To explore the role of IL4+ and TCRγδ+ cells as markers of mucosa damage in celiac disease

Background

Celiac disease (CD) is characterized by a large spectrum of intestinal inflammatory lesions and clinical manifestations. The overt-CD presents a massive lymphocyte infiltration, in particular of CD8+ and TCRγδ+ T cells, high production of inflammatory cytokines, as IFNγ and IL21, and atrophy of small intestinal villi. By contrast, the potential-CD condition presents a morphologically normal mucosa and mild signs of tissue inflammation. A recent study reported an expansion of IL4+ T cells that inversely correlated with the infiltration of TCRγδ+ T cells in potential-CD compared to overt-CD, suggesting the involvement of these two cell subsets in the evolution of the mucosal damage from potential- to overt-CD.

Hypothesis, Rationale and Aims

Subjects with potential-CD and on gluten diet are at high risk to develop the overt-CD, most likely due to the disruption of a balance between inflammatory and regulatory pathways that counteract the harmful process induced by gluten. However, to date, biomarkers specific for the different celiac mucosa lesions are still lacking. It has been reported that TCRγδ+ and IL4+ T cells are differentially present in the intestinal tissue of overt- and potential-CD patients. Preliminary observations demonstrated that the mucosal densities of CD4+ T cells producing IL4 inversely correlate with the frequency of TCRγδ+ cells and with CD antibody titres, in all forms of disease. In order to dissect the mechanisms behind the change from mild to acute mucosa lesions, this project aims to investigate the role of TCRγδ+ and IL4+ T cell subsets in both potential- and overt-CD conditions, by analysing their function, antigen specificity, phenotypic profile and cytokine production patterns. The immunological changes of these two intestinal cell subsets observed in mucosa biopsies will be correlated with the score of intestinal mucosa lesions, according to the current histological classifications.

Research Plan (Experimental design and methodologies)

The IL4+ and TCRγδ+ T cells will be evaluated in intestinal mucosa of paediatric patients with potential or overt-CD. At least 20 subjects for each group will be enrolled. The phenotype and cytokine productions of mucosa lymphocytes will be analysed both ex-vivo, in fresh biopsies, and in short-term T cell lines by flow cytometry. IL4+ and TCRγδ+ cells will be also isolated by fluorescence-activated cell sorting for an extensive phenotype and functional analysis, and to perform coculture experiments with gluten-reactive Th1 cells. Bioactivity will be evaluated by ELISA/ELISpot and proliferation assay by flow cytometry. (Task 1) The serum level of IL4 will be measured in all enrolled subjects by ELISA. (Task 2) In order to dissect the immunomodulatory role of IL4 on reactivity to gluten, st-TCLs will be generated in presence of gliadin with or without exogenous IL4. Changes in cell density, phenotype and function of both TCRαβ+ and TCRγδ+ T cell subsets will be monitored in short-term cultures. (Task 3) The findings of IL4+ and TCRγδ+ T cells will be correlated with the disease scores evaluated by biopsy histological classification and serum levels of CD-associated antibodies. (Task 4)

Expected results and Impact

This study could lead to the identification of specific cellular markers of mucosa damage that can help pathologists in the diagnosis of doubted cases of celiac disease. If it will be confirmed that the TCRγδ+ and IL4+ T cells play a relevant role in the transition of CD lesion from potential- to overt-CD form, the analysis of these two cell subsets will provide additional tool to determine the histological score of mucosa lesions. Thus, the combined detection of TCRγδ+ and IL4+ T cells in gut biopsy could represent a useful approach not only to better characterize the different CD forms, but also to predict the disease progression, and to support clinicians in the managing of potential-CD patients.