Project Summary/Abstract Celiac disease is a chronic immune disorder driven by gluten that typically develops in childhood. Over 1% of the world population is affected, many of whom are undiagnosed. Many more have an uncertain diagnosis because current diagnostic tests are not reliable in individuals following a gluten-free diet. Therefore, there is a strong need for better diagnostic methods that reflect the underlying causes of celiac disease and overcome the limitations of existing tests. Previous studies have shown that an increase in a cytokine called interleukin 2 (IL- 2) is the earliest and most sensitive marker of acute gluten ingestion in adults with celiac disease who are on a gluten-free diet. This method is not practical for diagnosis because patients who are on a gluten-free diet do not want to suffer symptoms of gluten ingestion and it is impractical for patients who are eating gluten to go on a gluten-free diet treatment just to confirm the diagnosis. An alternative to challenging patients with gluten is to challenge the immune cells in blood with gluten. In over 300 adults with celiac disease on a gluten-free diet, an ex vivo whole blood gluten challenge assay in which IL-2 is measured after blood has been incubated with gluten had near perfect specificity and high sensitivity for adults with celiac disease treated with a gluten-free diet. There is a need to conduct studies in children because celiac disease typically develops in this age group. As well, there may be age-related differences in immune responses. Therefore, the main goal of this research grant is to further investigate the immune response to gluten in children with celiac disease using the ex vivo gluten challenge whole blood assay. A cohort of 80 children with celiac disease (40 treated and 40 untreated) will be recruited. In Aim 1, the sensitivity of IL-2 rise in the ex vivo gluten challenge whole blood assay will be determined in the overall cohort. In a pre-planned secondary analysis, sensitivity in treated and untreated celiac disease will be compared. In Aim 2, an immune response signature of ex vivo gluten challenge will be determined using a panel of cytokines known to be elevated following oral gluten challenge (CCL20, CXCL9, IFN-γ, IL-2, IL-8, IL-10, IL-17A, IL-22, IP-10, and TNF-α). The sensitivity of each cytokine for detecting celiac disease will be determined as well as whether using a combination of cytokines improves the accuracy of the test. This novel study will determine whether IL-2 release in ex vivo gluten challenge whole blood assay is a sensitive marker of celiac disease in children. Critically, it will also be determined whether ex vivo gluten challenge evokes an immune response in children with untreated celiac disease. Results of this study will guide protocol development for a multi-center clinical trial to determine the diagnostic accuracy of cytokine release following gluten challenge for diagnosis of celiac disease. Results of this tri...