Isolation and characterization of circulating tissue transglutaminase-specific T cells in coeliac disease.
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.
Ciccocioppo
R
R
Center for the Study and Cure of Coeliac Disease, IRCCS Policlinico San Matteo Foundation, University of Pavia, Italy. rachele.ciccocioppo@unipv.it
Finamore
A
A
Mengheri
E
E
Millimaggi
D
D
Esslinger
B
B
Dieterich
W
W
Papola
F
F
Colangeli
S
S
Tombolino
V
V
Schuppan
D
D
Corazza
G R
GR
D000328:Adult; D002446:Celiac Disease / Q000209:etiology / Q000276:immunology*; D002469:Cell Separation; D005260:Female; D019204:GTP-Binding Proteins / Q000276:immunology*; D006683:HLA-DQ Antigens / Q000235:genetics; D006801:Humans; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000502:physiology; D008213:Lymphocyte Activation; D008297:Male; D008875:Middle Aged; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D013601:T-Lymphocytes / Q000276:immunology*; D011503:Transglutaminases / Q000276:immunology*
D006683:HLA-DQ Antigens; C053186:HLA-DQ2 antigen; D007371:Interferon-gamma; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins