Whole blood interleukin-2 release test to detect and characterize rare circulating gluten-specific T cell responses in coeliac disease.

Whole blood cytokine release assays (CRA) assessing cellular immunity to gluten could simplify the diagnosis and monitoring of coeliac disease (CD). We aimed to determine the effectiveness of electrochemiluminescence CRA to detect responses to immunodominant gliadin peptides. HLA-DQ2·5+ CD adults (cohort 1, n = 6; cohort 2, n = 12) and unaffected controls (cohort 3, n = 9) were enrolled. Cohort 1 had 3-day gluten challenge (GC). Blood was collected at baseline, and for cohort 1 also at 3 h, 6 h and 6 days after commencing 3-day GC. Gliadin peptide-stimulated proliferation, interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and 14- and 3-plex electrochemiluminescence CRA were performed. Poisson distribution analysis was used to estimate responding cell frequencies. In cohort 1, interleukin (IL)-2 dominated the gliadin peptide-stimulated cytokine release profile in whole blood. GC caused systemic IL-2 release acutely and increased gliadin peptide-stimulated IFN-γ ELISPOT and whole blood CRA responses. Whole blood CRA after GC was dominated by IL-2, but also included IFN-γ, C-X-C motif chemokine ligand 10/IFN-γ-induced protein 10 (CXCL10/IP-10), CXCL9/monokine induced by IFN-γ (MIG), IL-10, chemokine (C-C motif) ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP-1α), TNF-α and IL-8/CXCL8. In cohorts 2 and 3, gliadin peptide-stimulated whole blood IL-2 release was 100% specific and 92% sensitive for CD patients on a gluten-free diet; the estimated frequency of cells in CD blood secreting IL-2 to α-gliadin peptide was 0·5 to 11 per ml. Whole blood IL-2 release successfully mapped human leucocyte antigen (HLA)-DQ2·5-restricted epitopes in an α-gliadin peptide library using CD blood before and after GC. Whole blood IL-2 release assay using electrochemiluminescence is a sensitive test for rare gliadin-specific T cells in CD, and could aid in monitoring and diagnosis. Larger studies and validation with tetramer-based assays are warranted.

Resolving incomplete single nucleotide polymorphism tagging of HLA-DQ2.2 for coeliac disease genotyping using digital droplet PCR.

A hallmark of coeliac disease (CD) is the exceptionally strong genetic association with HLA-DQ2.5, DQ8, and DQ2.2. HLA typing provides information on CD risk important to both clinicians and researchers. A method that enables simple and fast detection of all CD risk genotypes is particularly desirable for the study of large populations. Single nucleotide polymorphism (SNP)-based HLA typing can detect the CD risk genotypes by detecting a combination of six SNPs but this approach can struggle to resolve HLA-DQ2.2, seen in 4% of European CD patients, because of the low resolution of one negatively predicting SNP. We sought to optimise SNP-based HLA typing by harnessing the additional resolution of digital droplet PCR to resolve HLA-DQ2.2. Here we test this two-step approach in an unselected sample of Mexican DNA and compare its accuracy to DNA typed using traditional exon detection. The addition of digital droplet PCR for samples requiring negative prediction of HLA-DQ2.2 enabled HLA-DQ2.2 to be accurately typed. This technique is a simple addition to a SNP-based typing strategy and enables comprehensive definition of all at-risk HLA genotypes in CD in a timely and cost-effective manner.

Assessing of Celiac Disease and Nonceliac Gluten Sensitivity.

The publication of papers on the topic of gluten related disorders has substantially increased over the last few years. This has motivated healthcare professionals to pay attention not only to celiac disease and wheat allergy but also to a condition termed nonceliac gluten sensitivity (NCGS). Until now this condition has been diagnosed clinically on the basis of exclusion criteria and clinical response to gluten withdrawal. In addition, recent research in this field has shown that other food components distinct from gluten are implicated in NCGS cases, thereby changing our general understanding of NCGS diagnosis in either individuals on gluten containing diets or those already following a gluten-free diet with no proper diagnostic work-up of celiac disease. With this in mind, the assessment of NCGS will require extensive knowledge of celiac disease manifestations and the laboratory tests commonly performed during diagnosis of celiac disease.

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.