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

## TL;DR

A new non-invasive test detects gliadin-reactive CD4+ T cells in blood to help diagnose celiac disease and monitor treatment.

## Contribution

A novel assay, G.A.T.CD4, detects gliadin-activated CD4+ T cells in peripheral blood for celiac disease diagnosis.

## Key findings

- Gliadin-specific CD4+ T cells with OX40 and 4-1BB markers are more frequent in untreated celiac patients.
- The frequency of these cells correlates with anti-tTG2 antibody levels in celiac disease.
- G.A.T.CD4 can distinguish treated patients from untreated ones by detecting pathogenic T cells in blood.

## Abstract

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 (Gsliadin‐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.