Treatment of both native and deamidated gluten peptides with an endo-peptidase from Aspergillus niger prevents stimulation of gut-derived gluten-reactive T cells from either children or adults with celiac disease.

Celiac disease (CD) is characterized by an inappropriate immunological reaction against gluten driven by gluten-specific CD4+ T cells. We screened 25 proteases and tested 10 for their potential to degrade gluten in vitro. Five proteases were further tested for their ability to prevent the proliferative response by a gluten-specific CD4+ T cell clone and seven gluten-reactive T cell lines to protease-digested gluten peptides. A proline-specific endo-peptidase from Aspergillus niger (AnP2) was particularly efficient at diminishing proliferation after stimulation with cleaved antigen, and could completely block the response against both native and deamidated gluten peptides. We found that AnP2 was efficient down to a 1:64 protease:substrate ratio (w:w). When AnP2 was tested in assays using seven gluten-reactive T cell lines from individual CD patients (three adults and four children), the response to gluten was diminished in all cases. Our study indicates a therapeutic benefit of AnP2 to CD patients.

The composition of T cell subtypes in duodenal biopsies are altered in coeliac disease patients.

One of the hallmarks of Celiac disease (CD) is intraepithelial lymphocytosis in the small intestine. Until now, investigations to characterize the T cell subpopulations within the epithelial layer have not discriminated between the heterodimeric co-receptor molecule, CD8αß, and the possibly immunoregulatory CD8αα homodimer molecule. Besides TCRαß+ CD4+ cells, no other phenotypes have been shown to be gluten-reactive. Using flow cytometry on lymphocytes from duodenal biopsies, we determined that the number of B cells (CD3- CD19+) and the number of CD3+ CD4- CD8- double-negative (DN) T cells were elevated 6-7 fold in children with CD. We next isolated and quantified intraepithelial lymphocytes (IELs) from biopsies obtained from patients (both children and adults) with CD, potential CD and non-CD controls. Flow cytometric analysis of the duodenal T cell subpopulations was performed including the markers TCRαß, TCRγδ, CD4, CD8α and CD8ß. Proportions of γδ T cells and CD8αß+ cells among IELs were increased in CD patients, whereas proportions of CD4+ CD8αα+ and CD4+ single-positive T cells were decreased. Additionally, two gluten-reactive T cell lines (TCLs) derived from CD biopsies were analyzed for changes in proportions of T cell subsets before and after gluten stimulation. In a proliferation assay, dividing cells were tracked with carboxyfluorescein succinimidyl ester (CFSE), and both αß and γδ T cells proliferated in response to gluten. Changes in duodenal T cell subpopulations in potential CD patients followed the same pattern as for CD patients, but with less pronounced effect.