NKG2D Expression and Function of T Cells Differs in Inflammatory Bowel Disease

Abstract

Purpose: Analyse role of NKG2D in IBD. NKG2D is an activating receptor on human CD8+ T and NK cells, and in chronic inflammatory conditions on CD4+ T cells. NKG2D expression is increased by TNF-α and IL-15, which are up regulated in inflammatory bowel disease (IBD). In mouse colitis, significantly more CD4+NKG2D+ cells in blood, mesenteric lymph nodes, colon, and spleen is observed in diseased mice compared to control mice, and NKG2D-antibody blockade attenuates the development of colitis. Therefore, this study aimed to further examine the expression and role of NKG2D and its ligands in IBD. Methods: Blood and intestinal biopsies from 32 IBD patients: 11 patients with quiescent ulcerative colitis (UC, a Mayo score 0-1); 12 patients with active UC (Mayo score ≥ 2), 9 patients with Crohn's disease (CD, 6 with active disease, a Harvey-Bradshaw score ≥ 5), and 21 controls. PBMCs and intestinal lymphocytes were analyzed by multicolor flow cytometry. Proliferation was analyzed by a CFSE dilution assay. Immunohistochemistry (ICH) was performed on intestinal biopsies for NKG2D. mRNA for NKG2D ligands + cytokines were analyzed by qPCR in intestinal biopsies. Results: An increase in PBMC CD4+ T cells from IBD patients lacking CD28 expression was seen as compared to controls (p<0.03), and an increase of NKG2D expressing CD4+ T cells was found in 4/12 patients with active UC, 1/11 of quiescent UC patients, and 3/6 patients with active CD. This phenotype was not observed in controls. More PBMC CD4+CD28-NKG2D+ T cells were seen in patients with active IBD (p<0.04 and p<0.01) compared to controls. Assessed by IHC, NKG2D+ cells were increased in inflamed as compared to non-inflamed intestine, and CD4+ NKG2D+ T-cells were found in inflamed intestinal biopsies from 2 out of 4 CD samples, whereas none was detected in non-inflamed tissue. Low levels of NKG2D ligands (MICA/B and ULBP1-4) mRNA were detected in control and non-inflamed intestinal biopsies, whereas up regulation of ligand was seen in UC patients (p<0.04) and CD patients (p<0.04). A higher level of IL-17 mRNA was found in active IBD compared to controls (p<0.05). Furthermore, NKG2D co-stimulated lymphocyte proliferation in UC patients PBMCs but not in control PBMCs in vitro. Conclusion: This study shows that NKG2D is significantly up regulated in circulating CD4+CD28- T-cells in IBD patients as compared to controls, and in inflamed intestine. Furthermore, NKG2D can co-stimulate proliferation, and an up regulation of NKG2D ligands is observed in the inflamed intestine. Taken together, these data suggest that a changed expression pattern and function of NKG2D and ligands exist in IBD and that co-stimulation via NKG2D may have a role in driving the inflammatory process in this disorder. Disclosure: Jacob Tveiten Bjerrum - Grant/Research Support. Ole Haagen Nielsen - Grant/Research Support.