Testing the role of P2X7 receptors in the development of type 1 diabetes in nonobese diabetic mice

Although P2rx7 has been proposed as a type 1 diabetes (T1D) susceptibility gene in NOD mice, its potential pathogenic role has not been directly determined. To test this possibility, we generated a new NOD stock deficient in P2X(7) receptors. T1D development was not altered by P2X(7) ablation. Previous studies found CD38 knockout (KO) NOD mice developed accelerated T1D partly because of a loss of CD4(+) invariant NKT (iNKT) cells and Foxp3(+) regulatory T cells (Tregs). These immunoregulatory T cell populations are highly sensitive to NAD-induced cell death activated by ADP ribosyltransferase-2 (ART2)-mediated ADP ribosylation of P2X(7) receptors. Therefore, we asked whether T1D acceleration was suppressed in a double-KO NOD stock lacking both P2X(7) and CD38 by rescuing CD4(+) iNKT cells and Tregs from NAD-induced cell death. We demonstrated that P2X(7) was required for T1D acceleration induced by CD38 deficiency. The CD38 KO-induced defects in homeostasis of CD4(+) iNKT cells and Tregs were corrected by coablation of P2X(7). T1D acceleration in CD38-deficient NOD mice also requires ART2 expression. If increased ADP ribosylation of P2X(7) in CD38-deficient NOD mice underlies disease acceleration, then a comparable T1D incidence should be induced by coablation of both CD38 and ART2, or CD38 and P2X(7). However, a previously established NOD stock deficient in both CD38 and ART2 expression is T1D resistant. This study demonstrated the presence of a T1D resistance gene closely linked to the ablated Cd38 allele in the previously reported NOD stock also lacking ART2, but not in the newly generated CD38/P2X(7) double-KO line.     

CD38 is required for the peripheral survival of immunotolerogenic CD4+ invariant NK T cells in nonobese diabetic mice

T cell-mediated autoimmune type-1 diabetes (T1D) in NOD mice partly results from this strain's numerical and functional defects in invariant NK T (iNKT) cells. T1D is inhibited in NOD mice treated with the iNKT cell superagonist alpha-galactosylceramide through a process involving enhanced accumulation of immunotolerogenic dendritic cells in pancreatic lymph nodes. Conversely, T1D is accelerated in NOD mice lacking CD38 molecules that play a role in dendritic cell migration to inflamed tissues. Unlike in standard NOD mice, alpha-galactosylceramide pretreatment did not protect the CD38-deficient stock from T1D induced by an adoptively transferred pancreatic beta cell-autoreactive CD8 T cell clone (AI4). We found that in the absence of CD38, ADP-ribosyltransferase 2 preferentially activates apoptotic deletion of peripheral iNKT cells, especially the CD4+ subset. Therefore, this study documents a previously unrecognized role for CD38 in maintaining survival of an iNKT cell subset that preferentially contributes to the maintenance of immunological tolerance.     

NAD+ released during inflammation participates in T cell homeostasis by inducing ART2-mediated death of naive T cells in vivo

Mono ADP-ribosyltransferase 2 (ART2) is an ectoenzyme expressed on mouse T lymphocytes, which catalyze the transfer of ADP-ribose groups from NAD(+) onto several target proteins. In vitro, ADP-ribosylation by ART2 activates the P2X7 ATP receptor and is responsible for NAD(+)-induced T cell death (NICD). Yet, the origin of extracellular NAD(+) and the role of NICD in vivo remain elusive. In a model of acute inflammation induced by polyacrylamide beads, we demonstrate release of NAD(+) into exudates during the early phase of the inflammatory response. This leads to T cell depletion in the draining lymph nodes from wild-type and, more severely, from mice lacking the CD38 NAD(+) glycohydrolase, whereas no effect is observed in ART2-deficient animals. Intravenous injection of NAD(+) used to exacerbate NICD in vivo results in fast and dramatic ART2- and P2X7-dependent depletion of CD4+ and CD8+ T lymphocytes, which can affect up to 80% of peripheral T cells in CD38(-/-) mice. This affects mainly naive T cells as most cells surviving in vivo NAD+ treatment exhibit the phenotype of recently activated/memory cells. Consistently, treatment with NAD(+) abolishes primary Ab response to a T-dependent Ag in NICD-susceptible CD38(-/-) mice but has no effect on the secondary response when given several days after priming. Unexpectedly NAD+ treatment improves the response in their wild-type BALB/c counterparts. We propose that NAD(+) released during early inflammation facilitates the expansion of primed T cells, through ART2-driven death of resting cells, thus contributing to the dynamic regulation of T cell homeostasis.     

TNF receptor 1 deficiency increases regulatory T cell function in nonobese diabetic mice

TNF has been implicated in the pathogenesis of type 1 diabetes. When administered early in life, TNF accelerates and increases diabetes in NOD mice. However, when administered late, TNF decreases diabetes incidence and delays onset. TNFR1-deficient NOD mice were fully protected from diabetes and only showed mild peri-insulitis. To further dissect how TNFR1 deficiency affects type 1 diabetes, these mice were crossed to β cell-specific, highly diabetogenic TCR transgenic I-A(g7)-restricted NOD4.1 mice and Kd-restricted NOD8.3 mice. TNFR1-deficient NOD4.1 and NOD8.3 mice were protected from diabetes and had significantly less insulitis compared with wild type NOD4.1 and NOD8.3 controls. Diabetic NOD4.1 mice rejected TNFR1-deficient islet grafts as efficiently as control islets, confirming that TNFR1 signaling is not directly required for β cell destruction. Flow cytometric analysis showed a significant increase in the number of CD4(+)CD25(+)Foxp3(+) T regulatory cells in TNFR1-deficient mice. TNFR1-deficient T regulatory cells were functionally better at suppressing effector cells than were wild type T regulatory cells both in vitro and in vivo. This study suggests that blocking TNF signaling may be beneficial in increasing the function of T regulatory cells and suppression of type 1 diabetes.     

Defective CD8+ T cell peripheral tolerance in nonobese diabetic mice.

Nonobese diabetic (NOD) mice develop spontaneous autoimmune diabetes that involves participation of both CD4+ and CD8+ T cells. Previous studies have demonstrated spontaneous reactivity to self-Ags within the CD4+ T cell compartment in this strain. Whether CD8+ T cells in NOD mice achieve and maintain tolerance to self-Ags has not previously been evaluated. To investigate this issue, we have assessed the extent of tolerance to a model pancreatic Ag, the hemagglutinin (HA) molecule of influenza virus, that is transgenically expressed by pancreatic islet beta cells in InsHA mice. Previous studies have demonstrated that BALB/c and B10.D2 mice that express this transgene exhibit tolerance of HA and retain only low-avidity CD8+ T cells specific for the dominant peptide epitope of HA. In this study, we present data that demonstrate a deficiency in peripheral tolerance within the CD8+ T cell repertoire of NOD-InsHA mice. CD8+ T cells can be obtained from NOD-InsHA mice that exhibit high avidity for HA, as measured by tetramer (K(d)HA) binding and dose titration analysis. Significantly, these autoreactive CD8+ T cells can cause diabetes very rapidly upon adoptive transfer into NOD-InsHA recipient mice. The data presented demonstrate a retention in the repertoire of CD8+ T cells with high avidity for islet Ags that could contribute to autoimmune diabetes in NOD mice.     

Pancreatic infiltration but not diabetes occurs in the relative absence of MHC class II-restricted CD4 T cells: studies using NOD/CIITA-deficient mice

The NOD (nonobese diabetic) mouse is a good animal model for human IDDM. MHC class II-restricted CD4 T cells are necessary for the onset of diabetes in NOD mice. Here, we demonstrate that NOD mice lacking the CIITA (class II transactivator) molecule, and hence deficient in MHC class II expression and peripheral CD4 T cells, show significant pancreatic infiltration but do not develop diabetes. CD4 T cell deficiency, then, does not prevent initial pancreatic infiltration, but does stop progression to insulitis. Adoptive transfer studies show that the paucity of CD4 T cells in NOD-CIITA knockout mice is responsible for the absence of diabetes, since the CD8 T cell and B cell compartments are functional. An autoaggressive CD8+ T cell clone can, however, transfer diabetes in CIITA knockout recipient mice without CD4 T cell help, albeit with some delay compared with that in CIITA-sufficient recipients. This highlights the fact that a high number of in vitro activated autoaggressive CD8 T cells can over-ride the requirement for CD4 T cell help for the onset of diabetes.     

Helminth protection against autoimmune diabetes in nonobese diabetic mice is independent of a type 2 immune shift and requires TGF-β

Leading hypotheses to explain helminth-mediated protection against autoimmunity postulate that type 2 or regulatory immune responses induced by helminth infections in the host limit pathogenic Th1-driven autoimmune responses. We tested these hypotheses by investigating whether infection with the filarial nematode Litomosoides sigmodontis prevents diabetes onset in IL-4-deficient NOD mice and whether depletion or absence of regulatory T cells, IL-10, or TGF-β alters helminth-mediated protection. In contrast to IL-4-competent NOD mice, IL-4-deficient NOD mice failed to develop a type 2 shift in either cytokine or Ab production during L. sigmodontis infection. Despite the absence of a type 2 immune shift, infection of IL-4-deficient NOD mice with L. sigmodontis prevented diabetes onset in all mice studied. Infections in immunocompetent and IL-4-deficient NOD mice were accompanied by increases in CD4(+)CD25(+)Foxp3(+) regulatory T cell frequencies and numbers, respectively, and helminth infection increased the proliferation of CD4(+)Foxp3(+) cells. However, depletion of CD25(+) cells in NOD mice or Foxp3(+) T cells from splenocytes transferred into NOD.scid mice did not decrease helminth-mediated protection against diabetes onset. Continuous depletion of the anti-inflammatory cytokine TGF-β, but not blockade of IL-10 signaling, prevented the beneficial effect of helminth infection on diabetes. Changes in Th17 responses did not seem to play an important role in helminth-mediated protection against autoimmunity, because helminth infection was not associated with a decreased Th17 immune response. This study demonstrates that L. sigmodontis-mediated protection against diabetes in NOD mice is not dependent on the induction of a type 2 immune shift but does require TGF-β.     

A mechanism for IL-10-mediated diabetes in the nonobese diabetic (NOD) mouse: ICAM-1 deficiency blocks accelerated diabetes

Neonatal islet-specific expression of IL-10 in nonobese diabetic (NOD) mice accelerates the onset of diabetes, whereas systemic treatment of young NOD mice with IL-10 prevents diabetes. The mechanism for acceleration of diabetes in IL-10-NOD mice is not known. Here we show, by adoptive transfers, that prediabetic or diabetic NOD splenocytes upon encountering IL-10 in the pancreatic islets readily promoted diabetes. This outcome suggests that the compartment of exposure, not the timing, confers proinflammatory effects on this molecule. Moreover, injection of IL-10-deficient NOD splenocytes into transgenic IL-10-NOD.scid/scid mice elicited accelerated disease, demonstrating that pancreatic IL-10 but not endogenous IL-10 is sufficient for the acceleration of diabetes. Immunohistochemical analysis revealed hyperexpression of ICAM-1 on the vascular endothelium of IL-10-NOD mice. The finding suggests that IL-10 may promote diabetes via an ICAM-1-dependent pathway. We found that introduction of ICAM-1 deficiency into IL-10-NOD mice as well as into NOD mice prevented accelerated insulitis and diabetes. Failure to develop insulitis and diabetes was preceded by the absence of GAD65-specific T cell responses. The data suggest that ICAM-1 plays a role in the formation of the "immunological synapse", thereby affecting the generation and/or expansion of islet-specific T cells. In addition, ICAM-1 also played a role in the effector phase of autoimmune diabetes because adoptive transfer of diabetogenic BDC2.5 T cells failed to elicit clinical disease in ICAM-1-deficient IL-10-NOD and NOD mice. These findings provide evidence that pancreatic IL-10 is sufficient to drive pathogenic autoimmune responses and accelerates diabetes via an ICAM-1-dependent pathway.     

TCR gamma delta intraepithelial lymphocytes are required for self-tolerance

Neonatal thymectomy (NTX) impairs T cell regulation and leads to organ-specific autoimmune disease in susceptible mouse strains. In the NOD mouse model of spontaneous type 1 diabetes, we observed that NTX dramatically accelerated autoimmune pancreatic beta cell destruction and diabetes. NTX had only a minor effect in NOD mice protected from diabetes by transgenic expression of the beta cell autoantigen proinsulin in APCs, inferring that accelerated diabetes after NTX is largely due to failure to regulate proinsulin-specific T cells. NTX markedly impaired the development of intraepithelial lymphocytes (IEL), the number of which was already reduced in euthymic NOD mice compared with control strains. IEL purified from euthymic NOD mice, specifically CD8alphaalpha TCRgammadelta IEL, when transferred into NTX-NOD mice, trafficked to the small intestinal epithelium and prevented diabetes. Transfer of prototypic CD4+CD25+ regulatory T cells also prevented diabetes in NTX-NOD mice; however, the induction of these cells by oral insulin in euthymic mice depended on the integrity of TCRgammadelta IEL. We conclude that TCRgammadelta IEL at the mucosal interface between self and nonself play a key role in maintaining peripheral tolerance both physiologically and during oral tolerance induction.     

Beta-cell apoptosis in an accelerated model of autoimmune diabetes.

BACKGROUND: The non-obese diabetic (NOD) mouse is a model of human type 1 diabetes in which autoreactive T cells mediate destruction of pancreatic islet beta cells. Although known to be triggered by cytotoxic T cells, apoptosis has not been unequivocally localized to beta cells in spontaneously diabetic NOD mice. We created a model of accelerated beta-cell destruction mediated by T cells from spontaneously diabetic NOD mice to facilitate the direct detection of apoptosis in beta cells. MATERIALS AND METHODS: NOD.scid (severe combined immunodeficiency) mice were crossed with bm1 mice transgenically expressing the costimulatory molecule B7-1 (CD80) in their beta cells, to generate B7-1 NOD.scid mice. Apoptosis in islet cells was measured as DNA strand breakage by the TdT-mediated-dUTP-nick end labeling (TUNEL) technique. RESULTS: Adoptive transfer of splenocytes from spontaneously diabetic NOD mice into B7-1 NOD.scid mice caused diabetes in recipients within 12-16 days. Mononuclear cell infiltration and apoptosis were significantly greater in the islets of B7-1 NOD.scid mice than in nontransgenic NOD.scid mice. Dual immunolabeling for TUNEL and either B-7 or insulin, or the T cell markers CD4 and CD8, and colocalization by confocal microscopy clearly demonstrated apoptosis in beta cells as well in a relatively larger number of infiltrating T cells. The clearance time of apoptotic beta cells was estimated to be less than 6 min. CONCLUSIONS: B7-1 transgenic beta cells undergo apoptosis during their accelerated destruction in response to NOD mouse effector T cells. Rapid clearance implies that beta cells undergoing apoptosis would be detected only rarely during more protracted disease in spontaneously diabetic NOD mice.     

Autoimmunity to both proinsulin and IGRP is required for diabetes in nonobese diabetic 8.3 TCR transgenic mice

T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) induce diabetes in nonobese diabetic (NOD) mice. TCR transgenic mice with CD8(+) T cells specific for IGRP(206-214) (NOD8.3 mice) develop accelerated diabetes that requires CD4(+) T cell help. We previously showed that immune responses against proinsulin are necessary for IGRP(206-214)-specific CD8(+) T cells to expand. In this study, we show that diabetes development is dramatically reduced in NOD8.3 mice crossed to NOD mice tolerant to proinsulin (NOD-PI mice). This indicates that immunity to proinsulin is even required in the great majority of NOD8.3 mice that have a pre-existing repertoire of IGRP(206-214)-specific cells. However, protection from diabetes could be overcome by inducing islet inflammation either by a single dose of streptozotocin or anti-CD40 agonist Ab treatment. This suggests that islet inflammation can substitute for proinsulin-specific CD4(+) T cell help to activate IGRP(206-214)-specific T cells.     

Granulocyte-macrophage colony-stimulating factor prevents diabetes development in NOD mice by inducing tolerogenic dendritic cells that sustain the suppressive function of CD4+CD25+ regulatory T cells

Autoimmune diabetes results from a breakdown of self-tolerance that leads to T cell-mediated beta-cell destruction. Abnormal maturation and other defects of dendritic cells (DCs) have been associated with the development of diabetes. Evidence is accumulating that self-tolerance can be restored and maintained by semimature DCs induced by GM-CSF. We have investigated whether GM-CSF is a valuable strategy to induce semimature DCs, thereby restoring and sustaining tolerance in NOD mice. We found that treatment of prediabetic NOD mice with GM-CSF provided protection against diabetes. The protection was associated with a marked increase in the number of tolerogenic immature splenic DCs and in the number of Foxp3+CD4+CD25+ regulatory T cells (Tregs). Activated DCs from GM-CSF-protected mice expressed lower levels of MHC class II and CD80/CD86 molecules, produced more IL-10 and were less effective in stimulating diabetogenic CD8+ T cells than DCs of PBS-treated NOD mice. Adoptive transfer experiments showed that splenocytes of GM-CSF-protected mice did not transfer diabetes into NOD.SCID recipients. Depletion of CD11c+ DCs before transfer released diabetogenic T cells from the suppressive effect of CD4+CD25+ Tregs, thereby promoting the development of diabetes. These results indicated that semimature DCs were required for the sustained suppressive function of CD4+CD25+ Tregs that were responsible for maintaining tolerance of diabetogenic T cells in NOD mice.     

Combined insulin B:9-23 self-peptide and polyinosinic-polycytidylic acid accelerate insulitis but inhibit development of diabetes by increasing the proportion of CD4+Foxp3+ regulatory T cells in the islets in non-obese diabetic mice

Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes. Combined treatment with B:9-23 peptide and polyinosinic-polycytidylic acid (poly I:C), but neither alone, induce insulitis in normal BALB/c mice. In contrast, the combined treatment accelerated insulitis, but prevented diabetes in NOD mice. Our immunofluorescence study with anti-CD4/anti-Foxp3 revealed that the proportion of Foxp3 positive CD4⁺CD25⁺ regulatory T cells (Tregs) was elevated in the islets of NOD mice treated with B:9-23 peptide and poly I:C, as compared to non-treated mice. Depletion of Tregs by anti-CD25 antibody hastened spontaneous development of diabetes in non-treated NOD mice, and abolished the protective effect of the combined treatment and conversely accelerated the onset of diabetes in the treated mice. These results indicate that poly I:C combined with B:9-23 peptide promotes infiltration of both pathogenic T cells and predominantly Tregs into the islets, thereby inhibiting progression from insulitis to overt diabetes in NOD mice.     

TNF receptor 1-dependent beta cell toxicity as an effector pathway in autoimmune diabetes

Autoimmune diabetes is characterized by a chronic progressive inflammatory autoimmune reaction that ultimately causes the selective elimination of pancreatic beta cells. To address the question of whether the cell death-inducing cytokines TNF and lymphotoxin alpha are involved in this process, we generated nonobese diabetic (NOD) mice that are deficient for TNF receptor 1 (TNFR1 or TNFRp55). Insulitis developed in these mice similarly to that in normal control NOD mice, but progression to diabetes was completely abrogated. Since this was probably due to the complex immunomodulatory effects of TNF and lymphotoxin alpha signaled via TNFR1 on lymphohemopoietic cells, adoptive transfer experiments with spleen cells from diabetic NOD mice were conducted. It was found that the absence of TNFR1 in recipients delayed diabetes induced by normal control and precluded diabetes induced by perforin-deficient spleen cells. In a CD8+ T cell-mediated model of diabetes, however, diabetes induced by adoptive transfer of TCR transgenic lymphocytic choriomeningitis virus glycoprotein-specific CD8+ T cells was not delayed by the absence of TNFR1 in recipient mice. Together with the described expression patterns of perforin and TNF in the mononuclear islet infiltrates of NOD mice, these results indicate that two diabetogenic effector mechanisms are delivered by distinct cell populations: CD8+ T cells lyse beta cells via perforin-dependent cytotoxicity, whereas CD4+ T cells, macrophages, and dendritic cells contribute to diabetes development via TNFR1-dependent beta cell toxicity.     

I-Ag7-mediated antigen presentation by B lymphocytes is critical in overcoming a checkpoint in T cell tolerance to islet beta cells of nonobese diabetic mice.

B cell-deficient nonobese diabetic (NOD) mice are protected from the development of spontaneous autoimmune diabetes, suggesting a requisite role for Ag presentation by B lymphocytes for the activation of a diabetogenic T cell repertoire. This study specifically examines the importance of B cell-mediated MHC class II Ag presentation as a regulator of peripheral T cell tolerance to islet beta cells. We describe the construction of NOD mice with an I-Ag7 deficiency confined to the B cell compartment. Analysis of these mice, termed NOD BCIID, revealed the presence of functionally competent non-B cell APCs (macrophages/dendritic cells) with normal I-Ag7 expression and capable of activating Ag-reactive T cells. In addition, the secondary lymphoid organs of these mice harbored phenotypically normal CD4+ and CD8+ T cell compartments. Interestingly, whereas control NOD mice harboring I-Ag7-sufficient B cells developed diabetes spontaneously, NOD BCIID mice were resistant to the development of autoimmune diabetes. Despite their diabetes resistance, histologic examination of pancreata from NOD BCIID mice revealed foci of noninvasive peri-insulitis that could be intentionally converted into a destructive process upon treatment with cyclophosphamide. We conclude that I-Ag7-mediated Ag presentation by B cells serves to overcome a checkpoint in T cell tolerance to islet beta cells after their initial targeting has occurred. Overall, this work indicates that the full expression of the autoimmune potential of anti-islet T cells in NOD mice is intimately regulated by B cell-mediated MHC class II Ag presentation.     

CD38 controls ADP-ribosyltransferase-2-catalyzed ADP-ribosylation of T cell surface proteins

ADP-ribosyltransferase-2 (ART2), a GPI-anchored, toxin-related ADP-ribosylating ectoenzyme, is prominently expressed by murine T cells but not by B cells. Upon exposure of T cells to NAD, the substrate for ADP-ribosylation, ART2 catalyzes ADP-ribosylation of the P2X7 purinoceptor and other functionally important cell surface proteins. This in turn activates P2X7 and induces exposure of phosphatidylserine and shedding of CD62L. CD38, a potent ecto-NAD-glycohydrolase, is strongly expressed by most B cells but only weakly by T cells. Following incubation with NAD, CD38-deficient splenocytes exhibited lower NAD-glycohydrolase activity and stronger ADP-ribosylation of cell surface proteins than their wild-type counterparts. Depletion of CD38(high) cells from wild-type splenocytes resulted in stronger ADP-ribosylation on the remaining cells. Similarly, treatment of total splenocytes with the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide increased the level of cell surface ADP-ribosylation. Furthermore, the majority of T cells isolated from CD38-deficient mice "spontaneously" exposed phosphatidylserine and lacked CD62L, most likely reflecting previous encounter with ecto-NAD. Our findings support the notion that ecto-NAD functions as a signaling molecule following its release from cells by lytic or nonlytic mechanisms. ART2 can sense and translate the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins, whereas CD38 controls the level of cell surface protein ADP-ribosylation by limiting the substrate availability for ART2.     

Pancreas-infiltrating Th1 cells and diabetes develop in IL-12-deficient nonobese diabetic mice.

IL-12 and IL-12 antagonist administration to nonobese diabetic (NOD) mice accelerates and prevents insulin-dependent diabetes mellitus (IDDM), respectively. To further define the role of endogenous IL-12 in the development of diabetogenic Th1 cells, IL-12-deficient NOD mice were generated and analyzed. Th1 responses to exogenous Ags were reduced by approximately 80% in draining lymph nodes of these mice, and addition of IL-12, but not IL-18, restored Th1 development in vitro, indicating a nonredundant role of IL-12. Moreover, spontaneous Th1 responses to a self Ag, the tyrosine phosphatase-like IA-2, were undetectable in lymphoid organs from IL-12-deficient, in contrast to wild-type, NOD mice. Nevertheless, wild-type and IL-12-deficient NOD mice developed similar insulitis and IDDM. Both in wild-type and IL-12-deficient NOD mice, approximately 20% of pancreas-infiltrating CD4+ T cells produced IFN-gamma, whereas very few produced IL-10 or IL-4, indicating that IDDM was associated with a type 1 T cell infiltrate in the target organ. T cell recruitment in the pancreas seemed favored in IL-12-deficient NOD mice, as revealed by increased P-selectin ligand expression on pancreas-infiltrating T cells, and this could, at least in part, compensate for the defective Th1 cell pool recruitable from peripheral lymphoid organs. Residual Th1 cells could also accumulate in the pancreas of IL-12-deficient NOD mice because Th2 cells were not induced, in contrast to wild-type NOD mice treated with an IL-12 antagonist. Thus, a regulatory pathway seems necessary to counteract the pathogenic Th1 cells that develop in the absence of IL-12 in a spontaneous chronic progressive autoimmune disease under polygenic control, such as IDDM.     

Granzyme B is dispensable in the development of diabetes in non-obese diabetic mice

Pancreatic beta cell destruction in type 1 diabetes is mediated by cytotoxic CD8(+) T lymphoctyes (CTL). Granzyme B is an effector molecule used by CTL to kill target cells. We previously showed that granzyme B-deficient allogeneic CTL inefficiently killed pancreatic islets in vitro. We generated granzyme B-deficient non-obese diabetic (NOD) mice to test whether granzyme B is an important effector molecule in spontaneous type 1 diabetes. Granzyme B-deficient islet antigen-specific CD8(+) T cells had impaired homing into islets of young mice. Insulitis was reduced in granzyme B-deficient mice at 70 days of age (insulitis score 0.043±0.019 in granzyme B-deficient versus 0.139±0.034 in wild-type NOD mice p<0.05), but was similar to wild-type at 100 and 150 days of age. We observed a reduced frequency of CD3(+)CD8(+) T cells in the islets and peripheral lymphoid tissues of granzyme B-deficient mice (p<0.005 and p<0.0001 respectively), but there was no difference in cell proportions in the thymus. Antigen-specific CTL developed normally in granzyme B-deficient mice, and were able to kill NOD islet target cells as efficiently as wild-type CTL in vitro. The incidence of spontaneous diabetes in granzyme B-deficient mice was the same as wild-type NOD mice. We observed a delayed onset of diabetes in granzyme B-deficient CD8-dependent NOD8.3 mice (median onset 102.5 days in granzyme B-deficient versus 57.50 days in wild-type NOD8.3 mice), which may be due to the delayed onset of insulitis or inefficient priming at an earlier age in this accelerated model of diabetes. Our data indicate that granzyme B is dispensable for beta cell destruction in type 1 diabetes, but is required for efficient early activation of CTL.