Assessment of activated gut-homing CD8⁺ T cells in blood by flow cytometry during a 3-day gluten challenge

Accurate celiac disease (CD) diagnosis must be performed in individuals following a gluten containing diet. Diagnostic procedures for individuals already on a gluten-free diet (GFD) avoiding long gluten reintroductions are still challenging. To deal with this issue, we developed an accurate but simple method that requires only a 3-day gluten challenge and circumvents the main limitations of previously suggested proposals such as requirement of specific peptides and unusual specialized lab facilities or high cost. In an attempt to standardize this methodology to be used in daily clinical practice, we describe here an optimized protocol for assessing activated gut-homing CD8⁺ T cells in blood combined with a short gluten challenge. Details about the amount and type of gluten antigen and the starting material are included, as well as the strategy to easily characterize and identify the cells of interest using flow cytometry. This methodology constitutes a diagnostic tool for CD diagnosis of high specificity and sensitivity for seropositive disease (>95%) as an alternative to long-term gluten challenge and open new possibilities to test the response to gluten in research and clinical trials.