Bart O. Roep

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.

Coxsackie B4 virus infection of β cells and natural killer cell insulitis in recent-onset type 1 diabetic patients

Type 1 diabetes is characterized by T cell-mediated autoimmune destruction of pancreatic β cells. Several studies have suggested an association between Coxsackie enterovirus seroconversion and onset of disease. However, a direct link between β cell viral infection and islet inflammation has not been established. We analyzed pancreatic tissue from six type 1 diabetic and 26 control organ donors. Immunohistochemical, electron microscopy, whole-genome ex vivo nucleotide sequencing, cell culture, and immunological studies demonstrated Coxsackie B4 enterovirus in specimens from three of the six diabetic patients. Infection was specific of β cells, which showed nondestructive islet inflammation mediated mainly by natural killer cells. Islets from enterovirus-positive samples displayed reduced insulin secretion in response to glucose and other secretagogues. In addition, virus extracted from positive islets was able to infect β cells from human islets of nondiabetic donors, causing viral inclusions and signs of pyknosis. None of the control organ donors showed signs of viral infection. These studies provide direct evidence that enterovirus can infect β cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.

Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients

In situ tetramer staining reveals the presence of islet antigen-reactive CD8+ T cells in pancreatic islets from deceased type 1 diabetes patients.

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.

The role of T-cells in the pathogenesis of Type 1 diabetes: From cause to cure

Type 1 diabetes mellitus results from a T-cell mediated autoimmune destruction of the pancreatic beta cells in genetically predisposed individuals. The knowledge of the immunopathogenesis has increased enormously in the last two decades. The contribution of T-cells in the pathogenesis is beyond doubt. Therapies directed against T-cells have been shown to halt the disease process and prevent recurrent beta-cell destruction after islet transplantation. Less is known about the nature and function of these T-cells, the cause of the loss of tolerance to islet autoantigens, why the immune system apparently fails to suppress autoreactivity, and whether (or which) autoantigen(s) are critically involved in the initiation or progression of the disease. The contribution of dendritic cells in directing the immune response is clear, while the contribution of B-cells and autoantibodies is subject to reconsideration. Autoreactive T-cells have proven to be valuable tools to study pathogenic or diabetes-related processes. Measuring T-cell autoreactivity has also provided critical information to determine the fate of islet allografts transplanted to Type 1 diabetic patients. Cellular autoimmunity is a difficult study subject, but it has been a worthwhile quest to unravel the role of T-cells in the pathogenesis of Type 1 diabetes. The challenge for the future is to determine which factors contribute to the loss of tolerance to beta-cell antigens, and to define what measures T-cells can provide to suppress autoreactivity, since it is becoming increasingly evident that T-cells provide a two-edged sword: some T-cells could be pathogenic, but others can regulate the disease process and thus form new targets for immunointervention.

Induction of Treg by monocyte-derived DC modulated by vitamin D3 or dexamethasone: differential role for PD-L1.

Specific therapy with modulated DC may restore immunological tolerance, thereby obviating the need for chronic immunosuppression in transplantation or autoimmunity. In this study we compared the tolerizing capacity of dexamethasone (Dex)‐ and 1α,25‐dihydroxyvitamin D3 (VD3)‐modulated DC. Treatment of monocytes with either VD3 or Dex resulted in DC with stable, semi‐mature phenotypes compared with standard DC, with intermediate levels of co‐stimulatory and MHC class II molecules, which remained unaltered after subsequent pro‐inflammatory stimulation. IL‐12p70 secretion was lost by VD3‐ and Dex‐DC, whereas IL‐10 secretion was unaffected. VD3‐DC distinctly produced large amounts of TNF‐α. Both VD3‐ and Dex‐DC possessed the capacity to convert CD4 T cells into IL‐10‐secreting Treg potently suppressing the proliferation of responder T cells. However, only Treg induced by VD3‐DC exhibited antigen specificity. VD3‐, but not Dex‐, DC expressed significant high levels of PD‐L1 (programmed death‐1 ligand), upon activation. Blockade of PD‐L1 during priming redirected T cells to produce IFN‐γ instead of IL‐10 and abolished acquisition of regulatory capacity. Our findings demonstrate that both VD3‐ and Dex‐DC possess durable but differential tolerogenic features, acting via different mechanisms. Both are potentially useful to specifically down‐regulate unwanted immune responses and induce immune tolerance. These modulated DC appear suitable as adjuvant in antigen‐specific clinical vaccination intervention strategies.

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.

Novel association in chromosome 4q27 region with rheumatoid arthritis and confirmation of type 1 diabetes point to a general risk locus for autoimmune diseases.

Recently, association of celiac disease with common single-nucleotide polymorphism (SNP) variants in an extensive linkage-disequilibrium block of 480 kb containing the KIAA1109, Tenr, IL2, and IL21 genes has been demonstrated in three independent populations (rs6822844P combined=1.3 x 10(-14)). The KIAA1109/Tenr/IL2/IL21 block corresponds to the Idd3 locus in the nonobese diabetic mouse model of type 1 diabetes (T1D). This block was recently found to be associated with T1D in a genomewide association study, although this finding lacks unequivocal confirmation. We therefore aimed to investigate whether the KIAA1109/Tenr/IL2/IL21 region is involved in susceptibility to multiple autoimmune diseases. We tested SNP rs6822844 for association with disease in 350 T1D-affected and 1,047 rheumatoid arthritis (RA)-affected Dutch patients and in 929 controls. We replicated the association with T1D (P=.0006; OR 0.64 [95% CI 0.50-0.83]), and revealed a similar novel association with RA (P=.0002; OR 0.72 [95% CI 0.61-0.86]). Our results replicate and extend the association found in the KIAA1109/Tenr/IL2/IL21 gene region with autoimmune diseases, implying that this locus is a general risk factor for multiple autoimmune diseases.

Satisfaction (not) guaranteed: re-evaluating the use of animal models of type 1 diabetes

Without a doubt, rodent models have been instrumental in describing pathways that lead to pancreatic β-cell destruction, evaluating potential causes of type 1 diabetes and providing proof-of-principle for the potential of immune-based interventions. However, despite more than two decades of productive research, we are still yet to define an initiating autoantigen for the human disease, to determine the precise mechanisms of β-cell destruction in humans and to design interventions that prevent or cure type 1 diabetes. In this Perspective article, we propose that a major philosophical change would benefit this field, a proposition that is based on evaluation of situations in which rodent models have provided useful guidance and in which they have led to disappointments.

Cellular islet autoimmunity associates with clinical outcome of islet cell transplantation

Background Islet cell transplantation can cure type 1 diabetes (T1D), but only a minority of recipients remains insulin–independent in the following years. We tested the hypothesis that allograft rejection and recurrent autoimmunity contribute to this progressive loss of islet allograft function. Methodology/Principal Findings Twenty-one T1D patients received cultured islet cell grafts prepared from multiple donors and transplanted under anti-thymocyte globulin (ATG) induction and tacrolimus plus mycophenolate mofetil (MMF) maintenance immunosuppression. Immunity against auto- and alloantigens was measured before and during one year after transplantation. Cellular auto- and alloreactivity was assessed by lymphocyte stimulation tests against autoantigens and cytotoxic T lymphocyte precursor assays, respectively. Humoral reactivity was measured by auto- and alloantibodies. Clinical outcome parameters - including time until insulin independence, insulin independence at one year, and C-peptide levels over one year- remained blinded until their correlation with immunological parameters. All patients showed significant improvement of metabolic control and 13 out of 21 became insulin-independent. Multivariate analyses showed that presence of cellular autoimmunity before and after transplantation is associated with delayed insulin-independence (p = 0.001 and p = 0.01, respectively) and lower circulating C-peptide levels during the first year after transplantation (p = 0.002 and p = 0.02, respectively). Seven out of eight patients without pre-existent T-cell autoreactivity became insulin-independent, versus none of the four patients reactive to both islet autoantigens GAD and IA-2 before transplantation. Autoantibody levels and cellular alloreactivity had no significant association with outcome. Conclusions/Significance In this cohort study, cellular islet-specific autoimmunity associates with clinical outcome of islet cell transplantation under ATG-tacrolimus-MMF immunosuppression. Tailored immunotherapy targeting cellular islet autoreactivity may be required. Monitoring cellular immune reactivity can be useful to identify factors influencing graft survival and to assess efficacy of immunosuppression. Trial Registration Clinicaltrials.gov NCT00623610