P0001 Single-cell profiling reveals distinct γδ T cell subsets with protective and pathogenic roles in Ulcerative Colitis

Abstract Background Comprising about 40% of mucosal lymphocytes, γδ T cells represent a significant lineage in the gut, and thus are key players in the intestinal immune homeostasis. In contrast to αβ TCRs, γδ TCRs are able to recognize antigen independent from MHC-restricted antigen presentation. While antigens recognized by γδ TCRs are largely undefined, activation of γδ T cells is initiated by phospho-antigens and members of the butyrophilin (BTN) family. Data from both murine and human studies suggest a protective role of γδ T cells in the pathogenesis of inflammatory bowel diseases (IBD). However, significant species-specific variations of γδ T cells demand more detailed investigation of human intestinal γδ T cells. To date, no single-cell study has comprehensively assessed subset-related roles of γδ T cells in ulcerative colitis (UC). Methods In this study, we performed multimodal profiling of intestinal γδ T cells in ulcerative colitis (UC) at single-cell resolution using FLASH-seq (healthy donors (HD) n=2, active UC n=5) and cytometry by time of flight (CyTOF, HD n=29, inactive UC n=26, active UC n=24). FLASH-seq analysis of additional samples is ongoing. Peripheral blood samples were analyzed in a subset of patients. Results Our analysis revealed that healthy gut epithelium is predominantly populated by CD103+ intraepithelial γδ lymphocytes (γδ IELs). However, in the inflamed intestinal environment, two distinct although clonally related γδ T cell subsets emerge: one TCF-1+ PD-1+ memory-like subset, resembling stem-like αβ T cells previously described in chronic infections and cancer, and the other exhibiting a cytotoxic phenotype, characterized by increased expression of GZMB, PRF1, and TBX21. The decline in CD103+ γδ T cells, coupled with the expansion of these two subsets, was associated with reduced expression of butyrophilin-like (BTNL) molecules, BTNL3 and BTNL8, and increased expression of BTN3A1 and BTN3A3, along with transcriptional and metabolic changes associated with enhanced γδ T cell-mediated killing in intestinal epithelial cells. Patients responding to therapies showed a return to a healthy γδ T cell subset composition, with the contraction of these subsets after remission, further supporting their pathogenic role. Additionally, significant opposing phenotypic changes were observed in peripheral blood γδ T cell subsets in UC. Conclusion Overall, our data provide a comprehensive single-cell landscape of γδ T cells in UC, identifying novel subsets that differentially carry out protective and pathogenic roles. These subsets may serve as valuable biomarkers, and may represent novel therapeutic targets.