Circulating beta cell-specific CD8(+) T cells restricted by high-risk HLA class I molecules show antigen experience in children with and at risk of type 1 diabetes

In type 1 diabetes (T1D), autoreactive cytotoxic CD8(+) T cells are implicated in the destruction of insulin-producing beta cells. The HLA-B*3906 and HLA-A*2402 class I genes confer increased risk and promote early disease onset, suggesting that CD8(+) T cells that recognize peptides presented by these class I molecules on pancreatic beta cells play a pivotal role in the autoimmune response. We examined the frequency and phenotype of circulating preproinsulin (PPI)-specific and insulin B (InsB)-specific CD8(+) T cells in HLA-B*3906(+) children newly diagnosed with T1D and in high-risk HLA-A*2402(+) children before the appearance of disease-specific autoantibodies and before diagnosis of T1D. Antigen-specific CD8(+) T cells were detected using human leucocyte antigen (HLA) class I tetramers and flow cytometry was used to assess memory status. In HLA-B*3906(+) children with T1D, we observed an increase in PPI5-12-specific transitional memory CD8(+) T cells compared to non-diabetic, age- and HLA-matched subjects. Furthermore, PPI5-12-specific CD8(+) T cells in HLA-B*3906(+) children with T1D showed a significantly more antigen-experienced phenotype compared to polyclonal CD8(+) T cells. In longitudinal samples from high-risk HLA-A*2402(+) children, the percentage of terminal effector cells within the InsB(15-24)-specific CD8(+) T cells was increased before diagnosis relative to samples taken before the appearance of autoantibodies. This is the first study, to our knowledge, to report HLA-B*3906-restricted autoreactive CD8(+) T cells in T1D. Collectively, our results provide evidence that beta cell-reactive CD8(+) T cells restricted by disease-associated HLA class I molecules display an antigen-experienced phenotype and acquire enhanced effector function during the period leading to clinical diagnosis, implicating these cells in driving disease.Peer reviewe

Erratum. Blood and Islet Phenotypes Indicate Immunological Heterogeneity in Type 1 Diabetes. Diabetes 2014;63:3835–3845

The article to which this is the erratum is available in ORE at: http://hdl.handle.net/10871/17968In the article, there are two errors in the research design and methods section. In the section with the heading “Studies on Islet-Infiltrating Leukocytes,” the antibody listed as #M0701 should be attributed to Dako and not to Abcam and the Abcam rabbit anti-CD8 catalogue number should read #ab4055 and not #GR404-4. The online version reflects these changes

Circulating β cell-specific CD8+ T cells restricted by high-risk HLA class I molecules show antigen experience in children with and at risk of type 1 diabetes

In type 1 diabetes (T1D), autoreactive cytotoxic CD8+ T cells are implicated in the destruction of insulin‐producing β cells. The HLA‐B*3906 and HLA‐A*2402 class I genes confer increased risk and promote early disease onset, suggesting that CD8+ T cells that recognize peptides presented by these class I molecules on pancreatic β cells play a pivotal role in the autoimmune response. We examined the frequency and phenotype of circulating preproinsulin (PPI)‐specific and insulin B (InsB)‐specific CD8+ T cells in HLA‐B*3906+ children newly diagnosed with T1D and in high‐risk HLA‐A*2402+ children before the appearance of disease‐specific autoantibodies and before diagnosis of T1D. Antigen‐specific CD8+ T cells were detected using human leucocyte antigen (HLA) class I tetramers and flow cytometry was used to assess memory status. In HLA‐B*3906+ children with T1D, we observed an increase in PPI5–12‐specific transitional memory CD8+ T cells compared to non‐diabetic, age‐ and HLA‐matched subjects. Furthermore, PPI5–12‐specific CD8+ T cells in HLA‐B*3906+ children with T1D showed a significantly more antigen‐experienced phenotype compared to polyclonal CD8+ T cells. In longitudinal samples from high‐risk HLA‐A*2402+ children, the percentage of terminal effector cells within the InsB15–24‐specific CD8+ T cells was increased before diagnosis relative to samples taken before the appearance of autoantibodies. This is the first study, to our knowledge, to report HLA‐B*3906‐restricted autoreactive CD8+ T cells in T1D. Collectively, our results provide evidence that β cell‐reactive CD8+ T cells restricted by disease‐associated HLA class I molecules display an antigen‐experienced phenotype and acquire enhanced effector function during the period leading to clinical diagnosis, implicating these cells in driving disease.</p

ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function

The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins

Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

Fluorochrome-conjugated peptide–major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin–biotin-based ‘tetramers’, can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR–pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology

A reference human induced pluripotent stem cell line for large-scale collaborative studies

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate human iPSC lines and deeply characterized their genetic properties using whole genome sequencing, their genomic stability upon CRISPR-Cas9-based gene editing, and their phenotypic properties including differentiation to commonly used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field

A distinct immunogenic region of glutamic acid decarboxylase 65 is naturally processed and presented by human islet cells to cytotoxic CD8 T cells

CD8 T cells specific for islet autoantigens are major effectors of β cell damage in type 1 diabetes, and measurement of their number and functional characteristics in blood represent potentially important disease biomarkers. CD8 T cell reactivity against glutamic acid decarboxylase 65 (GAD65) in HLA-A*0201 subjects has been reported to focus on an immunogenic region 114–123 (VMNILLQYVV), with studies demonstrating both 114–123 and 114–122 epitopes being targeted. However, the fine specificity of this response is unclear and the key question as to which epitope(s) β cells naturally process and present and, therefore, the pathogenic potential of CD8 T cells with different specificities within this region has not been addressed.We generated human leucocyte antigen (HLA)-A*0201-restricted CD8 T cell clones recognizing either 114–122 alone or both 114–122 and 114–123. Both clone types show potent and comparable effector functions (cytokine and chemokine secretion) and killing of indicator target cells externally pulsed with cognate peptide. However, only clones recognizing 114–123 kill target cells transfected with HLA-A*0201 and GAD2 and HLA-A*0201+ human islet cells. We conclude that the endogenous pathway of antigen processing by HLA-A*0201-expressing cells generates GAD65114–123 as the predominant epitope in this region. These studies highlight the importance of understanding β cell epitope presentation in the design of immune monitoring for potentially pathogenic CD8 T cells

Molecular pathways for immune recognition of preproinsulin signal peptide in type 1 diabetes

This is the author accepted manuscript. The final version is available from American Diabetes Association via the DOI in this record.The signal peptide region of preproinsulin (PPI) contains epitopes targeted by human leucocyte antigen-A (HLA-A)-restricted (HLA-A0201, A2402) cytotoxic T-cells as part of the pathogenesis of β-cell destruction in type 1 diabetes. We extended PPI epitope discovery to disease-associated HLA-B*1801 and HLA-B*3906 (risk) and HLA-A*1101 and HLA-B*3801 (protective) alleles revealing that 4/6 alleles present epitopes derived from the signal peptide region. During co-translational translocation of PPI, its signal peptide is cleaved and retained within the endoplasmic reticulum (ER) membrane, implying it is processed for immune recognition outside of the canonical, proteasome-directed pathway. Using in vitro translocation assays with specific inhibitors and gene knockout in PPI-expressing target cells we show that PPI signal peptide antigen processing requires signal peptide peptidase (SPP). The intramembrane protease SPP generates cytoplasm-proximal epitopes, which are transporter-associated-with–antigen-processing (TAP)-dependent, and ER-luminal (TAP-independent) epitopes, each presented by different HLA class I molecules, and N-terminal trimmed by ER aminopeptidase 1 (ERAP1) for optimal presentation. In vivo, TAP expression is significantly up-regulated and correlated with HLA class I hyper-expression in insulin-containing islets of patients with type 1 diabetes. Thus, PPI signal peptide epitopes are processed by SPP and loaded for HLA-guided immune recognition via pathways that are enhanced during disease pathogenesis.This study was supported by the NIHR Biomedical Research Centre at Guy’s and St Thomas’ Hospital Trusts and King’s College London, a Centre Grant from the Juvenile Diabetes Research Foundation (JDRF; 1-2007-1803 to MP), a JDRF Career Development Award to SJR (5-CDA-2014-221-A-N), a project grant 15/0005156 from Diabetes UK (to NGM & SJR) and a project grant FOR2290-TP1 from the Deutsche Forschungsgemeinschaft (to MKL)

Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

Fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin-biotin-based 'tetramers', can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR-pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology

Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes

Studies 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