Islet-infiltrating Lymphocytes Research Articles

Mechanism of Action of Oral Salmonella-Based Vaccine to Prevent and Reverse Type 1 Diabetes in NOD Mice.

A combination therapy of preproinsulin (PPI) and immunomodulators (TGFβ+IL10) orally delivered via genetically modified Salmonella and anti-CD3 promoted glucose balance in in NOD mice with recent onset diabetes. The Salmonella bacteria were modified to express the diabetes-associated antigen PPI controlled by a bacterial promoter in conjunction with over-expressed immunomodulating molecules. The possible mechanisms of action of this vaccine to limit autoimmune diabetes remained undefined. In mice, the vaccine prevented and reversed ongoing diabetes. The vaccine-mediated beneficial effects were associated with increased numbers of antigen-specific CD4+CD25+Foxp3+ Tregs, CD4+CD49b+LAG3+ Tr1-cells, and tolerogenic dendritic-cells (tol-DCs) in the spleens and lymphatic organs of treated mice. Despite this, the immune response to Salmonella infection was not altered. Furthermore, the vaccine effects were associated with a reduction in islet-infiltrating lymphocytes and an increase in the islet beta-cell mass. This was associated with increased serum levels of the tolerogenic cytokines (IL10, IL2, and IL13) and chemokine ligand 2 (CCL2) and decreased levels of inflammatory cytokines (IFNγ, GM-CSF, IL6, IL12, and TNFα) and chemokines (CXCL1, CXCL2, and CXCL5). Overall, the data suggest that the Salmonella-based vaccine modulates the immune response, reduces inflammation, and promotes tolerance specifically to an antigen involved in autoimmune diabetes.

Lymphocyte metabolism in the islet microenvironment

Abstract Type 1 diabetes (T1D) results in immune-mediated destruction of insulin-producing beta cells in the pancreas. Immune intervention to prevent or reverse T1D is an appealing therapeutic approach; however, selective targeting of autoreactive lymphocytes remains an elusive goal. Recently, cellular metabolism has emerged as a potent modulator of immune cell function. The metabolic state of lymphocytic infiltrate within the highly vascularized islet, termed insulitis, is not known. To test the hypothesis that pathogenic, islet-infiltrating lymphocytes have a distinct metabolic phenotype that has the potential to be selectively targeted, we compared the metabolic signatures of islet infiltrating lymphocytes in two transgenic mouse models. VH125SD.NOD mice develop accelerated diabetes and robust insulitis. BDC2.5.NOD develop insulitis but are protected from T1D. These models were used as examples of pathogenic and protective insulitis, respectively. Mitochondrial polarity was significantly increased in VH125SD.NOD islet-infiltrating B cells compared to BDC2.5.NOD islet-infiltrating B cells. Mitochondrial polarity was also increased in insulin-binding B cells compared to non-insulin-binding B cells in the pancreas, but not in the spleen or pancreatic lymph nodes. Assessment of hypoxia in islet-infiltrating lymphocytes revealed that the oxygen-sensing transcription factor, hypoxia-inducible factor 1α was increased in islet-infiltrating B cells specifically. Together, these findings suggest that tissue specific changes drive metabolic reprogramming at the site of autoimmune attack in the islet and that these metabolic pathways have the potential to be targeted for selective therapeutic interventions in T1D.

1726-P: MDA5, a Janus-Faced dsRNA Sensor in Coxsackievirus-Accelerated Autoimmune Diabetes

While microbial infections can trigger type 1 diabetes (T1D), innate immune activation and the synthesis of free radicals, proinflammatory cytokines, and type 1 interferons contribute to pancreatic β-cell destruction. We previously demonstrated that Coxsackievirus B3 (CB3) infection of Non-Obese Diabetic (NOD) mice can accelerate T1D, partly due to the induction of antiviral signaling pathways including melanoma differentiation-associated protein 5 (MDA5). Single-nucleotide polymorphisms within Ifih1, the gene encoding for MDA5, is associated with multiple autoimmune diseases including T1D, but the molecular mechanism contributing to innate immune dysregulation is not known. Since T1D is a chronic proinflammatory autoimmune disease involving MDA5, we hypothesized that attenuated MDA5 expression and signaling can delay diabetogenic viral-accelerated T1D. We generated NOD mice mutated in Ifih1 that exhibited a delay in spontaneous T1D due to the loss of MDA5 expression (NOD.Ifih1-m1) or an in-frame deletion in the helicase domain that decreased MDA5 synthesis (NOD.Ifih1-m4). Interestingly, CB3-infected NOD and NOD.Ifih1-m1 mice displayed virus-accelerated T1D, but NOD.Ifih1-m4 (n=9 per group) mice were significantly (p < 0.001) delayed. CB3-infected NOD.Ifih1-m4 mice exhibited a 5-fold decrease in IFN-β synthesis in the pancreas, the reduction in inflammation may be a driving factor to prevent the induction of autoimmunity. The Ifih1-m4 mutation did not compromise pancreatic viral clearance, and this was complemented by a ∼34% and ∼32% reduction in pancreas-infiltrating CD8 T cells and activated macrophages (F4/80+, I-Ag7+), respectively, in comparison to CB3-infected NOD mice. The protective Ifih1-m4 mutation may establish an acute level of type 1 interferons and islet-infiltrating lymphocytes to sufficiently inhibit CB3 replication without mediating bystander activation of autoreactive T cells, preventing virus-accelerated autoimmune diabetes. Disclosure S.I. Blum: None. Y. Chen: None. H.M. Tse: None. Funding American Diabetes Association (7-12-CD-11 to H.M.T.)

Aberrant MDA5 signaling and IFN-β synthesis elicit protection from Coxsackievirus-accelerated Type 1 diabetes

Abstract Viral infections can trigger the synthesis of proinflammatory cytokines and free radicals that contribute to pancreatic β-cell destruction leading to Type 1 diabetes (T1D). We previously demonstrated that Coxsackievirus B3 (CB3) infection of Non-Obese Diabetic (NOD) mice can accelerate T1D, due to the induction of antiviral signaling pathways including melanoma differentiation-associated protein 5 (MDA5). SNPs within Ifih1, the gene encoding for MDA5, is associated with T1D pathogenesis, but the molecular mechanism contributing to innate immune dysregulation is not known. We hypothesized that attenuated MDA5 expression and signaling can delay diabetogenic viral-accelerated T1D. We generated NOD mice mutated in Ifih1 that displayed a loss in MDA5 expression (NOD.Ifih1-m1) or an in-frame deletion in the helicase domain that decreased MDA5 synthesis (NOD.Ifih1-m4). Interestingly, CB3-infected NOD and NOD.Ifih1-m1 mice displayed virus-accelerated T1D, but NOD.Ifih1-m4 (n=9 per group) mice were significantly delayed in both spontaneous and CB3-accelerated T1D (p=0.0036 and p=0.0004, respectively). CB3-infected NOD.Ifih1-m4 mice exhibited a 4-fold decrease in pancreas IFN-β levels and may partly explain the delay in CB3-induction of T1D. The Ifih1-m4 mutation did not compromise pancreatic viral clearance, and this was complemented by a ~34% and ~32% reduction in pancreas-infiltrating CD8 T cells and activated macrophages (F4/80+, I-Ag7+), respectively, in comparison to CB3-infected NOD mice. The protective Ifih1-m4 mutation may establish an acute level of IFN-β and islet-infiltrating lymphocytes to inhibit CB3 replication without mediating bystander activation and virus-accelerated autoimmune diabetes.

Characterization of Islet-Infiltrating Lymphocytes in Type 1 Diabetes

Type 1 diabetes (T1D) results in the immune-mediated destruction of insulin-producing beta cells in the pancreas. As such, immune intervention to prevent or delay T1D is an appealing therapeutic approach. Development of an antigen-specific intervention that targets the diabetogenic immune response without compromising systemic immunity is complicated by an expanding list of antigenic targets, including post-translationally modified peptides generated at the site of autoimmune attack in the islet. Thus, development of effective antigen-based therapy requires not only antigen identification, but also understanding of the unique antigen processing environment facilitated by the islet itself. Recently, cellular metabolism has emerged as a potent modulator of immune cell function. However, the metabolic state lymphocytic infiltrate in the islet, termed insulitis, is not known. Given the highly vascularized nature of the islet needed to accommodate the metabolic demand associated with insulin secretion, we sought to characterize the islet’s distinct metabolic environment and its influence on T-B cell interactions at the site of autoimmune attack. Using a transgenic mouse model that develops accelerated diabetes (VH125SD.NOD), we assessed the metabolic signature of islet-infiltrating lymphocytes compared to those in the spleen and pancreatic lymph nodes. We determined that islet-infiltrating lymphocytes are characterized by decreased glucose uptake in CD4 and CD8 T cell subsets. Mitochondrial mass and polarity were decreased in all subsets compared to secondary lymphoid organs, suggesting reduced metabolic fitness. These studies reveal a metabolic distinction between islet-infiltrating and secondary lymphoid organ lymphocytes and suggest that metabolic features that differentiate immune cells at the site of autoimmune attack are a potential target for selective therapeutic interventions in T1D. Disclosure J.L. Felton: None. R.H. Bonami: None. C. Hulbert: None. J.W. Thomas: None.

Emerging evidence for the role of CD40/TNFR5 in pancreatic ß-cell death induced by glucolipotoxicity

D is a multifactorial disease whose pathogenesis results from insulin deficiency caused by a reduction in pancreatic β-cell mass. In type 1 diabetes this is associated with autoimmunity, whereas type 2 diabetes (T2D) is due to premature aging and impairment of β-cell function. The latter is largely due to toxicity induced by high levels of glucose and lipid. T2D is also characterized by systemic inflammation, with pro-inflammatory cytokines produced by islet-infiltrating macrophages and lymphocytes contributing to β-cell destruction. In addition, glucolipotoxicity also activates the islet inflammasome, NALP3, triggering IL-1β activation and release from pancreatic β-cells. This then serves to activate the transcription factor NF-κB through an autocrine feedback loop that results in increased β-cell expression and release of cytokines and chemokines, with the former serving to trigger β-cell apoptotic cascades and the latter to induce islet immune cell invasion. In order to better understand the intracellular β-cell signaling mechanisms associated with glucolipotoxicity we performed Affymetrix microarray experiments to identify genes whose expression was sensitive to extracellular glucose and lipid environments. Importantly, of all the TNF receptor superfamily members that have been shown to be linked to NF-βB activation in other cell types, we found the greatest change in expression of TNFR5/CD40. Independent qPCR analysis confirmed >3-fold increase in TNFR5/CD40 expression, with Western blot data supporting these findings at the protein level. Ongoing studies are now underway to fully elucidate the signaling mechanisms associated with this novel pathway of glucolipotoxic islet cell death.

Islet lymphocyte subsets in male and female NOD mice are qualitatively similar but quantitatively distinct

Islet-infiltrating lymphocytes of individual male and female non-obese diabetic (NOD) mice were examined with the purpose of determining the differences that lead to a predominance of diabetes in female versus males NOD mice. When normalized for the amount of islet lymphocytes recovered, the infiltrating lymphocytes of female NOD mice were indistinguishable from those of male NOD mice. The only observed difference was that islet inflammation progressed at an increased rate in female compared to male NOD mice. There was no difference in the composition of islet infiltrates in male and female NOD mice. Unexpectedly, the ratio of CD4+:CD8+ T cells was tightly controlled in the islets throughout diabetogenesis. The frequency of IL-4+ CD4+ T cells started high but quickly fell to 3% of the population that was maintained with increasing inflammation. A significant portion of the CD8+ T cells were islet-specific glucose-6-phosphatase catalytic subunit-related protein specific in both male and female NOD mice and this population was antigen experienced and increased at high levels of islet inflammation. Surprisingly, a large pool of antigen inexperienced naïve T cells was detected in the islets. We conclude the underlying immunological processes in both male and female NOD mice are similar while the rates differ and the presence of naïve T cell in the islets may contribute to epitope spreading.

Equivalent Specificity of Peripheral Blood and Islet-Infiltrating CD8+ T Lymphocytes in Spontaneously Diabetic HLA-A2 Transgenic NOD Mice

CD8(+) T cells play an important role in the initiation of insulitis and in the destructive stage leading to insulin-dependent diabetes mellitus. A string of recent studies has led to the identification of numerous HLA-A2-restricted epitopes derived from pancreatic beta cell Ags. It is hoped that assays detecting responses of patient PBMC to such epitopes might be instrumental for early diagnosis of beta cell-directed autoimmunity and for monitoring trials of immunointervention. However, it remains unclear whether the results of assays studying PBMC reflect responses of islet-infiltrating lymphocytes, and to what extent they correlate with disease risk and/or activity. We have used female and male humanized NOD mice expressing HLA-A2 in addition to murine MHC class I molecules to study spontaneous responses of islet-infiltrating blood, spleen, and lymph node lymphocytes of various age groups to a panel of 16 epitopes. Twelve of these are restricted by HLA-A2, have previously been shown to be recognized by patient CTL, and have identical sequences in human and murine autoantigens. Using an IFN-gamma ELISPOT assay, we find highly similar hierarchies of epitope immunodominance in the different T cell compartments, including peripheral blood and pancreatic islets. Moreover, we demonstrate that most of the epitopes eliciting dominant responses in humans display similar status in the mouse model. These results emphasize the potential of humanized mice as tools for studying spontaneous autoimmune CTL responses, and they provide a strong rationale for the development and use of assays monitoring responses of CD8(+) PBMC in human type 1 diabetes.

Fas Stimulation Results in Selective Islet Infiltrate Apoptosis in Situ and Reversal of Diabetes

Breakdown of peripheral T cell regulation due to defective antigen-activated apoptosis may lead to autoimmunity. In the nonobese diabetic (NOD) mouse model of type 1 diabetes mellitus, we have demonstrated defects in T cell activation and peripheral apoptosis. Stimulation of the Fas pathway by a Fas receptor agonist led to enhanced in vitro apoptosis and in vivo selective apoptosis of islet-infiltrating lymphocytes. Administration of the Fas agonist immediately after onset of diabetes led to reversal of diabetes in NOD mice. Inducing peripheral T cell apoptosis may be a potential method for reversal of autoimmune diabetes.

The pathogenicity of islet-infiltrating lymphocytes in the non-obese diabetic (NOD) mouse.

The aim of the present study was to investigate the pathogenic properties of islet-infiltrating lymphocytes related to the severity of the autoimmune destruction of islet beta-cells in the NOD mouse. We analysed the development of insulin-dependent diabetes mellitus (IDDM) produced by adoptive transfer of islet lymphocytes from NOD into NOD.scid mice. Here we show that the transfer was most effective when both CD4+ and CD8+ T cells were present in the infiltrate, but CD4+ T cells alone were sufficient to cause the disease. Islet lymphocytes from both females and males transferred diabetes effectively, but the severity of IDDM was higher when female islet lymphocytes were used. Unexpectedly, the sensitivity of male islets to beta-cell damage was greater than that of female islets. Treatment of NOD females with a peptide of heat shock protein (hsp)60, p277, known to protect NOD mice from IDDM, reduced the pathogenicity of the islet lymphocytes. In contrast, administration of cyclophosphamide to males, a treatment that accelerates the disease, rendered the islet lymphocytes more pathogenic. More severe disease in the recipient NOD.scid mice was associated with more interferon-gamma (IFN-gamma)-secreting islet T cells of the NOD donor. The disease induced by islet lymphocytes was strongly inhibited by co-transfer of spleen cells from prediabetic mice, emphasizing the regulatory role of peripheral lymphocytes. Thus, the cellular characteristics of the islet infiltrate and the pathogenicity of the cells are subject to complex regulation.

The role of the gut in beta-cell autoimmunity and type 1 diabetes: a hypothesis.

The origin of autoimmunity leading to the destruction of insulin-producing beta-cells is not known. Several studies suggest that a link exists between the gut immune system and the islets infiltrating lymphocytes. Inflamed pancreatic islets express the same adhesion molecules involved with the homing of gut-associated lymphocytes. The manifestation of autoimmune diabetes in the animal models can be modified by dietary factors, which cause changes in the cytokine production by islet-infiltrating lymphocytes. Increased risk of type 1 diabetes has been associated with an early introduction of cows' milk formula in infancy, indicating that triggering of the gut immune system in early infancy may contribute to the later development of beta-cell autoimmunity. Enhanced immune reactivity to cow milk (CM) proteins in the patients with type 1 diabetes suggests aberrant regulation of the gut immune system in this disease. In the patients with newly diagnosed type 1 diabetes, anti-glutamate decarboxylase (GAD)-reactivity was found in the subpopulation of lymphocytes expressing gut-associated homing receptor alpha 4 beta 7. Based on these findings, the hypothesis that aberrant function of the gut immune system would lead to the development of beta-cell autoimmunity and type 1 diabetes has recently received a lot of attention. The possibility that regulation of the gut immune system is not normal in subjects at risk of autoimmune diabetes should be considered when treatments interfering with mucosal immunity for the prevention of type 1 diabetes are planned.

The Role of CD8+ Cells, Cell Degeneration, and Fas Ligand in Insulitis After Intraperitoneal Transfer of NOD Splenocytes

Cells expressing CD4, CD8, CD18, CD49d/CD29, CD44, CD54, CD80, CD86, CD106, CD11b/CD18 or DNA breaks were stained in the pancreases of female or male scid/ scid mice after adoptive transfer of lymphocytes from older than 12-week female nonobese diabetic (NOD) or Balb/c mice. After intraperitoneal adoptive transfer of NOD splenocytes to female severe combined immunodeficiency (scid)/scid mice, in situ end labeling (ISEL)+ as well as CD80+ and CD86+ cell infiltrates appeared first in the blood vessel walls and pancreatic interstitial tissue at 2-3 weeks after transfer. CD4+, CD8+, CD18+, CD44+, CD54+, and CD106+ cells then encircled and invaded some islets of the scid/scid mice 2 to 7 weeks after transfer. Cells expressing these surface components, except CD8, were present also in the Balb/c mice, but as individual cells, not as infiltrates. CD8+ cells were observed in the pancreases of all NOD splenocyte-injected scid mice at 2, 3, 4, 6, and 7 weeks after transfer, but in none of the Balb/c splenocyte-injected scid mice. Some scid/scid mice injected with NOD splenocytes also developed severe noninfectious diarrhea and cachexia 4 weeks after transfer. ISEL+ cells were observed in the pancreases of NOD splenocyte-injected female scid mice at all times after transfer, especially in the blood vessel walls and in the islets. Fas ligand was not present in Western blotting. It is proposed that apoptosis commonly occurs in islet-infiltrating lymphocytes and that in the scid/scid adoptive-transfer model, the first islet-infiltrating cells are destroyed by programmed cell death independent of Fas ligand. Further, CD8+ T lymphocytes inevitably play a central role in intraperitoneal adoptive transfer of insulitis.

Retardation or acceleration of diabetes in NOD/Lt mice mediated by intrathymic administration of candidate beta-cell antigens.

A single injection of syngeneic islet cells into the thymus of 4-week-old NOD/Lt female mice strongly retards diabetogenesis. The present study used the intrathymic route of antigen administration to compare the relative efficacy of peptides/proteins derived from two major candidate pancreatic beta-cell autoantigens, insulin and GAD65, to modulate diabetogenesis. Intrathymic administration of insulin B chain or recombinant human GAD65 significantly suppressed diabetogenesis during a 20-week follow-up period, whereas no protection was mediated by either insulin A chain or a synthetic peptide (A2) derived from it. Quite unexpectedly, two GAD65-derived peptides near the COOH-terminus (p34 and p35) accelerated diabetes onset. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed on cDNAs from isolated islets or whole pancreases of NOD/Lt females 4 weeks after intrathymic injections. Protection mediated by intrathymic administration with either intact islet cells or GAD65 were correlated with an upregulation of mRNA for T-helper 2 (Th2)-associated cytokines (interleukin [IL]-4, IL-10), concomitant with downregulation of Th1-associated interferon (IFN) transcripts (all normalized to T-cell receptor Cbeta transcripts) in islet-infiltrating lymphocytes. Protection mediated by the intrathymic administration of insulin B chain, however, correlated only with a modest upregulation of IL-4 and IL-10 transcript levels, and no diminution in IFN-gamma transcripts. In contrast, the diabetes-accelerating GAD65 p34 and p35 peptides were not associated with an immune deviation, expressing levels of IFN-gamma characteristic of islet-infiltrating lymphocytes in vehicle-injected NOD controls. Hence, Th1-to-Th2 immune deviation provides only a partial explanation for peptide immunotherapy of diabetes in NOD mice. The finding that certain peptides can accelerate rather than retard diabetogenesis as a function of route and age of administration adds a cautionary note to this type of therapy.

Mucosa-associated (beta 7-integrinhigh) lymphocytes accumulate early in the pancreas of NOD mice and show aberrant recirculation behavior.

The mucosal addressin cell adhesion molecule-1 (MAdCAM-1) becomes expressed on islet vessels of NOD mice early during lymphocyte accumulation in islets. Because MAdCAM-1 preferentially mediates the homing of mucosal lymphocytes, islet-associated MAdCAM-1 could favor the accumulation of mucosal lymphocytes in pancreatic islets. Therefore, we investigated the relative frequency of islet-infiltrating lymphocytes with a mucosal phenotype (alpha 4/beta 7-integrinhigh and L-selectinlow) at early and advanced stages of insulitis. We found that until the age of 12 weeks, lymphocytes with a mucosal phenotype were particularly frequent in the pancreas (percentage of beta 7-integrinhigh-lymphocytes was 48% at 8 weeks and 73% at 12 weeks), whereas in diabetic mice older than 16 weeks, their relative number was smaller (26% of pancreas-infiltrating lymphocytes). To define the origin and homing behavior of beta 7-integrinhigh-lymphocytes before their accumulation in pancreatic islets in NOD mice, we sought for such lymphocytes in different lymphoid organs and studied their recirculation. We found that compared with lymphocytes in several other strains, in NOD mice such lymphocytes were most frequent in nonmucosal lymphoid tissues (peripheral and pancreatic lymph nodes, spleen) from an early age. When injected to blood circulation, mucosal beta 7high-lymphocytes failed to home efficiently back to mucosal lymphoid tissue in NOD mice but homed aberrantly to nonmucosal lymphoid tissues. After adoptive transfer of diabetogenic splenocytes, the first lymphocytes accumulating in the pancreas were predominantly beta 7-integrinhigh. We conclude that mucosa-associated lymphocytes are involved in the early phases of islet-inflammation in NOD mice and that the aberrant homing behavior of lymphocytes expressing high levels of beta 7-integrin may associate with their accumulation in pancreatic islets early during insulitis.

Coexpression of B7-1 and viral (“self”) transgenes in pancreatic β cells can break peripheral ignorance and lead to spontaneous autoimmune diabetes

We evaluated the role of the costimulatory molecule B7-1 in overcoming peripheral ignorance in transgenic mice, which expressed the glycoprotein (GP) or nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) as the self-antigen in pancreatic β cells. The viral transgenes or B7-1 alone did not induce autoimmune diabetes (IDDM). However, in bigenic mice expressing B7-1 and LCMV-GP, anti-self (viral) cytotoxic T lymphocytes (CTL) were activated without viral infection and spontaneous IDDM occurred. In contrast, bigenic RIP-B7-1 × RIP-NP mice with thymic expression of the self (viral-NP) antigen deleted the majority of their autoreactive CTL and did not develop spontaneous IDDM. However, these mice developed fast-onset IDDM 14 days after LCMV Infection, whereas single-transgenic RIP-NP littermates developed IDDM only within 4–5 months. Rapid IDDM was associated with increased numbers of anti-self CTL and a predominance of IFNγ produced by islet-infiltrating lymphocytes, whereas single transgenic RIP-NP littermates with slow-onset IDDM displayed less anti-self CTL and more IL-4- and IL-10-producing T lymphocytes in pancreatic infiltrates.

Islet-infiltrating lymphocytes from prediabetic NOD mice rapidly transfer diabetes to NOD-scid/scid mice.

In an effort to study the development of diabetes in NOD mice, our laboratory developed a novel adoptive transfer model using NOD-scid/scid (NOD-scid) mice as recipients of islet-infiltrating lymphocytes from donor prediabetic female NOD mice. We first confirmed previous results that demonstrated that splenocytes of diabetic and prediabetic female NOD mice could transfer diabetes to NOD-scid mice. We demonstrated that the kinetics of disease transfer were dependent on the age of transferred lymphocytes and reiterated the kinetics of diabetes in conventional female NOD mice. We then demonstrated that islet-infiltrating lymphocytes from prediabetic female NOD mice could transfer diabetes. In contrast with the age-dependent transfer of diabetes seen using splenocytes, islet-infiltrating lymphocytes obtained from prediabetic female NOD mice aged > or = 40 days rapidly transferred diabetes to NOD-scid recipients. The time required to transfer insulin-dependent diabetes mellitus (IDDM) using islet-infiltrating lymphocytes from young prediabetic mice (25 +/- 9 days) was not statistically different from the time required to transfer IDDM using splenocytes from overtly diabetic mice (32 +/- 5 days). Cotransfer of splenocyte cells or CD4+, but not CD8+ spleen cells, from 60- to 80-day-old prediabetic female NOD mice together with either splenocytes from diabetic mice or islet-infiltrating lymphocytes from prediabetic NOD mice delayed the rapid transfer of IDDM, suggesting that CD4+ cells mediated immunoregulation. Use of the NOD-scid islet-infiltrating lymphocyte-adoptive transfer model should help elucidate the pathophysiology of the early inflammatory events leading to insulitis and subsequent beta-cell destruction.(ABSTRACT TRUNCATED AT 250 WORDS)

Islet-infiltrating lymphocytes from prediabetic NOD mice rapidly transfer diabetes to NOD-scid/scid mice

Islet-infiltrating lymphocytes from prediabetic NOD mice rapidly transfer diabetes to NOD-scid/scid mice

New approach for in vivo detection of insulitis in type I diabetes: activated lymphocyte targeting with 123I-labelled interleukin 2

Insulitis is considered the histopathological hallmark of type I (insulin-dependent) diabetes. In the non-obese diabetic (NOD) mouse, diabetes has never been observed in the absence of insulitis. The in vivo detection of insulitis could be of relevance for early prediction of diabetes. As approximately 15% of islet-infiltrating lymphocytes express interleukin 2 receptors, we have labelled recombinant interleukin 2 with 123I and used this radiopharmaceutical to detect insulitis by gamma camera imaging. We studied 71 prediabetic NOD and 27 normal Balb/c mice. Labelled alpha-lactalbumin was used as the control protein. In the first set of experiments we studied the tissue distribution of radiolabelled interleukin 2 in isolated organs from animals sacrificed at different time points. Higher radioactivity was detected in the pancreas of NOD mice injected with labelled interleukin 2, as compared to NOD mice receiving labelled alpha-lactalbumin (p < 0.003 at 20 min: p < 0.001 at 40 min; p < 0.001 at 60 min) or Balb/c mice injected with labelled interleukin 2 (p < 0.05 at 40 min; p < 0.001 at 60 min). In another set of experiments, gamma camera images have been acquired after injection of 123I-labelled interleukin 2. Radioactivity in the pancreatic region of prediabetic NOD and Balb/c mice showed similar kinetics to those observed by single organ counting, with higher accumulation in the pancreatic region of NOD mice (p < 0.04 after 22-45 min in NOD mice vs Balb/c mice).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-specific T cells are a predominant component of islet infiltrates in pre-diabetic NOD mice.

Numerous investigation have demonstrated that T cells are involved in destruction of beta cells in the NOD mouse, a widely studied model of type I diabetes. In this report we describe a series of islet-specific T cell lines established from islet-infiltrating lymphocytes obtained from individual pre-diabetic NOD mice as well as a large panel of clones derived from these lines. Proliferation assays indicated that these nominally islet-specific lines responded vigorously to porcine insulin. Furthermore, of 40 islet-specific clones derived from lines established from 12-week-old mice, 22 (55%) responded to insulin. A similar analysis of islet-specific clones established from 7-week-old mice indicated that 2 of 14 (14%) were insulin specific. These findings demonstrate that insulin-specific T cells can comprise a major portion of the spontaneously arising T cell response to islets in NOD mice.

Role of infiltrating T cells for impaired glucose metabolism in pancreatic islets isolated from non-obese diabetic mice.

Pancreatic islets isolated from non-obese diabetic (NOD) mice, all of which have insulitis, exhibit an impaired glucose metabolism. In order to investigate the role of infiltrating lymphocytes for this altered metabolism, we injected 12- to 13-week-old female NOD mice with monoclonal antibodies directed against either the alpha beta-T cell receptor, CD4+ or CD8+ T cells. Control NOD mice were injected with normal rat IgG or with the vehicle (phosphate buffered saline) alone. Injection of the three different monoclonal antibodies markedly reduced the mononuclear cell infiltration. An intravenous glucose tolerance test showed no differences between the groups. Islet insulin release in response to glucose was similar in all groups. In contrast, islets isolated from the control NOD mice with insulitis showed a high basal (1.7 mmol/l glucose) glucose oxidation rate and a small increase in the glucose oxidation rate in response to a high glucose concentration (16.7 mmol/l glucose). The monoclonal antibodies counteracted the elevated basal glucose oxidation rate of the islets. Parallel studies of stimulated mononuclear cells suggested that the contribution of glucose oxidized by islet-infiltrating lymphocytes could only partially explain the observed alterations in NOD mouse islet metabolism. Culture of islets obtained from NOD mice in the presence of the cytokine interleukin-1 beta induced a similar pattern of glucose metabolism as seen earlier in IgG or phosphate-buffered saline treated control NOD mice. In conclusion, alterations in the glucose oxidation rates seem to be an early sign of disturbance in islets isolated from NOD mice. These early alterations in glucose metabolism can be reversed in vivo by monoclonal antibodies directed against effector lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)