Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes

This is an author-created, uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association (ADA), publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version is available in Diabetes in print and online at http://diabetes.diabetesjournals.orgThe erratum to this article is available in ORE at http://hdl.handle.net/10871/40335Studies in type 1 diabetes indicate potential disease heterogeneity, notably in the rate of β-cell loss, responsiveness to immunotherapies, and, in limited studies, islet pathology. We sought evidence for different immunological phenotypes using two approaches. First, we defined blood autoimmune response phenotypes by combinatorial, multiparameter analysis of autoantibodies and autoreactive T-cell responses in 33 children/adolescents with newly diagnosed diabetes. Multidimensional cluster analysis showed two equal-sized patient agglomerations characterized by proinflammatory (interferon-γ-positive, multiautoantibody-positive) and partially regulated (interleukin-10-positive, pauci-autoantibody-positive) responses. Multiautoantibody-positive nondiabetic siblings at high risk of disease progression showed similar clustering. Additionally, pancreas samples obtained post mortem from a separate cohort of 21 children/adolescents with recently diagnosed type 1 diabetes were examined immunohistologically. This revealed two distinct types of insulitic lesions distinguishable by the degree of cellular infiltrate and presence of B cells that we termed "hyper-immune CD20Hi" and "pauci-immune CD20Lo." Of note, subjects had only one infiltration phenotype and were partitioned by this into two equal-sized groups that differed significantly by age at diagnosis, with hyper-immune CD20Hi subjects being 5 years younger. These data indicate potentially related islet and blood autoimmune response phenotypes that coincide with and precede disease. We conclude that different immunopathological processes (endotypes) may underlie type 1 diabetes, carrying important implications for treatment and prevention strategies.JDRFNational Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College LondonEuropean Union (EU FP7) award - Persistent Virus Infection in Diabetes Network Study Group (PEVNET)EU FP7 Large-Scale Focused Collaborative Research Project on Natural Immunomodulators as Novel Immunotherapies for Type 1 Diabetes (NAIMIT)EU FP7 Large-Scale Focused Collaborative Research Project on β-cell preservation through antigen-specific immunotherapy in Type 1 Diabetes: Enhanced Epidermal Antigen Delivery Systems (EE-ASI)National Institutes of Health (NIH)National Institute of Diabetes and Digestive and Kidney DiseasesNational Institute of Allergy and Infectious DiseasesEunice Kennedy Shriver National Institute of Child Health and Human DevelopmentNational Center for Research ResourcesGeneral Clinical Research CenterAmerican Diabetes Association (ADA

Studies On Mycobacterium tuberculosis Transcriptional Regulators Involved in Intracellular Function.

Tuberculosis kills an estimated 2 million people each year. The causative organism, Mycobacterium tuberculosis, has evolved the ability to survive within human macrophages and avoid the destructive action of the host immune response. Understanding the molecular mechanisms of intracellular survival will uncover new targets for effective drug and vaccine design. High-throughput screening of M. tuberculosis transposon mutants selected during macrophage infection was used to identify mutants which were unable to inhibit phagosome-lysosome fusion. One of the mutants carried an insertion in a gene, Rv2745c, encoding a putative transcriptional regulator. Targeted gene knock-out of Rv2745c and two other putative regulators (Rv3058c and Rv2548) was successfully achieved in an M. tuberculosis H37Rv background. These mutant strains were complemented by reintroduction of the wild-type gene and each strain was characterised for in vitro growth and growth in J774 macrophages. All the mutants had reduced survival following heat shock. Only ΔRv2745c showed any evidence of attenuation for intracellular growth. Microarray analysis was performed to generate transcriptional profiles of each mutant strain grown in culture compared with the wild-type H37Rv strain. Gene expression differences in the ΔRv3058c strain appeared to be associated with differences in growth phase of the mutant and wild-type strain. No direct regulon for Rv3058c could be identified from the transcriptional profile of the mutant. Thirty eight gene transcripts were significantly more abundant and none less abundant in the ΔRv2548 strain. This is consistent with Rv2548 functioning as the Ribonuclease component of a toxin-antitoxin system. Transcriptional profiling of the ΔRv2745c mutant demonstrated that 31 genes were up-regulated compared to the wild-type and 9 genes were down-regulated. Bioinformatic analysis revealed Rv2745c to be similar to the transcriptional activator ClgR from the related bacterial species Streptomyces and Corynebacterium. The putative binding site of ClgR is CGC-N5-GCG shown to be involved in cell cycle and growth regulation in Streptomyces. A bioinformatic analysis of those genes down-regulated in the ΔRv2745c mutant revealed the following genes to have ClgR-like binding motifs: acr2, Rv1043c, ptrB, clpP and clpC. Expression changes of these genes in the mutant were confirmed using quantitative real-time PCR (QRTPCR). Recombinant GST-Rv2745c (ClgR) was expressed and purified in Escherichia coli. Electrophoretic mobility shift assays demonstrated recombinant GST-Rv2745c to bind ClgR-like promoter regions of ptrB and acr2 genes. Together, this project demonstrates that M. tuberculosis controls expression of stress induced chaperones Acr2, ClpC and proteases ClpP, Rv1043c and PtrB using a transcriptional activator, Rv2745c (ClgR). These chaperones and proteases are likely to be important in regulation of the protein content of the cell and this study provides preliminary evidence that correct coordination of this function is required for the survival within the macrophage

Studies on mycobacterium tuberculosis transcriptional regulators involved in intracellular infection

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Studies On Mycobacterium tuberculosis Transcriptional Regulators Involved in Intracellular Function.

Tuberculosis kills an estimated 2 million people each year. The causative organism, Mycobacterium tuberculosis, has evolved the ability to survive within human macrophages and avoid the destructive action of the host immune response. Understanding the molecular mechanisms of intracellular survival will uncover new targets for effective drug and vaccine design. High-throughput screening of M. tuberculosis transposon mutants selected during macrophage infection was used to identify mutants which were unable to inhibit phagosome-lysosome fusion. One of the mutants carried an insertion in a gene, Rv2745c, encoding a putative transcriptional regulator. Targeted gene knock-out of Rv2745c and two other putative regulators (Rv3058c and Rv2548) was successfully achieved in an M. tuberculosis H37Rv background. These mutant strains were complemented by reintroduction of the wild-type gene and each strain was characterised for in vitro growth and growth in J774 macrophages. All the mutants had reduced survival following heat shock. Only ΔRv2745c showed any evidence of attenuation for intracellular growth. Microarray analysis was performed to generate transcriptional profiles of each mutant strain grown in culture compared with the wild-type H37Rv strain. Gene expression differences in the ΔRv3058c strain appeared to be associated with differences in growth phase of the mutant and wild-type strain. No direct regulon for Rv3058c could be identified from the transcriptional profile of the mutant. Thirty eight gene transcripts were significantly more abundant and none less abundant in the ΔRv2548 strain. This is consistent with Rv2548 functioning as the Ribonuclease component of a toxin-antitoxin system. Transcriptional profiling of the ΔRv2745c mutant demonstrated that 31 genes were up-regulated compared to the wild-type and 9 genes were down-regulated. Bioinformatic analysis revealed Rv2745c to be similar to the transcriptional activator ClgR from the related bacterial species Streptomyces and Corynebacterium. The putative binding site of ClgR is CGC-N5-GCG shown to be involved in cell cycle and growth regulation in Streptomyces. A bioinformatic analysis of those genes down-regulated in the ΔRv2745c mutant revealed the following genes to have ClgR-like binding motifs: acr2, Rv1043c, ptrB, clpP and clpC. Expression changes of these genes in the mutant were confirmed using quantitative real-time PCR (QRTPCR). Recombinant GST-Rv2745c (ClgR) was expressed and purified in Escherichia coli. Electrophoretic mobility shift assays demonstrated recombinant GST-Rv2745c to bind ClgR-like promoter regions of ptrB and acr2 genes. Together, this project demonstrates that M. tuberculosis controls expression of stress induced chaperones Acr2, ClpC and proteases ClpP, Rv1043c and PtrB using a transcriptional activator, Rv2745c (ClgR). These chaperones and proteases are likely to be important in regulation of the protein content of the cell and this study provides preliminary evidence that correct coordination of this function is required for the survival within the macrophage

A novel approach to tracking antigen-experienced CD4 T cells into functional compartments via tandem deep and shallow TCR clonotyping

Abstract Extensive diversity in the human repertoire of TCRs for Ag is both a cornerstone of effective adaptive immunity that enables host protection against a multiplicity of pathogens and a weakness that gives rise to potential pathological self-reactivity. The complexity arising from diversity makes detection and tracking of single Ag-specific CD4 T cells (ASTs) involved in these immune responses challenging. We report a tandem, multistep process to quantify rare TCRβ-chain variable sequences of ASTs in large polyclonal populations. The approach combines deep high-throughput sequencing (HTS) within functional CD4 T cell compartments, such as naive/memory cells, with shallow, multiple identifier–based HTS of ASTs identified by activation marker upregulation after short-term Ag stimulation in vitro. We find that clonotypes recognizing HLA class II–restricted epitopes of both pathogen-derived Ags and self-Ags are oligoclonal and typically private. Clonotype tracking within an individual reveals private AST clonotypes resident in the memory population, as would be expected, representing clonal expansions (identical nucleotide sequence; “ultraprivate”). Other AST clonotypes share CDR3β amino acid sequences through convergent recombination and are found in memory populations of multiple individuals. Tandem HTS-based clonotyping will facilitate studying AST dynamics, epitope spreading, and repertoire changes that arise postvaccination and following Ag-specific immunotherapies for cancer and autoimmune disease.</jats:p