Combination Treatment With Anti-CD20 and Oral Anti-CD3 Prevents and Reverses Autoimmune Diabetes

Type 1 diabetes (T1D) is a T cell–mediated autoimmune disease, although B cells also play an important role in T1D development. Both T cell– and B cell–directed immunotherapies have shown efficacy in the prevention and reversal of T1D. However, whether the combined strategy of targeting both T and B cells could further improve therapeutic efficacy remains to be explored. We show that combined treatment with intravenous antihuman CD20 (hCD20) and oral anti-CD3 significantly delays diabetes development in prediabetic hCD20 transgenic NOD mice. More importantly, the combined treatment reverses diabetes in >60% of mice newly diagnosed with diabetes. Further mechanistic studies demonstrated that the addition of oral anti-CD3 to the B-cell depletion therapy synergistically enhances the suppressive function of regulatory T cells. Of note, the oral anti-CD3 treatment induced a fraction of interleukin (IL)-10–producing CD4 T cells in the small intestine through IL-10– and IL-27–producing dendritic cells. Thus, the findings demonstrate that combining anti-CD20 and oral anti-CD3 is superior to anti-CD20 monotherapy for restoring normoglycemia in diabetic NOD mice, providing important preclinical evidence for the optimization of B cell–directed therapy for T1D.Type 1 diabetes (T1D) is an autoimmune disease characterized by selective destruction of insulin-secreting β-cells in genetically predisposed individuals (1,2). T1D has been demonstrated to be a T cell–mediated disease. Therapeutic targeting of T cells by CD3-specific antibody prevented and reversed new-onset T1D in NOD mice (3,4). Clinical trials also suggested efficacy of anti-CD3 for patients with recent-onset T1D (5,6). Although these T cell–targeted therapies have been efficacious in newly diagnosed patients, side effects such as fever, rash, and anemia (5) as well as Epstein-Barr virus reactivation (6,7) were reported. Recently, oral administration of CD3-specific antibody has proven to be an effective strategy to treat autoimmune diseases (8,9). Of note, the side effects of intravenous anti-CD3 treatment were not observed when anti-CD3 was given orally (10), and important therapeutic effects of oral administration of anti-CD3 monoclonal antibody were demonstrated in both streptozotocin-induced and spontaneous diabetes mouse models (8,11).In addition to the pivotal role of T cells in T1D development, the contribution of B cells to the pathogenesis of T1D has increasingly been recognized. B cells are essential for the development of T1D in the NOD mouse model (12–14). B cell–deficient μMT^−/− NOD mice fail to develop diabetes (12,13). Furthermore, several preclinical studies demonstrated that B cell–targeted therapies can prevent and reverse autoimmune diabetes (15–17). In line with these findings, a clinical trial of the anti-B-cell antibody rituximab in patients with T1D has further confirmed the critical role of B cells in the development of T1D (18). Although the clinical trial report for patients with T1D was promising, rituximab therapy only partially preserved islet β-cell function. Thus, further improvement of therapeutic efficacy, together with reduction of potential side effects of treatment, is still needed. In addition, phase III clinical trials of anti-CD3 therapy suggested that targeting the T-cell arm of the immune response alone is not sufficient to block T1D progression (19), and both the phase III clinical trials of teplizumab and otelixizumab were terminated because of failure to reach their primary end points. Given that both T and B cells are essential in the development of T1D, it is likely that combined therapy targeting both T- and B-cell compartments may further improve the therapeutic efficacy for patients with T1D. Thus, we tested the effect of intravenous anti-CD20 and oral anti-CD3 combined treatment for the prevention and reversal of T1D in the human CD20 transgenic NOD (hCD20/NOD) animal model.Herein, we show that oral administration of anti-CD3 together with intravenous injection of anti-CD20 has a synergistic effect on the prevention and reversal of T1D in the hCD20/NOD mouse. Mechanistic studies demonstrated that the combined therapy enhanced immune tolerance by improving the Foxp3⁺ regulatory T cell (Treg) compartment quantitatively and qualitatively as well as by inducing interleukin (IL)-10– and IL-27–producing dendritic cells (DCs) to promote the induction of IL-10⁺ CD4 T cells in the small intestine.     

Adoptive Transfer of Immunomodulatory M2 Macrophages Prevents Type 1 Diabetes in NOD Mice

Macrophages are multifunctional immune cells that may either drive or modulate disease pathogenesis depending on their activation phenotype. Autoimmune type 1 diabetes (T1D) is a chronic proinflammatory condition characterized by unresolved destruction of pancreatic islets. Adoptive cell transfer of macrophages with immunosuppressive properties represents a novel immunotherapy for treatment of such chronic autoimmune diseases. We used a panel of cytokines and other stimuli to discern the most effective regimen for in vitro induction of immunosuppressive macrophages (M2r) and determined interleukin (IL)-4/IL-10/transforming growth factor-β (TGF-β) to be optimal. M2r cells expressed programmed cell death 1 ligand-2, fragment crystallizable region γ receptor IIb, IL-10, and TGF-β, had a potent deactivating effect on proinflammatory lipopolysaccharide/interferon-γ–stimulated macrophages, and significantly suppressed T-cell proliferation. Clinical therapeutic efficacy was assessed after adoptive transfer in NOD T1D mice, and after a single transfer of M2r macrophages, >80% of treated NOD mice were protected against T1D for at least 3 months, even when transfer was conducted just prior to clinical onset. Fluorescent imaging analyses revealed that adoptively transferred M2r macrophages specifically homed to the inflamed pancreas, promoting β-cell survival. We suggest that M2r macrophage therapy represents a novel intervention that stops ongoing autoimmune T1D and may have relevance in a clinical setting.Macrophages have critical functions in both innate and adaptive immune responses. They are present in almost every tissue, recognize exogenous/endogenous danger signals through pattern-recognition receptors, produce various cytokines/chemokines that orchestrate immune responses at the site of inflammation, and function as professional antigen-presenting cells (APCs).Two macrophage activation states have been defined in rodents and humans: “classically activated” (M1) cells have proinflammatory effector functions, and “alternatively activated” (M2) cells have anti-inflammatory properties. The existence of these different activation states implies prominent roles in different phases of an immunological response, i.e., inflammation versus resolution and tissue remodeling. M1 cells are identified by high expression of the enzyme inducible nitric oxide (NO) synthase, a potent respiratory burst, and secretion of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-12. The activation is induced by two signals, one toll-like receptor agonist, such as lipopolysaccharide (LPS), and one cytokine receptor–mediated signal, e.g., interferon-γ (IFN-γ) (1,2).We now understand that there are subpopulations of M2 macrophages, different types of activation leading to different functional phenotypes. M2 cells are generally characterized by secretion of anti-inflammatory cytokines such as IL-10 and by low or no secretion of proinflammatory cytokines. IL-4 was initially identified as an inducer of M2 macrophages (3), and it was later discerned that IL-4, in combination with IL-13, enhanced induction of wound-healing macrophages (M2a) (4). Further investigations determined that stimulation with glucocorticoids (e.g., dexamethasone), IL-10, immune complexes in combination with toll-like receptor agonists, and transforming growth factor-β (TGF-β) could induce at least two further distinct M2 polarization states with immunoregulatory properties (M2b and M2c) (1,5). We have previously studied macrophage phenotypes from autoimmune-resistant and -susceptible rodent strains and have determined that there is a diversity of M1 activation phenotypes; autoimmune-susceptible strains have a common phenotype that contributes to prolongation of inflammation instead of its resolution (6). Similar genetically determined aberrant macrophage phenotypes have also been reported for NOD mice (7,8) and in human type 1 diabetes (T1D) (9,10). This indicates that there is a genetic predisposition for autoimmune susceptibility regarding macrophage phenotype and implies that individuals with autoimmune diseases may lack the ability to generate a cellular phenotype important in the resolution of inflammation.Evidence of “immune regulatory” macrophage activity derives from cancer models in which tumor-associated macrophages have been reported to both suppress tumor immunity and promote tumor survival (5). Several in vitro (11,12) and in vivo disease studies (13–16) have investigated the regulatory role of macrophages in suppressing inflammation, the latter including models of multiple sclerosis, kidney disease, and spinal cord injury. Although these findings clearly indicate the important role of macrophages in the resolution of inflammation, there is no definitive consensus about their mechanism of action or optimal protocols for their induction.T1D is an autoimmune disease in which insulin-producing β-cells in the pancreas are attacked by leukocytes (macrophages and cluster of differentiation [CD]4⁺ and CD8⁺ T cells) (17), which leads to a subsequent loss of glucose control and acute complications. The most widely used animal model for investigation of T1D is the NOD mouse (18) in which diabetes spontaneously develops between 12 and 30 weeks of age after initial leukocyte infiltration into the pancreas (insulitis) between 3 and 5 weeks of age. Macrophages have been demonstrated to have a prominent role in T1D pathogenesis (19). Early M1 macrophage infiltration induces β-cell death and activates cytotoxic CD8⁺ T cells (20,21). Although many studies have provided convincing evidence of the destructive role of M1 macrophages in T1D, it was recently reported that transgenic NOD mice that did not spontaneously develop T1D expressed M2 macrophage–associated genes in the pancreas (22), and that the 10–20% of NOD mice that do not develop T1D possess protective macrophages with a phagocytic/immunosuppressive phenotype (23).Previous studies have investigated the use of cell transfer as an immunomodulatory therapy in NOD mice, with most reports using highly suppressive regulatory T cells (Tregs) (24,25). One drawback of this approach is that Treg antigen specificity is important for their suppressive ability (26), inferring that many Treg specificities might be required for the many pancreatic β-cell antigens implicated in T1D development. Adoptive cell transfer of tolerogenic dendritic cells has also been used in young NOD mice (5–8 weeks of age) during the early period of insulitis (27). For an optimal effect in a clinical setting, it would be advantageous to use cell therapy at the clinical debut of disease.The role of M2 macrophages in T1D and adoptive transfer of regulatory M2 macrophages as a cell therapy in T1D have never been reported before. In this study, we elucidated the ability of IL-4/IL-10/TGF-β to induce an immunosuppressive M2 NOD macrophage phenotype (M2r). When M2r macrophages were adoptively transferred into late-stage prediabetic NOD mice, the onset of T1D was significantly reduced; advanced imaging analysis revealed protection of residual pancreatic islets concomitant with infiltration of the transferred M2r cells.     

Virtual Optimization of Nasal Insulin Therapy Predicts Immunization Frequency to Be Crucial for Diabetes Protection

OBJECTIVE

Development of antigen-specific strategies to treat or prevent type 1 diabetes has been slow and difficult because of the lack of experimental tools and defined biomarkers that account for the underlying therapeutic mechanisms.

RESEARCH DESIGN AND METHODS

The type 1 diabetes PhysioLab platform, a large-scale mathematical model of disease pathogenesis in the nonobese diabetic (NOD) mouse, was used to investigate the possible mechanisms underlying the efficacy of nasal insulin B:9-23 peptide therapy. The experimental aim was to evaluate the impact of dose, frequency of administration, and age at treatment on Treg induction and optimal therapeutic outcome.

RESULTS

In virtual NOD mice, treatment efficacy was predicted to depend primarily on the immunization frequency and stage of the disease and to a lesser extent on the dose. Whereas low-frequency immunization protected from diabetes atrributed to Treg and interleukin (IL)-10 induction in the pancreas 1–2 weeks after treatment, high-frequency immunization failed. These predictions were confirmed with wet-lab approaches, where only low-frequency immunization started at an early disease stage in the NOD mouse resulted in significant protection from diabetes by inducing IL-10 and Treg.

CONCLUSIONS

Here, the advantage of applying computer modeling in optimizing the therapeutic efficacy of nasal insulin immunotherapy was confirmed. In silico modeling was able to streamline the experimental design and to identify the particular time frame at which biomarkers associated with protection in live NODs were induced. These results support the development and application of humanized platforms for the design of clinical trials (i.e., for the ongoing nasal insulin prevention studies).Type 1 diabetes is a complex and multifactorial autoimmune disease, in which, (pro-) insulin-specific T-cell responses have been described in lymphocytes obtained from nonobese diabetic (NOD) mice and (pre-)diabetic patients (1–4). In NOD mice, the insulin B:9-23 peptide sequence is a dominant epitope, and a single amino acid substitution in position 16 (B16:A) confers protection from the disease (5–7). Antigen-specific immunotherapies with whole insulin and B:9-23 peptide have been successful in preventing diabetes in NOD mice when administered via the subcutaneous, oral, or nasal route or via intramuscular DNA vaccination (8–18). The success in halting disease progression in prediabetic mice prompted physicians to establish similar protocols to test safety and efficacy in human prediabetic or diabetic subjects. To this end, clinical trials with nasal or oral whole insulin were conducted, which proved to be safe. However, diabetes progression was only slightly affected in a subset of insulin antibody–positive patients treated with oral insulin in the Diabetes Prevention Trial–Type 1 (19–24). In contrast, nasal insulin phase I–II trials in Finland (21,24) and in Australia (20), during which insulin was administered daily, failed to provide therapeutic efficacy. Highlighting the difficulties we are facing to rationally translate antigen-specific therapies to humans, there are conflicting reports on the efficacy of nasal B:9-23 peptide immunization in the NOD mouse. Taken together, these studies suggest that the manner by which insulin therapy is administered is important (25,26). Many variables may influence efficacy, including dose, frequency of administration, and the stage of the disease. Systematic investigation of each of these variables and combinations thereof is experimentally impractical because of the time constraints of in vivo studies, therefore necessitating biosimulation approaches.The type 1 diabetes PhysioLab platform is a top-down, outcome-focused, large-scale mathematical model composed of ordinary differential and algebraic equations (27,28). This model reproduces type 1 diabetes pathogenesis in female NOD mice from birth until disease onset, with extensive representation of critical biological processes that were described in the literature and take place in the pancreas, the pancreatic-draining lymph nodes (PDLN), the gut, the nasal-associated lymphoid tissue (NALT), and the peripheral blood.In the present study, we used a cohort of virtual NOD mice (VM) with diversity in underlying pathophysiology to investigate how variations in dose, frequency, and age at treatment initiation may impact the efficacy of nasal B:9-23 peptide therapy. The VM program was designed based on the following assumptions: 1) induction of Treg is beneficial for disease prevention, 2) induction of Th1 responses (interferon [IFN] γ) is detrimental for diabetes progression, whereas 3) induction of Th2 responses (interleukin [IL]-10 and IL-4) favor disease protection. In silico investigation of the underlying mechanisms predicted that too frequent nasal B:9-23 immunization would inhibit IL-10 induction and the generation of adaptive Treg (aTreg), which should be critical in mediating protection. The model also aided in defining the optimal time frame in which Treg and IL-10 would be induced after immunization, thereby mapping the timing for both as crucial biomarkers.Laboratory experiments confirmed many of these predictions, establishing immunization frequency and induction of IL-10⁺ Treg as important considerations for the design of future clinical trials. Interestingly, in the initial and follow-up Peakman studies (2,29), it became evident that naturally occurring circulating islet-specific IL-10–producing cells regulate proinflammatory Th1 responses in healthy individuals by linked suppression. This suggests than in certain circumstances, given the heterogeneity of human type 1 diabetes, IL-10 production could be sufficient in sustaining prolonged antigen-specific tolerance. Future and ongoing trials might benefit from less frequent immunization, for example the ongoing nasal insulin studies in Australia (20). Moving forward, better defining the immunological parameters that could serve as biomarkers is of particular importance for improving the previously failed clinical trials (19–23,30) and for increasing the chance of success (31,32).     

Interleukin-21 Is Required for the Development of Type 1 Diabetes in NOD Mice

OBJECTIVE

Interleukin (IL)-21 is a type 1 cytokine that has been implicated in the pathogenesis of type 1 diabetes via the unique biology of the nonobese diabetic (NOD) mouse strain. The aim of this study was to investigate a causal role for IL-21 in type 1 diabetes.

RESEARCH DESIGN AND METHODS

We generated IL-21R–deficient NOD mice and C57Bl/6 mice expressing IL-21 in pancreatic β-cells, allowing the determination of the role of insufficient and excessive IL-21 signaling in type 1 diabetes.

RESULTS

Deficiency in IL-21R expression renders NOD mice resistant to insulitis, production of insulin autoantibodies, and onset of type 1 diabetes. The lymphoid compartment in IL-21R^−/− NOD is normal and does not contain an increased regulatory T-cell fraction or diminished effector cytokine responses. However, we observed a clear defect in autoreactive effector T-cells in IL-21R^−/− NOD by transfer experiments. Conversely, overexpression of IL-21 in pancreatic β-cells induced inflammatory cytokine and chemokines, including IL-17A, IL17F, IFN-γ, monocyte chemoattractant protein (MCP)-1, MCP-2, and interferon-inducible protein-10 in the pancreas. The ensuing leukocytic infiltration in the islets resulted in destruction of β-cells and spontaneous type 1 diabetes in the normally diabetes-resistant C57Bl/6 and NOD × C57Bl/6 backgrounds.

CONCLUSIONS

This work provides demonstration of the essential prodiabetogenic activities of IL-21 on diverse genetic backgrounds (NOD and C57BL/6) and indicates that IL-21 blockade could be a promising strategy for interventions in human type 1 diabetes.The nonobese diabetic (NOD) mouse model is the most well-characterized animal model of human type 1 diabetes and has provided important insights into the etiology and pathogenesis of this increasingly prevalent autoimmune disease (1). Rigorous genetic analysis of the NOD background has revealed the existence of multiple defined chromosomal regions known as insulin-dependent diabetes (idd) loci that confer susceptibility to or protection from the development of type 1 diabetes (2). Of the ∼15 regions identified, idd3 is of particular importance, because congenic NOD lines containing alleles from protected strains at this locus are significantly less susceptible to diabetes. To date, idd3 is the most potent disease modifying the non–major histocompatibility complex (MHC) locus (3). Therefore, some of the genes within the idd3 interval must play a crucial role in regulating immune destruction of pancreatic β-cells.Among the several candidate genes within the idd3 locus, interleukin (IL)-21 is of particular interest, because dysregulated IL-21 production and signaling has been found in the NOD mouse (4). IL-21 belongs to the type 1 cytokine family, which includes potent immune modulators such as IL-2, IL-4, IL-7, and IL-15, whose high-affinity receptor complexes all use the common γc receptor subunit (5,6). The specificity of IL-21 signaling is achieved through its specific interaction with the IL-21 receptor subunit, which forms a heterodimer with the γc subunit (7). This receptor complex delivers IL-21 signals to a variety of immune cells including CD4⁺ and CD8⁺ T-cells, B-cells, NK cells, NKT cells, and dendritic cells (8–13), all of which can play some role in the pathogenesis of type 1 diabetes in the NOD mouse (14–20). Therefore, the aim of our present study was to better understand the role of IL-21 in type 1 diabetes. We demonstrate that loss of IL-21 signaling, via knockout of the IL-21 receptor, completely abrogates diabetes development on the NOD background. In addition, we demonstrate that overexpression of IL-21 in pancreatic β-cells induces a high incidence of spontaneous type 1 diabetes on the normally diabetes-resistant C57Bl/6 genetic background. Together, these findings clearly underline the potent prodiabetogenic activity of IL-21.     

Interleukin-21 is critically required in autoimmune and allogeneic responses to islet tissue in murine models

OBJECTIVE

Type 1 diabetes is an incurable chronic autoimmune disease. Although transplantation of pancreatic islets may serve as a surrogate source of insulin, recipients are subjected to a life of immunosuppression. Interleukin (IL)-21 is necessary for type 1 diabetes in NOD mice. We examined the efficacy of an IL-21–targeted therapy on prevention of diabetes in NOD mice, in combination with syngeneic islet transplantation. In addition, we assessed the role of IL-21 responsiveness in islet allograft rejection in mouse animal models.

RESEARCH DESIGN AND METHODS

NOD mice were treated with IL-21R/Fc, an IL-21–neutralizing chimeric protein. This procedure was combined with syngeneic islet transplantation to treat diabetic NOD mice. Survival of allogeneic islet grafts in IL-21R–deficient mice was also assessed.

RESULTS

Evidence is provided that IL-21 is continually required by the autoimmune infiltrate, such that insulitis was reduced and reversed and diabetes inhibited by neutralization of IL-21 at a late preclinical stage. Recovery from autoimmune diabetes was achieved by combining neutralization of IL-21 with islet transplantation. Furthermore, IL-21–responsiveness by CD8+ T-cells was sufficient to mediate islet allograft rejection.

CONCLUSIONS

Neutralization of IL-21 in NOD mice can inhibit diabetes, and when paired with islet transplantation, this therapeutic approach restored normoglycemia. The influence of IL-21 on a graft-mounted immune response was robust, since the absence of IL-21 signaling prevented islet allograft rejection. These findings suggest that therapeutic manipulation of IL-21 may serve as a suitable treatment for patients with type 1 diabetes.In type 1 diabetes, activated immune cells lead to the destruction of the insulin-producing β-cells in the islets of Langerhans of the pancreas (1). Clinical diabetes occurs in the nonobese diabetic (NOD) model after months of chronic pancreatic inflammation, progressing from peri-insulitis to destructive insulitis (2,3). Importantly, therapies designed to modulate lymphocyte activation are compatible with the prevention of the destructive form of insulitis, i.e., the movement of immune cells into the islet, and subsequent loss of insulin production from β-cells (4,5).Insulin replacement is the current standard for treating type 1 diabetes, however, transplantation of pancreatic islets has the potential to serve as a viable alternative (6). Challenges of islet transplantation include finding alternatives to broad-spectrum immunosuppression (7) while preventing graft rejection and recurrence of the underlying autoimmune destruction of pancreatic islets. As T-cells constitute an integral component in both autoimmune responses of diabetes and the rejection of transplanted islet allografts (8,9), therapies addressing modulation of T-cell function may provide an appropriate strategy.IL-21 is a member of the common γ-chain signaling family of cytokines that is necessary for the development of diabetes in the NOD mouse (10–12). The receptor for IL-21, comprising the α unit (IL-21Rα) and the common γ chain, is expressed on immune cells including T-, B-, NK, and dendritic cells, whereas IL-21 expression is largely limited to CD4+ T-cells (13). Several studies demonstrate that IL-21 acts as a lymphocyte costimulator, enhancing the proliferation and effector function of CD8+ T-cells (14,15), and transgenic over-expression of murine IL-21 revealed that IL-21 predominantly expands memory phenotype CD8+ T-cells (16). The prosurvival effect of IL-21 is important for CD8+ T-cells during chronic viral infection (17–19), with IL-21 also potently effecting the activation and differentiation of numerous CD4+ T-helper subsets, including Th17 cells (20,21).Consistent with its actions on lymphocyte populations, IL-21 has been found to contribute to the development of autoimmune diseases in several animal models (22). Likewise, IL-21 has the potential to influence the outcome of islet graft transplantation (23–25). For instance, IL-21 has a well-documented ability to promote the production of granzyme and perforin in differentiating CD8+ cytotoxic T-cells. Direct killing of islet cells by antigen-specific cytotoxic T-cells is an important component of both allograft rejection and the autoimmune destruction of β-cells (26,27). Secondly, IL-21 costimulates the activation and differentiation of antigen specific CD4+ T-cells, and these cells can produce proinflammatory cytokines that are toxic to the islets, such as IL-1β, tumor necrosis factor-α, and γ-interferon (IFNγ) (28–31).In this study, we demonstrate that IL-21 acts on immune cells to elicit autoimmune destruction of endogenous pancreatic islet tissue in autoimmune diabetes and islet graft rejection caused by both autoimmune and allogeneic immune responses. We provide evidence that through the modulation of IL-21, a potential therapeutic intervention for type 1 diabetes may be attainable.     

A Novel Clinically Relevant Strategy to Abrogate Autoimmunity and Regulate Alloimmunity in NOD Mice

OBJECTIVE

To investigate a new clinically relevant immunoregulatory strategy based on treatment with murine Thymoglobulin mATG Genzyme and CTLA4-Ig in NOD mice to prevent allo- and autoimmune activation using a stringent model of islet transplantation and diabetes reversal.

RESEARCH DESIGN AND METHODS

Using allogeneic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, we addressed the therapeutic efficacy and immunomodulatory mechanisms associated with a new immunoregulatory protocol based on prolonged low-dose mATG plus CTLA4-Ig.

RESULTS

BALB/c islets transplanted into hyperglycemic NOD mice under prolonged mATG+CTLA4-Ig treatment showed a pronounced delay in allograft rejection compared with untreated mice (mean survival time: 54 vs. 8 days, P < 0.0001). Immunologic analysis of mice receiving transplants revealed a complete abrogation of autoimmune responses and severe downregulation of alloimmunity in response to treatment. The striking effect on autoimmunity was confirmed by 100% diabetes reversal in newly hyperglycemic NOD mice and 100% indefinite survival of syngeneic islet transplantation (NOD.SCID into NOD mice).

CONCLUSIONS

The capacity to regulate alloimmunity and to abrogate the autoimmune response in NOD mice in different settings confirmed that prolonged mATG+CTLA4-Ig treatment is a clinically relevant strategy to translate to humans with type 1 diabetes.Successful islet transplantation can improve metabolic control and re-establish normoglycemia in subjects with type 1 diabetes (1). Unfortunately, transplanted islets are subject to both alloimmune responses and the recurrence of autoimmunity; either of which, when left uncontrolled, is capable of jeopardizing long-term islet function (1–4). Nonobese diabetic (NOD) mice represent the most commonly used animal model for human type 1 diabetes, and when used in settings of islet transplantation, the results obtained can be used to predict the results in type 1 diabetic patients (5–9). Indeed, several of the immunoregulatory defects observed in type 1 diabetic patients parallel those in NOD mice (10).Thymoglobulin (ATG), a widely used induction therapy in solid organ transplantation (11), as well as in islet transplantation (12–14), depletes peripheral T cells and inhibits T-effector cell (Teff) expansion, but spares T regulatory cells (Treg) (15). ATG may be used for this purpose in immunoregulatory protocols. Moreover, murine ATG (mATG), similar to other depleting agents with immunoregulatory function (9), has been shown to be effective in downregulating the autoimmune response and in providing therapeutic efficacy in terms of preventing and reverting type 1 diabetes in NOD mice (16,17); mATG was recently shown to be particularly effective when combined with granulocyte colony-stimulating factor therapy (18). We have used a new mATG-based protocol in which low chronic doses were administered to continually promote Treg expansion and maintain low numbers of Teffs. A major issue when using T-cell–depleting strategies, such as ATG, in the clinical setting is the robust proliferation of residual or new emerging lymphocytes in the process of homeostatic proliferation (19), which can create a barrier to tolerance induction because re-emerging lymphocytes typically have an activated phenotype and are more resistant to regulation compared with naïve cells (19). In this regard, CD28, which is expressed constitutively on T cells and ligates either B7-1 (CD80) or B7-2 (CD86) on antigen-presenting cells (APC), thus resulting in delivery of signals that promote clonal expansion and the effector function of T cells (20,21), may play a pivotal role in regulating homeostatic proliferation (19). Targeting CD28 signaling may thus be an effective strategy to control homeostatic proliferation during T-cell depletion. The most widely used reagent to target CD28-CD80/CD86 costimulation is CTLA4-Ig, a fusion protein modeled on the structure of CTLA4 (22). CTLA4 is a coinhibitory molecule expressed on T cells after activation that inhibits the T-cell response after CD80/CD86 engagement (23,24). Moreover, because the affinity for CD80/CD86 is greater in CTLA4 than CD28, the use of CTLA4-Ig prevents CD28 engagement on T cells and thus prevents T-cell activation (20). CTLA4-Ig is currently used in clinical settings for the treatment of autoimmune disorders (25–27).Based on this information, we hypothesized that combining short-term CTLA4-Ig treatment with prolonged low-dose mATG could prevent the homeostatic proliferation that follows mATG treatment, as well as synergizing with mATG to downregulate the allo- and autoimmune responses, thereby promoting both islet allograft survival and reversal of type 1 diabetes.     

Inflammatory Tendencies and Overproduction of IL-17 in the Colon of Young NOD Mice Are Counteracted With Diet Change

OBJECTIVE

Dietary factors influence diabetes development in the NOD mouse. Diet affects the composition of microbiota in the distal intestine, which may subsequently influence intestinal immune homeostasis. However, the specific effects of antidiabetogenic diets on gut immunity and the explicit associations between intestinal immune disruption and type 1 diabetes onset remain unclear.

RESEARCH DESIGN AND METHODS

Gut microbiota of NOD mice fed a conventional diet or ProSobee formula were compared using gas chromatography. Colonic lamina propria immune cells were characterized in terms of activation markers, cytokine mRNA and Th17 and Foxp3⁺ T-cell numbers, using real-time PCR and flow cytometry. Activation of diabetogenic CD4 T-cells by purified B-cells was assessed in both groups. Immune tolerance to autologous commensal bacteria was evaluated in vitro using thymidine-incorporation tests.

RESULTS

Young NOD mice showed a disturbed tolerance to autologous commensal bacteria. Increased numbers of activated CD4 T-cells and (CD11b⁺CD11c⁺) dendritic cells and elevated levels of Th17 cells and IL23 mRNA were moreover observed in colon lamina propria. These phenomena were abolished when mice were fed an antidiabetogenic diet. The antidiabetogenic diet also altered the expression levels of costimulatory molecules and the capacity of peritoneal B-cells to induce insulin-specific CD4 T-cell proliferation.

CONCLUSIONS

Young NOD mice show signs of subclinical colitis, but the symptoms are alleviated by a diet change to an antidiabetogenic diet. Disrupted immune tolerance in the distal intestine may influence peritoneal cell pools and B-cell–mediated activation of diabetogenic T-cells.Dietary and microbial factors may be partly responsible for the increase in type 1 diabetes incidence. The intestinal mucosa is constantly exposed to these factors, and it is therefore important to thoroughly understand how these factors affect the intestinal immune system.Evidence suggesting that gut immune disruptions may trigger type 1 diabetes originated from studies that showed correlations between a high prevalence of cow-milk antibodies, brief breastfeeding in infancy, and an increased risk of type 1 diabetes (1,2). This hypothesis gained further support from the discovery that lymphocytes accumulating in the islets share homing characteristics with gut-associated lymphocytes (3–5). Research in both humans and animals has thereafter lead to an understanding that type 1 diabetes is associated with increased permeability and enteropathy in the small intestine (6–9). The impaired barrier functions of the small intestine may subsequently cause alterations in antigen responses and thus disrupt the immunological homeostasis of the intestine. This in turn could cause intestinal inflammation and induce immune responses that lead to autoimmunity (7,10,11).Considerably less attention has been paid to the potential role of the large intestinal immune system in type 1 diabetes development. The large intestine differs immunologically in several aspects from the small intestine: The disruptions in small intestinal immunity is linked foremost to ingested antigens or allergens such as insulin from cow milk, cereal-based allergens, and other food derivatives. The most immediate sources of immune disruption in the large intestine, however, are the vast quantities of bacteria residing therein. Moreover, large intestinal lamina propria lymphocytes differ markedly in terms of population dynamics and cytokine expression from the lamina propria lymphocytes of the small intestine (12).There is some evidence that directly associates immune responses in the large intestine with the pancreas; lymph from the transverse colon has been reported to drain specifically to the pancreatic lymph nodes (13). This could allow innate immune stimuli to interfere with induction of peripheral immune tolerance to antigens. Accordingly, studies in BDC2.5/NOD mice have indicated that dextran sodium sulfate, which disrupts the barrier functions of the colonic epithelium, enhances the activation of islet reactive T-cells in pancreatic lymph nodes of NOD mice (14). It is therefore of interest to further investigate the role of the colonic immune system in type 1 diabetes.Dendritic cells as well as macrophages play a major part in large intestinal mucosal immune counterbalance. Macrophages and CD11b⁻ dendritic cells have been reported to secrete anti-inflammatory cytokines, such as IL-10, while CD11b⁺ dendritic cells elicit the production of proinflammatory IL-17 (15,16). Macrophages, which are capable of suppressing dendritic cell-induced IL-17 secretion (17), are reduced in numbers in the lamina propria of mice suffering from colitis, concomitant with a substantial increase of lamina propria CD11b⁺ dendritic cells (15). Intestinal macrophages in humans have moreover been described as inflammatorily anergic, producing only low levels of proinflammatory cytokines (18).The cytokine IL-23 has importance in several inflammatory disorders (19). IL-23 is capable of promoting both Th17 and Th1 responses in the intestinal lamina propria, because its absence decreases both Th1- and Th17-type cytokines in the intestine (20). The exact effects of IFN-γ and IL-17 on type 1 diabetes are nevertheless unclear. Though increased production of IFN-γ has been associated with type 1 diabetes, NOD mice lacking IFN-γ or IFN-γ receptor develop type 1 diabetes to a degree equal to wild-type NOD mice (21,22). IL-17 promotes pancreatic inflammation (23) and is upregulated in diabetic mice (24,25). Furthermore, treatment with IL-25, which inhibits the Th17 cell population, and with IL-17 neutralizing antibody, prevent diabetes in NOD mice (26). Treatment with IFN-γ can likewise prevent diabetes in NOD mice, probably by decreasing the production of IL-17 in the spleen and pancreas (27). Th17 cells can moreover transform to Th1-like cells under the influence of IL-12, demonstrating a considerable degree of plasticity between the Th17 and the Th1 cell lineages (28).The results presented in this study indicate that newly weaned NOD mice suffer from a mild level of colitis, which alters the colonic immune cell standing toward a proinflammatory status. This is moreover associated with a disruption of immune tolerance toward autologous intestinal microbiota. As recently reported, NOD peritoneal B cells show a significantly higher efficiency in activating insulin-specific T-cell reactivity than spleen-derived conventional B-cells (29). Remarkably, most of the abnormalities in the colon and peritoneal B-cell antigen-presenting activity of young NOD mice can be abrogated when NOD mice are fed an antidiabetogenic diet from the time of weaning. The substantial effects of the antidiabetogenic diet on the colonic and peritoneal immune system call attention to the importance of colon immune homeostasis in the development of type 1 diabetes in NOD mice.     

Induction of mixed chimerism with MHC-mismatched but not matched bone marrow transplants results in thymic deletion of host-type autoreactive T-cells in NOD mice

OBJECTIVE

Induction of mixed or complete chimerism via hematopoietic cell transplantation (HCT) from nonautoimmune donors could prevent or reverse type 1 diabetes (T1D). In clinical settings, HLA-matched HCT is preferred to facilitate engraftment and reduce the risk for graft versus host disease (GVHD). Yet autoimmune T1D susceptibility is associated with certain HLA types. Therefore, we tested whether induction of mixed chimerism with major histocompatibility complex (MHC)-matched donors could reverse autoimmunity in the NOD mouse model of T1D.

RESEARCH DESIGN AND METHODS

Prediabetic wild-type or transgenic BDC2.5 NOD mice were conditioned with a radiation-free GVHD preventative anti-CD3/CD8 conditioning regimen and transplanted with bone marrow (BM) from MHC-matched or mismatched donors to induce mixed or complete chimerism. T1D development and thymic deletion of host-type autoreactive T-cells in the chimeric recipients were evaluated.

RESULTS

Induction of mixed chimerism with MHC-matched nonautoimmune donor BM transplants did not prevent T1D in wild-type NOD mice, although induction of complete chimerism did prevent the disease. However, induction of either mixed or complete chimerism with MHC-mismatched BM transplants prevented T1D in such mice. Furthermore, induction of mixed chimerism in transgenic BDC2.5-NOD mice with MHC-matched or -mismatched MHC II^−/− BM transplants failed to induce thymic deletion of de novo developed host-type autoreactive T-cells, whereas induction of mixed chimerism with mismatched BM transplants did.

CONCLUSIONS

Induction of mixed chimerism with MHC-mismatched, but not matched, donor BM transplants re-establishes thymic deletion of host-type autoreactive T-cells and prevents T1D, with donor antigen-presenting cell expression of mismatched MHC II molecules being required.Type 1 diabetes (T1D) is an autoimmune disease in which autoreactive T-cells attack the insulin-secreting islet β-cells and result in insulin deficiency and hyperglycemia (1–3). NOD mouse is still the best animal model for T1D, although the autoimmune abnormality in NOD mice does not totally reflect the abnormality in T1D patients (4–6). The autoimmunity in NOD mice and T1D patients is associated with particular major histocompatibility complex (MHC) or HLA loci such as IAβ^g7 or HLA-DR (7–9). This particular genetic background is associated with central tolerance defects, in which autoreactive thymocytes are resistant to negative selection (10–12), as well as peripheral tolerance defects (13–17).Transgenic expression of protective MHC II molecules in the thymus has been shown to prevent T1D development in mice (18–20). However, this approach cannot readily be translated to humans. Immunomodulation therapies such as administration of anti-CD3 have been shown to reverse new-onset T1D in mouse or ameliorate new-onset T1D in patients via induction of regulatory T-cells (21–25). However, the therapeutic benefit in patients appears to be limited in terms of duration (25). This indicates modulation of peripheral tolerance may not be sufficient for stable re-establishment of immune tolerance in T1D patients, because the defective thymus may constantly export autoreactive T-cells, which can overwhelm peripheral tolerance mechanisms. Therefore, a therapy that can re-establish both central and peripheral tolerance in T1D patients would appear optimal as a means to reverse the autoimmunity associated with T1D.Indeed, autoimmune diseases such as T1D arise from abnormality in the immuno-hematological compartment, and a replacement of the system from a nonautoimmune individual can cure autoimmune T1D or vice versa (26). Therefore, previous studies have proposed that induction of mixed chimerism via hematopoietic cell transplantation (HCT) may provide a curative therapy for autoimmune diseases such as T1D (27). Although it was reported induction of mixed chimerism with bone marrow (BM) transplants from MHC-mismatched or MHC-matched nonautoimmune donors was able to prevent T1D development in NOD recipients conditioned with myelo- or nonmyeloablative total body irradiation (TBI) (28–32), as well as in recipients conditioned with a radiation-free anti-CD3-based regimen (33,34), the mechanisms whereby mixed chimerism reverses such autoimmunity remain largely unknown. So-called mixed chimerism has been defined by the coexistence of donor- and host-type lymphocytes in the periphery such as in the blood or spleen, but it remains unclear whether the host-type cells in the mixed chimeric recipients are de novo developed after HCT or residual mature lymphocytes developed before HCT. In other words, it is not clear whether mixed chimerism can mediate deletion of de novo developed autoreactive T-cells. In addition, although MHC-matched HCT is preferred in clinical settings, it is not yet clear whether induction of mixed chimerism with MHC-matched donor transplants can mediate thymic deletion of de novo developed host-type autoreactive T-cells, because the defect in negative selection is associated with particular MHC II loci (7–9).In the current study, we identified true mixed chimeras by measuring the donor and host-type T-cell precursors in the thymus as well as immature B and myeloid cells in the BM and we evaluated the impact of mixed and complete chimerism with MHC-matched or mismatched donor BM transplants.     

Natural killer cells from children with type 1 diabetes have defects in NKG2D-dependent function and signaling

OBJECTIVE

Natural killer (NK) cells from NOD mice have numeric and functional abnormalities, and restoration of NK cell function prevents autoimmune diabetes in NOD mice. However, little is known about the number and function of NK cells in humans affected by type 1 diabetes. Therefore, we evaluated the phenotype and function of NK cells in a large cohort of type 1 diabetic children.

RESEARCH DESIGN AND METHODS

Peripheral blood mononuclear blood cells were obtained from subjects whose duration of disease was between 6 months and 2 years. NK cells were characterized by flow cytometry, enzyme-linked immunosorbent spot assays, and cytotoxicity assays. Signaling through the activating NK cell receptor, NKG2D, was assessed by immunoblotting and reverse-phase phosphoprotein lysate microarray.

RESULTS

NK cells from type 1 diabetic subjects were present at reduced cell numbers compared with age-matched, nondiabetic control subjects and had diminished responses to the cytokines interleukin (IL)-2 and IL-15. Analysis before and after IL-2 stimulation revealed that unlike NK cells from nondiabetic control subjects, NK cells from type 1 diabetic subjects failed to downregulate the NKG2D ligands, major histocompatibility complex class I–related chains A and B, upon activation. Moreover, type 1 diabetic NK cells also exhibited decreased NKG2D-dependent cytotoxicity and interferon-γ secretion. Finally, type 1 diabetic NK cells showed clear defects in NKG2D-mediated activation of the phosphoinositide 3-kinase–AKT pathway.

CONCLUSIONS

These results are the first to demonstrate that type 1 diabetic subjects have aberrant signaling through the NKG2D receptor and suggest that NK cell dysfunction contributes to the autoimmune pathogenesis of type 1 diabetes.Type 1 diabetes is a multifactorial autoimmune disease characterized by T-cell destruction of insulin-producing β-cells and the eventual loss of glucose homeostasis (1). Although both genetic and environmental factors contribute to the breakdown of immunological self-tolerance, and many of the hallmarks of disease in humans are recapitulated in the NOD mouse (2), the precise mechanisms driving pathogenesis remain unclear. Current evidence suggests that natural killer (NK) cells may be both important regulators and inducers of autoimmune diseases (3–8), and several reports (9–13) have documented that NK cells in NOD mice are impaired compared with those in healthy mice. Although investigations of human subjects with type 1 diabetes have described NK cell alterations, these studies have been limited in size, and the mechanisms underlying the phenotype have not been identified (14–20).NK cells are well known to have critical roles against viral, bacterial, and parasitic pathogens through the direct killing of infected cells and the production of proinflammatory cytokines such as interferon (IFN)-γ and tumor necrosis factor-α (21). A balance of signals received through a diverse array of activating and inhibitory surface receptors determines whether NK cells evoke their potent effector functions toward a target (22). Some activating receptors are known to bind foreign viral proteins, whereas others recognize self-proteins that are induced upon cellular stress (23). A prominent activating receptor involved in the recognition of stressed, infected, or transformed cells is the C-type lectin NKG2D (24). Signaling by NKG2D is mediated through its association with the transmembrane adaptor protein DNAX-activating protein of 10 kDa (DAP10). Although the NKG2D-DAP10–signaling complex is unusual because it lacks an immunoreceptor tyrosine–based activation motif, DAP10 does contain a “YxxM” motif that functions to recruit the p85 subunit of phosphoinositide3-kinase (PI3K) upon tyrosine phosphorylation (25,26).Recent work (27) has shown that NOD NK cells exhibit decreased NKG2D-dependent functioning and that this deficit may contribute to disease in this murine model. Activated NOD NK cells, but not C57BL/6 NK cells, were found to maintain NKG2D ligand expression, resulting in the downmodulation of the NKG2D receptor through a mechanism dependent on the “YxxM” motif of DAP10 (27). Reduced NKG2D expression on NOD NK cells was mirrored by decreased cytotoxic and cytokine-secreting functions (27). Notably, we have previously shown that administration of complete Freund adjuvant (CFA) to NOD mice causes NK cells to downregulate NKG2D ligand expression and that the phenomenon is correlated with increased NKG2D receptor expression and heightened NK cell functions (28,29). In addition, NK cells rejuvenated by CFA treatment were able to protect NOD SCID (severe combined immune deficiency) mice from the development of autoimmune diabetes following the adoptive transfer of these hosts with diabetogenic splenocytes (28,29). Collectively, these findings suggest that the chronic exposure of NOD NK cells to NKG2D ligands results in their desensitization and also that augmentation of NK cell function protects NOD mice from disease.Given the important regulatory role of NK cells in diabetes of the NOD mouse, we sought to determine whether numeric or functional deficits also are present among human type 1 diabetic NK cells. Here, we report that NK cells from children with type 1 diabetes constitute a significantly reduced fraction of peripheral mononuclear cells relative to age-matched nondiabetic control subjects and that these NK cells are poorly responsive to interleukin (IL)-2/IL-15 stimulation. Analogous to findings in the NOD mouse (27–29), dysregulated expression of the NKG2D ligands on activated type 1 diabetic NK cells is present and associated with both impaired NKG2D-mediated effector function and signaling. These results suggest that NK cell dysfunction and aberrant NKG2D signaling may be a consequence of, or contribute to, the pathogenesis of type 1 diabetes.     

B-Cell Cross-Presentation of Autologous Antigen Precipitates Diabetes

For autoimmune conditions like type 1 diabetes to progress, self-reactive CD8⁺ T cells would need to interact with peptide–antigen cross-presented on the surface of antigen-presenting cells in a major histocompatibility complex (MHC) class I-restricted fashion. However, the mechanisms by which autoantigen is cross-presented remain to be identified. In this study, we show cross-presentation of islet-derived autoantigens by B cells. B cells engage self-reactive CD8⁺ T cells in the pancreatic lymph node, driving their proliferative expansion and differentiation into granzyme B⁺interferon-γ⁺lysosomal-associated membrane protein 1⁺ effector cells. B-cell cross-presentation of insulin required proteolytic cleavage and endosomal localization and was sensitive to inhibitors of protein trafficking. Absent B-cell MHC class I, or B-cell receptor restriction to an irrelevant specificity, blunted the expansion of self-reactive CD8⁺ T cells, suggesting B-cell antigen capture and presentation are critical in vivo events for CD8 activation. Indeed, the singular loss of B-cell MHC class I subverted the conversion to clinical diabetes in NOD mice, despite the presence of a pool of activated, and B cell–dependent, interleukin-21–expressing Vβ4⁺CD4⁺ T cells. Thus, B cells govern the transition from clinically silent insulitis to frank diabetes by cross-presenting autoantigen to self-reactive CD8⁺ T cells.There is good evidence that CD8⁺ cytotoxic T lymphocytes (CTL) subsequently kill β-cells, resulting in type 1 diabetes (T1D). CD8⁺ T-cell clones isolated from pancreatic infiltrates of NOD mice, a spontaneous diabetes model (1), recognize defined islet autoantigens including insulin (2), the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) (3), glutamic acid decarboxylase proteins (4), and dystrophia myotonica kinase (5). Defined CD8⁺ T-cell clones are sufficient to precipitate diabetes (6–9); conversely, CD8⁺ T-cell depletion (10), inhibition of CD8⁺ T-cell maturation (11), disabling CD8–effector pathways (12), or deleting surface major histocompatibility complex (MHC) class I from β-cells (13,14) prevents clinical diabetes. Understanding CTL responses is of high clinical relevance, as MHC class I-restricted T cells reactive to insulin, IGRP, and glutamic acid decarboxylase have been identified in human subjects with T1D (4,15,16). Therefore, the steps governing the activation of self-reactive CTL would represent critical nodes for intervention; nevertheless, the physiological processes that drive these events remain poorly understood.In the NOD mouse model, B-cell depletion prevents diabetes (17–21). We were intrigued by the observation that in some studies, B-cell depletion postinsulitis was protective, but also that B-cell depletion coincided with decreased CD8⁺ T cell activation (17,22). These observations suggested that targeting B cells prevented a late pathogenic event, such as CD8⁺ T cell–mediated β-cell destruction, and raised the possibility of a direct link between B cells and activation of self-reactive CD8⁺ T cells (23). B-cell depletion delays diabetes in man (24), indicating B cells as therapeutic targets for the treatment of type 1 diabetes (18); however, the mechanisms of action by which B-cell reduction effects diabetes progression are unclear. This background led us to investigate whether there was a requirement for B cells in the activation, expansion, and effector development of pathogenic CD8⁺ T cells and the subsequent transition to overt diabetes in the nonobese diabetic (NOD) model of spontaneous diabetes.     

Immune Depletion With Cellular Mobilization Imparts Immunoregulation and Reverses Autoimmune Diabetes in Nonobese Diabetic Mice

OBJECTIVE

The autoimmune destruction of β-cells in type 1 diabetes results in a loss of insulin production and glucose homeostasis. As such, an immense interest exists for the development of therapies capable of attenuating this destructive process through restoration of proper immune recognition. Therefore, we investigated the ability of the immune-depleting agent antithymocyte globulin (ATG), as well as the mobilization agent granulocyte colony–stimulating factor (GCSF), to reverse overt hyperglycemia in the nonobese diabetic (NOD) mouse model of type 1 diabetes.

RESEARCH DESIGN AND METHODS

Effects of each therapy were tested in pre-diabetic and diabetic female NOD mice using measurements of glycemia, regulatory T-cell (CD4+CD25+Foxp3+) frequency, insulitis, and/or β-cell area.

RESULTS

Here, we show that combination therapy of murine ATG and GCSF was remarkably effective at reversing new-onset diabetes in NOD mice and more efficacious than either agent alone. This combination also afforded durable reversal from disease (>180 days postonset) in animals having pronounced hyperglycemia (i.e., up to 500 mg/dl). Additionally, glucose control improved over time in mice subject to remission from type 1 diabetes. Mechanistically, this combination therapy resulted in both immunological (increases in CD4-to-CD8 ratios and splenic regulatory T-cell frequencies) and physiological (increase in the pancreatic β-cell area, attenuation of pancreatic inflammation) benefits.

CONCLUSIONS

In addition to lending further credence to the notion that combination therapies can enhance efficacy in addressing autoimmune disease, these studies also support the concept for utilizing agents designed for other clinical applications as a means to expedite efforts involving therapeutic translation.Type 1 diabetes is characterized by the autoimmune destruction of β-cells, resulting in a loss of insulin production and glucose control (1,2). In both humans and the nonobese diabetic (NOD) mouse model of type 1 diabetes, the disorder''s pathogenesis appears dependent on aberrant immune regulation (3–6). A reversal of type 1 diabetes in NOD mice has been achieved, with varying levels of success, through administration of a limited number of immunosuppressive and immunomodulatory agents, some of which are controversial with respect to their translational capabilities (7–19).Antithymocyte globulin (ATG) is currently in clinical use for a variety of purposes, including the treatment of acute rejection, graft versus host disease, and conditioning for stem-cell transplantation (20–22). It has been shown to target >40 epitopes and serves to induce lymphocyte depletion, the extent of which depends upon the dose administered. Previously, we have shown that murine ATG is capable of late prevention of diabetes in NOD mice and, importantly, that this agent was capable of inducing a regulatory T-cell population (16). With this, we questioned whether the efficacy of this therapy could be improved through the use of a second immunomodulatory agent differing in its presumed mechanism of therapeutic activity. To that regard, we elected to evaluate granulocyte colony–stimulating factor (GCSF).GCSF was initially developed as a means of mobilizing neutrophils (23,24), but recent reports (25) have also indicated a GCSF-induced immunoregulatory impact. These studies indicated the ability of GCSF to induce an immunoregulatory shift from a T_H1 to a T_H2 cytokine phenotype (26), the induction of tolerogenic dendritic cells (27), and the mobilization of regulatory T-cells. In regards to type 1 diabetes, GCSF has successfully prevented the onset of disease in the NOD mouse via the induction of both tolerogenic dendritic and regulatory T-cells (28) and prevented the cyclophosphamide-mediated acceleration of diabetes (29).Hence, in this report, we examined the therapeutic efficacy of these two agents, ATG and GCSF, subject to clinical use in settings outside of type 1 diabetes, for the purpose of testing their ability to reverse disease in NOD mice as well as to monitor their ability to reinstill self tolerance. In this study, we also tested the hypothesis that combination therapy will be more effective than either monotherapy for the purposes of treating type 1 diabetes in NOD mice.     

Loss of intra-islet CD20 expression may complicate efficacy of B-cell-directed type 1 diabetes therapies

OBJECTIVE

Consistent with studies in NOD mice, early clinical trials addressing whether depletion of B cells by the Rituximab CD20-specific antibody provides an effective means for type 1 diabetes reversal have produced promising results. However, to improve therapeutic efficacy, additional B-cell–depleting agents, as well as attempts seeking diabetes prevention, are being considered.

RESEARCH DESIGN AND METHODS

Autoantibodies, including those against insulin (IAAs), are used to identify at-risk subjects for inclusion in diabetes prevention trials. Therefore, we tested the ability of anti-CD20 to prevent diabetes in NOD mice when administered either before or after IAA onset.

RESULTS

The murine CD20-specific 18B12 antibody that like Rituximab, depletes the follicular (FO) but not marginal zone subset of B cells, efficiently inhibited diabetes development in NOD mice in a likely regulatory T-cell–dependent manner only when treatment was initiated before IAA detection. One implication of these results is that the FO subset of B cells preferentially contributes to early diabetes initiation events. However, most important, the inefficient ability of anti-CD20 treatment to exert late-stage diabetes prevention was found to be attributable to downregulation of CD20 expression upon B cell entry into pancreatic islets.

CONCLUSIONS

These findings provide important guidance for designing strategies targeting B cells as a potential means of diabetes intervention.While the autoimmune destruction of pancreatic β-cells that results in type 1 diabetes is ultimately mediated by both CD4 and CD8 T cells, in the NOD mouse model and potentially in humans, disease pathogenesis also requires contributions from B cells (reviewed in Silveira et al. [1]). Studies in NOD mice indicate B cells likely contribute to diabetes by serving as a subset of antigen presenting cells (APCs) that most efficiently support the expansion of pathogenic CD4 T-cell responses (2–4). This is because unlike other APC subsets, B cells express plasma membrane-bound Ig molecules, allowing for their specific and efficient capture of pancreatic β-cell proteins (5,6). Indeed, some diabetes susceptibility genes in NOD mice mechanistically contribute to disease pathogenesis by impairing immunological tolerance induction mechanisms normally deleting or inactivating B cells expressing autoreactive Ig specificities (7–9). Secreted autoreactive Ig molecules may also contribute to diabetes pathogenesis in NOD mice (10,11). In addition, B cells may contribute to diabetes in NOD mice by supporting development in the vicinity of pancreatic islets of tertiary lymphoid structures where pathogenic T cells might be activated (12).Eliminating B cells from birth by either genetic or antibody-mediated approaches inhibits diabetes development in NOD mice (13,14). Partly on the basis of these findings, early phase clinical trials were initiated to determine whether depletion of B cells using the human CD20-specific Rituximab antibody provided beneficial effects, including preservation of C-peptide production, for recent-onset diabetes patients (15,16). Hope for these trials was bolstered by several reports suggesting that in addition to a capacity to block progression to overt diabetes when initiated at an early prodromal stage of disease development, anti-CD20–mediated B-cell depletion (and in one case, using anti-CD22) can also reverse recently established hyperglycemia in at least a subset of NOD mice (17–19). However, it is unclear if CD20- and CD22-specific antibodies with a reported ability to reverse recent-onset diabetes in NOD mice exert the same pattern of B-cell subset deletion as Rituximab. In this regard, it should be noted that Rituximab efficiently depletes the follicular (FO) but not the marginal zone (MZ) subset of mature B cells (20). Such a characteristic is of potential importance given reports that MZ subset B cells can exert potent APC activity and may preferentially contribute to diabetes development in NOD mice (21,22). Furthermore, the capacity of anti-CD20 treatment to eliminate B cells that become activated within pancreatic insulitic infiltrates during diabetes development is also unknown.Another factor to consider is the short time frame after onset of overt hyperglycemia in which anti-CD20–mediated B-cell depletion can reportedly exert a disease reversal effect in NOD mice (18). It is unclear how frequently anti-CD20 treatment could be undertaken in an analogous time frame after diabetes onset in humans. Furthermore, the first reports from human diabetes intervention trials indicate Rituximab treatment retards the rate but does not eliminate the further erosion of residual pancreatic β-cell mass in recent disease onset patients (23). With this result, while promising, it has been questioned whether anti-CD20 treatment might prove more effective in preventing the progression to overt diabetes when initiated in individuals at late prodromal stages of disease development. Here, such trials would take advantage of a continual refinement of genetic and immunological susceptibility markers (24,25).One key marker considered predictive for future diabetes development in humans is the appearance of insulin autoantibodies (IAAs) (26). The presence of IAAs also reportedly marks individual NOD mice that will first develop overt diabetes (27). Hence, to model a potential clinical use setting, we determined if when first initiated in already IAA-positive NOD mice, treatment with a murine CD20-specific antibody sharing B-cell deletional characteristics similar to Rituximab retained a capacity to inhibit diabetes development. We also assessed whether during progression of diabetes development, anti-CD20 treatment could eliminate B cells within pancreatic islet leukocytic infiltrates. Diabetes resistance elicited in NOD mice treated with a B-cell activating factor (BAFF)-blocking reagent depleting all mature B cells reportedly results from an enhanced ability of residual remaining myeloid-type APCs to support a regulatory T-cell (Treg) expansion (28). Hence, we also evaluated if diabetes protection resulting from the possible partial pattern of anti-CD20–mediated B-cell depletion was due to a Treg expansion.     

PD-1, but Not PD-L1, Expressed by Islet-Reactive CD4+ T Cells Suppresses Infiltration of the Pancreas During Type 1 Diabetes

The inhibitory receptor programmed death-1 (PD-1) constrains type 1 diabetes (T1D) in the nonobese diabetic (NOD) mouse. However, how PD-1 influences diabetogenic CD4⁺ T cells during natural diabetes is not fully understood. To address this question, we developed a novel model to investigate antigen-specific CD4⁺ T cells under physiological conditions in vivo. We transferred a low number of naïve CD4⁺ T cells from the BDC2.5 mouse into prediabetic NOD mice to mimic a physiological precursor frequency and allowed the cells to become primed by endogenous autoantigen. Transferred BDC2.5 T cells became activated, differentiated into T-bet⁺ IFN-γ–producing cells, and infiltrated the pancreas. In this model, loss of PD-1, but not programmed death ligand-1 (PD-L1), on the antigen-specific CD4⁺ T cell resulted in increased cell numbers in the spleen, pancreas-draining lymph node, and pancreas. PD-1 deficiency also increased expression of the chemokine receptor CXCR3. Lastly, histological data showed that a loss of PD-1 caused BDC2.5 cells to penetrate deep into the islet core, resulting in conversion from peri-insulitis to destructive insulitis. These data support a model by which PD-1 regulates islet-reactive CD4⁺ T cells in a cell intrinsic manner by suppressing proliferation, inhibiting infiltration of the pancreas, and limiting diabetes.Type 1 diabetes (T1D) is an autoimmune disease mediated by T-cell destruction of the insulin-producing β-cells in the pancreatic islets of Langerhans (1). The nonobese diabetic (NOD) mouse is a classic model for studying T1D because it shares many similarities with human T1D, including the requirement of CD4⁺ T cells for disease (2–4). However, knowledge of how diabetogenic CD4⁺ T cells are regulated and how this regulation fails, causing T1D, is limited owing to a lack of tools to monitor endogenous diabetogetic CD4⁺ T cells.Common models used to study diabetogenic CD4⁺ T cells in NOD mice include adoptive transfer of high numbers of naïve or in vitro activated T-cell receptor (TCR) transgenic cells into wild-type (WT) or lymphopenic NOD recipients (5–10). While informative, these approaches fail to recapitulate the natural inflammatory environment present in NOD mice and the timing associated with T1D progression. Previous work in other systems showed that transferring lower numbers of naïve T cells allowed greater clonal expansion on a per cell basis and more efficient effector cell differentiation (11–14). Since we speculate that endogenous autoantigen in the NOD mouse is low, we predicted that limiting the diabetogenic precursor frequency would be essential for autoantigen encounter and activation. Therefore, in this study we developed a new model by transferring a small number of islet-specific BDC2.5 transgenic CD4⁺ T cells (15,16) into prediabetic NOD mice to mimic an endogenous preimmune repertoire.The inhibitory receptor programmed death-1 (PD-1) interacting with programmed death ligand-1 (PD-L1) is critical for suppressing diabetes, since disrupting PD-1/PD-L1 interactions accelerates T1D in NOD mice (7,17–19) and polymorphisms in PD-1 have been associated with human T1D (20). Previous studies demonstrated roles for the PD-1 pathway by inhibiting CD4⁺ T-cell survival, proliferation, and cytokine production using in vitro and in vivo systems (5,7,21–24). However, since many of the in vivo studies relied on adoptive transfer of nonphysiologically high numbers of TCR transgenic T cells, the cellular mechanisms by which PD-1 constrains diabetogenic CD4⁺ T cells in hosts with a normal T-cell repertoire remain unclear. We therefore reexamined the role of PD-1 in regulating CD4⁺ T cells in vivo using a new adoptive transfer model that more closely mimics the normal naïve preimmune repertoire. Our results show that PD-1 expressed by the BDC2.5 T cell is required to regulate proliferation, chemokine receptor CXCR3 expression, infiltration of the pancreas, and diabetes pathogenesis.     

TSG-6 Produced by hMSCs Delays the Onset of Autoimmune Diabetes by Suppressing Th1 Development and Enhancing Tolerogenicity

Genetic and immunological screening for type 1 diabetes has led to the possibility of preventing disease in susceptible individuals. Here, we show that human mesenchymal stem/stromal cells (hMSCs) and tumor necrosis factor-α–stimulated gene 6 (TSG-6), a protein produced by hMSCs in response to signals from injured tissues, delayed the onset of spontaneous autoimmune diabetes in NOD mice by inhibiting insulitis and augmenting regulatory T cells (Tregs) within the pancreas. Importantly, hMSCs with a knockdown of tsg-6 were ineffective at delaying insulitis and the onset of diabetes in mice. TSG-6 inhibited the activation of both T cells and antigen-presenting cells (APCs) in a CD44-dependent manner. Moreover, multiple treatments of TSG-6 rendered APCs more tolerogenic, capable of enhancing Treg generation and delaying diabetes in an adoptive transfer model. Therefore, these results could provide the basis for a novel therapy for the prevention of type 1 diabetes.Recent advances in the use of genetic and immunological screening for identification of prediabetic patients (1–3) have opened up the opportunity to prevent, delay, or halt disease progression before the diagnosis of diabetes. Based on the success in animal models (4–6), clinical trials of oral or nasal insulin (7,8) and nicotinamide (9,10) have been conducted in humans to prevent type 1 diabetes. However, despite all efforts, these clinical trials have failed to show any improvement in the prevention of type 1 diabetes.Recently, we found that intravenously administered human mesenchymal stem/stromal cells (hMSCs) were activated to express the anti-inflammatory protein tumor necrosis factor (TNF)-α–stimulated gene 6 (TSG-6), which reduced excessive inflammatory response in the myocardial-infarcted heart in mice (11), chemically and mechanically injured cornea in rodent models (12,13), and zymosan-induced peritonitis in mice (14). Specifically, our recent observation revealed that TSG-6 attenuated zymosan-induced mouse peritonitis by decreasing TLR2-mediated NF-κB signaling in resident macrophages (14). This suppressive effect of TSG-6 on NF-κB signaling could provide the rationale for TSG-6 as a potential therapy for the prevention of type 1 diabetes, since several studies have already shown that inflammation and the innate immune system contribute to induction, amplification, and maintenance of the immune cell infiltrate as well as β-cell destruction during this preclinical period (15–17). Particularly, antigen-presenting cells (APCs) from NOD mice, mainly dendritic cells (DCs) and macrophages, have been shown to secrete substantially elevated levels of interleukin-12 (IL-12) and TNF-α (18,19) due to NF-κB hyperactivity (18,20), which leads to T-helper 1 (Th1) development and overt diabetes (21).Here, we tested whether a new treatment for the prevention of type 1 diabetes could be developed using TSG-6, which hMSCs produce in response to signals from injured tissues. Our data showed that systemic administration of hMSCs to prediabetic mice delayed the onset of type 1 diabetes in NOD mice in part by secreting TSG-6.