P0163NKG2A upregulation on natural killer cells contributes to immune dysregulation in Ulcerative Colitis

Abstract Background Ulcerative colitis (UC) is a chronic inflammatory disease primarily mediated by overactivated CD4+ T cells. Previous studies have suggested that natural killer (NK) cells may exert immunoregulatory effects by lysing autologous overactivated CD4+ T cells1-4. However, the precise regulatory and effector mechanisms of NK cells in UC remain unclear. Methods Colon biopsy samples were collected from 6 newly diagnosed UC patients with active disease and 6 healthy volunteers. Using single-cell RNA sequencing, we identified NK and CD4+ T cell populations and analyzed their differentially expressed genes and associated enriched pathways. Flow cytometry was employed to detect the expression of NK cell surface receptors. Multiplex immunofluorescence was used to examine the expression and tissue distribution of NK and CD4+ T cells. Results Single-cell transcriptomic analysis revealed that KLRC1, the gene encoding the inhibitory receptor NKG2A, was highly expressed in NK cells from the intestinal tissue of active UC patients. Flow cytometry further confirmed a significant upregulation of NKG2A surface expression on NK cells derived from patient intestinal tissues. NKG2A is a transmembrane protein that, upon ligand binding, activates inhibitory signaling pathways within NK cells, thereby reducing their cytotoxic activity and impairing their ability to lyse target cells. These findings suggest that the capacity of intestinal NK cells to lyse activated CD4+ T cells may be suppressed in UC. Further analysis showed an increased abundance of CD4+ T cells in active UC intestinal tissues compared to healthy controls. Differentially expressed genes in these CD4+ T cells were significantly enriched in pathways related to cell proliferation, differentiation, and immune activation. Consistent with this, multiplex immunofluorescence demonstrated a marked infiltration of CD4+ T cells in patient tissues, with spatial co-localization observed between NK and CD4+ T cells. Conclusion In the intestinal tissue of active ulcerative colitis (UC) patients, upregulation of the inhibitory receptor NKG2A on NK cells compromises their cytotoxicity against activated CD4+ T cells. This impairment facilitates the accumulation of pathogenic CD4+ T cells within the local immune microenvironment, thereby exacerbating intestinal inflammation. Our integrated analysis, combining single-cell RNA sequencing, flow cytometry, and multiplex immunofluorescence, elucidates a possible mechanism through which NK cell dysfunction contributes to immune dysregulation in UC. References: 1. Mora-Bitria L, Asquith B. Germline natural killer cell receptors modulating the T cell response. Front Immunol. 2024;15:1477991. 2. Wu X. Innate Lymphocytes in Inflammatory Arthritis. Front Immunol. 2020;11:565275. 3. Yang Y, Day J, Souza-Fonseca Guimaraes F, Wicks IP, Louis C. Natural killer cells in inflammatory autoimmune diseases. Clin Transl Immunology. 2021;10(2):e1250. 4. Zhao ZB, Lu FT, Ma HD, et al. Liver-resident NK cells suppress autoimmune cholangitis and limit the proliferation of CD4+ T cells. Cell Mol Immunol. 2020;17(2):178-189. Conflict of interest: Dr. Zhang, Yingzhi: No conflict of interest Guo, Hong: No conflict of interest