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Oxysterols are expressed in T lymphocytes and impair type 1 regulatory CD4 T-cell differentiation
206
Abstracts
b) subsequent isolation of the relevant TCC from the patient’s cerebrospinal fluid (CSF). An actively demyelinating lesion based on reduced density of myelinated fibers and myelin degradation products within infiltrating macrophages was identified. Using deep sequencing approaches, we sequenced the TCR variable beta (V-beta) chains expressed by T cells infiltrating this lesion. We identified 312 different lesion-infiltrating TCC. Both CD4 (32%) and CD8 (68%) TCC were present and clonally expanded with frequencies up to 30%. In a following step, we were able to isolate 14 of the most frequent CD4+ and CD8+ TCC using as a cell source in vitro PHA-expanded CSF T cells. T cells expressing V-beta families of interest were sorted and cloned by limiting dilution. These TCC are currently being characterized for functional phenotype and antigen specificity by using a combination of two complementary unbiased methods, synthetic combinatorial peptide libraries and an autologous brain-derived cDNA library, which is expressed in suitable antigen presenting cells. doi:10.1016/j.jneuroim.2014.08.552
69 Oxysterols are expressed in T lymphocytes and impair type 1 regulatory CD4 T-cell differentiation Fanny Chalmina, Nishta Budoob, Rochemont Vivianeb, Doron Merklerb, Caroline Pota a
Department of Neurology, Geneva University Hospitals, Geneva, Switzerland; bDepartment of Pathology and Immunology, Geneva University, Geneva, Switzerland Oxysterols, oxidised forms of cholesterol, have recently been assigned with novel functions in modulating the immune response. More specifically, the enzyme cholesterol 25 hydroxylase (ch25h), the rate-limiting step to synthetize 25-hydroxycholesterol (25-OHC) from cholesterol, has been proposed to control viral infection and Immunoglobulin secretion. However the function of oxysterols in CD4+ T lymphocytes and their role during autoimmune diseases has not been assessed. The development and progression of multiple sclerosis results in part from the balance between pathogenicity of effector CD4+ T cells and negative regulation imposed by regulatory cells. Since the original classification of CD4+ T lymphocytes into TH1 and TH2 subsets, the repertoire of effector CD4+ T cell subsets has expanded to include additional effector T cell subsets like TH17 cells and regulatory T cell subsets, in particular Foxp3+ regulatory Tcells (Tregs) and IL-10-producing T regulatory type 1 (TR1). Regulatory IL-10-producing-type 1 (Tr1) T cells are instrumental in the prevention of autoimmune diseases and multiple sclerosis and IL-27 is a critical factor for Tr1 cell differentiation. We assessed the expression of Ch25h and 25-OHC in subset of CD4+ T cells differentiated in vitro. We found that Ch25h expression was specifically induced by IL-27 in Tr1 cells. We further demonstrated that 25-OHC prevented TR1 cell development both in vitro and in vivo and dissected the underlying signaling pathways. Together, our findings show that Ch25h and 25-OHC act as negative regulators of TR1 cell both in vitro and in vivo. Not only these findings unravel novel molecular mechanisms accounting for the generation of Tr1 cells, but they also provide oxysterols as critical players to regulate differentiation of Tr1 cells and to inhibit development of autoimmunity.
doi:10.1016/j.jneuroim.2014.08.553
607 Investigating the Orexin/Hypocretin Specific T Cell Response in Patients with Narcolepsy with Cataplexy Melanie Ramberger, Birgit Högl, Thomas Mitterling, Birgit Frauscher, Markus Reindl, Andreas Lutterotti Clinical Department of Neurology, Innsbruck Medical University, Innsbruck, Austria Narcolepsy with cataplexy is caused by a selective loss of orexin/ hypocretin producing neurons in the lateral hypothalamus. A strong genetic association with HLA DQB1*06:02 (95% in patients vs. 12-38% in controls) is well-known and further genes involved in immune modulation such as T cell receptor alpha, OX40L, cathepsin H, DNMT1 and P2RY11 were shown to be associated with narcolepsy in genomewide association studies. Although these associations strongly suggest an involvement of immune cells, the contribution of T cells in pathogenesis is largely unknown. We therefore investigated the orexin specific T cell reactivity in peripheral blood mononuclear cells (PBMC) of narcolepsy patients by a CFSE-based proliferation assay. The study aim was to analyse narcolepsy patients with cataplexy and healthy controls for T cell proliferation in response to stimulation with an orexin peptide library. Orexin specific CD3+CD4+ T cell reactivity was determined by flow cytometry and cytokine (IFN-γ and GM-CSF) analysis. So far, 13 patients and 11 healthy controls have been included. Individuals were considered as responders, if the stimulation index exceeded 3 in either CD3+CD4+ T cell proliferation, IFN-γ or GM-CSF secretion. Using these inclusion criteria, 6 patients (46.2%) and 2 controls (18.2%) showed orexin specific proliferation (p N 0.05). Furthermore, CD4+ reactivity to orexin was observed more frequently in individuals positive for HLA DQB1*06:02 compared to individuals negative for this risk allele (43.8% vs. 12.5%, p N 0.05). Although we observed a higher orexin specific CD4+ T cell proliferation in narcolepsy patients and in HLA DQB1*06:02 positive individuals, these preliminary data indicate no significant difference in CD4+ T cell reactivity to orexin between the groups by using autologous PBMC. In the near future we plan to compare CD4+ T cell responses to orexin using DQB1*06:02 expressing antigen presenting cells. Melanie Ramberger is enrolled in the graduate program SPIN supported by the Austrian Research Foundation (FWF DK W1206). doi:10.1016/j.jneuroim.2014.08.554
115 Central nervous system repair requires both effector and regulatory T cells with distinct temporal and spatial profiles Catarina Raposo, Michal Schwartz Weizmann Institute of Science, Department of Neurobiology, Rehovot, Israel Healing of the central nervous system (CNS) following traumatic injury was considered for decades to be a tissue autonomous process independent of outside intervention. It is now evident that CNS healing shares many of the properties and requirements of its peripheral counterparts. Monocyte-derived macrophages and T cells have been shown to contribute to spinal cord repair. Recently, the remote brain choroid plexus epithelium (CP) was identified as a portal for monocyte recruitment, and its activation for leukocyte trafficking was found to be IFN-gamma-dependent. Here, we addressed how the need for effector T cells can be reconciled with the role of inflammation-resolving immune cells in the repair process. Using an acute spinal cord injury model, we
Abstracts
b) subsequent isolation of the relevant TCC from the patient’s cerebrospinal fluid (CSF). An actively demyelinating lesion based on reduced density of myelinated fibers and myelin degradation products within infiltrating macrophages was identified. Using deep sequencing approaches, we sequenced the TCR variable beta (V-beta) chains expressed by T cells infiltrating this lesion. We identified 312 different lesion-infiltrating TCC. Both CD4 (32%) and CD8 (68%) TCC were present and clonally expanded with frequencies up to 30%. In a following step, we were able to isolate 14 of the most frequent CD4+ and CD8+ TCC using as a cell source in vitro PHA-expanded CSF T cells. T cells expressing V-beta families of interest were sorted and cloned by limiting dilution. These TCC are currently being characterized for functional phenotype and antigen specificity by using a combination of two complementary unbiased methods, synthetic combinatorial peptide libraries and an autologous brain-derived cDNA library, which is expressed in suitable antigen presenting cells. doi:10.1016/j.jneuroim.2014.08.552
69 Oxysterols are expressed in T lymphocytes and impair type 1 regulatory CD4 T-cell differentiation Fanny Chalmina, Nishta Budoob, Rochemont Vivianeb, Doron Merklerb, Caroline Pota a
Department of Neurology, Geneva University Hospitals, Geneva, Switzerland; bDepartment of Pathology and Immunology, Geneva University, Geneva, Switzerland Oxysterols, oxidised forms of cholesterol, have recently been assigned with novel functions in modulating the immune response. More specifically, the enzyme cholesterol 25 hydroxylase (ch25h), the rate-limiting step to synthetize 25-hydroxycholesterol (25-OHC) from cholesterol, has been proposed to control viral infection and Immunoglobulin secretion. However the function of oxysterols in CD4+ T lymphocytes and their role during autoimmune diseases has not been assessed. The development and progression of multiple sclerosis results in part from the balance between pathogenicity of effector CD4+ T cells and negative regulation imposed by regulatory cells. Since the original classification of CD4+ T lymphocytes into TH1 and TH2 subsets, the repertoire of effector CD4+ T cell subsets has expanded to include additional effector T cell subsets like TH17 cells and regulatory T cell subsets, in particular Foxp3+ regulatory Tcells (Tregs) and IL-10-producing T regulatory type 1 (TR1). Regulatory IL-10-producing-type 1 (Tr1) T cells are instrumental in the prevention of autoimmune diseases and multiple sclerosis and IL-27 is a critical factor for Tr1 cell differentiation. We assessed the expression of Ch25h and 25-OHC in subset of CD4+ T cells differentiated in vitro. We found that Ch25h expression was specifically induced by IL-27 in Tr1 cells. We further demonstrated that 25-OHC prevented TR1 cell development both in vitro and in vivo and dissected the underlying signaling pathways. Together, our findings show that Ch25h and 25-OHC act as negative regulators of TR1 cell both in vitro and in vivo. Not only these findings unravel novel molecular mechanisms accounting for the generation of Tr1 cells, but they also provide oxysterols as critical players to regulate differentiation of Tr1 cells and to inhibit development of autoimmunity.
doi:10.1016/j.jneuroim.2014.08.553
607 Investigating the Orexin/Hypocretin Specific T Cell Response in Patients with Narcolepsy with Cataplexy Melanie Ramberger, Birgit Högl, Thomas Mitterling, Birgit Frauscher, Markus Reindl, Andreas Lutterotti Clinical Department of Neurology, Innsbruck Medical University, Innsbruck, Austria Narcolepsy with cataplexy is caused by a selective loss of orexin/ hypocretin producing neurons in the lateral hypothalamus. A strong genetic association with HLA DQB1*06:02 (95% in patients vs. 12-38% in controls) is well-known and further genes involved in immune modulation such as T cell receptor alpha, OX40L, cathepsin H, DNMT1 and P2RY11 were shown to be associated with narcolepsy in genomewide association studies. Although these associations strongly suggest an involvement of immune cells, the contribution of T cells in pathogenesis is largely unknown. We therefore investigated the orexin specific T cell reactivity in peripheral blood mononuclear cells (PBMC) of narcolepsy patients by a CFSE-based proliferation assay. The study aim was to analyse narcolepsy patients with cataplexy and healthy controls for T cell proliferation in response to stimulation with an orexin peptide library. Orexin specific CD3+CD4+ T cell reactivity was determined by flow cytometry and cytokine (IFN-γ and GM-CSF) analysis. So far, 13 patients and 11 healthy controls have been included. Individuals were considered as responders, if the stimulation index exceeded 3 in either CD3+CD4+ T cell proliferation, IFN-γ or GM-CSF secretion. Using these inclusion criteria, 6 patients (46.2%) and 2 controls (18.2%) showed orexin specific proliferation (p N 0.05). Furthermore, CD4+ reactivity to orexin was observed more frequently in individuals positive for HLA DQB1*06:02 compared to individuals negative for this risk allele (43.8% vs. 12.5%, p N 0.05). Although we observed a higher orexin specific CD4+ T cell proliferation in narcolepsy patients and in HLA DQB1*06:02 positive individuals, these preliminary data indicate no significant difference in CD4+ T cell reactivity to orexin between the groups by using autologous PBMC. In the near future we plan to compare CD4+ T cell responses to orexin using DQB1*06:02 expressing antigen presenting cells. Melanie Ramberger is enrolled in the graduate program SPIN supported by the Austrian Research Foundation (FWF DK W1206). doi:10.1016/j.jneuroim.2014.08.554
115 Central nervous system repair requires both effector and regulatory T cells with distinct temporal and spatial profiles Catarina Raposo, Michal Schwartz Weizmann Institute of Science, Department of Neurobiology, Rehovot, Israel Healing of the central nervous system (CNS) following traumatic injury was considered for decades to be a tissue autonomous process independent of outside intervention. It is now evident that CNS healing shares many of the properties and requirements of its peripheral counterparts. Monocyte-derived macrophages and T cells have been shown to contribute to spinal cord repair. Recently, the remote brain choroid plexus epithelium (CP) was identified as a portal for monocyte recruitment, and its activation for leukocyte trafficking was found to be IFN-gamma-dependent. Here, we addressed how the need for effector T cells can be reconciled with the role of inflammation-resolving immune cells in the repair process. Using an acute spinal cord injury model, we