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Generation and Characterization of Insulin Peptide-specific Regulatory T Cells
S18
Abstracts
F.6 Plasmacytoid Dendritic Cells Induction by a Self-peptide Ep1.B Derived from Apolipoprotein E Prevent Autoimmune Diabetes
Regulation Lab DI-3, La Jolla Institute for Allergy and Immunology, La Jolla, CA
Enayat Nikoopour, Postdoctoral Fellow, Department of Microbiology and Immunology and Robarts Research Institute, London, ON, Canada, Tracey A. Stephens, Graduate Student, Department of Microbiology and Immunology and Robarts Research Institute, London, ON, Canada, Beverley J. Rider, Graduate Student, Department of Microbiology and Immunology and Robarts Research Institute, London, ON, Canada, Edwin Lee-Chan, Lab Technician/Lab Manager, Department of Microbiology and Immunology and Robarts Research Institute, London, ON, Canada, Bhagirath Singh, Professor, Department of Microbiology and Immunology and Robarts Research Institute, London, ON, Canada
Our goal is to generate insulin peptide-specific regulatory T cells (Tregs) in vitro, which suppress overt diabetes in prediabetic and reverse already ongoing disease in diabetic mice. Furthermore we aim to analyze the suppressive properties and mechanisms of these Tregs in vitro and in vivo. In order to generate insulin peptide-specific Tregs, we took advantage of insTCR transgenic mice, which express T cell receptor (TCR) transgenic CD4+ T cells specific for the insulinB9–23 peptide (insB) presented on MHC class II H-2IAg7/d molecules. The in vitro generation of insBspecific Tregs involved purification of either CD4+CD25+ or CD4+CD25− insTCR T cells, which were cultured for 1– 2 weeks with the insB peptide, syngeneic antigen presenting cells and high doses of IL-2 yielding 25+ and 25− cultures, respectively. Using the classical in vitro suppression assay, only cells derived from the 25+ cultures were able to suppress while cells from the 25− cultures enhanced proliferation and cytokine secretion of CD8+ effector T cells. In vivo, however, cells from both cultures were unable to suppress lymphocytic choriomeningitis virus-induced diabetes. Interestingly, freshly isolated as well as IL-10cultured CD4+CD25− but not freshly isolated CD4+CD25+ insTCR T cells suppressed spontaneous diabetes in NOD females. We are currently investigating the mechanisms underlying the in vivo suppressive potential of these CD4 +CD25− T cells.
Dendritic cells (DC) are potent inducers of T cell tolerance. The mechanism by which this is achieved is not well understood. We postulated that in vivo induction of plasmacytoid dendritic cells (PDC) could prevent autoimmunity through induction of T cell tolerance. We report that a novel self-peptide Ep1.B of mouse Apolipoprotein E (ApoE) induced differentiation of bone marrow derived monocytes (BMM) into PDC. In vitro Ep1.B induced PDCs are B220+, Ly6C+, CD11c+, mPDCA+, CD62L+ and CD8a+. Footpad injection of Ep1.B in diabetes prone NOD mice increased the level of CD11c+, mPDCA and B220+ cells in the draining lymph nodes (LN) and these CD11c+ cells have an increased expression of tolerogenic programmed death ligand (PD-L1). Since PD-1PD-L1 pathway has been shown to be important in maintaining tolerance, PDC induced by Ep1.B may inhibit the effector T cells in disease process. In addition, we found increased number of CD4+CD25+ T cells with elevated glucocorticoidinduced tumor necrosis factor receptor related protein (GITR) in the LN of these animals. We found that injection of Ep1.B in young NOD mice significantly prevented type 1 diabetes development in these mice. In summary, we suggest that in vivo Ep1.B induced differentiation of BMM into PDC that in turn prevented autoimmunity and induced regulatory T cells in type 1 diabetes prone NOD mice.
doi:10.1016/j.clim.2007.03.219
F.7 Generation and Characterization of Insulin Peptide-specific Regulatory T Cells Marianne Martinic, Postdoctoral Fellow, Immune Regulation Lab DI-3, La Jolla Institute for Allergy and Immunology, La Jolla, CA, Lisa Togher, Technician, Immune Regulation Lab DI-3, La Jolla Institute for Allergy and Immunology, La Jolla, CA, Christophe Filippi, Postdoctoral Fellow, Immune Regulation Lab DI-3, La Jolla Institute for Allergy and Immunology, La Jolla, CA, Jean Jasinski, PhD Student, Barbara Davis Center for Childhood Diabetes, Aurora, CO, Damien Bresson, Postdoctoral Fellow, Immune Regulation Lab DI-3, La Jolla Institute for Allergy and Immunology, La Jolla, CA, George Eisenbarth, Executive Director, Barbara Davis Center for Childhood Diabetes, Aurora, CO, Matthias von Herrath, Division Head, Immune
doi:10.1016/j.clim.2007.03.220
F.8 The Insulitis Reporter Mouse Jennifer Ondr, Postdoctoral Fellow, Cincinnati Children’s Hospital Medical Center, Division of Endocrinology, Diabetes Research Center, Cincinnati, OH, Robert Opoka, Research Assistant, Cincinnati Children’s Hospital Medical Center, Division of Endocrinology, Diabetes Research Center, Cincinnati, OH, Sankarannand Vukkadapu, Postdoctoral Fellow, Cincinnati Children’s Research Foundation, Cincinnati, OH, Jonathan Katz, Associate Professor, Cincinnati Children’s Hospital Medical Center, Division of Endocrinology, Diabetes Research Center, Cincinnati, OH Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of insulin producing beta cells by leukocytes that infiltrate the pancreas in a prolonged and clinically silent process termed insulitis. Inability to detect insulitis prior to the onset of overt disease symptoms stymies progress in T1DM research and treatment. In humans, detection of antibody to islet cell antigens is the only means to diagnose pre-diabetic insulitis, however, no relationship between sero-conversion and insulitis progression is established. In NOD mice, insulitis can be measured, but only as an end-stage pancreatectomy. An early, accurate diagnosis of insulitis would allow the possibility of therapeutic intervention, thus there remains an urgent need for non-invasive and real-time measures of insulitis. To this end, we are generating a gene-targeted NOD mouse in which production of a
Abstracts
F.6 Plasmacytoid Dendritic Cells Induction by a Self-peptide Ep1.B Derived from Apolipoprotein E Prevent Autoimmune Diabetes
Regulation Lab DI-3, La Jolla Institute for Allergy and Immunology, La Jolla, CA
Enayat Nikoopour, Postdoctoral Fellow, Department of Microbiology and Immunology and Robarts Research Institute, London, ON, Canada, Tracey A. Stephens, Graduate Student, Department of Microbiology and Immunology and Robarts Research Institute, London, ON, Canada, Beverley J. Rider, Graduate Student, Department of Microbiology and Immunology and Robarts Research Institute, London, ON, Canada, Edwin Lee-Chan, Lab Technician/Lab Manager, Department of Microbiology and Immunology and Robarts Research Institute, London, ON, Canada, Bhagirath Singh, Professor, Department of Microbiology and Immunology and Robarts Research Institute, London, ON, Canada
Our goal is to generate insulin peptide-specific regulatory T cells (Tregs) in vitro, which suppress overt diabetes in prediabetic and reverse already ongoing disease in diabetic mice. Furthermore we aim to analyze the suppressive properties and mechanisms of these Tregs in vitro and in vivo. In order to generate insulin peptide-specific Tregs, we took advantage of insTCR transgenic mice, which express T cell receptor (TCR) transgenic CD4+ T cells specific for the insulinB9–23 peptide (insB) presented on MHC class II H-2IAg7/d molecules. The in vitro generation of insBspecific Tregs involved purification of either CD4+CD25+ or CD4+CD25− insTCR T cells, which were cultured for 1– 2 weeks with the insB peptide, syngeneic antigen presenting cells and high doses of IL-2 yielding 25+ and 25− cultures, respectively. Using the classical in vitro suppression assay, only cells derived from the 25+ cultures were able to suppress while cells from the 25− cultures enhanced proliferation and cytokine secretion of CD8+ effector T cells. In vivo, however, cells from both cultures were unable to suppress lymphocytic choriomeningitis virus-induced diabetes. Interestingly, freshly isolated as well as IL-10cultured CD4+CD25− but not freshly isolated CD4+CD25+ insTCR T cells suppressed spontaneous diabetes in NOD females. We are currently investigating the mechanisms underlying the in vivo suppressive potential of these CD4 +CD25− T cells.
Dendritic cells (DC) are potent inducers of T cell tolerance. The mechanism by which this is achieved is not well understood. We postulated that in vivo induction of plasmacytoid dendritic cells (PDC) could prevent autoimmunity through induction of T cell tolerance. We report that a novel self-peptide Ep1.B of mouse Apolipoprotein E (ApoE) induced differentiation of bone marrow derived monocytes (BMM) into PDC. In vitro Ep1.B induced PDCs are B220+, Ly6C+, CD11c+, mPDCA+, CD62L+ and CD8a+. Footpad injection of Ep1.B in diabetes prone NOD mice increased the level of CD11c+, mPDCA and B220+ cells in the draining lymph nodes (LN) and these CD11c+ cells have an increased expression of tolerogenic programmed death ligand (PD-L1). Since PD-1PD-L1 pathway has been shown to be important in maintaining tolerance, PDC induced by Ep1.B may inhibit the effector T cells in disease process. In addition, we found increased number of CD4+CD25+ T cells with elevated glucocorticoidinduced tumor necrosis factor receptor related protein (GITR) in the LN of these animals. We found that injection of Ep1.B in young NOD mice significantly prevented type 1 diabetes development in these mice. In summary, we suggest that in vivo Ep1.B induced differentiation of BMM into PDC that in turn prevented autoimmunity and induced regulatory T cells in type 1 diabetes prone NOD mice.
doi:10.1016/j.clim.2007.03.219
F.7 Generation and Characterization of Insulin Peptide-specific Regulatory T Cells Marianne Martinic, Postdoctoral Fellow, Immune Regulation Lab DI-3, La Jolla Institute for Allergy and Immunology, La Jolla, CA, Lisa Togher, Technician, Immune Regulation Lab DI-3, La Jolla Institute for Allergy and Immunology, La Jolla, CA, Christophe Filippi, Postdoctoral Fellow, Immune Regulation Lab DI-3, La Jolla Institute for Allergy and Immunology, La Jolla, CA, Jean Jasinski, PhD Student, Barbara Davis Center for Childhood Diabetes, Aurora, CO, Damien Bresson, Postdoctoral Fellow, Immune Regulation Lab DI-3, La Jolla Institute for Allergy and Immunology, La Jolla, CA, George Eisenbarth, Executive Director, Barbara Davis Center for Childhood Diabetes, Aurora, CO, Matthias von Herrath, Division Head, Immune
doi:10.1016/j.clim.2007.03.220
F.8 The Insulitis Reporter Mouse Jennifer Ondr, Postdoctoral Fellow, Cincinnati Children’s Hospital Medical Center, Division of Endocrinology, Diabetes Research Center, Cincinnati, OH, Robert Opoka, Research Assistant, Cincinnati Children’s Hospital Medical Center, Division of Endocrinology, Diabetes Research Center, Cincinnati, OH, Sankarannand Vukkadapu, Postdoctoral Fellow, Cincinnati Children’s Research Foundation, Cincinnati, OH, Jonathan Katz, Associate Professor, Cincinnati Children’s Hospital Medical Center, Division of Endocrinology, Diabetes Research Center, Cincinnati, OH Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of insulin producing beta cells by leukocytes that infiltrate the pancreas in a prolonged and clinically silent process termed insulitis. Inability to detect insulitis prior to the onset of overt disease symptoms stymies progress in T1DM research and treatment. In humans, detection of antibody to islet cell antigens is the only means to diagnose pre-diabetic insulitis, however, no relationship between sero-conversion and insulitis progression is established. In NOD mice, insulitis can be measured, but only as an end-stage pancreatectomy. An early, accurate diagnosis of insulitis would allow the possibility of therapeutic intervention, thus there remains an urgent need for non-invasive and real-time measures of insulitis. To this end, we are generating a gene-targeted NOD mouse in which production of a