355 Immune Tolerance Induced By Peptide Immunotherapy in An HLA Dq2-Dependent Mouse Model of Gluten Immunity

Abstract

Patients with Coeliac disease (CD) mount inflammatory T-cell responses to deamidated gluten peptides. Encounter of harmless protein via mucosal surfaces generally induces mucosal tolerance. Using animal models we have shown that oral tolerance to the harmless protein ovalbumin (OVA) depends on differentiation of regulatory T-cells (Treg) in the mucosa draining lymphoid tissue. How this translates to the uptake of gluten is difficult to predict as little is known with respect to localization and presentation of gluten after uptake via the gastrointestinal tract. The aim of this study was to identify the phenotype and localization of the gliadin specific T-cell response In Vivo. Mice transgenically expressing disease predisposing human HLA-DQ2 and human gliadin-specific TCR (CD-TCR) on an MHC-II-/background were used. CD-TCR+CD4+ T-cells from double transgenic mice were isolated and labelled with CFSE. After labelling, the cells were i.v. injected into HLA-DQ2tg x MHC-II-/mice. The acceptor mice received non-deamidated or deamidated chymotrypsin digested gliadin by gavage or deamidated gliadin in the thigh muscle (i.m.). After 72h the response of the gliadin-specific T-cells was analyzed in the gut-draining mesenteric lymph node (MLN), spleen and peripheral lymph nodes (PLN) and compared to the known tolerogenic T-cell response to OVA. At 72h after gavage T-cell response to non-deamidated gliadin was very low suggesting that during homeostasis gliadin is not deamidated by murine tissue transglutaminase. Conversely, a vigorous T-cell response was detected in MLN after gavage of deamidated gliadin. Unlike the response to harmless OVA, deamidated gliadin induced more proliferation in spleen than in MLN implying that the adaptive response to gliadin is not confined to the mucosa draining lymphoid tissue. Proliferating gliadin-specific T-cells in MLN contained minimal numbers of Foxp3+ Treg. By contrast, the cells phenotypically resembled activated T-cells and were comparable to differentiating effector T-cells in the PLN of mice that received deamidated gliadin i.m.. Culture of naive CD4+CD-TCR+ T-cells with HLADQ2+ APC in the presence of deamidated gliadin yielded phenotypically identical effector T-cells. Upon addition of factors that mediate mucosal Treg differentiation, Foxp3+CD4+CD-TCR+ T-cells did differentiate, indicating that the cells do have the intrinsic capacity to develop into adaptive mucosal Treg. Dissecting gliadin-specific T-cell responses in this novel murine system provides important opportunities to study the pathogenesis of CD, investigate efficacy of compounds and treatment strategies but also safety of novel dietary products.