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Mechanisms that underlie loss of antigen tolerance in HLA-associated autoimmune diseases

Final Report Summary - IMMUNE TOLERANCE (Mechanisms that underlie loss of antigen tolerance in HLA-associated autoimmune diseases)

Our project had the following aims:

Aim #1: Identification of the functional roles of HLA-DR*0405 (class II)-restricted CD4+ T cells in disease induction/prevention in a novel humanized mouse model of autoimmune pancreatitis (AIP). Work on this subproject started in 2009, with crossbreedings of mouse strains. Lymphocyte-deficient HLA-DR*0405/-DQ8 transgenic Rag1-/- Ab0 NOD mice served as recipients in lymphocyte adoptive transfer experiments from immunocompetent HLA-DR*0405/huCD4 Ab0 NOD donor mice. In a series of experiments, by transferring either CD4+ T cells, CD8+ T cells or CD19+ B cells, we identified the pathogenic lymphocyte population causing AIP in recipient mice (unpublished results). Mice in the affected group lost body weight, and had reduced pancreas weights at necropsy, consistent with the development of pancreatic atrophy. This was confirmed by histological analyses.

Having reached this project milestone, we can now proceed towards an investigation of the function of HLA-DR*0405-restricted regulatory T (Treg) cells in vivo. Co-transfer experiments may allow a direct comparison between the regulatory function of HLA-DR*0405-restricted Treg cells (vs. HLA-DQ8-restricted Treg control) in preventing the development of AIP in recipient mice. Based on the results obtained in our project, we expect that HLA-DR*0405-restricted Treg cells fail to protect from AIP in recipient mice. This would suggest that AIP develops in mice due to a functional defect of the HLA-DR*0405 allele in inducing tissue-specific Treg. This mechanism could also explain why autoimmune diseases are frequently associated with specific HLA class II alleles.

Aim #2: Identification of humoral autoantigens in HLA-DR*0405 transgenic mice that can aid in the diagnosis of human AIP. Due to a lack of co-funding over the duration of the project, unfortunately this part of the project could not be addressed.

Aim #3: Enhancement of a novel mouse model of human celiac disease (CD) by introduction of the disease associated HLA-DQ8 (class II) allele. Work on this subproject started in 2010. We performed adoptive transfers of gliadin-primed memory T cells into HLA-DQ8 transgenic Rag1-/- Abo NOD recipient mice, following a published protocol for the induction of gluten-sensitive enteropathy. Surprisingly, recipient mice did not show any signs of intestinal inflammation, neither in the small nor the large bowel. Spleen sizes remained small, suggesting a failure of T cell reconstitution in lymphopenic recipients after adoptive transfers, possibly a result of the use of mice on the NOD genetic background (vs. C57BL/6). Since it was not feasible to obtain higher numbers of Treg-depleted memory T cells for adoptive transfers, the aim of humanizing the celiac disease model by use of HLA-DQ8/huCD4 transgenic Ab0 NOD mice had to be abandoned. Instead, we are now using HLA-DR3/-DQ2/huCD4 transgenic, mouse MHC class II deficient C57BL/6 mice for the same purpose. The fact that this HLA-DQ2 transgenic mouse strain is on the same genetic background as the established celiac model (C57BL/6) will increase the chances to obtain a relevant humanized celiac model.

Aim #4: Pre-clinical testing of oral enzyme therapy of CD in mice. Work on this project started in 2013. Groups of lymphocyte-deficient mice received adoptive transfers of gliadin-primed CD4+ memory T cells, and were treated with oral gluten (positive control), oral gluten + 2-enzyme glutenase, or gfd (negative control). The glutenase consisted of a combination aspergillopepsin I and a prolyl-endopeptidase. Glutenase and/or gluten were mixed into gluten-free diet, and fed continuously over 8 weeks. The results of this study are currently being analyzed. They will determine whether a therapeutic effect of the 2-enzyme glutenase can be demonstrated in celiac mice.

(submitted 12/2013)