Isolation and characterization of circulating tissue transglutaminase- specific T cells in coeliac disease

Rachele Ciccocioppo, A. Finamore, E. Mengheri, D. Millimaggi, B. Esslinger, W. Dieterich, F. Papola, S. Colangeli, V. Tombolino, D. Schuppan, G. R. Corazza

Research output: Contribution to journalArticlepeer-review

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-1β, IL-4, IL-6, IL-10, IL-12, TGF-β, IFN-γ and TNF-α 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%) were CD3/CD4+ and only a small percentage (14.3%) were CD3/CD8+, all carried the TCR αβ, and had a memory phenotype. The cytokine profile showed high levels of IFN-γ 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.

Original languageEnglish
Pages (from-to)179-191
Number of pages13
JournalInternational Journal of Immunopathology and Pharmacology
Volume23
Issue number1
Publication statusPublished - Jan 2010

Keywords

  • Coeliac disease
  • Cytokines
  • T cells
  • Tissue transglutaminase

ASJC Scopus subject areas

  • Pharmacology
  • Immunology
  • Immunology and Allergy

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