Chromogranin A Deficiency Confers Protection From Autoimmune Diabetes via Multiple Mechanisms

Recognition of β-cell antigens by autoreactive T cells is a critical step in the initiation of autoimmune type1 diabetes. A complete protection from diabetes development in NOD mice harboring a point mutation in the insulin B-chain 9–23 epitope points to a dominant role of insulin in diabetogenesis. Generation of NOD mice lacking the chromogranin A protein (NOD.ChgA^−/−) completely nullified the autoreactivity of the BDC2.5 T cell and conferred protection from diabetes onset. These results raised the issue concerning the dominant antigen that drives the autoimmune process. Here we revisited the NOD.ChgA^−/− mice and found that their lack of diabetes development may not be solely explained by the absence of chromogranin A reactivity. NOD.ChgA^−/− mice displayed reduced presentation of insulin peptides in the islets and periphery, which corresponded to impaired T-cell priming. Diabetes development in these mice was restored by antibody treatment targeting regulatory T cells or inhibiting transforming growth factor-β and programmed death-1 pathways. Therefore, the global deficiency of chromogranin A impairs recognition of the major diabetogenic antigen insulin, leading to broadly impaired autoimmune responses controlled by multiple regulatory mechanisms.     

ZnT8-Reactive T Cells Are Weakly Pathogenic in NOD Mice but Can Participate in Diabetes Under Inflammatory Conditions

Autoantibodies to the islet-specific Zn transporter ZnT8 (Slc30a8), as well as CD4 T cells, have been identified in patients with type 1 diabetes. Here we examined for CD4 T-cell reactivity to ZnT8 epitopes in the NOD mouse. Immunization with a cytoplasmic domain of the protein or with peptides predicted to bind to I-A^g7 resulted in a CD4 T-cell response, indicating a lack of deletional tolerance. However, presentation by intraislet antigen-presenting cells (APC) to the T cells was not detectable in prediabetic mice. Presentation by islet APC was found only in islets of mice with active diabetes. In accordance, a culture assay indicated the weak transfer of ZnT8 reactivity from insulinomas or primary β-cells to APC for presentation to T cells. A T cell directed to one peptide (345–359) resulted in the transfer of diabetes, but only in conditions in which the recipient NOD mice or NOD.Rag1^−/− mice were subjected to light irradiation. In late diabetic NOD mice, CD4 T cells were found as well as a weak antibody response. We conclude that in NOD mice, ZnT8 is a minor diabetogenic antigen that can participate in diabetes in conditions in which the islet is first made receptive to immunological insults.     

Gene Targeting in NOD Mouse Embryos Using Zinc-Finger Nucleases

Studies in NOD mice have provided important insight into the genetics and pathogenesis of type 1 diabetes (T1D). Our goal was to further explore novel methods of genetic manipulation in this mouse model. We tested the feasibility of using zinc-finger nucleases (ZFNs) to knock out a gene directly in a pure NOD background, bypassing the need of embryonic stem cells. We report here the successful application of ZFN pairs to specifically and efficiently knock out Tnfrsf9 (encoding CD137/4–1BB) directly in the NOD mouse by embryo microinjection. Histology and T1D incidence studies indicated that CD137 was dispensable for the development of insulitis but played a role to promote progression to overt diabetes in NOD mice. We also demonstrated that CD137-deficient T-cells were less diabetogenic than their wild-type counterpart when adoptively transferred into NOD.Rag1^−/− recipients, even when CD25⁺ cells were predepleted. In vitro assays suggested that CD137 deficiency had a limited effect on the suppressive function of CD4⁺CD25⁺ regulatory T-cells (Tregs). Therefore, CD137 deficiency predominately affected effector T-cells rather than Tregs. Our study demonstrates the ability to generate gene-targeted knockouts in a pure NOD background by using ZFNs without potential confounding factors introduced by contaminating genetic materials obtained from other strains.     

Exosomes Released by Islet-Derived Mesenchymal Stem Cells Trigger Autoimmune Responses in NOD Mice

Exosomes (EXOs) are secreted, nano-sized membrane vesicles that contain potent immunostimulatory materials. We have recently demonstrated that insulinoma-released EXOs can stimulate the autoimmune responses in nonobese diabetic (NOD) mice, a spontaneous disease model for type 1 diabetes. To investigate whether primary islet cells can produce EXOs, we isolated cells from the islet of Langerhans of NOD mice and cultured them in vitro. Interestingly, cultured islets release fibroblast-like, fast-replicating cells that express mesenchymal stem cell (MSC) markers, including CD105 and stem-cell antigen-1. These islet MSC–like cells release highly immunostimulatory EXOs that could activate autoreactive B and T cells endogenously primed in NOD mice. Serum EXO levels and EXO-induced interferon-γ production were positively correlated with disease progression at the early prediabetic stage. Consistent with these observations, immunohistological analysis of pancreata showed that CD105⁺ cells are restricted to the peri-islet area in normal islets but penetrate into the β-cell area as lymphocyte infiltration occurs. Immunization with EXOs promoted expansion of transferred diabetogenic T cells and accelerated the effector T cell–mediated destruction of islets. Thus, EXOs could be the autoantigen carrier with potent adjuvant activities and may function as the autoimmune trigger in NOD mice.     

Loss of NADPH Oxidase–Derived Superoxide Skews Macrophage Phenotypes to Delay Type 1 Diabetes

Macrophages are early islet-infiltrating cells seen in type 1 diabetes (T1D). While proinflammatory M1 macrophages induce T1D, M2 macrophages have been shown to delay this autoimmune disease in nonobese diabetic (NOD) mice, but the environmental cues that govern macrophage polarization and differentiation remain unresolved. We previously demonstrated the importance of reactive oxygen species (ROS) in T1D, as NOD mice deficient in NADPH oxidase (NOX)-derived superoxide (Ncf1^m1J) were protected against T1D partly because of blunted Toll-like receptor–dependent macrophage responses. We provide evidence that NOX-derived ROS contribute to macrophage differentiation in T1D. During spontaneous diabetes progression, T1D-resistant NOD.Ncf1^m1J islet-resident macrophages displayed a dampened M1 and increased M2 phenotype. The transfer of diabetogenic T cells into NOX-deficient NOD.Rag.Ncf1^m1J recipients resulted in decreased TNF-α⁺ and IL-1β⁺ islet-infiltrating M1 macrophages and a concomitant enhancement in arginase-1⁺ M2 macrophages. Mechanistic analysis of superoxide-deficient bone marrow–derived macrophages revealed a marked diminution in a proinflammatory M1 phenotype due to decreased P-STAT1 (Y701) and interferon regulatory factor 5 compared with NOD mice. We have therefore defined a novel mechanistic link between NOX-derived ROS and macrophage phenotypes, and implicated superoxide as an important factor in macrophage differentiation. Thus, targeting macrophage redox status may represent a promising therapy in halting human T1D.     

TLR2- and Dectin 1–Associated Innate Immune Response Modulates T-Cell Response to Pancreatic β-Cell Antigen and Prevents Type 1 Diabetes

The progression of autoimmune diseases is dictated by deviations in the fine balance between proinflammatory versus regulatory responses, and pathogen recognition receptors (PRRs) play a key role in maintaining this balance. Previously, we have reported that ligation of Toll-like receptor 2 (TLR2) and Dectin 1 on antigen-presenting cells by zymosan results in a regulatory immune response that prevents type 1 diabetes (T1D). Here, we show that TLR2 and Dectin 1 engagement by zymosan promotes regulatory T-cell (Treg) responses against the pancreatic β-cell–specific antigen (Ag). Unlike the TLR4 ligand, bacterial lipopolysaccharide, which induced proinflammatory cytokines and pathogenic T cells, zymosan induced a mixture of pro- and anti-inflammatory factors and Tregs, both in vitro and in vivo. Ag-specific T cells that are activated using zymosan-exposed dendritic cells (DCs) expressed Foxp3 and produced large amounts of IL-10, TGF-β1, and IL-17. NOD mice that received β-cell-Ag–loaded, zymosan-exposed DCs showed delayed hyperglycemia. Injection of NOD mice at the prediabetic age and early hyperglycemic stage with β-cell-Ag, along with zymosan, results in a superior protection of the NOD mice from diabetes as compared with mice that received zymosan alone. This therapeutic effect was associated with increased frequencies of IL-10–, IL-17–, IL-4–, and Foxp3-positive T cells, especially in the pancreatic lymph nodes. These results show that zymosan can be used as an immune regulatory adjuvant for modulating the T-cell response to pancreatic β-cell-Ag and reversing early-stage hyperglycemia in T1D.     

Local Autoantigen Expression as Essential Gatekeeper of Memory T-Cell Recruitment to Islet Grafts in Diabetic Hosts

It is generally believed that inflammatory cues can attract noncognate, “bystander” T-cell specificities to sites of inflammation. We have shown that recruitment of naive and in vitro activated autoreactive CD8⁺ T cells into endogenous islets requires local autoantigen expression. Here, we demonstrate that absence of an autoantigen in syngeneic extrapancreatic islet grafts in diabetic hosts renders the grafts “invisible” to cognate memory (and naive) T cells. We monitored the recruitment of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)_206–214-reactive CD8⁺ T cells into IGRP_206–214-competent and IGRP_206–214-deficient islet grafts in diabetic wild-type or IGRP_206–214^−/− nonobese diabetic hosts (harboring either naive and memory T cells or only naive IGRP_206–214-specific T-cells, respectively). All four host–donor combinations had development of recurrent diabetes within 2 weeks. Wild-type hosts recruited IGRP_206–214-specific T cells into IGRP_206–214^+/+ but not IGRP_206–214^−/− grafts. In IGRP_206–214^−/− hosts, there was no recruitment of IGRP_206–214-specific T cells, regardless of donor type. Graft-derived IGRP_206–214 activated naive IGRP_206–214-specific T cells, but graft destruction invariably predated their recruitment. These results indicate that recurrent diabetes is exclusively driven by autoreactive T cells primed during the primary autoimmune response, and demonstrate that local antigen expression is a sine qua non requirement for accumulation of memory T cells into islet grafts. These findings underscore the importance of tackling autoreactive T-cell memory after β-cell replacement therapy.Nonobese diabetic (NOD) mice have development of a form of type 1 diabetes that results from destruction of β cells by CD4⁺ and CD8⁺ T cells recognizing many autoantigenic peptides (1). A significant fraction of islet-associated CD8⁺ cells recognize the mimotope NRP-V7 in the context of the major histocompatibility complex (MHC) molecule K^d (2). These cells are a significant component of the earliest NOD islet CD8⁺ infiltrates (2,3), are diabetogenic (4,5), and target residues 206–214 of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) (6). The peripheral IGRP_206–214-reactive CD8⁺ T-cell pool is sizeable (7) and, on recruitment into islets, undergoes a local avidity maturation process that contributes to disease progression (8).Studies in infection and autoimmune disease models have suggested that recruitment of T cells into sites of extralymphoid inflammation does not require local expression of cognate peptide–MHC (pMHC) (9–11). However, we recently have shown that cues emanating from pancreatic islets undergoing spontaneous autoimmune inflammation in NOD mice cannot recruit naive or newly activated bystander T-cell specificities. This was established by monitoring the recruitment of naive or in vitro activated IGRP_206–214-specific CD8⁺ T cells in gene-targeted NOD mice expressing a T-cell “invisible” IGRP_206–214 sequence. These mice had development of diabetes with normal incidence, but their insulitic lesions could not recruit either cell type. These results indicated that recruitment of naive T cells or effector cytotoxic T lymphocytes to a site of autoimmune inflammation results from an active process that is strictly dependent on local display of cognate pMHC (12).Here, we asked whether this revised paradigm also applies to recruitment of memory (autoantigen-experienced) autoreactive T cells and/or recruitment of naive and memory T cells to syngeneic islet grafts. We reasoned that the “nonphysiological” lymphatic and vascular anatomy of islets grafts transplanted under the kidney capsule (13–15), coupled with a high rate of graft cell death (16), should allow recruitment of “graft-irrelevant” (i.e., nonautoreactive) memory T cells to the site in response to local inflammatory cues, including those caused by grafting. We demonstrate that recruitment of CD8⁺ T cells to islet grafts during disease recurrence exclusively involves autoantigen-specific T cells from the memory pool, excluding a role for bystander T-cell specificities or graft antigen-activated autoreactive T cells.     

A Humanized Mouse Model of Autoimmune Insulitis

Many mechanisms of and treatments for type 1 diabetes studied in the NOD mouse model have not been replicated in human disease models. Thus, the field of diabetes research remains hindered by the lack of an in vivo system in which to study the development and onset of autoimmune diabetes. To this end, we characterized a system using human CD4⁺ T cells pulsed with autoantigen-derived peptides. Six weeks after injection of as few as 0.5 × 10⁶ antigen-pulsed cells into the NOD-Scid Il2rg^−/− mouse expressing the human HLA-DR4 transgene, infiltration of mouse islets by human T cells was seen. Although islet infiltration occurred with both healthy and diabetic donor antigen-pulsed CD4⁺ T cells, diabetic donor injections yielded significantly greater levels of insulitis. Additionally, significantly reduced insulin staining was observed in mice injected with CD4⁺ T-cell lines from diabetic donors. Increased levels of demethylated β-cell–derived DNA in the bloodstream accompanied this loss of insulin staining. Together, these data show that injection of small numbers of autoantigen-reactive CD4⁺ T cells can cause a targeted, destructive infiltration of pancreatic β-cells. This model may be valuable for understanding mechanisms of induction of human diabetes.     

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.     

Antiaging Gene Klotho Attenuates Pancreatic β-Cell Apoptosis in Type 1 Diabetes

Apoptosis is the major cause of death of insulin-producing β-cells in type 1 diabetes mellitus (T1DM). Klotho is a recently discovered antiaging gene. We found that the Klotho gene is expressed in pancreatic β-cells. Interestingly, halplodeficiency of Klotho (KL^+/−) exacerbated streptozotocin (STZ)-induced diabetes (a model of T1DM), including hyperglycemia, glucose intolerance, diminished islet insulin storage, and increased apoptotic β-cells. Conversely, in vivo β-cell–specific expression of mouse Klotho gene (mKL) attenuated β-cell apoptosis and prevented STZ-induced diabetes. mKL promoted cell adhesion to collagen IV, increased FAK and Akt phosphorylation, and inhibited caspase 3 cleavage in cultured MIN6 β-cells. mKL abolished STZ- and TNFα-induced inhibition of FAK and Akt phosphorylation, caspase 3 cleavage, and β-cell apoptosis. These promoting effects of Klotho can be abolished by blocking integrin β1. Therefore, these cell-based studies indicated that Klotho protected β-cells by inhibiting β-cell apoptosis through activation of the integrin β1-FAK/Akt pathway, leading to inhibition of caspase 3 cleavage. In an autoimmune T1DM model (NOD), we showed that in vivo β-cell–specific expression of mKL improved glucose tolerance, attenuated β-cell apoptosis, enhanced insulin storage in β-cells, and increased plasma insulin levels. The beneficial effect of Klotho gene delivery is likely due to attenuation of T-cell infiltration in pancreatic islets in NOD mice. Overall, our results demonstrate for the first time that Klotho protected β-cells in T1DM via attenuating apoptosis.     

Protective role of programmed death 1 ligand 1 (PD-L1)in nonobese diabetic mice: the paradox in transgenic models

OBJECTIVE—Coinhibitory signals mediated via programmed death 1 (PD-1) receptor play a critical role in downregulating immune responses and in maintaining peripheral tolerance. Programmed death 1 ligand 1 (PD-L1), the interacting ligand for PD-1, widely expressed in many cell types, acts as a tissue-specific negative regulator of pathogenic T-cell responses. We investigated the protective potential of PD-L1 on autoimmune diabetes by transgenically overexpressing PD-L1 in pancreatic β-cells in nonobese diabetic (NOD) mice.RESEARCH DESIGN AND METHODS—We established an insulin promoter–driven murine PD-L1 transgenic NOD mouse model to directly evaluate the protective effect of an organ-specific PD-L1 transgene against autoimmune diabetes. Transgene expression, insulitis, and diabetic incidence were characterized in these transgenic NOD mice. Lymphocyte development, Th1 cells, and regulatory T-cells were analyzed in these transgenic mice; and T-cell proliferation, adoptive transfer, and islet transplantation were performed to evaluate the PD-L1 transgene–mediated immune-protective mechanisms.RESULTS—The severity of insulitis in these transgenic mice is significantly decreased, disease onset is delayed, and the incidence of diabetes is markedly decreased compared with littermate controls. NOD/SCID mice that received lymphocytes from transgenic mice became diabetic at a slower rate than mice receiving control lymphocytes. Moreover, lymphocytes collected from recipients transferred by lymphocytes from transgenic mice revealed less proliferative potential than lymphocytes obtained from control recipients. Transgenic islets transplanted in diabetic recipients survived moderately longer than control islets.CONCLUSIONS—Our results demonstrate the protective potential of transgenic PD-L1 in autoimmune diabetes and illustrate its role in downregulating diabetogenic T-cells in NOD mice.Programmed death 1 (PD-1) is an immunoreceptor of the CD28/CTLA-4 family whose expression is induced in activated T- and B-cells and in macrophages (1,2). PD-1 has two cytoplasmic tyrosine motifs: one an immunoreceptor tyrosine-based inhibition motif and the other an immunoreceptor tyrosine-based switch motif (ITSM). On interaction of PD-1 with its ligands PD-L1 (B7-H1) or PD-L2 (B7-DC), the tyrosine-phosphorylated ITSM of PD-1 recruits a src homology 2 domain–containing tyrosine phosphatase 2, which mediates the dephosphorylation signaling and reduces lymphocyte activation (3). PD-1^−/− mice on different genetic backgrounds develop distinct autoimmune phenotypes, such as lupus-like glomerulonephritis/arthritis in C57Bl/6 (B6) mice or anticardiac troponin I–mediated dilated cardiomyopathy in Balb/c mice (4,5). These observations indicate that PD-1 is a critical negative regulator of lymphocyte activation and that the phenotype of PD-1 deficiency–induced autoimmunity is highly influenced by other genetic factors.Murine PD-L1 is expressed on many cell types, including stromal cells within many organs, but PD-L2 expression is much more restricted, occurring mainly in dendritic cells, activated monocytes, and macrophages (6,7). Although accumulating data indicate that both PD-L1/PD-1 and PD-L2/PD-1 signals can suppress T-cell proliferation and effector function by blocking cell cycle progression and cytokine production, signaling through PD-L1 interaction is more potent than that through PD-L2 (8). This is consistent with the observations that cytokine production, cytotoxic activity, and clonal expansion were significantly enhanced in T-cells and antigen-presenting cells from PD-L1^−/− mice, compared with cells from wild-type or PD-L2^−/− mice (9,10). Moreover, the PD-L1^−/− mice revealed an increased susceptibility to the induction of autoimmune diseases, such as experimental allergic encephalomyelitis (9), strongly suggesting a protective role of tissue PD-L1 in the maintenance of immune tolerance. Furthermore, treatment of nonobese diabetic (NOD) mice with a combination of agonistic PD-L1.Ig fusion protein and monoclonal antibodies (mAbs) to CD154 induced long-term islet allograft survival, whereas the inhibition of PD-L1–mediated signals by blocking antibody exacerbated autoimmune diabetes (11). Based on these findings, we hypothesize that transgenic expression of PD-L1 in an islet-specific manner may help in preventing T-cell–mediated islet destruction in NOD mice.To investigate the preventive and/or therapeutic potential of PD-L1 in autoimmune diabetes, we generated transgenic NOD mice overexpressing PD-L1 under control of an insulin promoter. Although a recent report demonstrated that local expression of transgenic PD-L1 on β-cells of B6 mice unexpectedly promotes organ-specific autoimmunity and transplant rejection (12), we hypothesized that overexpression of PD-L1 on islet cells in NOD mice would enhance inhibitory signaling through the PD-1–PD-L1 interaction and protect islet cells from lymphocyte attack. Our results demonstrate that transgenic PD-L1 on islet cells significantly ameliorates the severity of insulitis and incidence of diabetes in NOD mice. Interestingly, our results also indicate that local transgene expression not only protects islets in situ but also mediates a peripheral tolerance. Moreover, transgenic islets transplanted in diabetic recipients survived moderately longer than control islets. Overall, we demonstrate for the first time the preventive potential of transgenic PD-L1 in autoimmune diabetes and provide a theoretical basis for organ-specific genetic manipulation for disease prevention.     

TLR9 Deficiency in B Cells Promotes Immune Tolerance via Interleukin-10 in a Type 1 Diabetes Mouse Model

Toll-like receptor 9 (TLR9) is highly expressed in B cells, and B cells are important in the pathogenesis of type 1 diabetes (T1D) development. However, the intrinsic effect of TLR9 in B cells on β-cell autoimmunity is not known. To fill this knowledge gap, we generated NOD mice with a B-cell–specific deficiency of TLR9 (TLR9^fl/fl/CD19-Cre+ NOD). The B-cell–specific deletion of TLR9 resulted in near-complete protection from T1D development. Diabetes protection was accompanied by an increased proportion of interleukin-10 (IL-10)–producing B cells. We also found that TLR9-deficient B cells were hyporesponsive to both innate and adaptive immune stimuli. This suggested that TLR9 in B cells modulates T1D susceptibility in NOD mice by changing the frequency and function of IL-10–producing B cells. Molecular analysis revealed a network of TLR9 with matrix metalloproteinases, tissue inhibitor of metalloproteinase-1, and CD40, all of which are interconnected with IL-10. Our study has highlighted an important connection of an innate immune molecule in B cells to the immunopathogenesis of T1D. Thus, targeting the TLR9 pathway, specifically in B cells, may provide a novel therapeutic strategy for T1D treatment.     

Soluble Factors Secreted by T Cells Promote β-Cell Proliferation

Type 1 diabetes is characterized by infiltration of pancreatic islets with immune cells, leading to insulin deficiency. Although infiltrating immune cells are traditionally considered to negatively impact β-cells by promoting their death, their contribution to proliferation is not fully understood. Here we report that islets exhibiting insulitis also manifested proliferation of β-cells that positively correlated with the extent of lymphocyte infiltration. Adoptive transfer of diabetogenic CD4⁺ and CD8⁺ T cells, but not B cells, selectively promoted β-cell proliferation in vivo independent from the effects of blood glucose or circulating insulin or by modulating apoptosis. Complementary to our in vivo approach, coculture of diabetogenic CD4⁺ and CD8⁺ T cells with NOD.RAG1^−/− islets in an in vitro transwell system led to a dose-dependent secretion of candidate cytokines/chemokines (interleukin-2 [IL-2], IL-6, IL-10, MIP-1α, and RANTES) that together enhanced β-cell proliferation. These data suggest that soluble factors secreted from T cells are potential therapeutic candidates to enhance β-cell proliferation in efforts to prevent and/or delay the onset of type 1 diabetes.     

Differentiating MHC-Dependent and -Independent Mechanisms of Lymph Node Stromal Cell Regulation of Proinsulin-Specific CD8+ T Cells in Type 1 Diabetes

Lymph node stromal cells (LNSC) are essential for providing and maintaining peripheral self-tolerance of potentially autoreactive cells. In type 1 diabetes, proinsulin-specific CD8⁺ T cells, escaping central and peripheral tolerance, contribute to β-cell destruction. Using G9Cα^−/−CD8⁺ T cells specific for proinsulin, we studied the mechanisms by which LNSC regulate low-avidity autoreactive cells in the NOD mouse model of type 1 diabetes. Whereas MHC-matched NOD-LNSC significantly reduced G9Cα^−/−CD8⁺ T-cell cytotoxicity and dendritic cell–induced proliferation, they failed to sufficiently regulate T cells stimulated by anti-CD3/CD28. In contrast, non-MHC–matched, control C57BL/6 mouse LNSC suppressed T-cell receptor engagement by anti-CD3/CD28 via MHC-independent mechanisms. This C57BL/6-LNSC suppression was maintained even after removal of the LNSC, demonstrating a direct effect of LNSC on T cells, modifying antigen sensitivity and effector function. Thus, our results suggest that a loss of NOD-LNSC MHC-independent suppressive mechanisms may contribute to diabetes development.     

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.     

Global Biochemical Profiling Identifies β-Hydroxypyruvate as a Potential Mediator of Type 2 Diabetes in Mice and Humans

Glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 are incretins secreted by respective K and L enteroendocrine cells after eating and amplify glucose-stimulated insulin secretion (GSIS). This amplification has been termed the “incretin response.” To determine the role(s) of K cells for the incretin response and type 2 diabetes mellitus (T2DM), diphtheria toxin–expressing (DT) mice that specifically lack GIP-producing cells were backcrossed five to eight times onto the diabetogenic NONcNZO10/Ltj background. As in humans with T2DM, DT mice lacked an incretin response, although GLP-1 release was maintained. With high-fat (HF) feeding, DT mice remained lean but developed T2DM, whereas wild-type mice developed obesity but not diabetes. Metabolomics identified biochemicals reflecting impaired glucose handling, insulin resistance, and diabetes complications in prediabetic DT/HF mice. β-Hydroxypyruvate and benzoate levels were increased and decreased, respectively, suggesting β-hydroxypyruvate production from d-serine. In vitro, β-hydroxypyruvate altered excitatory properties of myenteric neurons and reduced islet insulin content but not GSIS. β-Hydroxypyruvate–to–d-serine ratios were lower in humans with impaired glucose tolerance compared with normal glucose tolerance and T2DM. Earlier human studies unmasked a neural relay that amplifies GIP-mediated insulin secretion in a pattern reciprocal to β-hydroxypyruvate–to–d-serine ratios in all groups. Thus, K cells may maintain long-term function of neurons and β-cells by regulating β-hydroxypyruvate levels.     

Long-Term Remission of Diabetes in NOD Mice Is Induced by Nondepleting Anti-CD4 and Anti-CD8 Antibodies

Residual β-cells found at the time of clinical onset of type 1 diabetes are sufficient to control hyperglycemia if rescued from ongoing autoimmune destruction. The challenge, however, is to develop an immunotherapy that not only selectively suppresses the diabetogenic response and efficiently reverses diabetes, but also establishes long-term β-cell–specific tolerance to maintain remission. In the current study, we show that a short course of nondepleting antibodies (Abs) specific for the CD4 and CD8 coreceptors rapidly reversed clinical disease in recent-onset diabetic NOD mice. Once established, remission was maintained indefinitely and immunity to foreign antigens unimpaired. Induction of remission involved selective T-cell purging of the pancreas and draining pancreatic lymph nodes and upregulation of transforming growth factor (TGF)-β1 by pancreas-resident antigen-presenting cells. Neutralization of TGF-β blocked the induction of remission. In contrast, maintenance of remission was associated with tissue-specific immunoregulatory T cells. These findings demonstrate that the use of nondepleting Ab specific for CD4 and CD8 is a robust approach to establish long-term β-cell–specific T-cell tolerance at the onset of clinical diabetes.Type 1 diabetes is marked by the progressive infiltration of the islets (i.e., insulitis) by immune effectors and subsequent destruction of the β-cells (1,2). Clinical diabetes is diagnosed when ∼80–90% of β-cell mass has been destroyed or rendered nonfunctional. Notably, a sufficient number of residual β-cells typically exist at the time of diagnosis so that diabetes can be reversed if the autoimmune response is rapidly suppressed (3). Studies in NOD mice, and indirect evidence from diabetic patients, indicate that CD4⁺ and CD8⁺ T cells are the primary mediators of β-cell destruction (4–6). Pathogenic β-cell–specific CD4⁺ and CD8⁺ T cells often exhibit a type 1 phenotype marked by interferon-γ (IFN-γ) secretion. The differentiation and expansion of pathogenic autoreactive T cells in type 1 diabetes are partly due to dysregulation of immunoregulatory T cells (Treg). Foxp3-expressing CD25⁺CD4⁺ Treg (Foxp3⁺ Treg), for instance, have impaired survival and/or suppressor activity in NOD mice and type 1 diabetic patients (7–10).Efforts to prevent and treat type 1 diabetes have focused on immunotherapies that directly tolerize or deplete pathogenic T effectors and/or enhance Treg populations. Anti-CD3 antibodies (Abs) and antithymocyte globulin induce remission to varying degrees in recent-onset diabetic NOD mice by depleting the autoreactive T effectors and increasing the frequency of CD25⁺CD4⁺ Treg (11–13). Treatment of recent-onset diabetic patients with non-Fc receptor binding (NFB) anti-CD3 Abs also rescues residual β-cell mass; however, the protective effect is transient, and euglycemia and insulin independence are not achieved (14–16). Furthermore, T cell-depleting Abs may compromise normal protective immunity. Systemic albeit transient depletion of T cells following NFB anti-CD3 Ab treatment has been linked to recurrent viral infections in some patients (15).Nondepleting Abs specific for the CD4 and CD8 T-cell coreceptor molecules have been used to establish persistent T-cell tolerance (17). Waldmann and colleagues (17–19) demonstrated that nondepleting anti-CD4 and anti-CD8 coupled with donor-derived splenocytes induce long-lasting tolerance in allograft models. Systemic T-cell numbers are unaffected by the nondepleting Abs, and transplantation tolerance is mediated by alloantigen-specific Foxp3⁺ Treg (18,20). Nondepleting anti-CD4 or anti-CD8 has also been used to prevent type 1 diabetes. YTS105, a rat IgG2a anti-CD8α, blocks insulitis and diabetes in young NOD mice (21). Furthermore, YTS177, a rat IgG2a anti-CD4 prevents diabetes in NOD mouse adoptive transfer models (22,23). The nondepleting nature of YTS177 and YTS105 is attributed to these two rat IgG2a Abs exhibiting an inability to bind murine Fc receptors and fix complement efficiently. In this study, we tested whether tolerance induced by nondepleting Abs specific for CD4 and CD8 was sufficiently robust to elicit remission and long-term β-cell tolerance in recent-onset diabetic NOD mice.