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Myelin-specific Regulatory T Cells Accumulate in the Central Nervous System, but Fail to Suppress Pathogenic Effector T Cells at the Peak of Autoimmune Inflammation
S152 Trafficking of antigen presenting cells (APC) into the central nervous system (CNS) is essential for lymphocyte reactivation within the CNS compartment. The origin and function of the APC found in CNS autoimmune or inflammatory lesions remain, however, controversial. We demonstrate that blood CD14+ monocytes migrate across the inflammed blood–brain barrier (BBB) and differentiate into CD209+CD83+ (myeloid) or CD123+ (plasmacytoid) dendritic cells (DCs) under the influence of CNS endothelial-secreted transforming growth factor-beta (TGF-β) and granulocyte macrophage colony stimulating factor (GM-CSF). We also demonstrate that while CD123+ DCs secrete interleukin-12 p70 (IL-12p70) and promote the proliferation and expansion of interferon-γ-secreting Th1 CD4+ lymphocytes, CD209+ CD83+ DCs secrete high levels of TGF-β and IL-6 and skew CD4+ lymphocytes towards a Th17 phenotype. Using multiple sclerosis as a prototypical human CNS autoimmune disease, we confirmed by immunohistochemistry the abundance of such perivascular CD83+ and CD123+ DCs within acute lesions, closely associated with microvascular BBBendothelial cells. Our data support the notion that functional perivascular myeloid and plasmacytoid CNS DCs arise as a consequence of migration of blood CD14+ monocytes across the human BBB, through the concerted actions of TGF-β and GM-CSF. doi:10.1016/j.clim.2007.03.071
Su.31 Characterization of Single B Cell Immunoglobulin Variable Region Genes Derived from Infiltrated Muscle Tissue of Subjects with Inflammatory Myopathies Elizabeth Bradshaw, Postdoctoral Fellow, Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA, Ana Orihuela, Research Technician, Children’s Hospital Informatics Program at the Harvard-MIT Division of Health Sciences and Technology, Boston, MA, Shannon McArdel, Research Technician, Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA, David Hafler, Professor, Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA, Anthony Amato, Associate Professor, Department of Neurology, Division of Neuromuscular Disease, Brigham and Women’s Hospital and Harvard, Boston, MA, Kevin O’Connor, Assistant Professor, Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA The inflammatory myopathies (IM) are putative autoimmune disorders characterized by muscle weakness and the presence of inflammatory infiltrates in skeletal muscle. Varying numbers of both B cells and plasma cells are among the muscle tissue infiltrate found in these diseases. Through examination of immunoglobulin heavy chain variable regions, we previously demonstrated that there was significant oligoclonal expansion of B cells found in IM muscle. To extend these findings, we isolated single B cells from IM muscle by laser capture microdissection or FACS, then examined the paired immunoglobulin heavy and light chain variable region
Abstracts sequences. B cell immunoglobulin variable region cDNAs were sequenced affording identification of the isotype, CDR3 and the degree of somatic mutation in the entire V-region. A pattern of B cell affinity maturation was evident in clones that had accumulated varying numbers of common and unique somatic mutations. These data suggested that a local inflammatory response occurs within the muscle tissue of patients with IM that may indicate the presence of a B cell antigen-specific response. doi:10.1016/j.clim.2007.03.072
Su.32 Myelin-specific Regulatory T Cells Accumulate in the Central Nervous System, but Fail to Suppress Pathogenic Effector T Cells at the Peak of Autoimmune Inflammation Thomas Korn, Postdoctoral Fellow, Harvard Medical School, Center for Neurologic Diseases, Boston, MA, Mohamed Oukka, Instructor, Harvard Medical School, Center for Neurologic Diseases, Boston, MA, Jay, Reddy, Instructor, Harvard Medical School, Center for Neurologic Diseases, Boston, MA, Estelle Bettelli, Instructor, Harvard Medical School, Center for Neurologic Diseases, Boston, MA, Amit Awasthi, Postdoctoral Fellow, Harvard Medical School, Center for Neurologic Diseases, Boston, MA, Raymond Sobel, Associate Professor of Pathology, Stanford University, Department of Pathology, Stanford, CA, Kai Wucherpfennig, Associate Professor of Neurology, Harvard Medical School, Dana Farber Cancer Institute, Department of Cancer Immunology and AIDS, Boston, MA, Vijay Kuchroo, Professor of Neurology, Harvard Medical School, Center for Neurologic Diseases, Boston, MA Treatment with ex vivo generated regulatory T cells (Treg) has been regarded as highly attractive therapeutic approach for autoimmune diseases. However, the dynamics and function of T-reg in autoimmunity are not well understood. Thus, we developed Foxp3gfp “knock-in” mice and myelin oligodendrocyte glycoprotein (MOG)35–55/IAb tetramers to track autoantigen-specific effector T cells (T-eff) and T-reg in vivo during experimental autoimmune encephalomyelitis, an animal model for Multiple Sclerosis. Following immunization with the encephalitogenic peptide MOG35–55 emulsified in complete Freund's adjuvant, MOG35–55-tetramer-reactive, Foxp3+ T-reg expanded in the peripheral lymphoid compartment and readily accumulated in the central nervous system (CNS), but did not prevent the onset of disease. During disease onset, the MOGtetramer+ T-eff population in the CNS increased faster than the population of antigen-specific T-reg. At the peak of disease, the ratio of T-reg vs. T-eff was 1:17 which dramatically changed into 1:2 at the beginning of recovery. Foxp3+ T-reg isolated from the CNS were fully competent in suppressing naive MOG-specific T cells. However, Foxp3+ Treg failed to control encephalitogenic T-eff which in contrast to T-eff from the peripheral immune compartment, secreted IL-6 and TNF when they were isolated from the CNS at the peak of disease. Our data suggest that in the face of inflammation, the regulation of autoimmunity by CD4+Foxp3+ T-reg in situ may not be accomplished simply by changing the numerical balance of antigen-specific pathogenic vs.
Abstracts regulatory T cells, but may require the control of tissue inflammation as well. doi:10.1016/j.clim.2007.03.073
Su.34 Proteomic Profiling of Multiple Sclerosis Lesions Identify Potential Targets for Therapy May H. Han, Postdoctoral Fellow, Department of Neurology and Neurological Sciences Stanford University, Stanford, CA, Sunil Hwang, Postdoctoral Fellow, Department of Physiology, University of Conneticut Health Center, Farmington, CT, Dolly Roy, Neurologist, Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, Cedric Raine, Professor, Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, William H. Robinson, Assistant Professor, Departments of Medicine and Immunology, Stanford University, Stanford, CA, Raymond A. Sobel, Professor of Pathology, Department of Pathology, Stanford University, Stanford, CA, David Han, Associate Professor, Department of Physiology, University of Conneticut Health Center, Farmington, CT, Lawrence Steinman, Professor, Department of Neurology and Neurological Sciences, Stanford, CA Demyelinating lesions or plaques in multiple sclerosis (MS) have distinct histological features that reflect their stage of evolution. To identify key molecules involved at different stages during lesion progression, we performed proteomic profiling of three types of MS plaques using mass spectrometry and bioinformatics. Fresh frozen autopsy brain samples from MS patients (n=6) and age-matched controls (n=2) were classified as 1) acute plaque (AP, characterized by presence of inflammatory cells, ongoing demyelination), 2) chronic active plaque (CAP, characterized by chronic demyelination, astrogliosis with active rim) or 3) chronic plaque (CP, characterized by lack of inflammatory cells, dense astrogliosis). Samples from plaques were isolated by laser capture microdissection and global protein identification was carried out by SDS-PAGE, in-gel protease digestion, liquid chromatography and mass spectrometry. A total of 3894 proteins were identified out of which 2184 proteins were unique to MS lesions. Then we identified proteins unique to each type of lesion (AP (N=172), CAP (N=248) and CP (N=252), and proteins common to all three types (N=89) using bio-informatics software INTERSECT. Analysis by software PROTEOME 3D identified proteins of coagulation cascade and neural regeneration which may be potential targets for therapy. These results provide the first and most comprehensive information on global protein expression in MS plaques, enhance our understanding of the molecular pathogenesis of MS lesions and identify potential key molecules involved in disease progression that offer possibilities for therapeutic targets. doi:10.1016/j.clim.2007.03.075
Su.35 A Genome-wide Screen for Genetic Variants Affecting the Expression of Immunologically Relevant Cell Surface Markers Philip De Jager, Assistant Professor, Department of Neurology, Brigham and Women’s Hospital, Boston, MA,
S153 Roman Yelensky, Student, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, Elizabeth Rossin, Data Analyst, Medical and Population Genetics Program, Broad Institute, Boston, MA, Sasha Bonadkar, Research Assistant, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, Edwin Choy, Director, Sarcoma Research, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, Mark Daly, Assistant Professor, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, David Altshuler, Associate Professor, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, David Hafler, Professor, Department of Neurology, Brigham and Women's Hospital, Boston, MA While a few alleles affecting the expression level of immunologically relevant molecules have been identified and validated, our knowledge of human polymorphisms associated with differences in immune function remains very limited. We have therefore evaluated the use of the HapMap lymphoblastic cell lines (LCLs) as a platform to discover such polymorphisms on a large scale using a test panel of cell surface molecules that includes 15 cell-surface markers: CD19, CD20, CD21, CD40, CD58, CD80, CD86, CD95, CD227, HLA DQ, HLA DR, IgD, IgG, IgM, and IL6R. We then correlated the mean expression level of a particular molecule in each LCL with the over 2 million genotypes available in the HapMap database. Amongst several positive results, this analysis reveals that certain SNPs in the vicinity of the HLA DQA1 gene are correlated with both the level of expression of HLA DQ on the LCL surface and the level of mRNA expression for HLA DQA1. One of these SNPs, rs9272346, is a null allele of HLA DQA1 and is associated to this immunophenotype at a level that exceeds our predetermined level of genome-wide significance (P b 2.6 × 10−8), validating our approach. In exploring the mRNA data further within the MHC class I and class II clusters, it is clear that several different polymorphisms exist that affect the level of expression of these molecules. We are currently assessing the role of these functional variants in the association of the MHC with MS susceptibility. doi:10.1016/j.clim.2007.03.076
Su.36 Novel Computational Methods to Enhance the Analysis of Cytometric Immunophenotypes Philip De Jager, Assistant Professor, Department of Neurology, Brigham and Women’s Hospital, Boston, MA, Saumyadipta Pyne, Postdoctoral Fellow, Cancer Program, Broad Institute, Cambridge, MA, Elizabeth Rossin, Data Analyst, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, Jill Mesirov, Director, Cancer Program, Broad Institute, Cambridge, MA, Pablo Tamayo, Staff Scientist, Cancer Program, Broad Institute, Cambridge, MA, David Hafler, Professor, Department of Neurology, Brigham and Women’s Hospital, Boston, MA The current method for analyzing raw flow cytometry information involves gating, the process of first visualizing cell-surface marker expression values for a population of cells and then manually selecting distinct cell populations on
Su.31 Characterization of Single B Cell Immunoglobulin Variable Region Genes Derived from Infiltrated Muscle Tissue of Subjects with Inflammatory Myopathies Elizabeth Bradshaw, Postdoctoral Fellow, Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA, Ana Orihuela, Research Technician, Children’s Hospital Informatics Program at the Harvard-MIT Division of Health Sciences and Technology, Boston, MA, Shannon McArdel, Research Technician, Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA, David Hafler, Professor, Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA, Anthony Amato, Associate Professor, Department of Neurology, Division of Neuromuscular Disease, Brigham and Women’s Hospital and Harvard, Boston, MA, Kevin O’Connor, Assistant Professor, Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA The inflammatory myopathies (IM) are putative autoimmune disorders characterized by muscle weakness and the presence of inflammatory infiltrates in skeletal muscle. Varying numbers of both B cells and plasma cells are among the muscle tissue infiltrate found in these diseases. Through examination of immunoglobulin heavy chain variable regions, we previously demonstrated that there was significant oligoclonal expansion of B cells found in IM muscle. To extend these findings, we isolated single B cells from IM muscle by laser capture microdissection or FACS, then examined the paired immunoglobulin heavy and light chain variable region
Abstracts sequences. B cell immunoglobulin variable region cDNAs were sequenced affording identification of the isotype, CDR3 and the degree of somatic mutation in the entire V-region. A pattern of B cell affinity maturation was evident in clones that had accumulated varying numbers of common and unique somatic mutations. These data suggested that a local inflammatory response occurs within the muscle tissue of patients with IM that may indicate the presence of a B cell antigen-specific response. doi:10.1016/j.clim.2007.03.072
Su.32 Myelin-specific Regulatory T Cells Accumulate in the Central Nervous System, but Fail to Suppress Pathogenic Effector T Cells at the Peak of Autoimmune Inflammation Thomas Korn, Postdoctoral Fellow, Harvard Medical School, Center for Neurologic Diseases, Boston, MA, Mohamed Oukka, Instructor, Harvard Medical School, Center for Neurologic Diseases, Boston, MA, Jay, Reddy, Instructor, Harvard Medical School, Center for Neurologic Diseases, Boston, MA, Estelle Bettelli, Instructor, Harvard Medical School, Center for Neurologic Diseases, Boston, MA, Amit Awasthi, Postdoctoral Fellow, Harvard Medical School, Center for Neurologic Diseases, Boston, MA, Raymond Sobel, Associate Professor of Pathology, Stanford University, Department of Pathology, Stanford, CA, Kai Wucherpfennig, Associate Professor of Neurology, Harvard Medical School, Dana Farber Cancer Institute, Department of Cancer Immunology and AIDS, Boston, MA, Vijay Kuchroo, Professor of Neurology, Harvard Medical School, Center for Neurologic Diseases, Boston, MA Treatment with ex vivo generated regulatory T cells (Treg) has been regarded as highly attractive therapeutic approach for autoimmune diseases. However, the dynamics and function of T-reg in autoimmunity are not well understood. Thus, we developed Foxp3gfp “knock-in” mice and myelin oligodendrocyte glycoprotein (MOG)35–55/IAb tetramers to track autoantigen-specific effector T cells (T-eff) and T-reg in vivo during experimental autoimmune encephalomyelitis, an animal model for Multiple Sclerosis. Following immunization with the encephalitogenic peptide MOG35–55 emulsified in complete Freund's adjuvant, MOG35–55-tetramer-reactive, Foxp3+ T-reg expanded in the peripheral lymphoid compartment and readily accumulated in the central nervous system (CNS), but did not prevent the onset of disease. During disease onset, the MOGtetramer+ T-eff population in the CNS increased faster than the population of antigen-specific T-reg. At the peak of disease, the ratio of T-reg vs. T-eff was 1:17 which dramatically changed into 1:2 at the beginning of recovery. Foxp3+ T-reg isolated from the CNS were fully competent in suppressing naive MOG-specific T cells. However, Foxp3+ Treg failed to control encephalitogenic T-eff which in contrast to T-eff from the peripheral immune compartment, secreted IL-6 and TNF when they were isolated from the CNS at the peak of disease. Our data suggest that in the face of inflammation, the regulation of autoimmunity by CD4+Foxp3+ T-reg in situ may not be accomplished simply by changing the numerical balance of antigen-specific pathogenic vs.
Abstracts regulatory T cells, but may require the control of tissue inflammation as well. doi:10.1016/j.clim.2007.03.073
Su.34 Proteomic Profiling of Multiple Sclerosis Lesions Identify Potential Targets for Therapy May H. Han, Postdoctoral Fellow, Department of Neurology and Neurological Sciences Stanford University, Stanford, CA, Sunil Hwang, Postdoctoral Fellow, Department of Physiology, University of Conneticut Health Center, Farmington, CT, Dolly Roy, Neurologist, Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, Cedric Raine, Professor, Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, William H. Robinson, Assistant Professor, Departments of Medicine and Immunology, Stanford University, Stanford, CA, Raymond A. Sobel, Professor of Pathology, Department of Pathology, Stanford University, Stanford, CA, David Han, Associate Professor, Department of Physiology, University of Conneticut Health Center, Farmington, CT, Lawrence Steinman, Professor, Department of Neurology and Neurological Sciences, Stanford, CA Demyelinating lesions or plaques in multiple sclerosis (MS) have distinct histological features that reflect their stage of evolution. To identify key molecules involved at different stages during lesion progression, we performed proteomic profiling of three types of MS plaques using mass spectrometry and bioinformatics. Fresh frozen autopsy brain samples from MS patients (n=6) and age-matched controls (n=2) were classified as 1) acute plaque (AP, characterized by presence of inflammatory cells, ongoing demyelination), 2) chronic active plaque (CAP, characterized by chronic demyelination, astrogliosis with active rim) or 3) chronic plaque (CP, characterized by lack of inflammatory cells, dense astrogliosis). Samples from plaques were isolated by laser capture microdissection and global protein identification was carried out by SDS-PAGE, in-gel protease digestion, liquid chromatography and mass spectrometry. A total of 3894 proteins were identified out of which 2184 proteins were unique to MS lesions. Then we identified proteins unique to each type of lesion (AP (N=172), CAP (N=248) and CP (N=252), and proteins common to all three types (N=89) using bio-informatics software INTERSECT. Analysis by software PROTEOME 3D identified proteins of coagulation cascade and neural regeneration which may be potential targets for therapy. These results provide the first and most comprehensive information on global protein expression in MS plaques, enhance our understanding of the molecular pathogenesis of MS lesions and identify potential key molecules involved in disease progression that offer possibilities for therapeutic targets. doi:10.1016/j.clim.2007.03.075
Su.35 A Genome-wide Screen for Genetic Variants Affecting the Expression of Immunologically Relevant Cell Surface Markers Philip De Jager, Assistant Professor, Department of Neurology, Brigham and Women’s Hospital, Boston, MA,
S153 Roman Yelensky, Student, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, Elizabeth Rossin, Data Analyst, Medical and Population Genetics Program, Broad Institute, Boston, MA, Sasha Bonadkar, Research Assistant, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, Edwin Choy, Director, Sarcoma Research, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, Mark Daly, Assistant Professor, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, David Altshuler, Associate Professor, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, David Hafler, Professor, Department of Neurology, Brigham and Women's Hospital, Boston, MA While a few alleles affecting the expression level of immunologically relevant molecules have been identified and validated, our knowledge of human polymorphisms associated with differences in immune function remains very limited. We have therefore evaluated the use of the HapMap lymphoblastic cell lines (LCLs) as a platform to discover such polymorphisms on a large scale using a test panel of cell surface molecules that includes 15 cell-surface markers: CD19, CD20, CD21, CD40, CD58, CD80, CD86, CD95, CD227, HLA DQ, HLA DR, IgD, IgG, IgM, and IL6R. We then correlated the mean expression level of a particular molecule in each LCL with the over 2 million genotypes available in the HapMap database. Amongst several positive results, this analysis reveals that certain SNPs in the vicinity of the HLA DQA1 gene are correlated with both the level of expression of HLA DQ on the LCL surface and the level of mRNA expression for HLA DQA1. One of these SNPs, rs9272346, is a null allele of HLA DQA1 and is associated to this immunophenotype at a level that exceeds our predetermined level of genome-wide significance (P b 2.6 × 10−8), validating our approach. In exploring the mRNA data further within the MHC class I and class II clusters, it is clear that several different polymorphisms exist that affect the level of expression of these molecules. We are currently assessing the role of these functional variants in the association of the MHC with MS susceptibility. doi:10.1016/j.clim.2007.03.076
Su.36 Novel Computational Methods to Enhance the Analysis of Cytometric Immunophenotypes Philip De Jager, Assistant Professor, Department of Neurology, Brigham and Women’s Hospital, Boston, MA, Saumyadipta Pyne, Postdoctoral Fellow, Cancer Program, Broad Institute, Cambridge, MA, Elizabeth Rossin, Data Analyst, Medical and Population Genetics Program, Broad Institute, Cambridge, MA, Jill Mesirov, Director, Cancer Program, Broad Institute, Cambridge, MA, Pablo Tamayo, Staff Scientist, Cancer Program, Broad Institute, Cambridge, MA, David Hafler, Professor, Department of Neurology, Brigham and Women’s Hospital, Boston, MA The current method for analyzing raw flow cytometry information involves gating, the process of first visualizing cell-surface marker expression values for a population of cells and then manually selecting distinct cell populations on