A Glycosylation-Dependent Checkpoint Restrains Intestinal Intra-Epithelial Lymphocyte Activation

Abstract Intraepithelial lymphocytes (IELs) are abundant in the intestinal epithelium, where they maintain barrier integrity and provide immune defense. Because of their potent cytotoxic and effector potential, IEL activity must be tightly controlled to prevent tissue damage. However, the mechanisms that calibrate IEL responsiveness remain unclear. Here, we identify glucosaminyl (N-acetyl) transferase 2 ( Gcnt2 ) as a key restrainer of both natural and induced gut IEL. Among T cells, Gcnt2 is uniquely enriched in the intestine and partly dependent on retinoic acid signaling. GCNT2-mediated branched glycosylation marks IELs with signatures of tissue adaptation and reduced TCR responsiveness. Genetic ablation of Gcnt2 enhanced IEL degranulation, cytokine production, and cytotoxicity upon stimulation, improving bacterial clearance and limiting infection-induced disease, while aggravating the pathological consequences of strong T cell activation. Mechanistically, GCNT2-mediated glycosylation of CD45 reduced its phosphatase activity, thereby dampening TCR signaling and effector responses. Together, these findings reveal GCNT2 as a glycosylation-dependent checkpoint that fine-tunes IEL effector functions, uncovering a novel mechanism by which the intestinal immune system balances responsiveness and tolerance. Summary GCNT2-mediated I-branching glycosylation of CD45 restrains IEL activation.