T cell response to preproinsulin I and II in the nonobese diabetic mouse

Immunization against insulin, insulin B chain, or B chain peptide B(9-23) (preproinsulin peptide II(33-47)) prevents diabetes in the nonobese diabetic (NOD) mouse. Whether or not peptide II(33-47) is the only proinsulin determinant recognized by CD4 T cells remains unclear. Using two peptide libraries spanning the entire sequence of preproinsulin I and preproinsulin II, respectively, we identified T cells specific for four proinsulin epitopes within the islet cell infiltrate of prediabetic female NOD mice. These epitopes were among immunogenic epitopes to which a T cell response was detected after immunization of NOD mice with individual peptides in CFA. Immunogenic epitopes were found on both isoforms of insulin, especially proinsulin II, which is the isoform expressed in the thymus. The autoimmune response to proinsulin represented only part of the immune response to islet cells within the islet cell infiltrate in 15-wk-old NOD mice. This is the first systematic study of preproinsulin T cell epitopes in the NOD mouse model.     

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.     

Induction of diabetes in nonobese diabetic mice by Th2 T cell clones from a TCR transgenic mouse

We have produced a panel of cloned T cell lines from the BDC-2.5 TCR transgenic (Tg) mouse that exhibit a Th2 cytokine phenotype in vitro but are highly diabetogenic in vivo. Unlike an earlier report in which T cells obtained from the Tg mouse were cultured for 1 wk under Th2-promoting conditions and were found to induce disease only in NOD.scid recipients, we found that long-term T cell clones with a fixed Th2 cytokine profile can transfer disease only to young nonobese diabetic (NOD) mice and never to NOD.scid recipients. Furthermore, the mechanism by which diabetes is transferred by a Tg Th2 T cell clone differs from that of the original CD4+ Th1 BDC-2.5 T cell clone made in this laboratory. Whereas the BDC-2.5 clone rapidly causes disease in NOD.scid recipients less than 2 wk old, the Tg Th2 T cell clones can do so only when cotransferred with other diabetogenic T cells, suggesting that the Th2 T cell requires the presence of host T cells for initiation of disease.     

CD8+ T cell tolerance in nonobese diabetic mice is restored by insulin-dependent diabetes resistance alleles

Although candidate genes controlling autoimmune disease can now be identified, a major challenge that remains is defining the resulting cellular events mediated by each locus. In the current study we have used NOD-InsHA transgenic mice that express the influenza hemagglutinin (HA) as an islet Ag to compare the fate of HA-specific CD8+ T cells in diabetes susceptible NOD-InsHA mice with that observed in diabetes-resistant congenic mice having protective alleles at insulin-dependent diabetes (Idd) 3, Idd5.1, and Idd5.2 (Idd3/5 strain) or at Idd9.1, Idd9.2, and Idd9.3 (Idd9 strain). We demonstrate that protection from diabetes in each case is correlated with functional tolerance of endogenous islet-specific CD8+ T cells. However, by following the fate of naive, CFSE-labeled, islet Ag-specific CD8+ (HA-specific clone-4) or CD4+ (BDC2.5) T cells, we observed that tolerance is achieved differently in each protected strain. In Idd3/5 mice, tolerance occurs during the initial activation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes where CD25+ regulatory T cells (Tregs) effectively prevent their accumulation. In contrast, resistance alleles in Idd9 mice do not prevent the accumulation of islet Ag-specific CD8+ and CD4+ T cells in the pancreatic lymph nodes, indicating that tolerance occurs at a later checkpoint. These results underscore the variety of ways that autoimmunity can be prevented and identify the elimination of islet-specific CD8+ T cells as a common indicator of high-level protection.     

CD8+ T cell homing to the pancreas in the nonobese diabetic mouse is CD4+ T cell-dependent.

The adoptive transfer of type I diabetes in nonobese diabetic mice requires the contribution of both CD4+ and CD8+ T cells. To further elucidate the cellular pathway(s) of beta-cell destruction and the responsibility of each subset, high doses of committed T cells from diabetic mice purified to single subsets, were injected into syngeneic nonobese diabetic neonates. The recipients of single or mixed subsets were followed for clinical manifestations of diabetes and examined at 30 days of age for in situ lesions. None of the animals injected with either CD4+ or CD8+ T cells became overtly diabetic during the 30 days of observation whereas 8 of 23 mice inoculated with a mixture of the two subsets developed glycosuria and hyperglycemia. However, insulitis was found in 6 of the 13 mice injected with CD4+ T cells whereas only 1 of the 9 mice injected with CD8+ T cells showed marginal infiltration of the pancreas. The lesions initiated by CD4+ T cells alone were considerably less severe than those induced by the mixture of both subsets, corroborating the fact that overt disease did not occur in the former group. Together, these results suggest a distinct function for each diabetogenic T cell subset. CD4+ T cells, which have the capacity to home to the pancreas, promote in turn the influx of CD8+ effector T cells that do not by themselves accumulate in this organ. These results illustrate a novel form of T-T cell interactions leading to organ specific autoimmune lesions.     

Differential contributions of APC subsets to T cell activation in nonobese diabetic mice

Despite the pivotal role of dendritic cells (DC) in shaping immunity, little is known about their functionality in type 1 diabetes. Moreover, due to the paucity of DC in vivo, functional studies have relied largely upon in vitro-expanded cells to elucidate type 1 diabetes-associated functional abnormalities. In this study, we provide a comprehensive analysis of the functional capabilities of in vivo-derived DC subsets from NOD mice by comparing DC to other NOD APC types and to DC from autoimmune-resistant strains. NOD DC closely resemble those from nonautoimmune strains with respect to costimulation and cytokine production. The exception is the CD8alpha(+)CD11b(-)DC subset which is numerically reduced in NOD spleens, but not in the pancreatic lymph nodes, while DC from both tissues produce little IL-12 in this strain. This defect results in unusual deferral toward macrophage-derived IL-12 in NOD mice; NOD macrophages produce aberrantly high IL-12 levels that can overcompensate for the DC defect in Th1 polarization. APC subset use for autoantigen presentation also differs in NOD mice. NOD B cells overshadow DC at activating islet-reactive T cells, whereas DC and B cells in NOD-resistant mice are functionally comparable. Differential involvement of APC subsets in T cell activation and tolerance induction may prove to be a crucial factor in the selection and expansion of autoreactive T cells.     

T cell tolerance to germline-encoded antibody sequences in a lupus-prone mouse

The BCR V region has been implicated as a potential avenue of T cell help for autoreactive B cells in systemic lupus erythematosus. In principle, either germline-encoded or somatically generated sequences could function as targets of such help. Preceding studies have indicated that class II MHC-restricted T cells in normal mice attain a state tolerance to germline-encoded Ab diversity. In this study, we tested whether this tolerance is intact in systemic lupus erythematosus-prone (New Zealand Black x SWR)F1 mice (SNF1). Using a hybridoma sampling approach, we found that SNF1 T cells were tolerant to germline-encoded Ab sequences. Specifically, they were tolerant to germline-encoded sequences derived from a lupus anti-chromatin Ab that arose spontaneously in this strain. This was true both for diseased and prediseased mice. Thus, there does not appear to be a global defect in T cell tolerance to Ab V regions in this autoimmune-prone strain either before or during autoimmune disease.     

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.     

I-E-independent deletion of V beta 17a+ T cells by Mtv-3 from the nonobese diabetic mouse.

Analysis of TCR beta-chain V region (V beta) frequency among NOD lymphocytes reveals a profound depletion of V beta 3+ T cells, and a recent study has linked this phenomenon to the Mtv-3 insertion on chromosome 11. When the V beta 17a gene segment is introduced into mice with an nonobese diabetic mouse background, T cells bearing the TCR encoded by this gene segment are also dramatically reduced in frequency. Deletion of V beta 17a+ T cells segregates with deletion of T cells bearing V beta 3 and occurs in the absence of I-E, which had been shown in previous studies to be a major deleting element for V beta 17a+ thymocytes.     

A defect in cell death of macrophages is a conserved feature of nonobese diabetic mouse

Impaired apoptosis in immune effector cells such as macrophages has been implicated in the development of autoimmune disease by promoting the breakdown of self-tolerance and the sustained production of cytotoxic molecules. Macrophages from nonobese diabetic (NOD) mouse, an animal model of human autoimmune diabetes, exhibit several defects that are causally linked to the onset and progression of the disease. In this context, we investigated whether NOD macrophages have a defect in a cell death pathway, and if that is the case, the mechanism underlying such dysregulation of cell death. We found that NOD macrophages were resistant to treatment with a broad spectrum of cell death stimuli, triggering both apoptotic and non-apoptotic death. Through analysis of intracellular signaling pathways along with the expression of apoptosis-related proteins, we found that atypical resistance to cell death was associated with an elevated expression of anti-apoptotic Bcl-X(L) but not the NF-κB signaling pathway in NOD macrophages. Further, ABT-737, which can inhibit Bcl-X(L) function, sensitized NOD macrophages to apoptosis induced by diverse apoptotic stimuli, thus restoring sensitivity to cell death. Taken together, our results suggest a macrophage-intrinsic defect in cell death as a potential mechanism that promotes an immune attack towards pancreatic β-cells and the development of autoimmune diabetes in NOD mice.     

Genetic analysis of diabetes in the nonobese diabetic mouse. I. MHC and T cell receptor beta gene expression

Backcross nonobese diabetic (NOD) ((NOD x SWr)F1 x NOD) mice (108 females and 105 males) were typed for MHC, TCR V beta, and monitored for 350 days for the onset of diabetes. The presence of "antipolar" antibodies in the sera and the occurrence of insulitis was examined in a proportion of these backcross mice. There was no difference in the incidence of diabetes in mice heterozygous for TCR V beta b/a vs those homozygous for TCR V beta b/b. Among the 17 diabetics (all female) detected in this backcross, 14/17 were H-2nod/nod but 3/17 were H-2nod/q. This supports a previous observation suggesting that the MHC-linked diabetogenic gene originally thought to be recessive may rather be dominant but have a low penetrance in the heterozygous state. Antipolar autoantibodies were found in both female and male backcross mice, and were similarly distributed in diabetic and nondiabetic mice. There appeared to be no correlation between the level of these auto-antibodies and development of diabetes. The incidence and severity of insulitis was linked to MHC but no influence of TCR genes on insulitis nor an association between insulitis and antipolar antibodies could be demonstrated in this study. Further analyses of H-2nod/nod intercross mice homozygous for TCR V beta a or TCR V beta b are currently underway.     

Adoptive T cell transfer of autoimmune nonobese diabetic mouse diabetes does not require recruitment of host B lymphocytes

The autoimmune nonobese diabetic mouse, a model of human juvenile type I diabetes mellitus, exhibits features of both B and T cell autoreactivity against insulin-producing cells. Using the neonatal cell transfer model of the disease, which we have described previously, we have shown that B cell suppression of newborn recipients by anti-mu treatment did not affect the transfer of diabetes by means of T cells. B cell-depleted, purified T cells from diabetic adults were injected into newborns treated with either IR-52, a control rat myeloma protein, or LOMM.9, a rat anti-mouse mu-chain mAb. Both groups developed diabetes over a similar time scale. Although the pancreases in both groups showed massive infiltration by T lymphocytes, B lymphocytes, presumably recruited in the host, were present in the IR-52-treated group, whereas they were absent in the LOMM.9-treated group. Anti-mu-treated diabetic animals showed substantial B cell suppression in vivo and in vitro when compared with IR-52-treated controls. These results suggest that B cell autoreactivity is a secondary phenomenon that is unimportant during the effector phase of diabetes in nonobese diabetic mice.     

Evidence that beta cell death in the nonobese diabetic mouse is Fas independent.

Recent studies suggest that Fas expression on pancreatic beta cells may be important in the development of autoimmune diabetes in the nonobese diabetic (NOD) mouse. To address this, pancreatic islets from NOD mice were analyzed by flow cytometry to directly identify which cells express Fas and Fas ligand (FasL) ex vivo and after in vitro culture with cytokines. Fas expression was not detected on beta cells isolated from young (35 days) NOD mice. In vitro, incubation of NOD mouse islets with both IL-1 and IFN-gamma was required to achieve sufficient Fas expression and sensitivity for islets to be susceptible to lysis by soluble FasL. In islets isolated from older (>/=125 days) NOD mice, Fas expression was detected on a limited number of beta cells (1-5%). FasL was not detected on beta cells from either NOD or Fas-deficient MRLlpr/lpr islets. Also, both NOD and MRLlpr/lpr islets were equally susceptible to cytokine-induced cell death. This eliminates the possibility that cytokine-treated murine islet cells commit "suicide" due to simultaneous expression of Fas and FasL. Last, we show that NO is not required for cytokine-induced Fas expression and Fas-mediated apoptosis of islet cells. These findings indicate that beta cells can be killed by Fas-dependent cytotoxicity; however, our results raise further doubts about the clinical significance of Fas-mediated beta cell destruction because few Fas-positive cells were isolated immediately before the development of diabetes.     

More stringent conditions of plasmid DNA vaccination are required to protect grafted versus endogenous islets in nonobese diabetic mice

Recurrent autoimmune destruction of the insulin-producing beta cells is a key factor limiting successful islet graft transplantation in type I diabetic patients. In this study, we investigated the feasibility of using an Ag-specific plasmid DNA (pDNA)-based strategy to protect pro-islets that had developed from a neonatal pancreas implanted under the kidney capsule of nonobese diabetic (NOD) mice. NOD recipient mice immunized with pDNA encoding a glutamic acid decarboxylase 65 (GAD65)-IgFc fusion protein (JwGAD65), IL-4 (JwIL4), and IL-10 (pIL10) exhibited an increased number of intact pro-islets expressing high levels of insulin 15 wk posttransplant, relative to NOD recipient mice immunized with pDNA encoding a hen egg lysozyme (HEL)-IgFc fusion protein (JwHEL)+JwIL4 and pIL10 or left untreated. Notably, the majority of grafted pro-islets detected in JwGAD65+JwIL4- plus pIL10-treated recipients was free of insulitis. In addition, administration of JwGAD65+JwIL4+pIL10 provided optimal protection for engrafted islets compared with recipient NOD mice treated with JwGAD65+JwIL4 or JwGAD65+pIL10, despite effective protection of endogenous islets mediated by the respective pDNA treatments. Efficient protection of pro-islet grafts correlated with a marked reduction in GAD65-specific IFN-gamma reactivity and an increase in IL-10-secreting T cells. These results demonstrate that pDNA vaccination can be an effective strategy to mediate long-term protection of pro-islet grafts in an Ag-specific manner and that conditions are more stringent to suppress autoimmune destruction of grafted vs endogenous islets.     

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 activation of T suppressor cell function in nonobese diabetic mice. Potential relation to cytokine deficiencies

Nonobese diabetic (NOD) is an inbred mouse strain susceptible to development of T cell-mediated autoimmune diabetes. The strain is characterized by high percentages of T lymphocytes in lymphoid organs. The syngeneic mixed lymphocyte reaction (SMLR), a T cell response to self MHC class II Ag, is reportedly involved in the generation of a number of immunoregulatory cells, including suppressor inducers. A severely depressed SMLR characteristic of certain other autoimmune strains was found in NOD but not in nonautoimmune SWR/Bm mice. Moreover, IL-2 produced by NOD T cells at day 6 in an SMLR was at least one hundredfold reduced compared with SWR, and NOD T cells harvested from an SMLR at day 6 were functionally defective when tested for ability to induce suppression of an allogeneic MLR. However, functionally competent suppressor T cells were generated in NOD splenic leukocyte cultures in response to Con A, and IL-2 release from these was equivalent to that released by Con A-stimulated SWR splenocytes. A deficiency in cytokine release was not limited to IL-2, because peritoneal exudate cells from NOD exhibited a greatly diminished sensitivity to LPS-stimulated IL-1 release in comparison to SWR mice. IL-2 supplementation both in vitro and in vivo restored the ability of NOD T cells to respond in a SMLR, with production of cells capable of inducing suppression. Like SMLR-activated T cells from untreated SWR controls, SMLR blasts from IL-2-treated NOD mice were enriched for the L3T4 phenotype. IL-1 supplementation in vitro resulted in partial restoration of T suppressor activation in a SMLR. The depressed SMLR exhibited by NOD mice was apparently a stimulator cell dysfunction, because NOD stimulator cells failed to activate T cells from (SWR x NOD)F1 mice, whereas stimulators from SWR or F1 mice were capable of doing so. Collectively, these results suggest a defect in suppressor cell activation rather than an absence of this immunoregulatory cell population.     

Down-regulation of blood-brain glucose transport in the hyperglycemic nonobese diabetic mouse

The intracarotid injection method has been utilized to examine blood-brain barrier (BBB) glucose transport in hyperglycemic (4–6 days) mice. In anesthetized mice, Brain Uptake Indices were measured over a range of glucose concentrations from 0.010–50 mmol/l; glucose uptake was found to be saturable and kinetically characterized. The maximal velocity (V_max) for glucose transport was 989±214 nmol·min^–1·g^–1· and the half-saturation constant estimated to be 5.80±1.38 mmol/l. The unsaturated Permeability Surface are product (PS) is=171+8 l·min.^–1·g^–1. A rabbit polyclonal antiserum to a synthetic peptide encoding the 13 C-terminal amino acids of the human erythrocyte glucose transporter immunocytochemically confirmed the presence of the GLUT1 isoform in non-obese diabetic (NOD) mouse brain capillary endothelia. These studies indicate that a down-regulation of BBB glucose transport occurs in these spontaneously hyperglycemic mice; both BBB glucose permeability (as indicated by PS product) and transporter maximal velocity are reduced (in comparison to normoglycemic CD-1 mice), but the half-saturation constant remains unchanged.     

Homeostatic T cell proliferation as a barrier to T cell tolerance

The maintenance of T cell numbers in the periphery is mediated by distinct homeostatic mechanisms that ensure the proper representation of na?ve and memory T cells. Homeostatic proliferation refers to the process by which T cells in lymphopenic hosts divide in the absence of cognate antigen to reconstitute the peripheral lymphoid compartment. During this process T cells acquire effector-memory like properties, including the ability to respond to low doses of antigen in the absence of CD28 costimulation. Furthermore, this capacity is retained long after proliferation has ceased. Accumulating data implicates homeostatic proliferation in autoimmune diseases and transplant rejection, and suggests that it may represent a barrier to tolerance in protocols that use T cell depletion. Implementing combination therapies that aim to promote the development and expansion of regulatory T cell populations while specifically targeting alloresponsive T cells may be the soundest approach to attaining allograft tolerance in the aftermath of T cell depletion and homeostatic proliferation.