Cutting edge: homologous recombination of the MHC class I K region defines new MHC-linked diabetogenic susceptibility gene(s) in nonobese diabetic mice.

To localize the MHC-linked diabetogenic genes in the nonobese diabetic (NOD) mouse, a recombinational hotspot from the B10.A(R209) mouse was introduced to the region between the MHC class I K and class II A of the NOD mouse with the recombinational site centromeric to the Lmp2/Tap1 complex by breeding the two strains. Replacement of the NOD region centromeric to the recombinational site with the same region in R209 mice prevented the development of diabetes (from 71 to 3%) and insulitis (from 61 to 15%) in the N7 intra-MHC recombinant NOD mice. Similarly, the replacement of the NOD class II A, E and class I D region with the same region in R209 mice prevented the diseases (diabetes, from 71 to 0%; insulitis, from 61 to 3%). In addition to the MHC class II genes, there are at least two MHC-linked diabetogenic genes in the region centromeric to Lmp2.     

Destruction of pancreatic islet cells by cytotoxic T lymphocytes in nonobese diabetic mice

Proliferation of islet-associated leukocytes occurred when isolated islets from 20-wk-old female nonobese diabetic (NOD) mice were cultured with 10 U/ml rIL-2 for 7 days. Co-culture of these leukocytes with freshly isolated islets from 6- to 8-wk-old NOD donors in the presence of 1 U/ml rIL-2 produced islet structural deformation within 24 h and islet cytolysis within 48 h. Three lines of evidence suggest that these leukocytes were composed mainly of CTL specific for islet cells. First, morphologically, these proliferating cells adhered to NOD islets at 6 h and killed islets within 48 h of culture, but these phenomena could not be observed in the other tissues from NOD mice. These islet-derived cells were cytotoxic to NOD islet cells in a 51Cr-release assay, whereas no appreciable cytotoxicity was observed when NOD Con A-induced splenic blasts or fibroblasts were used as targets. Second, a flow cytometric analysis showed that these cells consisted of 97% Thy-1.2, 69% Lyt-2, 8% L3T4, and 4% asialo-GM1-positive cells, whereas Mac-1-positive cells could not be seen in these assays. After treatment with anti-Thy-1.2 or Lyt-2 mAb and C, these cells lost their activity to lyse NOD islet cells. However, these cells still had a full killing activity after the depletion of L3T4 or asialo GM1-positive cells. Third, islet cells from BALB/c, DBA/2, and B10.GD mice which share the same H-2K Ag with NOD mice were susceptible to cytolytic activity of these cells, whereas islet cells from NON, C57BL/6, C57BL/10, and C3H mice remained intact. Furthermore, anti-Kd antibody was capable of blocking this cytolysis. These results suggest that CTL expressing Thy-1.2 and Lyt-2 phenotypes appear to recognize the islet cell Ag with the restriction of MHC class I Kd, and then destroy NOD islet cells.     

Perturbed homeostasis of peripheral T cells elicits decreased susceptibility to anti-CD3-induced apoptosis in prediabetic nonobese diabetic mice

Activation-induced cell death (AICD) plays a key role in the homeostasis of the immune system. Autoreactive T cells are eliminated through AICD both from the thymus and periphery. In this study, we show that NOD peripheral T cells, especially CD8(+) T cells, display a decreased susceptibility to anti-CD3-induced AICD in vivo compared with T cells from diabetes-resistant B6, nonobese diabetes-resistant, and NOD.B6Idd4 mice. The susceptibility of NOD CD8(+) T cells to AICD varies in an age- and dose-dependent manner upon stimulation in vivo with either a mitogenic or nonmitogenic anti-CD3. NOD T cells preactivated by anti-CD3 in vivo are less susceptible than B6 T cells to TCR-induced AICD. Treatment of NOD mice with a mitogenic anti-CD3 depletes CD4(+)CD25(-)CD62L(+) but not CD4(+)CD25(+)CD62L(+) T cells, thereby resulting in an increase of the latter subset in the spleen. Treatment with a nonmitogenic anti-CD3 mAb delays the onset of T1D in 8.3 TCR transgenic NOD mice. These results demonstrate that the capacity of anti-CD3 to protect NOD mice from T1D correlates with its ability to perturb T cell homeostasis by inducing CD8(+) T cell AICD and increasing the number of CD4(+)CD25(+)CD62L(+) T cells in the periphery.     

Molecular mechanisms for gender differences in susceptibility to T cell-mediated autoimmune diabetes in nonobese diabetic mice

Nonobese diabetic (NOD) mice spontaneously develop diabetes with a strong female prevalence; however, the mechanisms for this gender difference in susceptibility to T cell-mediated autoimmune diabetes are poorly understood. This investigation was initiated to find mechanisms by which sex hormones might affect the development of autoimmune diabetes in NOD mice. We examined the expression of IFN-gamma, a characteristic Th1 cytokine, and IL-4, a characteristic Th2 cytokine, in islet infiltrates of female and male NOD mice at various ages. We found that the most significant difference in cytokine production between sexes was during the early stages of insulitis at 4 wk of age. IFN-gamma was significantly higher in young females, whereas IL-4 was higher in young males. CD4(+) T cells isolated from lymph nodes of female mice and activated with anti-CD3 and anti-CD28 Abs produced more IFN-gamma, but less IL-4, as compared with males. Treatment of CD4(+) T cells with estrogen significantly increased, whereas testosterone treatment decreased the IL-12-induced production of IFN-gamma. We then examined whether the change in IL-12-induced IFN-gamma production by treatment with sex hormones was due to the regulation of STAT4 activation. We found that estrogen treatment increased the phosphorylation of STAT4 in IL-12-stimulated T cells. We conclude that the increased susceptibility of female NOD mice to the development of autoimmune diabetes could be due to the enhancement of the Th1 immune response through the increase of IL-12-induced STAT4 activation by estrogen.     

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.     

Regulation of diabetes development by regulatory T cells in pancreatic islet antigen-specific TCR transgenic nonobese diabetic mice

Nonobese diabetic (NOD) mice carrying a transgenic TCR from an islet Ag-specific CD4 T cell clone, BDC2.5, do not develop diabetes. In contrast, the same transgenic NOD mice on the SCID background develop diabetes within 4 wk after birth. Using a newly developed mAb specific for the BDC2.5 TCR, we examined the interaction between diabetogenic T cells and regulatory T cells in NOD.BDC transgenic mice. CD4 T cells from NOD.BDC mice, expressing high levels of the clonotype, transfer diabetes to NOD.SCID recipients. In contrast, CD4 T cells expressing low levels due to the expression of both transgenic and endogenous TCR alpha-chains inhibit diabetes transfer. The clonotype-low CD4 T cells appear late in the ontogeny in the thymus and peripheral lymphoid organs, coinciding with resistance to cyclophosphamide-induced diabetes. These results demonstrate that diabetic processes in NOD.BDC mice are regulated by a balance between diabetogenic T cells and regulatory T cells. In the absence of specific manipulation, regulatory T cell function seems to be dominant and mice remain diabetes free. Understanding of mechanisms by which regulatory T cells inhibit diabetogenic processes would provide means to prevent diabetes development in high-risk human populations.     

A self MHC class II beta-chain peptide prevents diabetes in nonobese diabetic mice

We explored T cell responses to the self class II MHC (I-Ag7) beta-chain-derived peptides in diabetic and prediabetic nonobese diabetic (NOD) mice. We found that one of these immunodominant epitopes of the beta-chain of I-Ag7 molecule, peptide 54-76, could regulate autoimmunity leading to diabetes in NOD mice. T cells from prediabetic young NOD mice do not respond to the peptide 54-76, but T cells from diabetic NOD mice proliferated in response to this peptide. T cells from older nondiabetic mice or mice protected from diabetes do not respond to this peptide, suggesting a role for peptide 54-76-specific T cells in pathogenesis of diabetes. We show that this peptide is naturally processed and presented by the NOD APCs to self T cells. However, the peptide-specific T cells generated after immunization of young mice regulate autoimmunity in NOD mice by blocking the diabetogenic cells in adoptive transfer experiments. The NOD mice immunized with this peptide are protected from both spontaneous and cyclophosphamide-induced insulin-dependent diabetes mellitus. Immunization of young NOD mice with this peptide elicited T cell proliferation and production of Th2-type cytokines. In addition, immunization with this peptide induced peptide-specific Abs of IgG1 isotype that recognized native I-Ag7 molecule on the cell surface and inhibited the T cell proliferative responses. These results suggest that I-Abetag7(54-76) peptide-reactive T cells are involved in the pathogenesis of diabetes. However, immunization with this peptide at young age induces regulatory cells and the peptide-specific Abs that can modulate autoimmunity in NOD mice and prevent spontaneous and induced diabetes.     

Subcongenic analyses reveal complex interactions between distal chromosome 4 genes controlling diabetogenic B cells and CD4 T cells in nonobese diabetic mice

Autoimmune type 1 diabetes (T1D) in humans and NOD mice results from interactions between multiple susceptibility genes (termed Idd) located within and outside the MHC. Despite sharing ～88% of their genome with NOD mice, including the H2(g7) MHC haplotype and other important Idd genes, the closely related nonobese resistant (NOR) strain fails to develop T1D because of resistance alleles in residual genomic regions derived from C57BLKS mice mapping to chromosomes (Chr.) 1, 2, and 4. We previously produced a NOD background strain with a greatly decreased incidence of T1D as the result of a NOR-derived 44.31-Mb congenic region on distal Chr. 4 containing disease-resistance alleles that decrease the pathogenic activity of autoreactive B and CD4 T cells. In this study, a series of subcongenic strains for the NOR-derived Chr. 4 region was used to significantly refine genetic loci regulating diabetogenic B and CD4 T cell activity. Analyses of these subcongenic strains revealed the presence of at least two NOR-origin T1D resistance genes within this region. A 6.22-Mb region between rs13477999 and D4Mit32, not previously known to contain a locus affecting T1D susceptibility and now designated Idd25, was found to contain the main NOR gene(s) dampening diabetogenic B cell activity, with Ephb2 and/or Padi2 being strong candidates as the causal variants. Penetrance of this Idd25 effect was influenced by genes in surrounding regions controlling B cell responsiveness and anergy induction. Conversely, the gene(s) controlling pathogenic CD4 T cell activity was mapped to a more proximal 24.26-Mb region between the rs3674285 and D4Mit203 markers.     

ICOS-dependent homeostasis and function of Foxp3+ regulatory T cells in islets of nonobese diabetic mice

A progressive waning in Foxp3(+) regulatory T cell (Treg) functions is thought to provoke autoimmunity in the NOD model of type 1 diabetes (T1D). A deficiency in IL-2 is one of the main triggers for the defective function of Tregs in islets. Notably, abrogation of the ICOS pathway in NOD neonates or BDC2.5-NOD (BDC2.5) mice exacerbates T1D, suggesting an important role for this costimulatory pathway in tolerance to islet Ags. Thus, we hypothesize that ICOS selectively promotes Foxp3(+) Treg functions in BDC2.5 mice. We show that ICOS expression discriminates effector Foxp3(-) T cells from Foxp3(+) Tregs and specifically designates a dominant subset of intra-islet Tregs, endowed with an increased potential to expand, secrete IL-10, and mediate suppressive activity in vitro and in vivo. Consistently, Ab-mediated blockade or genetic deficiency of ICOS selectively abrogates Treg-mediated functions and T1D protection and exacerbates disease in BDC2.5 mice. Moreover, T1D progression in BDC2.5 mice is associated with a decline in ICOS expression in and expansion and suppression by intra-islet Foxp3(+) Tregs. We further show that the ICOS(+) Tregs, in contrast to their ICOS(-) counterparts, are more sensitive to IL-2, a critical signal for their survival and functional stability. Lastly, the temporal loss in ICOS(+) Tregs is readily corrected by IL-2 therapy or protective Il2 gene variation. Overall, ICOS is critical for the homeostasis and functional stability of Foxp3(+) Tregs in prediabetic islets and maintenance of T1D protection.     

Influence of Igh-C- and MHC-linked genes on the activity of immunoglobulin-recognizing T cells

BALB/c Lyt-1+ cells that proliferate in response to NP-modified BALB/c Ig fail to respond to BALB/c Ig modified by a different hapten, TNP. By contrast, when NP-modified BALB/c Ig is used as the immunogen in C.B-20 mice, a strain congenic with BALB/c but expressing the Ighb allotype of C57BL/6 (B6), a partial cross-reaction between NP- and TNP-BALB/c Ig is observed. Similarly, strains expressing different Igh haplotypes also show distinct reactivities toward NP-B6 Ig and NP-modified myeloma proteins. Thus, the proliferative response to NP-modified Ig is regulated by Igh-linked genes. In this paper, we also characterize the T cell proliferative response to IgG2a of the b allotype. We show that allotypic determinants on this molecule are recognized in association with self MHC-encoded gene products and that responsiveness is controlled by MHC-linked Ir genes. Thus, products of MHC-linked genes also influence the activity of Ig-recognizing T cells. Taken together, experiments described in this report suggest that T cells directed against immunogenic determinants on antibody molecules are governed by the same rules as T cells that respond to conventional antigens.     

Genetic control of T and B lymphocyte activation in nonobese diabetic mice.

Type 1 diabetes in nonobese diabetic (NOD) mice is characterized by the infiltration of T and B cells into pancreatic islets. T cells bearing the TCR Vbeta3 chain are disproportionately represented in the earliest stages of islet infiltration (insulitis) despite clonal deletion of most Vbeta3(+) immature thymocytes by the mammary tumor virus-3 (Mtv-3) superantigen (SAg). In this report we showed that a high frequency of NOD Vbeta3(+) T cells that escape deletion are activated in vivo and that this phenotype is linked to the Mtv-3 locus. One potential mechanism of SAg presentation to peripheral T cells is by activated B cells. Consistent with this idea, we found that NOD mice harbor a significantly higher frequency of activated B cells than nondiabetes-prone strains. These activated NOD B cells expressed cell surface molecules consistent with APC function. At the molecular level, the IgH repertoire of activated B cells in NOD mice was equivalent to resting B cells, suggesting a polyclonal response in vivo. Genetic analysis of the activated B cell phenotype showed linkage to Idd1, the NOD MHC haplotype (H-2(g7)). Finally, Vbeta3(+) thymocyte deletion and peripheral T cell activation did not require B cells, suggesting that other APC populations are sufficient to generate both Mtv-3-linked phenotypes. These data provide insight into the genetic regulation of NOD autoreactive lymphocyte activation that may contribute to failure of peripheral tolerance and the pathogenesis of type I diabetes.     

Reduced susceptibility of nonobese diabetic mice to TNF-alpha and D-galactosamine-mediated hepatocellular apoptosis and lethality

Nonobese diabetic (NOD/LtJ or NOD) mice are resistant to doses of LPS and D-galactosamine that uniformly produce lethality in C57BL/6J (B6) mice (p < 0.01). Liver caspase-3-like activity, serum transaminase levels (both p < 0.05), and the numbers of apoptotic liver nuclei were also reduced in NOD compared with B6 mice treated with LPS (100 ng) and D-galactosamine (8 mg). NOD mice were also at least 100-fold more resistant to recombinant human TNF-alpha and D-galactosamine treatment than B6 mice (p < 0.001). Binding of recombinant human TNF-alpha to splenocytes from NOD mice was similar to that seen in B6 mice, suggesting that the defect in responsiveness was not due to an inability of recombinant human TNF-alpha to bind the NOD TNF type 1 (p55) receptor. Because the TNF type 1 (p55) receptor shares a common signaling pathway with Fas (CD95), NOD and B6 mice were treated with the Fas agonist antibody, Jo-2. Surprisingly, NOD mice were as sensitive as B6 mice to Fas-induced lethality and hepatic injury. In addition, primary hepatocytes isolated from NOD mice and cultured in vitro in the presence of D-galactosamine with or without TNF-alpha were found to be resistant to apoptosis and cytotoxicity when compared with B6 mice. In contrast, Jo-2 treatment produced similar increases in caspase-3 activity and cytotoxicity in primary hepatocytes from NOD and B6 mice. The resistance to LPS- and TNF-alpha-mediated lethality and hepatic injury in D-galactosamine-sensitized NOD mice is apparently due to a post-TNFR binding defect, and independent of signaling pathways shared with Fas.     

Regulatory and effector CD4 T cells in nonobese diabetic mice recognize overlapping determinants on glutamic acid decarboxylase and use distinct V beta genes

The 524--543 region of glutamic acid decarboxylase (GAD65), GAD65(524--543), is one of the first fragments of this islet Ag to induce proliferative T cell responses in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes. Furthermore, NOD mice given tolerogenic doses of GAD65(524--543) are protected from spontaneous and cyclophosphamide-induced diabetes. In this study, we report that there are at least two I-A(g7)-restricted determinants present in the GAD65(524--543) sequence, each capable of recruiting unique T cell repertoires characterized by distinct TCR V beta gene use. CD4(+) T cells arise spontaneously in young NOD mice to an apparently dominant determinant found within the GAD65 peptide 530--543 (p530); however, T cells to the overlapping determinant 524-538 (p524) dominate the response only after immunization with GAD65(524--543). All p530-responsive T cells used the V beta 4 gene, whereas the V beta 12 gene is preferentially used to encode the TCR of p524-responsive T cell populations. T cell clones and hybridomas from both of these T cell groups were responsive to APC pulsed with GAD65(524--543) or whole rGAD65. p524-reactive cells appeared to be regulatory upon adoptive transfer into young NOD mice and could inhibit insulin-dependent diabetes mellitus development, although they were unable to produce IL-4, IL-10, or TGF beta upon antigenic challenge. Furthermore, we found that i.p. injection with p524/IFA was very effective in providing protection from cyclophosphamide-induced insulin-dependent diabetes mellitus. These data demonstrate that the regulatory T cells elicited by immunizing with GAD65(524--543) are unique and distinct from those that arise from spontaneous endogenous priming, and that T cells to this limited region of GAD65 may be either regulatory or pathogenic.     

Enhanced early expansion and maturation of semi-invariant NK T cells inhibited autoimmune pathogenesis in congenic nonobese diabetic mice

Semi-invariant NK T cell (iNKT) deficiency has long been associated with the pathogenesis of type 1 diabetes (T1D), but the linkage between this the deficiency and T1D susceptibility gene(s) remains unclear. We analyzed NOD mice subcongenic for resistant alleles of Idd9 locus in search for protective mechanisms against T1D, and found that iNKT cell development was significantly enhanced with a more advanced mature phenotype and function in mice containing Idd9.1 sublocus of B10 origin. The enhanced iNKT cell development and function suppressed effector function of diabetogenic T cells. Elimination of iNKT cells by CD1d deficiency almost abolished T1D protection in these mice. Interestingly, although the iNKT cells were responsible for a Th2 orientated cytokine profile that is often regarded as a mechanism of T1D prevention, our data suggests that the Th2 bias played little if any role for the protection. In addition, dendritic cells from the congenic NOD mice showed increased abilities to engage and potentiate iNKT cells, suggesting that a mechanism mediated by dendritic cells or other APCs may be critical for the enhanced development and maturation of iNKT cells. The products of T1D susceptibility gene(s) in Idd9.1 locus may be a key factor for this mechanism.     

Characterization of peripheral regulatory CD4+ T cells that prevent diabetes onset in nonobese diabetic mice

The period that precedes onset of insulin-dependent diabetes mellitus corresponds to an active dynamic state in which pathogenic autoreactive T cells are kept from destroying beta cells by regulatory T cells. In prediabetic nonobese diabetic (NOD) mice, CD4+ splenocytes were shown to prevent diabetes transfer in immunodeficient NOD recipients. We now demonstrate that regulatory splenocytes belong to the CD4+ CD62Lhigh T cell subset that comprises a vast majority of naive cells producing low levels of IL-2 and IFN-gamma and no IL-4 and IL-10 upon in vitro stimulation. Consistently, the inhibition of diabetes transfer was not mediated by IL-4 and IL-10. Regulatory cells homed to the pancreas and modified the migration of diabetogenic to the islets, which resulted in a decreased insulitis severity. The efficiency of CD62L+ T cells was dose dependent, independent of sex and disease prevalence. Protection mechanisms did not involve the CD62L molecule, an observation that may relate to the fact that CD4+ CD62Lhigh lymph node cells were less potent than their splenic counterparts. Regulatory T cells were detectable after weaning and persist until disease onset, sustaining the notion that diabetes is a late and abrupt event. Thus, the CD62L molecule appears as a unique marker that can discriminate diabetogenic (previously shown to be CD62L-) from regulatory T cells. The phenotypic and functional characteristics of protective CD4+ CD62L+ cells suggest they are different from Th2-, Tr1-, and NK T-type cells, reported to be implicated in the control of diabetes in NOD mice, and may represent a new immunoregulatory population.     

Insulinoma-released exosomes or microparticles are immunostimulatory and can activate autoreactive T cells spontaneously developed in nonobese diabetic mice

Exosomes (EXO) are secreted intracellular microparticles that can trigger inflammation and induce Ag-specific immune responses. To test possible roles of EXO in autoimmunity, we isolated small microparticles, mainly EXO, from mouse insulinoma and examined their activities to stimulate the autoimmune responses in NOD mice, a model for human type 1 diabetes. We demonstrate that the EXO contains strong innate stimuli and expresses candidate diabetes autoantigens. They can induce secretion of inflammatory cytokines through a MyD88-dependent pathway, and activate purified APC and result in T cell proliferation. To address whether EXO or the secreted microparticles are possible autoimmune targets causing islet-specific inflammation, we monitored the T cell responses spontaneously developed in prediabetic NOD mice for their reactivity to the EXO, and compared this reactivity between diabetes-susceptible and -resistant congenic mouse strains. We found that older NOD females, which have advanced islet destruction, accumulated more EXO-reactive, IFN-γ-producing lymphocytes than younger females or age-matched males, and that pancreatic lymph nodes from the prediabetic NOD, but not from the resistant mice, were also enriched with EXO-reactive Th1 cells. In vivo, immunization with the EXO accelerates insulitis development in nonobese diabetes-resistant mice. Thus, EXO or small microparticles can be recognized by the diabetes-associated autoreactive T cells, supporting that EXO might be a possible autoimmune target and/or insulitis trigger in NOD or congenic mouse strains.