Robert P. Anderson

Ex-vivo whole blood secretion of interferon (IFN)-γ and IFN-γ-inducible protein-10 measured by enzyme-linked immunosorbent assay are as sensitive as IFN-γ enzyme-linked immunospot for the detection of gluten-reactive T cells in human leucocyte antigen (HLA)-DQ2·5+-associated coeliac disease

T cell cytokine release assays are used to diagnose infectious diseases, but not autoimmune or allergic disease. Coeliac disease (CD) is a common T cell‐mediated disease diagnosed by the presence of gluten‐dependent intestinal inflammation and serology. Many patients cannot be diagnosed with CD because they reduce dietary gluten before medical workup. Oral gluten challenge in CD patients treated with gluten‐free diet (GFD) mobilizes gluten‐reactive T cells measurable by interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) or major histocompatibility complex (MHC) class II tetramers. Immunodominant peptides are quite consistent in the 90% of patients who possess HLA‐DQ2·5. We aimed to develop whole blood assays to detect gluten‐specific T cells. Blood was collected before and after gluten challenge from GFD donors confirmed to have CD (n = 27, all HLA‐DQ2·5+), GFD donors confirmed not to have CD (n = 6 HLA‐DQ2·5+, 11 HLA‐DQ2·5−) and donors with CD not following GFD (n = 4, all HLA‐DQ2·5+). Plasma IFN‐γ and IFN‐γ inducible protein‐10 (IP‐10) were measured by enzyme‐linked immunosorbent assay (ELISA) after whole blood incubation with peptides or gliadin, and correlated with IFN‐γ ELISPOT. No T cell assay could distinguish between CD patients and controls prior to gluten challenge, but after gluten challenge the whole blood IFN‐γ ELISA and the ELISPOT were both 85% sensitive and 100% specific for HLA‐DQ2·5+ CD patients; the whole blood IP‐10 ELISA was 94% sensitive and 100% specific. We conclude that whole blood cytokine release assays are sensitive and specific for detection of gluten‐reactive T cells in CD; further clinical studies addressing the utility of these tests in patients with an uncertain diagnosis of CD is warranted.

Circulating gluten-specific FOXP3 + CD39 + regulatory T cells have impaired suppressive function in patients with celiac disease

Background: Celiac disease is a chronic immune‐mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T‐cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood. Objective: We sought to define whether patients with celiac disease have a dysfunction or lack of gluten‐specific forkhead box protein 3 (FOXP3)+ Treg cells. Methods: Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten‐specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten‐specific CD4+ T‐cell response, we paired traditional IFN‐&ggr; ELISpot with an assay to detect antigen‐specific CD4+ T cells that does not rely on tetramers, antigen‐stimulated cytokine production, or proliferation but rather on antigen‐induced coexpression of CD25 and OX40 (CD134). Results: Numbers of circulating gluten‐specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten‐specific CD4+ T cells were FOXP3+CD39+ Treg cells, which reside within the pool of memory CD4+CD25+CD127lowCD45RO+ Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten‐specific FOXP3+CD39+ Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells. Conclusion: This study provides the first estimation of FOXP3+CD39+ Treg cell frequency within circulating gluten‐specific CD4+ T cells after oral gluten challenge of patients with celiac disease. FOXP3+CD39+ Treg cells comprised a major proportion of all circulating gluten‐specific CD4+ T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key contributor to disease pathogenesis.