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Type 1 diabetes is characterized by a loss of tolerance to pancreatic β-cell autoantigens and defects in T regulatory cell (Treg) function. In preclinical models, immunotherapy with MHC-selective, autoantigenic peptides restores immune tolerance, prevents diabetes and shows greater potency when multiple peptides are used. To translate this strategy into the clinical setting we administered a mixture of 6 HLA-DRB1*0401 -selective, β-cell peptides intradermally to patients with recent-onset type 1 diabetes possessing this genotype in a randomized placebo-controlled study at monthly doses of 10, 100 and 500µg for 24 weeks. Stimulated C-peptide (measuring insulin functional reserve) had declined in all placebo subjects at 24 weeks, but was maintained at ≥100% baseline levels in half of the treated group. Treatment was accompanied by significant changes in islet specific immune responses and a dose-dependent increase in Treg expression of the canonical transcription factor FoxP3 and changes in Treg gene expression. In this first-in-human study, multiple-peptide immunotherapy shows promise as a strategy to correct immune regulatory defects fundamental to the pathobiology of autoimmune diabetes.

BackgroundHeterogeneity in the rate of β-cell loss in newly diagnosed type 1 diabetes patients is poorly understood and creates a barrier to designing and interpreting disease-modifying clinical trials. Integrative analyses of complementary multi-omics data obtained after the diagnosis of T1D may provide mechanistic insight into the diverse rates of disease progression.MethodsWe collected samples in a pan-European consortium that enabled the concerted analysis of five different omics modalities in data from 97 newly diagnosed patients. In this study we used Multi-Omics Factor Analysis to identify molecular signatures correlating with post-diagnosis decline in β-cell mass measured as fasting C-peptide.ResultsTwo molecular signatures were significantly correlated with fasting C-peptide levels. One signature showed a correlation to neutrophil degranulation, cytokine signaling, lymphoid and non-lymphoid cell interactions and G-protein coupled receptor signaling events that were inversely associated with rapid decline in β-cell function. The second signature was related to translation and viral infection were inversely associated with change in β-cell function. In addition, the immunomics data revealed a Natural Killer cell signature associated with rapid β-cell decline.ConclusionFeatures that differ between individuals with slow and rapid decline in β-cell mass could be valuable in staging and prediction of the rate of disease progression and thus enable smarter (shorter and smaller) trial designs for disease modifying therapies, as well as offering biomarkers of therapeutic effect.FundingThis work is funded by the Innovative Medicine Initiative 2 Joint Undertaking (IMI2 JU) under grant agreement N° 115797 (INNODIA) and N° 945268 (INNODIA HARVEST). This Joint Undertaking receives support from the Union’s Horizon 2020 research and innovation program and ‘EFPIA’, ‘JDRF’ and ‘The Leona M. and Harry B. Helmsley Charitable Trust’.

Autoimmune diseases and in particular type 1 diabetes rely heavily on treatments that target the symptoms rather than prevent the underlying disease. One of the barriers to better therapeutic strategies is the inability to detect and efficiently target rare autoreactive T-cell populations that are major drivers of these conditions. Here, we develop a unique artificial antigen-presenting cell (aAPC) system from biocompatible polymer particles that allows specific encapsulation of bioactive ingredients. Using our aAPC, we demonstrate that we are able to detect rare autoreactive CD4 populations in human patients, and using mouse models, we demonstrate that our particles are able to induce desensitization in the autoreactive population. This system provides a promising tool that can be used in the prevention of autoimmunity before disease onset.

In the current study we aimed to evaluat T cell phenotypes and metabolic profiles in high-risk individuals who progressed to type 1 diabetes compared to those remaining disease free.A Fluorspot assay was used to examine T cell responses to a panel of islet autoantigen peptides in samples obtained 6- and 30-months preceding disease onset and at the same timepoints in non-progressors.We noted a significant increase in the magnitude of the proinflammatory interferon-γ response to proinsulin and insulin peptides in individuals who progressed to type 1 diabetes. In contrast, in the non-progressors, we observed an increase in the regulatory IL-10 response to proinsulin peptides. Furthermore, the T cell responses to the islet peptide panel predisposed towards a proinflammatory interferon-γ bias in the progressors.Collectively, these data suggest that a proinflammatory T cell response is prevalent in high-risk individuals who progress to type 1 diabetes and can be detected up to 6 months prior to onset of disease. This observation, albeit in a small cohort, can potentially be harnessed in disease staging, particularly in identifying autoantibody-positive individuals transitioning from stage 2 (dysglycemia present and pre-symptomatic) to stage 3 (dysglycemia present and symptomatic). The detection of these different T cell phenotypes in progressors and non-progressors suggests the presence of disease endotypes.

Follicular helper T (TFH) cells are implicated in type 1 diabetes (T1D), and their development has been linked to CD28 costimulation. We tested whether TFH cells were decreased by costimulation blockade using the CTLA-4–immunoglobulin (Ig) fusion protein (abatacept) in a mouse model of diabetes and in individuals with new-onset T1D. Unbiased bioinformatics analysis identified that inducible costimulatory molecule (ICOS)+ TFH cells and other ICOS+ populations, including peripheral helper T cells, were highly sensitive to costimulation blockade. We used pretreatment TFH profiles to derive a model that could predict clinical response to abatacept in individuals with T1D. Using two independent approaches, we demonstrated that higher frequencies of ICOS+ TFH cells at baseline were associated with a poor clinical response following abatacept administration. Therefore, TFH analysis may represent a new stratification tool, permitting the identification of individuals most likely to benefit from costimulation blockade. The CTLA-4–Ig fusion protein (abatacept) can have beneficial effects in autoimmune disease. Walker and colleagues show in mouse and human type 1 diabetes that abatacept targets pathogenic follicular helper T cells, and the frequencies of these cells at baseline can be used to stratify treatment responses in patients.

Aims/hypothesis Antigen-specific therapy aims to modify inflammatory T cell responses in type 1 diabetes and restore immune tolerance. One strategy employs GAD65 conjugated to aluminium hydroxide (GAD-alum) to take advantage of the T helper (Th)2-biasing adjuvant properties of alum and thereby regulate pathological Th1 autoimmunity. We explored the cellular and molecular mechanism of GAD-alum action in the setting of a previously reported randomised placebo-controlled clinical trial conducted by Type 1 Diabetes TrialNet. Methods In the clinical trial conducted by Type 1 Diabetes TrialNet, participants were immunised with 20 μg GAD-alum (twice or three times) or alum alone and peripheral blood mononuclear cell samples were banked at baseline and post treatment. In the present study, GAD-specific T cell responses were measured in these samples and GAD-specific T cell lines and clones were generated, which were then further characterised. Results At day 91 post immunisation, we detected GAD-specific IL-13+ CD4 T cell responses significantly more frequently in participants immunised with GAD-alum (71% and 94% treated twice or three times, respectively) compared with those immunised with alum alone (38%; p = 0.003 and p = 0.0002, respectively) accompanied by high secreted levels of IL-13, IL-4 and IL-5, confirming a GAD-specific, GAD-alum-induced Th2 response. Of note, GAD-specific, IL-13+ CD4 T cells observed after immunisation co-secreted IFN-γ, displaying a bifunctional Th1/Th2 phenotype. Single-cell transcriptome analysis identified IL13 and IFNG expression in concert with the canonical Th2 and Th1 transcription factor genes GATA3 and TBX21, respectively. T cell receptor β-chain (TCRB) CDR3 regions of GAD-specific bifunctional T cells were identified in circulating naive and central memory CD4 T cell pools of non-immunised participants with new-onset type 1 diabetes and healthy individuals, suggesting the potential for bifunctional responses to be generated de novo by GAD-alum immunisation or via expansion from an existing public repertoire. Conclusions/interpretation GAD-alum immunisation activates and propagates GAD-specific CD4 T cells with a distinctive bifunctional phenotype, the functional analysis of which might be important in understanding therapeutic responses.

The clinical diagnosis of new-onset type 1 diabetes has, for many years, been considered relatively straightforward. Recently, however, there is increasing awareness that within this single clinical phenotype exists considerable heterogeneity: disease onset spans the complete age range; genetic susceptibility is complex; rates of progression differ markedly, as does insulin secretory capacity; and complication rates, glycemic control, and therapeutic intervention efficacy vary widely. Mechanistic and immunopathological studies typically show considerable patchiness across subjects, undermining conclusions regarding disease pathways. Without better understanding, type 1 diabetes heterogeneity represents a major barrier both to deciphering pathogenesis and to the translational effort of designing, conducting, and interpreting clinical trials of disease-modifying agents. This realization comes during a period of unprecedented change in clinical medicine, with increasing emphasis on greater individualization and precision. For complex disorders such as type 1 diabetes, the option of maintaining the “single disease” approach appears untenable, as does the notion of individualizing each single patient’s care, obliging us to conceptualize type 1 diabetes less in terms of phenotypes (observable characteristics) and more in terms of disease endotypes (underlying biological mechanisms). Here, we provide our view on an approach to dissect heterogeneity in type 1 diabetes. Using lessons from other diseases and the data gathered to date, we aim to delineate a roadmap through which the field can incorporate the endotype concept into laboratory and clinical practice. We predict that such an effort will accelerate the implementation of precision medicine and has the potential for impact on our approach to translational research, trial design, and clinical management.

Aims/hypothesis The molecular basis for the pathological impact of specific HLA molecules on autoimmune diseases such as type 1 diabetes remains unclear. Recent natural history studies in children have indicated a link between specific HLA genotypes and the first antigenic target against which immune responses develop. We set out to examine this link in vivo by exploring the diabetogenicity of islet antigens on the background of a common diabetes-associated HLA haplotype. Methods We generated a novel HLA-transgenic mouse model that expresses high-risk genes for type 1 diabetes (DRB1*03:01-DQA1*05:01-DQB1*02:01) as well as human CD80 under the rat insulin promoter and human CD4, on a C57BL/6 background. Adjuvanted antigen priming was used to reveal the diabetogenicity of candidate antigens and peptides. Results HLA-DR3-DQ2+huCD4+IA/IE−/−RIP.B7.1+ mice spontaneously developed autoimmune diabetes (incidence 46% by 35 weeks of age), accompanied by numerous hallmarks of human type 1 diabetes (autoantibodies against GAD65 and proinsulin; pancreatic islet infiltration by CD4+, CD8+ B220+, CD11b+ and CD11c+ immune cells). Disease was markedly accelerated and had deeper penetrance after adjuvanted antigen priming with proinsulin (mean onset 11 weeks and incidence 100% by 20 weeks post challenge). Moreover, the diabetogenic effect of proinsulin located to the 15-residue B29-C11 region. Conclusions/interpretation Our study identifies a proinsulin-derived peptide region that is highly diabetogenic on the HLA-DR3-DQ2 background using an in vivo model. This approach and the peptide region identified may have wider implications for future studies of human type 1 diabetes.

Antigen specific immunotherapy aims to tolerise patients to specific autoantigens that are responsible for the pathology of an autoimmune disease. Immune tolerance is generated in conditions where the immune response is suppressed and thus gold nanoparticles (AuNPs) are an attractive drug delivery platform due to their anti-inflammatory effects and their potential to facilitate temporal and spatial delivery of a peptide autoantigen in conjunction with pro-tolerogenic elements. In this study we have covalently attached an autoantigen, currently under clinical evaluation for the treatment of type 1 diabetes (PIC19-A3 peptide), to AuNPs to create nanoscale (

AimsRecent studies highlight the potentially important role of neoepitopes in breaking immune tolerance in type 1 diabetes. T cell reactivity to these neoepitopes has been reported, but how this response compares quantitatively and phenotypically with previous reports on native epitopes is not known. Thus, an understanding of the relationship between native and neoepitopes and their role as tolerance breakers or disease drivers in type 1 diabetes is required. We set out to compare T cell reactivity and phenotype against a panel of neo- and native islet autoantigenic epitopes to examine how this relates to stages of type 1 diabetes development.MethodsFifty-four subjects comprising patients with T1D, and autoantibody-positive unaffected family members were tested against a panel of neo- and native epitopes by ELISPOT (IFN-γ, IL-10, and IL-17). A further subset of two patients was analyzed by Single Cell Immune Profiling (RNAseq and TCR α/β) after stimulation with pools of native and neoepitope peptides.ResultsT cell responses to native and neoepitopes were present in patients with type 1 diabetes and at-risk subjects, and overall, there were no significant differences in the frequency, magnitude, or phenotype between the two sets of peptide stimuli. Single cell RNAseq on responder T cells revealed a similar profile in T1D patients stimulated with either neo- or native epitopes. A pro-inflammatory gene expression profile (TNF-α, IFN-γ) was dominant in both native and neoepitope stimulated T cells. TCRs with identical clonotypes were found in T cell responding to both native and neoepitopes.Conclusion/InterpretationThese data suggest that in peripheral blood, T cell responses to both native and neoepitopes are similar in terms of frequency and phenotype in patients with type 1 diabetes and high-risk unaffected family members. Furthermore, using a combination of transcriptomic and clonotypic analyses, albeit using a limited panel of peptides, we show that neoepitopes are comparable to native epitopes currently in use for immune-monitoring studies.

Autoimmune diseases and in particular type 1 diabetes rely heavily on treatments that target the symptoms rather than prevent the underlying disease. One of the barriers to better therapeutic strategies is the inability to detect and efficiently target rare autoreactive T-cell populations that are major drivers of these conditions. Here, we develop a unique artificial antigen presenting cell (aAPC) system from biocompatible polymer particles that allows specific encapsulation of bioactive ingredients. Using our aAPC we demonstrate that we are able to detect rare autoreactive CD4 populations in human patients and using mouse models we demonstrate that our particles are able to induce desensitization in the autoreactive population. This system provides a promising tool that can be used in the prevention of autoimmunity before disease onset.

The Nuclear Factor Kappa B (NFκB) pathway is an important signalling pathway in the immune system. Single gene defects in the NFκB pathway are described in a number of immunodeficiency diseases. These conditions provide a unique opportunity to investigate the mechanisms of NFκB function and how genetic mutations that disrupt this function lead to human disease. Here we describe a robust method for quantifying small differences in the functional activity of the NFκB pathway. Peripheral blood mononuclear cells from healthy donors were stimulated over several days, with a combination of anti-IgM antibody and multimeric CD40 ligand. Nuclear proteins were thereafter extracted and tested for the ability of activated transcription factors, to bind known NFκB DNA binding motifs. Repeatability experiments showed that the DNA binding Activity can be quantified with an average inter and intra assay coefficient of variation of less than 10% (RelB and p52) and less than 15% (p50 and RelA). In healthy individuals there is a significant increase in the DNA binding activity of NFκB transcription factors in response to stimulation, although the magnitude of this response varies across individuals. The kinetics of the DNA binding activity also differs between the canonical and non-canonical transcription factors. P50 and RelA DNA binding activity responds within hours of stimulation, whilst RelB and p52 response was delayed to more than a day after stimulation. Activation of NFκB signalling in response to B cell specific stimulation, can be precisely measured to distinguish individuals with differences in the functional activity of this pathway. This test may prove to be an important biomarker for investigating the functional impact of genetic variants on NFκB signalling.

In this study, we sought to fill an important gap in fundamental immunology research by conducting a comprehensive systems immunology analysis of daily variation in the normal human peripheral immune system. Although variation due to circadian rhythmicity was not a significant source of variation in daily B-cell levels or any CD4+ functional subset, it accounted for more than 25% of CD4+ regulatory T-cell variation and over 50% of CD8+ central memory variation. Circadian rhythmicity demonstrated phase alignment within functional phenotypes. In addition, we observed that previously-described mechanistic relationships can also appear in the peripheral system as phase shifting in rhythmic patterns. We identified a set of immune factors which are ubiquitously correlated with other factors and further analysis also identified a tightly-correlated “core” set whose relational structure persisted after analytically removing circadian-related variation. This core set consisted of CD8+ and its subpopulations and the NK population. In sum, the peripheral immune system can be conceptualized as a dynamic, interconnected wave-field repeating its pattern on a daily basis. Our data provide a comprehensive inventory of synchronization and correlation within this wave-field and we encourage use of our data to discover unknown mechanistic relationships which can then be tested in the laboratory.

We previously reported that costimulation blockade by abatacept limits the decline of β-cell function and the frequency of circulating CD4+ central memory T cells (TCM) (CD45RO+CD62L+) in new-onset type 1 diabetes. In human subjects receiving placebo, we found a significant association between an increase in CD4+ TCM cells and the decline of β-cell function. To extend and refine these findings, we examined changes in human CD4+ and CD8+ naive and memory T cell subsets at greater resolution using polychromatic flow and mass cytometry. In the placebo group, we successfully reproduced the original finding of a significant association between TCM and β-cell function and extended this to other T cell subsets. Furthermore, we show that abatacept treatment significantly alters the frequencies of a majority of CD4+ conventional and regulatory T cell subsets; in general, Ag-naive subsets increase and Ag-experienced subsets decrease, whereas CD8+ T cell subsets are relatively resistant to drug effects, indicating a lesser reliance on CD28-mediated costimulation. Importantly, abatacept uncouples the relationship between changes in T cell subsets and β-cell function that is a component of the natural history of the disease. Although these data suggest immunological markers for predicting change in β-cell function in type 1 diabetes, the finding that abatacept blunts this relationship renders the biomarkers nonpredictive for this type of therapy. In sum, our findings point to a novel mechanism of action for this successful immunotherapy that may guide other disease-modifying approaches for type 1 diabetes.

The ready availability of human blood makes it the first choice for immuno-monitoring. However, this has been largely confined to static metrics, particularly resting T cell phenotypes. Conversely, dynamic assessments have mostly relied on cell stimulation in vitro which is subject to multiple variables. Here, immunodynamic insights from the peripheral blood are shown to be obtainable by applying a revised approach to cell-cycle analysis. Specifically, refined flow cytometric protocols were employed, assuring the reliable quantification of T cells in the S-G2/M phases of the cell-cycle (collectively termed “T Double S” for T cells in S-phase in Sanguine: in short “TDS” cells). Without protocol refinement, TDS could be either missed, as most of them layed out of the conventional lymphocyte gates, or confused with cell doublets artefactually displaying high DNA-content. To illustrate the nature of TDS cells, and their relationship to different immunodynamic scenarios, we examined them in healthy donors (HD); infectious mononucleosis (IM) patients versus asymptomatic EBV+ carriers; and recently-diagnosed T1D patients. TDS were reproducibly more abundant among CD8+ T cells and a defined subset of T-regulatory CD4+ T cells, and were substantially increased in IM and a subset of T1D patients. Of note, islet antigen-reactive TDS cell frequencies were associated with an aggressive T cell effector phenotype, suggesting that peripheral blood can reflect immune events within tissues in T1D, and possibly in other organ-specific autoimmune diseases. Our results suggest that tracking TDS cells may provide a widely applicable means of gaining insight into ongoing immune response dynamics in a variety of settings, including tissue immunopathologies where the peripheral blood has often not been considered insightful., Highlights • “TDS” cells (i.e. in S-G2/M phases of cell cycle) are present in peripheral blood. • TDS cells are represented to different degrees in different T cell subtypes. • TDS cells were associated with activated effector functions in T1D patients. • TDS analyses can potentially functionally refine immuno-monitoring in many settings. • Abstract.

Antigen-specific immunotherapy of autoimmune disease currently remains the only potentially curative approach. However, translation of promising pre-clinical results into successful clinical application has proven challenging. In part, this is because pre-clinical findings in mouse models have to be redesigned for human application due to differences in MHC II. To reduce the gap between pre-clinical and clinical studies, we have created a novel mouse model that expresses human HLA-DR4, but no endogenous MHC on antigen-presenting cells. Moreover, human B7.1 (CD80) is expressed in the pancreatic islets under the control of the rat insulin promoter. Although this model does not develop diabetes spontaneously, it is susceptible to the induction of type 1 diabetes by challenging mice with overlapping peptides derived from murine proinsulin-2 in adjuvant. Unlike the NOD model of spontaneous type 1 diabetes, but akin to the human condition, this model does not have a gender bias. Furthermore, similar to the human condition, the disease is characterised by a diverse leucocyte infiltration of the pancreatic islets and the formation of anti-proinsulin auto-antibodies. The model that we report here offers detailed insights into type-1 diabetes and is expected to prove instrumental when studying the mechanism of action in translational, antigen-specific immunotherapy.

Based on the success in animal models of type 1 diabetes (T1D), clinical trials of adoptive regulatory T cell (Treg) therapy are underway using ex vivo expanded polyclonal Tregs. However, pre-clinical data also demonstrate that islet-specific Tregs are more potent than polyclonal Tregs at reversing T1D. Translation of this approach into man will require methods to generate large populations of islet-specific Tregs which, to date, has proved to be a major hurdle. Here we demonstrate the feasibility of lentiviral-mediated T cell receptor (TCR) gene transfer to confer antigen specificity on polyclonal human Tregs. Targeting has been achieved using TCRs isolated from human islet-specific and viral-specific CD4+ T cell clones. Engineered T cells demonstrated expression of ectopically-delivered TCRs, resulting in endowment of cognate antigen-specific responses. This enabled antigen-specific suppression at increased potency compared to polyclonal Tregs. However, cells transduced with islet-specific TCRs were less responsive to cognate antigen than viral-specific TCRs, and in some cases, required additional methods to isolate functional antigen-specific Tregs. This study demonstrates the potential of TCR gene transfer to develop islet-specific Treg therapies for effective treatment of T1D, but also highlights that additional optimisation may be required to achieve its full potential.

We previously reported that co-stimulation blockade by abatacept limits the decline of β-cell function and the frequency of circulating CD4(+) central memory (CD45RO(+)CD62L(+)) T (T(CM)) cells in new-onset type 1 diabetes. In human subjects receiving placebo we found a significant association between increase in CD4(+) T(CM) cells and decline of β-cell function. To extend and refine these findings, we examined changes in human CD4(+) and CD8(+) naïve and memory T-cell subsets at greater resolution using polychromatic flow and mass cytometry. In the placebo group we successfully reproduced the original finding of a significant association between T(CM) and β-cell function, and extended this to other T-cell subsets. Furthermore, we show that abatacept treatment significantly alters the frequencies of a majority of CD4(+) conventional and regulatory T-cell subsets; in general, antigen-naïve subsets increase and antigen-experienced decrease, while CD8(+) T-cell subsets are relatively resistant to drug effects, indicating a lesser reliance on CD28-mediated co-stimulation. Importantly, abatacept uncouples the relationship between changes in T-cell subsets and β-cell function that is a component of the natural history of the disease. Although these data suggest immunological markers for predicting change in β-cell function in type 1 diabetes, the finding that abatacept blunts this relationship renders the biomarkers non-predictive for this type of therapy. In sum, our findings point to a novel mechanism of action for this successful immunotherapy that may guide other disease-modifying approaches for type 1 diabetes.

With the conceptual advance some four decades ago that type 1 diabetes (T1D) represents an autoimmune disease, hope emerged that immune-based therapies would quickly evolve as a means to prevent and reverse the disorder. However, despite dozens of clinical trials seeking to achieve that purpose, the goal remains unfulfilled, at least in a pragmatic form. With the benefit of hindsight, multiple reasons are likely to account for this unfortunate situation, and several stand out: failure to appreciate disease heterogeneity; inappropriate utilization of and insight from rodent models of disease; inadequacies in addressing the immunologic and metabolic contributions to the disease; suboptimal trial designs; and lack of a clear understanding of the disorder’s pathogenesis. This review conveys how recent knowledge gains in these areas, combined with efforts related to disease staging and emerging mechanistic data from clinical trials, provide cautious optimism that an immune-based means to prevent the loss of β-cells in T1D will emerge into clinical practice.

Background: Peptide immunotherapy (PIT) aims to modulate immune responses to specific antigens in order to restore immune tolerance. Recently, we reported outcomes of a phase 1b study of PIT at type 1 diabetes onset, showing safety, tolerability and changes in immune regulation pathways, following up to 12 doses of 10μg of a single proinsulin peptide that is naturally processed and presented by HLA-DR4 (DRB1*0401). Preclinical PIT studies show that multiple peptides in combination enhance tolerance induction. Aims: To evaluate the safety and clinical effects of PIT using a mix of multiple islet autoantigen peptides in adults with recent-onset type 1 diabetes. Methods: The MultiPepT1De study was a single centre, double-blind placebo-controlled, ascending dose, randomized trial recruiting adults aged 18-45 years within 4 years of diagnosis with HLA-DRB1*0401 genotype, positive islet autoantibodies and stimulated C-peptide >0.2pmol/mL. Study drug comprised of 6 naturally processed and presented, islet-derived peptides (MultiPepT1De). Participants were enrolled into 3 cohorts for monthly intradermal injections for 6 months. In each cohort, subjects were randomized to receive placebo (n=2) or MultiPepT1De (n=6) at 10µg (Cohort 1), 100µg (Cohort 2) or 500µg (Cohort 3) doses. Results: Twenty-seven participants were randomized; 3 participants withdrew for non-clinical reasons and were replaced. There were no episodes of systemic hypersensitivity, anaphylaxis or serious adverse reactions linked to MultiPepT1De administration. Analysis of changes in stimulated C-peptide, HbA1c and insulin dose per kg showed no evidence of disease acceleration following dosing compared with placebo. Mild transient injection site erythema was observed in all cohorts, with no increase on repeated dosing. Conclusions: Intradermal administration of multiple islet-derived peptides up to a cumulative dose of 3000µg, is well tolerated with no safety concerns. Disclosure Y. Liu: Other Relationship; Self; Novo Nordisk Limited. Research Support; Self; Bristol-Myers Squibb Company. J.K. Powrie: None. S. Arif: None. N. Fountoulakis: None. M. Joshi: None. E.L. Smith: Employee; Self; UCB, Inc. F. Strimenopoulou: Employee; Self; UCB, Inc. M. Thomson: Employee; Self; UCB, Inc. M. Peakman: Research Support; Self; Bristol-Myers Squibb Company. Advisory Panel; Self; Bristol-Myers Squibb Company. Consultant; Self; UCB, Inc.. Research Support; Self; UCB, Inc.. Advisory Panel; Self; T1D Exchange.

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.

Objectives To describe the characteristics of children and adults with incident type 1 diabetes in contemporary, multiethnic UK, focusing on differences between the islet autoantibody negative and positive. Design Observational cohort study. Setting 146 mainly secondary care centres across England and Wales. Participants 3312 people aged ≥5 years were recruited within 6 months of a clinical diagnosis of type 1 diabetes via the National Institute for Health Research Clinical Research Network. 3021 were of white European ethnicity and 291 (9%) were non-white. There was a small male predominance (57%). Young people

Blood-based biomarker discovery for psychotic disorders has yet to impact upon routine clinical practice. In physical disorders antibodies have established roles as diagnostic, prognostic and predictive (theranostic) biomarkers, particularly in disorders thought to have a substantial autoimmune or infective aetiology. Two approaches to antibody biomarker identification are distinguished: a “top-down” approach, in which antibodies to specific antigens are sought based on the known function of the antigen and its putative role in the disorder, and emerging “bottom-up” or “omics” approaches that are agnostic as to the significance of any one antigen, using high-throughput arrays to identify distinctive components of the antibody repertoire. Here we review the evidence for antibodies (to self-antigens as well as infectious organism and dietary antigens) as biomarkers of diagnosis, prognosis, and treatment response in psychotic disorders. Neuronal autoantibodies have current, and increasing, clinical utility in the diagnosis of organic or atypical psychosis syndromes. Antibodies to selected infectious agents show some promise in predicting cognitive impairment and possibly other symptom domains (eg, suicidality) within psychotic disorders. Finally, infectious antibodies and neuronal and other autoantibodies have recently emerged as potential biomarkers of response to anti-infective therapies, immunotherapies, or other novel therapeutic strategies in psychotic disorders, and have a clear role in stratifying patients for future clinical trials. As in nonpsychiatric disorders, combining biomarkers and large-scale use of “bottom-up” approaches to biomarker identification are likely to maximize the eventual clinical utility of antibody biomarkers in psychotic disorders.

In type 1 diabetes, cytotoxic CD8+ T cells with specificity for β cell autoantigens are found in the pancreatic islets, where they are implicated in the destruction of insulin-secreting β cells. In contrast, the disease relevance of β cell-reactive CD8+ T cells that are detectable in the circulation, and their relationship to β cell function, are not known. Here, we tracked multiple, circulating β cell-reactive CD8+ T cell subsets and measured β cell function longitudinally for 2 years, starting immediately after diagnosis of type 1 diabetes. We found that change in β cell-specific effector memory CD8+ T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8+ T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer-specific protein of 37 kDa, and CD16, and reduced expression of CD28) compared with their CD57-counterparts, and network association modeling indicated that the dynamics of β cell-reactive CD57+ effector memory CD8+ T cell subsets were strongly linked. Thus, coordinated changes in circulating β cell-specific CD8+ T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.

Coxsackie B Virus (CBV) infection has been linked to the aetiology of type 1 diabetes (T1D) and vaccination has been proposed as prophylaxis for disease prevention. Serum neutralising antibodies and the presence of viral protein and RNA in tissues have been common tools to examine this potential disease relationship, whilst the role of anti-CBV cytotoxic T cell responses and their targets have not been studied. To address this knowledge gap, we augmented conventional HLA-binding predictive algorithm-based epitope discovery by cross-referencing epitopes with sites of positive natural selection within the CBV3 viral genome, identified using mixed effects models of evolution. Eight epitopes for the common MHC class I allele HLA-A*0201 occur at sites that appear to be positively selected. Furthermore, such epitopes span the viral genome, indicating that effective anti-viral responses may not be restricted to the capsid region. To assess the spectrum of IFNy responses in non-diabetic subjects and recently diagnosed type 1 diabetes (T1D) patients, we stimulated PBMC ex vivo with pools of synthetic peptides based on component-restricted sequences identified in silico. We found responders were more likely to recognize multiple rather than a single CBV peptide pool, indicating that the natural course of infection results in multiple targets for effector memory responses, rather than immunodominant epitopes or viral components. The finding that anti-CBV CD8 T cell immunity is broadly targeted has implications for vaccination strategies and studies on the pathogenesis of CBV-linked diseases.

BACKGROUND. Neutrophils and their inflammatory mediators are key pathogenic components in multiple autoimmune diseases, while their role in human type 1 diabetes (T1D), a disease that progresses sequentially through identifiable stages prior to the clinical onset, is not well understood. We previously reported that the number of circulating neutrophils is reduced in patients with T1D and in presymptomatic at-risk subjects. The aim of the present work was to identify possible changes in circulating and pancreas-residing neutrophils throughout the disease course to better elucidate neutrophil involvement in human T1D. METHODS. Data collected from 389 subjects at risk of developing T1D, and enrolled in 4 distinct studies performed by TrialNet, were analyzed with comprehensive statistical approaches to determine whether the number of circulating neutrophils correlates with pancreas function. To obtain a broad analysis of pancreas-infiltrating neutrophils throughout all disease stages, pancreas sections collected worldwide from 4 different cohorts (i.e., nPOD, DiViD, Siena, and Exeter) were analyzed by immunohistochemistry and immunofluorescence. Finally, circulating neutrophils were purified from unrelated nondiabetic subjects and donors at various T1D stages and their transcriptomic signature was determined by RNA sequencing. RESULTS. Here, we show that the decline in β cell function is greatest in individuals with the lowest peripheral neutrophil numbers. Neutrophils infiltrate the pancreas prior to the onset of symptoms and they continue to do so as the disease progresses. Of interest, a fraction of these pancreas-infiltrating neutrophils also extrudes neutrophil extracellular traps (NETs), suggesting a tissue-specific pathogenic role. Whole-transcriptome analysis of purified blood neutrophils revealed a unique molecular signature that is distinguished by an overabundance of IFN-associated genes; despite being healthy, said signature is already present in T1D-autoantibody-negative at-risk subjects. CONCLUSIONS. These results reveal an unexpected abnormality in neutrophil disposition both in the circulation and in the pancreas of presymptomatic and symptomatic T1D subjects, implying that targeting neutrophils might represent a previously unrecognized therapeutic modality. FUNDING. Juvenile Diabetes Research Foundation (JDRF), NIH, Diabetes UK.

HLA-DQ2/8 heterozygous individuals are at far greater risk for type 1 diabetes (T1D) development by expressing HLA-DQ8trans on antigen-presenting cells compared with HLA-DQ2 or -DQ8 homozygous individuals. Dendritic cells (DC) initiate and shape adaptive immune responses by presenting HLA-epitope complexes to naïve T cells. To dissect the role of HLA-DQ8trans in presenting natural islet epitopes, we analyzed the islet peptidome of HLA-DQ2, -DQ8, and -DQ2/8 by pulsing DC with preproinsulin (PPI), IA-2, and GAD65. Quality and quantity of islet epitopes presented by HLA-DQ2/8 differed from -DQ2 or -DQ8. We identified two PPI epitopes solely processed and presented by HLA-DQ2/8 DC: an HLA-DQ8trans–binding signal-sequence epitope previously identified as CD8 T-cell epitope and a second epitope that we previously identified as CD4 T-cell epitope with increased binding to HLA-DQ8trans upon posttranslational modification. IA-2 epitopes retrieved from HLA-DQ2/8 and -DQ8 DC bound to HLA-DQ8cis/trans. No GAD65 epitopes were eluted from HLA-DQ. T-cell responses were detected against the novel islet epitopes in blood from patients with T1D but scantly detected in healthy donor subjects. We report the first PPI and IA-2 natural epitopes presented by highest-risk HLA-DQ8trans. The selective processing and presentation of HLA-DQ8trans–binding islet epitopes provides insight in the mechanism of excessive genetic risk imposed by HLA-DQ2/8 heterozygosity and may assist immune monitoring of disease progression and therapeutic intervention as well as provide therapeutic targets for immunotherapy in subjects at risk for T1D.

Analysis of antigen-specific T-cell populations by flow cytometry with peptide–MHC (pMHC) multimers is now commonplace. These reagents allow the tracking and phenotyping of T cells during infection, autoimmunity and cancer, and can be particularly revealing when used for monitoring therapeutic interventions. In 2009, we reviewed a number of ‘tricks’ that could be used to improve this powerful technology. More recent advances have demonstrated the potential benefits of using higher order multimers and of ‘boosting’ staining by inclusion of an antibody against the pMHC multimer. These developments now allow staining of T cells where the interaction between the pMHC and the T-cell receptor is over 20-fold weaker (KD > 1 mm) than could previously be achieved. Such improvements are particularly relevant when using pMHC multimers to stain anti-cancer or autoimmune T-cell populations, which tend to bear lower affinity T-cell receptors. Here, we update our previous work to include discussion of newer tricks that can produce substantially brighter staining even when using log-fold lower concentrations of pMHC multimer. We further provide a practical guide to using pMHC multimers that includes a description of several common pitfalls and how to circumvent them.

Type 1 diabetes is an autoimmune disease arising from the destruction of pancreatic insulin-producing beta cells. The disease represents a continuum, progressing sequentially at variable rates through identifiable stages prior to the onset of symptoms, through diagnosis and into the critical periods that follow, culminating in a variable depth of beta cell depletion. The ability to identify the very earliest of these presymptomatic stages has provided a setting in which prevention strategies can be trialled, as well as furnishing an unprecedented opportunity to study disease evolution, including intrinsic and extrinsic initiators and drivers. This niche opportunity is occupied by Type 1 Diabetes TrialNet, an international consortium of clinical trial centres that leads the field in intervention and prevention studies, accompanied by deep longitudinal bio-sampling. In this review, we focus on discoveries arising from this unique bioresource, comprising more than 70,000 samples, and outline the processes and science that have led to new biomarkers and mechanistic insights, as well as identifying new challenges and opportunities. We conclude that via integration of clinical trials and mechanistic studies, drawing in clinicians and scientists and developing partnership with industry, TrialNet embodies an enviable and unique working model for understanding a disease that to date has no cure and for designing new therapeutic approaches.

Defects in T cell receptor (TCR) repertoire are proposed to predispose to autoimmunity. Here we show, by analyzing >2 × 108 TCRB sequences of circulating naive, central memory, regulatory and stem cell-like memory CD4+ T cell subsets from patients with type 1 diabetes and healthy donors, that patients have shorter TCRB complementarity-determining region 3s (CDR3), in all cell subsets, introduced by increased deletions/reduced insertions during VDJ rearrangement. High frequency of short CDR3s is also observed in unproductive TCRB sequences, which are not subjected to thymic culling, suggesting that the shorter CDR3s arise independently of positive/negative selection. Moreover, TCRB CDR3 clonotypes expressed by autoantigen-specific CD4+ T cells are shorter compared with anti-viral T cells, and with those from healthy donors. Thus, early events in thymic T cell development and repertoire generation are abnormal in type 1 diabetes, which suggest that short CDR3s increase the potential for self-recognition, conferring heightened risk of autoimmune disease., T cell receptors are generated by somatic gene recombination, and are normally selected against autoreactivity. Here the authors show that CD4 T cells from patients with autoimmune type 1 diabetes have shorter TCRβ sequences, broader repertoire diversity, and more repertoire sharing than those from healthy individuals.

Interleukin (IL)-36α, IL-36β, and IL-36γ are innate mediators of acute epithelial inflammation. We sought to demonstrate that these cytokines are also required for the pathogenesis of plaque psoriasis, a common and chronic skin disorder, caused by abnormal T helper 17 (TH17) cell activation. To investigate this possibility, we first defined the genes that are induced by IL-36 cytokines in primary human keratinocytes. This enabled us to demonstrate a significant IL-36 signature among the transcripts that are up-regulated in plaque psoriasis and the susceptibility loci associated with the disease in genome-wide studies. Next, we investigated the impact of in vivo and ex vivo IL-36 receptor blockade using a neutralizing antibody or a recombinant antagonist. Both inhibitors had marked anti-inflammatory effects on psoriatic skin, demonstrated by statistically significant reductions in IL-17 expression, keratinocyte activation, and leukocyte infiltration. Finally, we explored the potential safety profile associated with IL-36 blockade by phenotyping 12 individuals carrying knockout mutations of the IL-36 receptor gene. We found that normal immune function was broadly preserved in these individuals, suggesting that IL-36 signaling inhibition would not substantially compromise host defenses. These observations, which integrate the results of transcriptomics and model system analysis, pave the way for early-stage clinical trials of IL-36 antagonists.

Immunotherapy using short immunogenic peptides of disease-related autoantigens restores immune tolerance in preclinical disease models. We studied safety and mechanistic effects of injecting human leukocyte antigen-DR4(DRB1*0401)-restricted immunodominant proinsulin peptide intradermally every 2 or 4 weeks for 6 months in newly diagnosed type 1 diabetes patients. Treatment was well tolerated with no systemic or local hypersensitivity. Placebo subjects showed a significant decline in stimulated C-peptide (measuring insulin reserve) at 3, 6, 9, and 12 months versus baseline, whereas no significant change was seen in the 4-weekly peptide group at these time points or the 2-weekly group at 3, 6, and 9 months. The placebo group's daily insulin use increased by 50% over 12 months but remained unchanged in the intervention groups. C-peptide retention in treated subjects was associated with proinsulin-stimulated interleukin-10 production, increased FoxP3 expression by regulatory T cells, low baseline levels of activated β cell-specific CD8 T cells, and favorable β cell stress markers (proinsulin/C-peptide ratio). Thus, proinsulin peptide immunotherapy is safe, does not accelerate decline in β cell function, and is associated with antigen-specific and nonspecific immune modulation.