and Crohn's disease patients with ileitis, isolated colonic involvement, or in remission. Despite tissue heterogeneity, our optimized preprocessing pipeline enabled robust tissue annotation, single-cell phenotyping, and neighbourhood-level analysis. While conventional analysis based on cell frequencies did not distinguish disease states, spatial analysis revealed disease-associated remodelling of lymphoid architecture. Biopsies from colonic Crohn's disease patients showed, within follicles, increased frequencies of activated CD8⁺ T cells and a reduction in naïve T cells, alongside enrichment of B cell-T cell interaction neighbourhoods. These alterations were most pronounced in smaller B-cell patches, suggesting more functionally dynamic immune-cell interactions in compact lymphoid structures. In contrast, Crohn's disease ileitis samples closely resembled healthy tissue, with minimal structural or immune cell perturbations. Our data support a model in which Peyer's patches and lymphoid follicles undergo structural and functional remodelling in response to colonic inflammation. These findings underscore the value of spatially resolved immune profiling to uncover tissue-specific immune dynamics in inflammatory bowel disease.