gamma delta Intraepithelial Lymphocytes Facilitate Pathological Epithelial Cell Shedding Via CD103-Mediated Granzyme Release

BACKGROUND & AIMS: Excessive shedding of apoptotic enterocytes into the intestinal lumen is observed in inflam-matory bowel disease and is correlated with disease relapse. Based on their cytolytic capacity and surveillance behavior, we investigated whether intraepithelial lymphocytes expressing the gamma delta T cell receptor (gamma delta IELs) are actively involved in the shedding of enterocytes into the lumen. METHODS: Intravital microscopy was performed on GFP gamma delta T cell reporter mice treated with intraperitoneal lipopolysaccharide (10 mg/kg) for 90 minutes to induce tumor necrosis factor-mediated apoptosis. Cell shedding in various knockout or transgenic mice in the presence or absence of blocking antibody was quantified by immunostaining for ZO-1 funnels and cleaved caspase-3 (CC3). Granzyme A and granzyme B release from ex vivo-stimulated gamma delta IELs was quantified by enzyme-linked immunosorbent assay. Immunostaining for gamma delta T cell receptor and CC3 was performed on duodenal and ileal biopsies from controls and patients with Crohn's disease. RESULTS: Intravital microscopy of lipopolysaccharide-treated mice revealed that gamma delta IELs make extended contact with shedding enterocytes. These prolonged interactions require CD103 engagement by E-cad-herin, and CD103 knockout or blockade significantly reduced lipopolysaccharide-induced shedding. Furthermore, we found that granzymes A and B, but not perforin, are required for cell shedding. These extracellular granzymes are released by gamma delta IELs both constitutively and after CD103/E-cadherin ligation. Moreover, we found that the frequency of gamma delta IEL localization to CC3-positive enterocytes is increased in Crohn's disease bi-opsies compared with healthy controls. CONCLUSIONS: Our results uncover a previously unrecognized role for gamma delta IELs in facilitating tumor necrosis factor-mediated shedding of apoptotic enterocytes via CD103-mediated extracellular gran-zyme release.