Detection of Gliadin-Activated CD4⁺ T Cells Is a New Assay to Reveal Pathogenic Lymphocytes in Celiac Disease

The diagnosis of celiac disease (CD) relies on the presence of serum antibodies against type 2-tissue transglutaminase (tTG2) and endomysium, which in adult patients must be confirmed by assessing small intestinal mucosa damage through esophagogastroduodenoscopy. We aim to establish a non-invasive method focused on detecting activated CD4⁺ T cells that are reactive to gliadin in the peripheral blood of patients with untreated CD. Peripheral blood mononuclear cells from 20 patients with untreated CD, 17 patients on a gluten-free diet and 10 healthy donors were stimulated in vitro with whole gliadin or immunodominant peptides. After 48 h, cells were stained with fluorochrome-conjugated monoclonal antibodies against OX40 and 4-1BB surface activation markers. Gliadin-specific, activated CD4⁺ T cells were detected through multiparametric flow cytometry. OX40 and 4-1BB activation markers are upregulated on CD4⁺ T cells following the engagement of the HLA-DQ2.5-gliadin complex with the T-cell receptor (TCR). The frequency of gliadin-specific, CD3⁺/CD4⁺/OX40⁺/4-1BB⁺ cells is significantly higher in untreated than in treated patients and healthy controls and shows a positive correlation with the anti-tTG2 antibody titers. This assay, defined as the G.A.T.CD4 (Gliadin activated CD4⁺ T cells) method, might support CD diagnosis, particularly in doubtful cases having low autoantibody serum titers. G.A.T.CD4 can be of help in monitoring disease progression, as the pathogenic T cells expressing OX40 and 4-1BB activation markers are undetectable in the blood of treated patients. In the future, we aim to propose the G.A.T.CD4 instead of esophagogastroduodenoscopy to perform accurate and less invasive diagnosis.