P1197 IMMUNE RESPONSES IN VITRO TO THE HLA CLASS I-RESTRICTED A-GLIADIN PEPTIDE 123???132 (PEP A2) IN CULTURES OF INTESTINAL MUCOSA FROM COELIAC DISEASE PATIENTS ON GLUTEN-FREE DIET:

Introduction: Coeliac disease (CD) is associated with HLA class II-restricted CD4+ T mediated gliadin-specific responses, albeit a massive infiltration by CD8+ T cells represents the disease hallmark. A 10mer gliadin peptide (A-gliadin 123–132) is recognized by class I-restricted CD8+ T lymphocytes from both peripheral blood and intestinal mucosa of HLA-A2+ CD patients (1). We have tested in the organ culture of treated coeliac mucosa the capacity of A-gliadin 123–132 peptide to activate the local immune response. Methods: Jejunal biopsies were obtained from 11 treated coeliac patients (6 HLA-A2 positive and 5 HLA-A2 negative), and in vitro cultured for 24 hours with A-gliadin 123–132 QLIPCMD-VVL (pep A2). A peptic-tryptic digest of gliadin (PT-gliadin), or the immunodominant, A2-restricted, HIVgag 17 peptide SLYNTVATL (pep C), were used as controls. Immunohistochemical analysis was carried out assessing lamina propria CD25+ cells, IEL CD3+ cells density and FAS epithelial expression. Percentage of CD8/CD25, CD8/CD69, CD8/FAS-L double positive cells were evaluated by FACS analysis on cells isolated from two different CD biopsies cultured with medium or pep A2. Results: In HLA A2+ coeliacs lamina propria CD25+ cells in biopsies cultured in the presence of pep A2 (median, range: 95, 38–107) or PT (122, 58–213), were significantly higher (p<0.05) than in biopsies cultured with medium alone (27, 18–48) or pep C (27, 15–39). Similarly, the density of IEL CD3+/mm epithelium was higher for pep A2 and PT (42, 27–48; and 50, 39–71 vs medium: 28, 18–55 or pep C: 26, 23–28). Increased FAS epithelial expression was also noted. No differences were observed with pep A2 compared to medium in cultures of HLA A2- biopsies. An enhanced frequency of CD8/CD25 and CD8/FAS-L double positive cells was observed in cell preparations from pep A2-cultured biopsies compared to medium-cultured biopsies. Conclusion: These data show that pep A2 is able to activate a peptide specific-immune response in HLA-A2+ coeliac patients and suggest the involvement of mucosa infiltrating CD8 T lymphocytes in CD pathogenesis. This work underlines also the importance of in vitro organ culture as a valuable tool to test the toxicity of gliadin peptides for coeliac patients.