Isolation and Characterization of Circulating Tissue Transglutaminase-Specific T Cells in Coeliac Disease

Abstract

Tissue transglutaminase (TG2) was identified as the humoral autoantigen in coeliac disease, but whether it can also serve as T cell autoantigen is still unknown. We aimed, therefore, to firstly explore the presence of TG2-specific T cells in peripheral blood of ten adult patients (four active, i.e. carrying both serological and histological features of the disease; four treated, i.e. with proven mucosal recovery and disappearance of specific antibodies after an adequate period of gluten free diet; and two potential coeliacs, i.e. carrying the serological stigmata of the disease, but not the intestinal lesions), and four healthy controls (two carrying the HLA-DQ2 haplotype of susceptibility to the disease), and secondly to carry out a detailed in vitro characterization of the isolated antigen-specific T cells. T cell lines were first established by means of weekly stimulation with human recombinant TG2 followed by generation of T cell clones through distribution of T cells on plates at one cell/well limiting dilution and further rounds of stimulation. Antigen specificity and HLA-DQ2 restriction were both assessed by evaluating the proliferative response to TG2 in the absence and presence of human sera blocking HLA-DQ2 molecules, after exclusion of impurities in the antigen preparation. Immune phenotyping of T cell clones was performed by flow cytometry, and the expression of IL-1â, IL-4, IL-6, IL-10, IL-12, TGF-beta, IFN-gamma and TNF-alpha was determined by ELISA assay on the supernatants of these clones. A total of 91 T cell clones were isolated from the three HLA-DQ2-positive, active patients, but none from the other patients and controls. The immune phenotyping showed that the majority of them (85.7 percent) were CD3/CD4+ and only a small percentage (14.3 percent) were CD3/CD8+, all carried the TCR alphabeta, and had a memory phenotype. The cytokine profile showed high levels of IFN-gamma and IL-6 that, together with the absence of IL-4, placed these T cell clones in the T helper type 1-like category. Further in vitro analysis was carried out on 32/91 CD4+ clones and showed a specific and dose-dependent proliferative response towards TG2 and an HLA-DQ2 restriction. Finally, when incubating duodenal mucosal specimens of treated patients with the supernatant of TG2-specific T cell clones, characteristic disease lesions were found, indicating a role for TG2-specific cellular immune response in the pathogenesis of coeliac disease.

Characterization of Gliadin-Specific Th17 Cells from the Mucosa of Celiac Disease Patients

“…Th is fi nding is fully consistent with previous data showing no detection of transglutaminase-reactive cells within the CD mucosa ( 31 ), although a recent study has found that transglutaminase-specifi c Th 1 cell clones derived from peripheral blood of some, but not all, active CD patients induce ex vivo typical lesion changes in the CD intestinal mucosa. Th e detection of these reactive cells was infl uenced by the clinical situation of patients and also required 8 -10 weeks of repeated stimulation, thus indicating that even a small percentage of transglutaminase-reactive cells that were undetectable in our short-term cultures are nevertheless enough to contribute to intestinal damage ( 32 ).…”

T Cells in Celiac Disease

“…Although some reports have indicated that TG2- reactive CD4 T cells do exist (49, 50), experiments from one of the author’s lab have failed to identify TG2-reactive T cells in T-cell lines made from intestinal biopsies (Ráki & Sollid, unpublished observations). Important theoretical considerations further speak against the existence of autoreactive TG2-specific T cells.…”

The Role of T-Cell Subsets in Chronic Inflammation in Celiac Disease and Inflammatory Bowel Disease Patients: More Common Mechanisms or More Differences?

“…All T-cell subsets involved in celiac disease seem to be antigen-specific: gliadin-specific CD4 + T cells [18], gliadin-specific Th17 cells [62], tissue transglutaminase-specific T cells [66,67], antigen-specific IEL [68]. Gliadin might be recognized by cytotoxic CD8 + T cells due to epitope spreading [69].…”