Mark Peakman

Autoreactive T cell responses show proinflammatory polarization in diabetes but a regulatory phenotype in health

According to the quality of response they mediate, autoreactive T cells recognizing islet beta cell peptides could represent both disease effectors in the development of type 1 diabetes (T1DM) and directors of tolerance in nondiabetic individuals or those undergoing preventative immunotherapy. A combination of the rarity of these cells, inadequate technology, and poorly defined epitopes, however, has hampered examination of this paradigm. We have identified a panel of naturally processed islet epitopes by direct elution from APCs bearing HLA-DR4. Employing these epitopes in a sensitive, novel cytokine enzyme-linked immunosorbent spot assay, we show that the quality of autoreactive T cells in patients with T1DM exhibits extreme polarization toward a proinflammatory Th1 phenotype. Furthermore, we demonstrate that rather than being unresponsive, the majority of nondiabetic, HLA-matched control subjects also manifest a response against islet peptides, but one that shows extreme T regulatory cell (Treg, IL-10-secreting) bias. We conclude that development of T1DM depends on the balance of autoreactive Th1 and Treg cells, which may be open to favorable manipulation by immune intervention.

CTLs are targeted to kill β cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

The final pathway of beta cell destruction leading to insulin deficiency, hyperglycemia, and clinical type 1 diabetes is unknown. Here we show that circulating CTLs can kill beta cells via recognition of a glucose-regulated epitope. First, we identified 2 naturally processed epitopes from the human preproinsulin signal peptide by elution from HLA-A2 (specifically, the protein encoded by the A*0201 allele) molecules. Processing of these was unconventional, requiring neither the proteasome nor transporter associated with processing (TAP). However, both epitopes were major targets for circulating effector CD8+ T cells from HLA-A2+ patients with type 1 diabetes. Moreover, cloned preproinsulin signal peptide-specific CD8+ T cells killed human beta cells in vitro. Critically, at high glucose concentration, beta cell presentation of preproinsulin signal epitope increased, as did CTL killing. This study provides direct evidence that autoreactive CTLs are present in the circulation of patients with type 1 diabetes and that they can kill human beta cells. These results also identify a mechanism of self-antigen presentation that is under pathophysiological regulation and could expose insulin-producing beta cells to increasing cytotoxicity at the later stages of the development of clinical diabetes. Our findings suggest that autoreactive CTLs are important targets for immune-based interventions in type 1 diabetes and argue for early, aggressive insulin therapy to preserve remaining beta cells.

A single autoimmune T cell receptor recognizes more than a million different peptides

Background: How does a limited pool of <108 T cell receptors (TCRs) provide immunity to >1015 antigens? Results: A single TCR can respond to >one million different decamer peptides. Conclusion: This unprecedented level of receptor promiscuity explains how the naïve TCR repertoire achieves effective immunity. Significance: TCR degeneracy has enormous potential to be the root cause of autoimmune disease. The T cell receptor (TCR) orchestrates immune responses by binding to foreign peptides presented at the cell surface in the context of major histocompatibility complex (MHC) molecules. Effective immunity requires that all possible foreign peptide-MHC molecules are recognized or risks leaving holes in immune coverage that pathogens could quickly evolve to exploit. It is unclear how a limited pool of <108 human TCRs can successfully provide immunity to the vast array of possible different peptides that could be produced from 20 proteogenic amino acids and presented by self-MHC molecules (>1015 distinct peptide-MHCs). One possibility is that T cell immunity incorporates an extremely high level of receptor degeneracy, enabling each TCR to recognize multiple peptides. However, the extent of such TCR degeneracy has never been fully quantified. Here, we perform a comprehensive experimental and mathematical analysis to reveal that a single patient-derived autoimmune CD8+ T cell clone of pathogenic relevance in human type I diabetes recognizes >one million distinct decamer peptides in the context of a single MHC class I molecule. A large number of peptides that acted as substantially better agonists than the wild-type “index” preproinsulin-derived peptide (ALWGPDPAAA) were identified. The RQFGPDFPTI peptide (sampled from >108 peptides) was >100-fold more potent than the index peptide despite differing from this sequence at 7 of 10 positions. Quantification of this previously unappreciated high level of CD8+ T cell cross-reactivity represents an important step toward understanding the system requirements for adaptive immunity and highlights the enormous potential of TCR degeneracy to be the causative factor in autoimmune disease.

Translational Mini‐Review Series on Type 1 Diabetes: Systematic analysis of T cell epitopes in autoimmune diabetes

T cell epitopes represent the molecular code words through which the adaptive immune system communicates. In the context of a T cell‐mediated autoimmune disease such as type 1 diabetes, CD4 and CD8 T cell recognition of islet autoantigenic epitopes is a key step in the autoimmune cascade. Epitope recognition takes place during the generation of tolerance, during its loss as the disease process is initiated, and during epitope spreading as islet cell damage is perpetuated. Epitope recognition is also a potentially critical element in therapeutic interventions such as antigen‐specific immunotherapy. T cell epitope discovery, therefore, is an important component of type 1 diabetes research, in both human and murine models. With this in mind, in this review we present a comprehensive guide to epitopes that have been identified as T cell targets in autoimmune diabetes. Targets of both CD4 and CD8 T cells are listed for human type 1 diabetes, for humanized [human leucocyte antigen (HLA)‐transgenic] mouse models, and for the major spontaneous disease model, the non‐obese diabetic (NOD) mouse. Importantly, for each epitope we provide an analysis of the relative stringency with which it has been identified, including whether recognition is spontaneous or induced and whether there is evidence that the epitope is generated from the native protein by natural antigen processing. This analysis provides an important resource for investigating diabetes pathogenesis, for developing antigen‐specific therapies, and for developing strategies for T cell monitoring during disease development and therapeutic intervention.

Resident CD141 (BDCA3)+ dendritic cells in human skin produce IL-10 and induce regulatory T cells that suppress skin inflammation

Human skin-resident IL-10+ regulatory dendritic cells induce T reg cells that suppress allogeneic skin graft inflammation.

Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers.

Flow cytometry with fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) tetramers has transformed the study of antigen-specific T-cells by enabling their visualization, enumeration, phenotypic characterization and isolation from ex vivo samples. Here, we demonstrate that the reversible protein kinase inhibitor (PKI) dasatinib improves the staining intensity of human (CD8+ and CD4+) and murine T-cells without concomitant increases in background staining. Dasatinib enhances the capture of cognate pMHC tetramers from solution and produces higher intensity staining at lower pMHC concentrations. Furthermore, dasatinib reduces pMHC tetramer-induced cell death and substantially lowers the T-cell receptor (TCR)/pMHC interaction affinity threshold required for cell staining. Accordingly, dasatinib permits the identification of T-cells with very low affinity TCR/pMHC interactions, such as those that typically predominate in tumour-specific responses and autoimmune conditions that are not amenable to detection by current technology.

Simultaneous detection of circulating autoreactive CD8+ T-cells specific for different islet cell-associated epitopes using combinatorial MHC multimers.

OBJECTIVE Type 1 diabetes results from selective T-cell–mediated destruction of the insulin-producing β-cells in the pancreas. In this process, islet epitope–specific CD8+ T-cells play a pivotal role. Thus, monitoring of multiple islet–specific CD8+ T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive. RESEARCH DESIGN AND METHODS Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B10–18, prepro-insulin (PPI)15–24, islet antigen (IA)-2797–805, GAD65114–123, islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP)265–273, and prepro islet amyloid polypeptide (ppIAPP)5–13–specific CD8+ T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients. RESULTS Using this kit, islet autoreactive CD8+ T-cells recognizing insulin B10–18, IA-2797–805, and IGRP265–273 were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI15–24 proved to be the most sensitive epitope. Applying the “Diab-Q-kit” to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell–derived epitopes that were associated with disease activity and correlated with clinical outcome. CONCLUSIONS A kit was developed that allows simultaneous detection of CD8+ T-cells reactive to multiple HLA-A2–restricted β-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.

Naturally processed and presented epitopes of the islet cell autoantigen IA-2 eluted from HLA-DR4

During immune responses, antigen-presenting cells (APCs) process antigens and present peptide epitopes complexed with human leukocyte antigen (HLA) molecules. CD4 cells recognize these naturally processed and presented epitopes (NPPEs) bound to HLA class II molecules. Epitope identification is important for developing diagnostic and therapeutic tools for immune-mediated diseases and providing insight into their etiology, but current approaches overlook effects of natural processing on epitope selection. We have developed a technique to identify NPPEs using mass spectrometry (MS) after antigen is targeted onto APCs using a lectin-based antigen delivery system (ADS). We applied the technique to identify NPPEs of the intracellular domain of the type 1 diabetes mellitus-associated (type 1 DM-associated) autoantigen insulinoma-associated-2 (IA-2ic), presented by HLA-DR4 (0401). IA-2ic-derived NPPEs eluted from HLA-DR4 constitute 6 sets of peptides nested around distinct core regions. Synthetic peptides based on these regions bind HLA-DR4 and elicit primary T-cell proliferation frequently in HLA-DR4-positive type 1 DM patients, but rarely in non-HLA-DR4 patients, and in none of the HLA-DR4 nondiabetic controls we tested. This flexible, direct approach identifies an HLA allele-specific map of NPPEs for any antigen, presented by any HLA class II molecule. This method should enable a greater understanding of epitope selection and lead to the generation of sensitive and specific reagents for detecting autoreactive T cells.

Translational Mini‐Review Series on Type 1 Diabetes: Immune‐based therapeutic approaches for type 1 diabetes

Type 1 diabetes (T1D) is often considered the prototype organ‐specific autoimmune disease in clinical immunology circles. The key disease features − precise destruction of a single endocrine cell type occurring on a distinct genetic and autoimmune background − have been unravelled in recent years to such an extent that there is a growing expectation that the disease should be curable. T1D is something of an orphan disease, currently managed by endocrinologists yet dependent upon the wit of immunologists, both basic and clinical, to find the best approaches to prevention and cure. Type 1 diabetes thus represents one of the most active arenas for translational research, as novel immune‐based interventions find their way to the clinic. The first serious attempt at immune‐based treatment for T1D was in 1984, the first at prevention in 1993; current and planned trials will take us into the next decade before reporting their results. This paper represents the first attempt at a comprehensive review of this quarter century of endeavour, documenting all the strategies that have emerged into clinical studies. Importantly, the intense clinical activity has established robust infrastructures for future T1D trials and frameworks for their design. The evident success of the monoclonal anti‐CD3 antibody trials in established T1D demonstrate that modulation of islet autoimmunity in humans after the onset of overt disease can be achieved, and give some reason to be cautiously optimistic for the ability of these and other agents, alone and in combination, to provide an effective immunotherapy for the disease.

Increased resistance to CD4+CD25hi regulatory T cell-mediated suppression in patients with type 1 diabetes

Type I diabetes (T1D) is a T cell‐mediated autoimmune disease characterized by loss of tolerance to islet autoantigens, leading to the destruction of insulin‐producing beta cells. Peripheral tolerance to self is maintained in health through several regulatory mechanisms, including a population of CD4+CD25hi naturally occurring regulatory T cells (Tregs), defects in which could contribute to loss of self‐tolerance in patients with T1D. We have reported previously that near to T1D onset, patients demonstrate a reduced level of suppression by CD4+CD25hi Tregs of autologous CD4+CD25‐ responder cells. Here we demonstrate that this defective regulation is also present in subjects with long‐standing T1D (> 3 years duration; P = 0·009). No difference was observed in forkhead box P3 or CD127 expression on CD4+CD25hi T cells in patients with T1D that could account for this loss of suppression. Cross‐over co‐culture assays demonstrate a relative resistance to CD4+CD25hi Treg‐mediated suppression within the CD4+CD25‐ T cells in all patients tested (P = 0·002), while there appears to be heterogeneity in the functional ability of CD4+CD25hi Tregs from patients. In conclusion, this work demonstrates that defective regulation is a feature of T1D regardless of disease duration and that an impaired ability of responder T cells to be suppressed contributes to this defect.