Joana R. F. Abreu

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.

Plasmid-encoded proinsulin preserves C-peptide while specifically reducing proinsulin-specific CD8 + T cells in type 1 diabetes

A plasmid encoding proinsulin reduces the frequency of CD8+ T cells reactive to proinsulin while preserving C-peptide. Diabetes Trial Hits Its Mark Type 1 diabetes is an autoimmune disease where an inflammatory response destroys the insulin-producing cells of the pancreas. One way to block this response is through immunosuppression; however, it has proven difficult to target the specific autoreactive cells without suppressing the rest of the immune response. Now, Roep et al. demonstrate that an engineered plasmid that expresses proinsulin can preserve β cell function in type 1 diabetes patients. The authors randomized patients recently diagnosed with type 1 diabetes to receive various doses of either a proinsulin-expressing engineered plasmid or PBS vehicle. They observed no serious adverse events. The subjects in the experimental group had improved C-peptide levels—a readout of β cell function. The authors then examined the immune responses in these patients and found that there was a decrease in proinsulin-specific CD8+ T cells, but not unrelated CD8+ T cells. No difference was observed in cytokine production by CD4+ T cells. If these data hold true in larger studies, a plasmid encoding proinsulin could serve as a targeted means of immunosuppression for type 1 diabetes. In type 1 diabetes (T1D), there is an intense inflammatory response that destroys the β cells in the pancreatic islets of Langerhans, the site where insulin is produced and released. A therapy for T1D that targets the specific autoimmune response in this disease while leaving the remainder of the immune system intact, has long been sought. Proinsulin is a major target of the adaptive immune response in T1D. We hypothesized that an engineered DNA plasmid encoding proinsulin (BHT-3021) would preserve β cell function in T1D patients through reduction of insulin-specific CD8+ T cells. We studied 80 subjects over 18 years of age who were diagnosed with T1D within the past 5 years. Subjects were randomized 2:1 to receive intramuscular injections of BHT-3021 or BHT-placebo, weekly for 12 weeks, and then monitored for safety and immune responses in a blinded fashion. Four dose levels of BHT-3021 were evaluated: 0.3, 1.0, 3.0, and 6.0 mg. C-peptide was used both as an exploratory efficacy measure and as a safety measure. Islet-specific CD8+ T cell frequencies were assessed with multimers of monomeric human leukocyte antigen class I molecules loaded with peptides from pancreatic and unrelated antigens. No serious adverse events related to BHT-3021 were observed. C-peptide levels improved relative to placebo at all doses, at 1 mg at the 15-week time point (+19.5% BHT-3021 versus −8.8% BHT-placebo, P < 0.026). Proinsulin-reactive CD8+ T cells, but not T cells against unrelated islet or foreign molecules, declined in the BHT-3021 arm (P < 0.006). No significant changes were noted in interferon-γ, interleukin-4 (IL-4), or IL-10 production in CD4 T cells. Thus, we demonstrate that a plasmid encoding proinsulin reduces the frequency of CD8+ T cells reactive to proinsulin while preserving C-peptide over the course of dosing.

Islet-Specific CTL Cloned from a Type 1 Diabetes Patient Cause Beta-Cell Destruction after Engraftment into HLA-A2 Transgenic NOD/SCID/IL2RG Null Mice

Despite increasing evidence that autoreactive CD8 T-cells are involved in both the initiation of type 1 diabetes (T1D) and the destruction of beta-cells, direct evidence for their destructive role in-vivo is lacking. To address a destructive role for autoreactive CD8 T-cells in human disease, we assessed the pathogenicity of a CD8 T-cell clone derived from a T1D donor and specific for an HLA-A2-restricted epitope of islet-specific glucose-6-phosphatase catalytic-subunit related protein (IGRP). HLA-A2/IGRP tetramer staining revealed a higher frequency of IGRP-specific CD8 T-cells in the peripheral blood of recent onset human individuals than of healthy donors. IGRP265–273-specific CD8 T-cells that were cloned from the peripheral blood of a recent onset T1D individual were shown to secrete IFNγ and Granzyme B after antigen-specific activation and lyse HLA-A2-expressing murine islets in-vitro. Lytic capacity was also demonstrated in-vivo by specific killing of peptide-pulsed target cells. Using the HLA-A2 NOD-scid IL2rγnull mouse model, HLA-A2-restricted IGRP-specific CD8 T-cells induced a destructive insulitis. Together, this is the first evidence that human HLA-restricted autoreactive CD8 T-cells target HLA-expressing beta-cells in-vivo, demonstrating the translational value of humanized mice to study mechanisms of disease and therapeutic intervention strategies.

Discovery of low-affinity preproinsulin epitopes and detection of autoreactive CD8 T-cells using combinatorial MHC multimers.

Autoreactive cytotoxic CD8 T-cells (CTLs) play a key pathogenic role in the destruction of insulin-producing beta-cells resulting in type 1 diabetes. However, knowledge regarding their targets is limited, restricting the ability to monitor the course of the disease and immune interventions. In a multi-step discovery process to identify novel CTL epitopes in human preproinsulin (PPI), PPI was digested with purified human proteasomes, and resulting COOH-fragments aligned with algorithm-predicted HLA-binding peptides to yield nine potential HLA-A1, -A2, -A3 or -B7-restricted candidates. An UV-exchange method allowed the generation of a repertoire of multimers including low-affinity HLA-binding peptides. These were labeled with quantum dot-fluorochromes and encoded in a combinatorial fashion, allowing parallel and sensitive detection of specific, low-avidity T-cells. Significantly increased frequencies of T-cells against four novel PPI epitopes (PPI(4-13)/B7, PPI(29-38)/A2, PPI(76-84)/A3 and PPI(79-88)/A3) were detected in stored blood of patients with recent onset diabetes but not in controls. Changes in frequencies of circulating CD8 T-cells against these novel epitopes were detected in blood of islet graft recipients at different time points after transplantation, which correlated with clinical outcome. In conclusion, our novel strategy involving a sensitive multiplex detection technology and requiring minimal volumes of stored blood represents a major improvement in the direct ex-vivo characterization and enumeration of immune cells in the pathogenesis of type 1 diabetes.

CD8 T cell autoreactivity to preproinsulin epitopes with very low human leucocyte antigen class I binding affinity.

Beta cells presenting islet epitopes are recognized and destroyed by autoreactive CD8 T cells in type 1 diabetes. These islet‐specific T cells are believed to react with epitopes binding with high affinity to human leucocyte antigen (HLA) expressed on beta cells. However, this assumption might be flawed in case of islet autoimmunity. We evaluated T cell recognition of the complete array of preproinsulin (PPI) peptides with regard to HLA binding affinity and T cell recognition. In a comprehensive approach, 203 overlapping 9–10mer PPI peptides were tested for HLA‐A2 binding and subjected to binding algorithms. Subsequently, a high‐throughput assay was employed to detect PPI‐specific T cells in patient blood, in which conditional HLA ligands were destabilized by ultraviolet irradiation and HLA molecules refolded with arrays of PPI peptides, followed by quantum‐dot labelling and T cell staining. Analysis of patient blood revealed high frequencies of CD8 T cells recognizing very low HLA binding peptides. Of 28 peptides binding to HLA‐A2, a majority was predicted not to bind. Unpredicted peptides bound mainly with low affinities. HLA binding affinity and immunogenicity may not correlate in autoimmunity. Algorithms used to predict high‐affinity HLA peptide binders discount the majority of low‐affinity HLA binding epitopes. Appreciation that peptides binding HLA with very low affinity can act as targets of autoreactive T cells may help to understand loss of tolerance and disease pathogenesis and possibly point to tissue‐specific immune intervention targets.

Immunological Balance Is Associated with Clinical Outcome after Autologous Hematopoietic Stem Cell Transplantation in Type 1 Diabetes

Autologous hematopoietic stem cell transplantation (AHSCT) increases C-peptide levels and induces insulin independence in patients with type 1 diabetes. This study aimed to investigate how clinical outcomes may associate with the immunological status, especially concerning the balance between immunoregulation and autoreactivity. Twenty-one type 1 diabetes patients were monitored after AHSCT and assessed every 6 months for duration of insulin independence, C-peptide levels, frequencies of islet-specific autoreactive CD8+ T cells (CTL), regulatory lymphocyte subsets, thymic function, and T-cell repertoire diversity. In median follow-up of 78 (range 15–106) months, all patients became insulin-independent, resuming insulin after median of 43 (range 6–100) months. Patients were retrospectively divided into short- or prolonged-remission groups, according to duration of insulin independence. For the entire follow-up, CD3+CD4+ T-cell numbers remained lower than baseline in both groups, whereas CD3+CD8+ T-cell levels did not change, resulting in a CD4/CD8 ratio inversion. Memory CTL comprehended most of T cells detected on long-term follow-up of patients after AHSCT. B cells reconstituted to baseline levels at 2–3 months post-AHSCT in both patient groups. In the prolonged-remission-group, baseline islet-specific T-cell autoreactivity persisted after transplantation, but regulatory T cell counts increased. Patients with lower frequencies of autoreactive islet-specific T cells remained insulin-free longer and presented greater C-peptide levels than those with lower frequencies of these cells. Therefore, immune monitoring identified a subgroup of patients with superior clinical outcome of AHSCT. Our study shows that improved immunoregulation may balance autoreactivity endorsing better metabolic outcomes in patients with lower frequencies of islet-specific T cells. Development of new strategies of AHSCT is necessary to increase frequency and function of T and B regulatory cells and decrease efficiently autoreactive islet-specific T and B memory cells in type 1 diabetes patients undergoing transplantation.

Alternative splicing and differential expression of the islet autoantigen IGRP between pancreas and thymus contributes to immunogenicity of pancreatic islets but not diabetogenicity in humans

Aims/hypothesisThymic expression of self-antigens during T-lymphocyte development is believed to be crucial for preventing autoimmunity. It has been suggested that G6PC2, the gene encoding islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), is differentially spliced between pancreatic beta cells and the thymus. This may contribute to incomplete elimination of IGRP-specific T lymphocytes in the thymus, predisposing individuals to type 1 diabetes. We tested whether specific splice variation in islets vs thymus correlates with loss of tolerance to IGRP in type 1 diabetes.MethodsExpression of G6PC2 splice variants was compared among thymus, purified medullary thymic epithelial cells and pancreatic islets by RT-PCR. Differential immunogenicity of IGRP splice variants was tested in patients and healthy individuals for autoantibodies and specific cytotoxic T lymphocytes using radiobinding assays and HLA class I multimers, respectively.ResultsPreviously reported G6PC2 splice variants, including full-length G6PC2, were confirmed, albeit that they occurred in both pancreas and thymus, rather than islets alone. Yet, their expression levels were profoundly greater in islets than in thymus. Moreover, three novel G6PC2 variants were discovered that occur in islets only, leading to protein truncations, frame shifts and neo-sequences prone to immunogenicity. However, autoantibodies to novel or known IGRP splice variants did not differ between patients and healthy individuals, and similar frequencies of IGRP-specific cytotoxic T lymphocytes could be detected in both patients with type 1 diabetes and healthy individuals.Conclusions/interpretationWe propose that post-transcriptional variation of tissue-specific self-proteins may affect negative thymic selection, although this need not necessarily lead to disease.

Targeting proinsulin-reactive CD8+ T cells: a new direction for Type 1 diabetes treatment

Type 1 diabetes (T1D) is a chronic autoimmune disease in which the b-cells in the pancreatic islets of Langerhans are destroyed by autoreactive T cells. Currently, there is no cure for T1D. Therefore, patients need life-long insulin replacement therapy to control blood glucose levels. In the past, various efforts have been undertaken to develop and assess strategies that halt disease progression [1]. Immunotherapeutic strategies have aimed either at systemic downmodulation of the immune system or targeting specific autoimmune mechanisms involved in the disease. Nonspecific immune suppressive approaches broadly inhibit the immune system and may therefore have undesirable risks and side effects. The calcineurin inhibitor cyclosporine A was among the first nonspecific immune suppressants to be used in T1D. Continuous treatment initiated soon after disease onset demonstrated that it was possible to preserve b-cell function, with patients experiencing insulin independency [1]. However, this effect was short lasting and associated with accelerated renal dysfunction [2]. The enthusiasm for the use of this and other non-specific immune suppressants faded with trials using azathioprine, prednisolone and anti-thymocyte globulin showing no lasting clinical efficacy [1]. Clearly, the development of an antigenspecific therapy selectively targeting pathogenic autoreactive T cells, while sparing the ability of the immune system to respond to pathogens would be highly desirable. In T1D, attention was focused mainly on CD4 T cells. However, it has become evident that CD8 T cells also play a crucial role in b-cell destruction. Studies on insulitic lesions showed large infiltrates of CD8 T cells and increased MHC-I expression in pancreata from recent onset T1D patients [3,4]. Recently, the presence of islet autoreactive CD8 T cells in human insulitic lesions could be demonstrated, providing the ultimate indication that T1D is indeed an autoimmune disease where islet autoreactive CD8 T cells selectively infiltrate pancreatic islets and cause b-cell destruction [5]. Importantly, this study also demonstrated that patients with disease for many years still possess insulin-positive b-cells, in spite of undetectable levels of C-peptide, as well as islet infiltration and autoimmunity. These findings have led to new opportunities: as long as b-cell mass is preserved and chronic islet inflammation lasts, interventional therapy is warranted. Indeed, recently diagnosed T1D patients treated with high-dose immunosuppression followed by autologous hematopoietic stem cell transplantation [6] became insulin independent, often for several years, without therapeutically targeting b-cell function directly. Yet, the in situ studies on insulitic lesions also revealed large heterogeneity between individual patients, even

PS2 - 11. Immune signatures defining graft acceptance in clinical islet transplantation

Islet transplantation offers great promise for brittle type 1 diabetes, but improved long term results are desired. Alternative immunosuppressive protocols are under investigation to improve efficacy and durability, and additional (immune)biomarkers would be useful to add to patient selection, efficacy and monitoring.