Invasive Insulitis Research Articles

Transient Depletion of Foxp3+ Regulatory T Cells Selectively Promotes Aggressive β Cell Autoimmunity in Genetically Susceptible DEREG Mice.

Type 1 diabetes (T1D) represents a hallmark of the fatal multiorgan autoimmune syndrome affecting humans with abrogated Foxp3+ regulatory T (Treg) cell function due to Foxp3 gene mutations, but whether the loss of Foxp3+ Treg cell activity is indeed sufficient to promote β cell autoimmunity requires further scrutiny. As opposed to human Treg cell deficiency, β cell autoimmunity has not been observed in non-autoimmune-prone mice with constitutive Foxp3 deficiency or after diphtheria toxin receptor (DTR)-mediated ablation of Foxp3+ Treg cells. In the spontaneous nonobese diabetic (NOD) mouse model of T1D, constitutive Foxp3 deficiency did not result in invasive insulitis and hyperglycemia, and previous studies on Foxp3+ Treg cell ablation focused on Foxp3DTR NOD mice, in which expression of a transgenic BDC2.5 T cell receptor (TCR) restricted the CD4+ TCR repertoire to a single diabetogenic specificity. Here we revisited the effect of acute Foxp3+ Treg cell ablation on β cell autoimmunity in NOD mice in the context of a polyclonal TCR repertoire. For this, we took advantage of the well-established DTR/GFP transgene of DEREG mice, which allows for specific ablation of Foxp3+ Treg cells without promoting catastrophic autoimmune diseases. We show that the transient loss of Foxp3+ Treg cells in prediabetic NOD.DEREG mice is sufficient to precipitate severe insulitis and persistent hyperglycemia within 5 days after DT administration. Importantly, DT-treated NOD.DEREG mice preserved many clinical features of spontaneous diabetes progression in the NOD model, including a prominent role of diabetogenic CD8+ T cells in terminal β cell destruction. Despite the severity of destructive β cell autoimmunity, anti-CD3 mAb therapy of DT-treated mice interfered with the progression to overt diabetes, indicating that the novel NOD.DEREG model can be exploited for preclinical studies on T1D under experimental conditions of synchronized, advanced β cell autoimmunity. Overall, our studies highlight the continuous requirement of Foxp3+ Treg cell activity for the control of genetically pre-installed autoimmune diabetes.

Hyaluronan deposition in islets may precede and direct the location of islet immune-cell infiltrates

Substantial deposition of the extracellular matrix component hyaluronan (HA) is characteristic of insulitis in overt type 1 diabetes. We investigated whether HA accumulation is detectable in islets early in disease pathogenesis and how this affects the development of insulitis and beta cell mass. Pancreas tissue from 15 non-diabetic organ donors who were positive for islet autoantibodies (aAbs) and from 14 similarly aged aAb- control donors were examined for the amount of islet HA staining and the presence of insulitis. The kinetics of HA deposition in islets, along with the onset and progression of insulitis and changes in beta cell mass, were investigated in BioBreeding DRLyp/Lyp rats (a model of spontaneous autoimmune diabetes) from 40days of age until diabetes onset. Abundant islet HA deposits were observed in pancreas tissues from n = 3 single- and n = 4 double-aAb+ donors (aAb+HAhigh). In these seven tissues, the HA-stained areas in islets measured 1000 ± 240μm2 (mean ± SEM) and were fourfold larger than those from aAb- control tissues. The aAb+HAhigh tissues also had a greater prevalence of islets that were highly rich in HA (21% of the islets in these tissues contained the largest HA-stained areas [>2000μm2] vs less than 1% in tissues from aAb- control donors). The amount of HA staining in islets was associated with the number of aAbs (i.e. single- or double-aAb positivity) but not with HLA genotype or changes in beta cell mass. Among the seven aAb+HAhigh tissues, three from single- and one from double-aAb+ donors did not show any islet immune-cell infiltrates, indicating that HA accumulates in aAb+ donors independently of insulitis. The three aAb+HAhigh tissues that exhibited insulitis had the largest HA-stained areas and, in these tissues, islet-infiltrating immune cells co-localised with the most prominent HA deposits (i.e. with HA-stained areas >2000μm2). Accumulation of HA in islets was evident prior to insulitis in 7-8-week-old presymptomatic DRLyp/Lyp rats, in which the islet HA-stained area measured 2370 ± 170μm2 (mean ± SEM), which was threefold larger than in 6-week-old rats. This initial islet HA deposition was not concurrent with beta cell loss. Insulitis was first detected in 9-10-week-old rats, in which the HA-stained areas were 4980 ± 500μm2. At this age, the rats also exhibited a 44% reduction in beta cell mass. Further enlargement of the HA-positive areas (mean ± SEM: 7220 ± 880μm2) was associated with invasive insulitis. HA deposits remained abundant in the islets of rats with destructive insulitis, which had lost 85% of their beta cells. This study indicates that HA deposition in islets occurs early in type 1 diabetes and prior to insulitis, and points to a potential role of HA in triggering islet immune-cell infiltration and the promotion of insulitis.

Dietary SCFAs, IL-22, and GFAP: The Three Musketeers in the Gut-Neuro-Immune Network in Type 1 Diabetes.

Microbial metabolites have a profound effect on the development of type 1 diabetes (T1D). The cross-talk between the gut microbiota, the nervous system, and immune system is necessary to establish and maintain immune and gut tolerance. As quoted by Hippocrates, “All disease begins in the gut.” Although this has been recognized for 2,000 years, the connection between the gut and autoimmune T1D is not yet well-understood. Here, we outline new advances supported by our research and others that have contributed to elucidate the impact of microbial metabolites on the physiology of the pancreas and the gut through their remarkable effect on the immune and nervous system. Among many of the mechanisms involved in the gut–beta-cell–immune cross-talk, glial fibrillary acidic protein (GFAP)-expressing cells are critical players in the development of invasive insulitis. Besides, this review reveals a novel mechanism for microbial metabolites by stimulating IL-22, an essential cytokine for gut homeostasis and beta-cell survival. The close connections between the gut and the pancreas are highlighted through our review as microbial metabolites recirculate through the whole body and intimately react with the nervous system, which controls essential disorders associated with diabetes. As such, we discuss the mechanisms of action of microbial metabolites or short-chain fatty acids (SCFAs), IL-22, and GFAP on beta-cells, gut epithelial cells, neurons, and glial cells via metabolite sensing receptors or through epigenetic effects. The fine-tuned gut–neuro–immune network may be profoundly affected by SCFA deficiency related to dysbiosis and diet alterations at very early stages of the initiation of the disease. Thus, dampening the initial immune response or preventing the perpetuation of the immune response by maintaining the integrity of the gut is among the alternative approaches to prevent T1D.

Pancreatic ductal cell antigens are important in the development of invasive insulitis in Non-Obese Diabetic mice

Type 1 Diabetes (T1D) is an autoimmune disease in which insulin producing beta cells of the pancreas are selectively destroyed. Glial Fibrillary Acidic Protein (GFAP) expressed in peri-islet Schwann cells (pSCs) and in the ductal cells of the pancreas is one of the candidate autoantigens for T1D. Immune responses to GFAP expressing cell types precede the islet autoimmunity in Non-Obese Diabetic (NOD) mice. By removing MHC class I from GFAP expressing cell types, we tested the role of autoantigens presented by these cell types in the development of invasive insulitis. Our findings indicate that antigens expressed by pancreatic ductal cells are important in the development of invasive insulitis in NOD mice.

Thinking Outside the Cell: A Key Role for Hyaluronan in the Pathogenesis of Human Type 1 Diabetes

The important thing in science is not so much to obtain new facts as to discover new ways of thinking about them.—William Lawrence Bragg For more than 50 years, chronic immunological processes have been considered central to type 1 diabetes pathogenesis. Studies in pancreata from patients with type 1 diabetes have revealed the presence of insulitis, identified histologically as immune cell infiltrates around and within the islets. Finding the insulitis lesion in a portion of patients with recent-onset type 1 diabetes indicates heterogeneity of the pathogenic process, while the uneven occurrence of the lesion within diabetic pancreata supports the view that the process of islet damage does not take place in all the islets concomitantly. The intermittent pattern of insulitis and the differential recruitment of islets into the pathological process despite the continuous presence of β-cell autoreactive immune cells in circulation suggest the islet pathological process may not be solely dependent on the presence of these cells. Changes in islet tissue-specific structural characteristics and in the local microenvironment may take place in the course of islet inflammation, which predisposes for islet invasion by the immune cells. Local tissue extracellular matrix (ECM) constituents are active participants in the regulation of in situ inflammatory processes during which the functional and structural properties of the local tissue components and of the immune cells themselves are continuously modulated. Recent studies in human diabetic pancreata have indicated the presence of greatly altered hyaluronan (HA), a major ECM component, in the diabetic islets, which is associated with the extent of invasive insulitis and β-cell loss. These novel observations led to the hypothesis that HA guides immune cell migration into the islets and regulates the immune cell phenotype and that alterations in islet HA contribute to the increased vulnerability of the β-cells to inflammatory insult. This …

The CD19 signalling molecule is elevated in NOD mice and controls type 1 diabetes development

Type 1 diabetes is characterised by early peri-islet insulitis and insulin autoantibodies, followed by invasive insulitis and beta cell destruction. The immunological events that precipitate invasive insulitis are not well understood. We tested the hypothesis that B cells in diabetes-prone NOD mice drive invasive insulitis through elevated expression of CD19 and consequent enhanced uptake and presentation of beta cell membrane-bound antigens to islet invasive T cells. CD19 expression and signalling pathways in B cells from NOD and control mice were compared. Expansion of CD8(+) T cells specific for insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) were compared in CD19-deficient and wild-type NOD mice and this was correlated with insulitis severity. The therapeutic potential of anti-CD19 treatment during the period of T cell activation was assessed for its ability to block invasive insulitis. CD19 expression and signalling in B cells was increased in NOD mice. CD19 deficiency significantly diminished the expansion of CD8(+) T cells with specificity for the membrane-bound beta cell antigen, IGRP. Conversely the reduction in CD8(+) T cells with specificity for the soluble beta cell antigen, insulin, was relatively small and not significant. Elevated CD19 on NOD B cells promotes presentation of the membrane-bound antigen, IGRP, mediating the expansion of autoreactive T cells specific for antigens integral to beta cells, which are critical for invasive insulitis and diabetes. Downregulating the CD19 signalling pathway in insulin autoantibody-positive individuals before the development of type 1 diabetes may prevent expansion of islet-invasive T cells and preserve beta cell mass.

Cutting Edge: Accelerated Autoimmune Diabetes in the Absence of LAG-3

Lymphocyte activation gene-3 (LAG-3; CD223) is a CD4 homolog that is required for maximal regulatory T cell function and for the control of CD4(+) and CD8(+) T cell homeostasis. Lag3(-)(/)(-) NOD mice developed substantially accelerated diabetes with 100% incidence. Adoptive transfer experiments revealed that LAG-3 was primarily responsible for limiting the pathogenic potential of CD4(+) T cells and, to a lesser extent, CD8(+) T cells. Lag3(-)(/)(-) mice exhibited accelerated, invasive insulitis, corresponding to increased CD4(+) and CD8(+) T cell islet infiltration and intraislet proliferation. The frequencies of islet Ag-reactive chromogranin A-specific CD4(+) T cells and islet specific glucose-6-phosphatase-specific CD8(+) T cells were significantly increased in the islets of Lag3(-)(/)(-) mice, suggesting an early expansion of pathogenic clones that is normally restrained by LAG-3. We conclude that LAG-3 is necessary for regulating CD4(+) and CD8(+) T cell function during autoimmune diabetes, and thus may contribute to limiting autoimmunity in disease-prone environments.

IL-13Rα1 Expression on β-Cell–Specific T Cells in NOD Mice

OBJECTIVEImmunotherapy using peptides from the β-cell antigen GAD65 can preserve glucose homeostasis in diabetes-prone NOD mice; however, the precise mechanisms that arrest islet-reactive T cells remain unresolved. Our previous work revealed that a dominant GAD65 epitope contained two overlapping I-Ag7–restricted determinants, 524-538 and 530-543, with the former associated with amelioration of hyperglycemia. Here, we sought to discover whether p524-538–specific T cells could directly regulate islet-reactive T cells.RESEARCH DESIGN AND METHODSPrediabetic NOD mice were used to determine the relationship between peptide p524-538–induced interleukin (IL)-13 and regulation of islet autoimmunity. Pancreatic lymph node (PLN) cells from mice at distinct stages of islet inflammation, peri-insulitis versus invasive insulitis, were harvested to establish the expression pattern of IL-13 receptor α1 (IL-13Rα1) on islet-associated T cells.RESULTSPeptide p524-538 preferentially induced IL-13–producing T cells that antagonized the release of γ-interferon by spontaneously arising GAD65 autoimmunity, and recombinant human IL-13 inhibited proliferation of islet-reactive clonotypic T cells. A subset of CD4+ T cells in NOD and NOD.BDC2.5 T cell receptor transgenic mice expressed functional IL-13Rα1, which induced phosphorylation of signal transducer and activator of transcription 6 in the presence of cognate cytokine. Notably, the number of IL-13Rα1+ T cells was heightened in the PLN of young NOD mice when compared with older female counterparts with advanced insulitis. Immunization with p524-538 preserved IL-13Rα1 expression on PLN T cells.CONCLUSIONSIL-13 may be important for regulating autoimmunity in the early stages of insulitis, and the loss of IL-13Rα1 on islet-reactive T cells may be a biomarker for fading regional immune regulation and progression to overt diabetes.

CD4+CD25+ Regulatory T Cells Prevent Type 1 Diabetes Preceded by Dendritic Cell-Dominant Invasive Insulitis by Affecting Chemotaxis and Local Invasiveness of Dendritic Cells

Development of type 1 diabetes (T1D) is preceded by invasive insulitis. Although CD4(+)CD25(+) regulatory T cells (nTregs) induce tolerance that inhibits insulitis and T1D, the in vivo cellular mechanisms underlying this process remain largely unclear. Using an adoptive transfer model and noninvasive imaging-guided longitudinal analyses, we found nTreg depletion did not affect systemic trafficking and tissue localization of diabetogenic CD4(+) BDC2.5 T (BDC) cells in recipient mice prior to development of T1D. In addition, neither the initial expansion/activation of BDC cells nor the number of CD11c(+) or NK cells in islets and pancreatic lymph nodes were altered. Unexpectedly, our results showed nTreg depletion led to accelerated invasive insulitis dominated by CD11c(+) dendritic cells (ISL-DCs), not BDC cells, which stayed in the islet periphery. Compared with control mice, the phenotype of ISL-DCs and their ability to stimulate BDC cells did not change during invasive insulitis development. However, ISL-DCs from nTreg-deficient recipient mice showed increased in vitro migration toward CCL19 and CCL21. These results demonstrated invasive insulitis dominated by DCs, not CD4(+) T cells, preceded T1D onset in the absence of nTregs, and suggested a novel in vivo function of nTregs in T1D prevention by regulating local invasiveness of DCs into islets, at least partly, through regulation of DC chemotaxis toward CCL19/CCL21 produced by the islets.

Neutralization of Interleukin-16 Protects Nonobese Diabetic Mice From Autoimmune Type 1 Diabetes by a CCL4-Dependent Mechanism

OBJECTIVEThe progressive infiltration of pancreatic islets by lymphocytes is mandatory for development of autoimmune type 1 diabetes. This inflammatory process is mediated by several mediators that are potential therapeutic targets to arrest development of type 1 diabetes. In this study, we investigate the role of one of these mediators, interleukin-16 (IL-16), in the pathogenesis of type 1 diabetes in NOD mice.RESEARCH DESIGN AND METHODSAt different stages of progression of type 1 diabetes, we characterized IL-16 in islets using GEArray technology and immunoblot analysis and also quantitated IL-16 activity in cell migration assays. IL-16 expression was localized in islets by immunofluorescence and confocal imaging. In vivo neutralization studies were performed to assess the role of IL-16 in the pathogenesis of type 1 diabetes.RESULTSThe increased expression of IL-16 in islets correlated with the development of invasive insulitis. IL-16 immunoreactivity was found in islet infiltrating T-cells, B-cells, NK-cells, and dendritic cells, and within an insulitic lesion, IL-16 was derived from infiltrating cells. CD4+ and CD8+ T-cells as well as B220+ B-cells were identified as sources of secreted IL-16. Blockade of IL-16 in vivo protected against type 1 diabetes by interfering with recruitment of CD4+ T-cells to the pancreas, and this protection required the activity of the chemokine CCL4.CONCLUSIONSIL-16 production by leukocytes in islets augments the severity of insulitis during the onset of type 1 diabetes. IL-16 and CCL4 appear to function as counterregulatory proteins during disease development. Neutralization of IL-16 may represent a novel therapy for the prevention of type 1 diabetes.

Loss of islet sympathetic nerves and impairment of glucagon secretion in the NOD mouse: relationship to invasive insulitis

We hypothesised that non-obese diabetic mice (NOD) mice have an autoimmune-mediated loss of islet sympathetic nerves and an impairment of sympathetically mediated glucagon responses. We aimed: (1) to determine whether diabetic NOD mice have an early impairment of the glucagon response to insulin-induced hypoglycaemia (IIH) and a coincident loss of islet sympathetic nerves; (2) to determine whether invasive insulitis is required for this nerve loss; and (3) to determine whether sympathetically mediated glucagon responses are also impaired. We measured glucagon responses to both IIH and tyramine in anaesthetised mice. We used immunohistochemistry to quantify islet sympathetic nerves and invasive insulitis. The glucagon response to IIH was markedly impaired in NOD mice after only 3 weeks of diabetes (change, -70%). Sympathetic nerve area within the islet was also markedly reduced at this time (change, -66%). This islet nerve loss was proportional to the degree of invasive insulitis. More importantly, blocking the infiltration prevented the nerve loss. Mice with autoimmune diabetes had an impaired glucagon response to sympathetic nerve activation, whereas those with non-autoimmune diabetes did not. The invasive insulitis seen in diabetic NOD mice causes early sympathetic islet neuropathy. Further studies are needed to confirm that early sympathetic islet neuropathy is responsible for the impaired glucagon response to tyramine.

Curative and β cell regenerative effects of α1-antitrypsin treatment in autoimmune diabetic NOD mice

Invasive insulitis is a destructive T cell-dependent autoimmune process directed against insulin-producing beta cells that is central to the pathogenesis of type 1 diabetes mellitus (T1DM) in humans and the clinically relevant nonobese diabetic (NOD) mouse model. Few therapies have succeeded in restoring long-term, drug-free euglycemia and immune tolerance to beta cells in overtly diabetic NOD mice, and none have demonstrably enabled enlargement of the functional beta cell mass. Recent studies have emphasized the impact of inflammatory cytokines on the commitment of antigen-activated T cells to various effector or regulatory T cell phenotypes and insulin resistance and defective insulin signaling. Hence, we tested the hypothesis that inflammatory mechanisms trigger insulitis, insulin resistance, faulty insulin signaling, and the loss of immune tolerance to islets. We demonstrate that treatment with alpha1-antitrypsin (AAT), an agent that dampens inflammation, does not directly inhibit T cell activation, ablates invasive insulitis, and restores euglycemia, immune tolerance to beta cells, normal insulin signaling, and insulin responsiveness in NOD mice with recent-onset T1DM through favorable changes in the inflammation milieu. Indeed, the functional mass of beta cells expands in AAT-treated diabetic NOD mice.

Image-Guided Analyses Reveal that Non-CD4 Splenocytes Contribute to CD4+T Cell–Mediated Inflammation Leading to Islet Destruction by Altering Their Local Function and Not Systemic Trafficking Patterns

Recruitment of CD4(+) T cells into islets is a critical component of islet inflammation (insulitis) leading to type 1 diabetes; therefore, determining if conditions used to treat diabetes change their trafficking patterns is relevant to the outcome. Cotransfer of CD4(+)BDC2.5 (BDC) cells with non-CD4 splenocytes obtained from newly diabetic NOD mice, but not when they are transferred alone, induces accelerated diabetes. It is unclear whether these splenocytes affect diabetes development by altering the systemic and/or local trafficking and proliferation patterns of BDC cells in target and nontarget tissues. To address these questions, we developed an animal model to visualize BDC cell trafficking and proliferation using whole-body in vivo bioluminescence imaging and used the images to direct tissue sampling for further analyses of the cell distribution within tissues. The whole-body, or macroscopic, trafficking patterns were not dramatically altered in both groups of recipient mice. However, the local patterns of cell distribution were distinct, which led to invasive insulitis only in cotransferred mice with an increased number of islet-infiltrating CD11b(+) and CD11c(+) cells. Taken together, the non-CD4 splenocytes act locally by promoting invasive insulitis without altering the systemic trafficking patterns or proliferation of BDC cells and thus contributing to diabetes by altering the localization within the tissue.

Suppression of Type 1 Diabetes in NOD Mice by Bifunctional Peptide Inhibitor: Modulation of the Immunological Synapse Formation

The aim of this work was to design and utilize a bifunctional peptide inhibitor called glutamic acid decarboxylase-bifunctional peptide inhibitor to suppress the progression of type 1 diabetes in non-obese diabetic mice. The hypothesis is that glutamic acid decarboxylase-bifunctional peptide inhibitor binds simultaneously to major histocompatibility complex-II and intercellular adhesion molecule type 1 on antigen-presenting cell and inhibits the immunological synapse formation during T-cell-antigen-presenting cell interactions. Glutamic acid decarboxylase-bifunctional peptide inhibitor was composed of a major epitope of the type 1 diabetes-associated antigen, glutamic acid decarboxylase 65 kDa, covalently linked to a peptide derived from CD11a of lymphocyte function-associated antigen-1. The suppression of insulitis and type 1 diabetes was evaluated using non-obese diabetic and non-obese diabetic severe combined immunodeficiency mice. Glutamic acid decarboxylase-bifunctional peptide inhibitor had the capacity to suppress invasive insulitis in non-obese diabetic mice. CD4+ T-cells isolated from glutamic acid decarboxylase-bifunctional peptide inhibitor treated mice also suppressed insulitis and hyperglycemia when transferred with diabetogenic non-obese diabetic spleen cells into non-obese diabetic severe combined immunodeficiency recipients. As predicted, the glutamic acid decarboxylase-bifunctional peptide inhibitor cross-linked a significant fraction of major histocompatibility complex class-II molecules to intercellular adhesion molecule type 1 molecules on the surface of live antigen-presenting cell. Intravenous injection of the glutamic acid decarboxylase-bifunctional peptide inhibitor elicited interleukin-4-producing T-cells in non-obese diabetic mice primed against the glutamic acid decarboxylase-epitope peptide. Together, the results indicate that glutamic acid decarboxylase-bifunctional peptide inhibitor induces interleukin-4-producing regulatory cells but does not expand the glutamic acid decarboxylase-specific Th2 population. Given that Th2 effector cells can cause pathology, the glutamic acid decarboxylase-bifunctional peptide inhibitor may represent a novel mechanism to induce interleukin-4 without Th2-associated pathology.

CD8 + T lymphocytes specific for glutamic acid decarboxylase 90–98 epitope mediate diabetes in NOD SCID mouse

During the past decade, glutamic acid decarboxylase (GAD) has been considered a crucial β-cell autoantigen involved in type 1 diabetes in the NOD mouse and human. Recently, the etiological role of GAD has remained controversy. In the NOD mouse, some previous studies argued in favor of a regulatory role for GAD-specific CD4 + T cells, and no diabetogenic CD8 + T cells specific for GAD have been identified so far, discrediting the importance of GAD in β-cell injury. Here, we identified, in the NOD model, a relevant GAD CD8 + T cell epitope (GAD 90–98) using immunization with a plasmid encoding GAD, a protocol relying on in vivo processing of peptides from the autoantigenic protein. In pancreatic lymph nodes of naïve female NOD mice, CD8 + T lymphocytes recognizing GAD 90–98 peptide were detected during the initial phase of invasive insulitis (between 4 and 8 weeks of age), suggesting an important role for these cells in the first stage of the disease. GAD 90–98 specific CD8 + lymphocytes lysed efficiently islet cells in vitro and transferred diabetes into NOD SCID mice (100%). Finally, diabetes was accelerated greatly in 3-week-old female NOD mice injected i.p. with GAD 90–98, strengthening the role of GAD-specific CTLs in diabetes pathogenesis.

β Cells cannot directly prime diabetogenic CD8 T cells in nonobese diabetic mice

Type 1 diabetes (T1D) is caused by the destruction of insulin-producing islet beta cells. CD8 T cells are prevalent in the islets of T1D patients and are the major effectors of beta cell destruction in nonobese diabetic (NOD) mice. In addition to their critical involvement in the late stages of diabetes, CD8 T cells are implicated in the initiation of disease. NOD mice, in which the beta2-microglobulin gene has been inactivated by gene targeting (NOD.beta2M-/-), have a deficiency in CD8 T cells and do not develop insulitis, which suggests that CD8 T cells are required for the initiation of T1D. However, neither in humans nor in NOD mice have the immunological requirements for diabetogenic CD8 T cells been precisely defined. In particular, it is not known in which cell type MHC class I expression is required for recruitment and activation of CD8 T cells. Here we have generated transgenic NOD mice, which lack MHC class I on mature professional antigen-presenting cells (pAPCs). These "class I APC-bald" mice developed periislet insulitis but not invasive intraislet insulitis, and they never became diabetic. Recruitment to the islet milieu does not therefore require cognate interaction between CD8 T cells and MHC class I on mature pAPCs. Conversely, such an interaction is critically essential to allow the crucial shift from periislet insulitis to invasive insulitis. Importantly, our findings demonstrate unequivocally that CD8 T cells cannot be primed to become diabetogenic by islet beta cells alone.

Activation of CD1d-restricted T cells protects NOD mice from developing diabetes by regulating dendritic cell subsets.

CD1d-restricted invariant NKT (iNKT) cells are immunoregulatory cells whose loss exacerbates diabetes in nonobese diabetic (NOD) female mice. Here, we show that the relative numbers of iNKT cells from the pancreatic islets of NOD mice decrease at the time of conversion from peri-insulitis to invasive insulitis and diabetes. Conversely, NOD male mice who have a low incidence of diabetes showed an increased frequency of iNKT cells. Moreover, administration of alpha-galactosylceramide, a potent activating ligand presented by CD1d, ameliorated the development of diabetes in NOD female mice and resulted in the accumulation of iNKT cells and myeloid dendritic cells (DC) in pancreatic lymph nodes (PLN), but not in inguinal lymph nodes. Strikingly, injection of NOD female mice with myeloid DC isolated from the PLN, but not those from the inguinal lymph nodes, completely prevented diabetes. Thus, the immunoregulatory role of iNKT cells is manifested by the recruitment of tolerogenic myeloid DC to the PLN and the inhibition of ongoing autoimmune inflammation.

Suppression of T(h)1 cell activation and prevention of autoimmune diabetes in NOD mice by local expression of viral IL-10.

Insulin-dependent diabetes mellitus in the NOD mouse model is caused by the T cell-mediated autoimmune destruction of pancreatic beta cells. Viral IL-10 (vIL-10), encoded in the Epstein-Barr virus genome, shares many of the anti-inflammatory properties of cellular IL-10, but lacks its immunostimulatory properties. In the present study, we generated transgenic (Tg) NOD mice in which vIL-10 was produced exclusively in pancreatic islets and investigated the effect of vIL-10 on the development of diabetes. The accumulation of lymphocytes around islets was more prominent, but the invasive insulitis decreased in the vIL-10 Tg mice. The incidence of diabetes was markedly reduced in the vIL-10 Tg mice, in clear contrast to the accelerated diabetes seen in the murine IL-10 Tg NOD mice. IL-12p40 and IFN-gamma mRNA levels were decreased in pancreata of the vIL-10 Tg mice, although CD4 mRNA level was markedly increased. These results suggest that locally produced vIL-10 induced leukocyte migration, but inhibited the activation of T(h)1, probably through suppressing the production of IL-12. They indicate that vIL-10 may well be superior to cellular IL-10 in the treatment of autoimmune diabetes. The vIL-10 Tg NOD mice should provide a useful tool for understanding the differential action of vIL-10 versus cellular IL-10.