Date Published: February 6, 2017
Publisher: Public Library of Science
Author(s): Janni V. Steenholt, Christian Nielsen, Leen Baudewijn, Anne Staal, Karina S. Rasmussen, Hardee J. Sabir, Torben Barington, Steffen Husby, Henrik Toft-Hansen, Jose C. Crispin.
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, CD8αβ, and the possibly immunoregulatory CD8αα homodimer molecule. Besides TCRαβ+ 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 TCRαβ, TCRγδ, CD4, CD8α and CD8β. Proportions of γδ T cells and CD8αβ+ 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 αβ 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.
Celiac disease (CD) is an immune-mediated disease that can develop in genetically predisposed individuals following ingestion of gluten . Gluten-dependent small intestinal epithelial damage as well as presence of CD-specific antibodies in serum characterize the disorder . The severity of epithelial affection may be graded in accordance with the Marsh classification , which in Oberhüber’s modification  ranges from grade 1 to 3(a-c) based on the level of intraepithelial lymphocytosis, crypt hyperplasia and villous atrophy. CD is estimated to affect about 1% of the population in western countries and appears to increase in prevalence [5–7]. About 95% percent of CD patients have the class II human leukocyte antigen (HLA)-DQ2 . Most of the remaining patients have either HLA-DQ8 or the α or β-subunit of the DQ2 molecule [9, 10]. These antigen-presenting molecules have high affinity for deamidated gluten peptides (DGP). The deamidation is caused by the enzyme tissue transglutaminase (tTG), which converts neutral glutamines into negatively charged glutamic acids . Cells with ability to present antigen on HLA class II molecules such as dendritic cells, macrophages, and possibly B cells, present DGP to CD4+ T cells in the lamina propria (LP), activating them and causing an inflammatory response to gluten, and eventually also leading to a destruction of epithelial cells by cytotoxic T cells [8, 12].
In the first round of experiments, we quantified lymphocyte populations in blood samples and duodenal biopsies from patients with CD (previously un-diagnosed) and compared to biopsies from non-CD subjects. We found that the number of B cells and the number of DN T cells were significantly higher (6–7 fold) in patients with CD. In a second round of experiments, we analyzed IELs and LPLs separately using an extended panel of phenotypic T cell markers. As expected, the previously observed increase in number of DN T cells reflected an increase in γδ T cells. We further showed that IEL T cell subtypes were altered in biopsies from CD patients, and that potential CD patients showed a pattern more similar to CD than to non-CD controls. Finally, both αβ T cells and γδ T cells in gluten-reactive TCLs were able to proliferate in response to gluten.