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DOP009. Ulcerative colitis: The αEβ7 integrin is associated with a high frequency of Th17, Th1 and Th17/Th1 CD4 lymphocytes

C.A. Lamb1,2, J.C. Mansfield2,3, G.W. Tew4, D.L. Gibbons5, A.K. Long6, P.M. Irving5,7, L. Diehl4, J. Eastham-Anderson4, G. O'Boyle8, D.E.J. Jones1, A.C. Hayday5, M.E. Keir4, J.G. Egen4, J.A. Kirby1, 1Newcastle University, Institute of Cellular Medicine, Newcastle upon Tyne, United Kingdom, 2Newcastle Upon Tyne Hospitals NHS Foundation Trust, Gastroenterology, Newcastle Upon Tyne, United Kingdom, 3Newcastle University, Institute of Genetic Medicine, Newcastle upon Tyne, United Kingdom, 4Genentech, Research and Early Development, South San Francisco, United States, 5King's College London, Peter Gorer Department of Immunobiology, London, United Kingdom, 6Newcastle upon Tyne Hospitals NHS Foundation Trust, Pathology, Newcastle upon Tyne, United Kingdom, 7Guy's & St Thomas' NHS Foundation Trust, Gastroenterology, London, United Kingdom, 8University of Sunderland, Department of Pharmacy Health and Well-being, Sunderland, United Kingdom

Background

T lymphocytes expressing the αEβ7 integrin are highly enriched within human intestinal epithelium and lamina propria. While αEβ7 is critical for lymphocyte retention, studies exploring pathogenic or protective functions of αEβ7 expressing cells, particularly in the context of human diseases, such as ulcerative colitis (UC), are lacking. Defining this phenotype is critical for our understanding of IBD pathogenesis and of translational importance with the development of etrolizumab, a humanized antibody specific to the β7 integrin that blocks α4β7:MAdCAM-1 and αEβ7:E-cadherin interactions. The aim of this study was to determine the differential phenotype of αEβ7+ and αEβ7− colonic T lymphocytes in normal colon and UC.

Methods

Lymphocytes within colonic biopsies from a total of 43 UC and 35 non-disease control patients were studied by flow cytometry (FACS), immunohistochemistry (IHC) and qPCR. Multi-colour FACS was optimised to determine surface and intracellular protein expression (CD45, CD3, CD4, CD8, αE, β7, CD161, IL-17A, TNFα, IFNγ & IL-10). qPCR was performed on TCRαβ+ lymphocytes, FACS sorted into CD4+αEβ7+, CD4+αEβ7−, CD8+αEβ7+ and CD8+αEβ7− prior to gene expression assay. Dual stain IHC for αE, plus CD3, CD4, CD8 and FOXP3 was performed using a Ventana Benchmark XT autostainer. Severity of UC was stratified using the Mayo endoscopic score for UC.

Results

Ulcerative colitis was associated with a significantly increased frequency of T lymphocytes in the intestinal mucosa (p < 0.05). IHC revealed the highest expression of αE on CD4 and CD8 intraepithelial lymphocytes, although a substantial number of lamina propria lymphocytes also expressed this integrin. In UC, FACS demonstrated CD4+αEβ7+ lymphocytes had a higher potential to produce the pro-inflammatory cytokines IFNγ (p < 0.01), TNFα (p < 0.001) and IL-17A (p < 0.0001) than CD4+αEβ7− lymphocytes. In addition, a mean of 31.5% of the CD4+αEβ7+ lymphocytes produced both IL-17A and IFNγ compared to a mean of only 7.7% in the CD4+αEβ7− compartment (p < 0.001). IL-10 was not differentially expressed between CD4+αEβ7+ and CD4+αEβ7− lymphocytes in controls or UC, and a low frequency of αEβ7+FOXP3+ cells was observed by IHC. qPCR array confirmed higher mRNA levels of IFNγ (p < 0.001), TNFα (p < 0.01) and IL-17A (p < 0.01), and lower transcription of FOXP3 (p < 0.0001) in CD4+αEβ7+ cells compared to CD4+αEβ7− cells.

Conclusion

αEβ7 expression was associated with an enrichment of pro-inflammatory Th17, Th1 and Th17/Th1 T lymphocytes, and not associated with a regulatory phenotype. These data suggest therapeutic interventions targeting αE expressing T cells and the αEβ7 integrin itself may be viable approaches for reducing aberrant inflammatory responses in UC.