A locus on mouse chromosome 13 inversely regulates CD1d expression and the development of invariant natural killer T-cells.

Invariant natural killer T (iNKT)-cell development is controlled by many polymorphic genes present in commonly used mouse inbred strains. Development of type 1 diabetes (T1D) in NOD mice partly results from their production of fewer iNKT-cells compared with non-autoimmune-prone control strains, including ICR. We previously identified several iNKT-cell quantitative trait genetic loci co-localized with known mouse and human T1D regions in a (NOD × ICR)F2 cross. To further dissect the mechanisms underlying the impaired iNKT-cell compartment in NOD mice, we carried out a series of bone marrow transplantation as well as additional genetic mapping studies. We found that impaired iNKT-cell development in NOD mice was mainly due to the inability of their double-positive (DP) thymocytes to efficiently select this T-cell population. Interestingly, we observed higher levels of CD1d expression by NOD than by ICR DP thymocytes. The genetic control of the inverse relationship between the CD1d expression level on DP thymocytes and the frequency of thymic iNKT-cells was further mapped to a region on chromosome 13 between 60.12 and 70.59 Mb. The NOD allele was found to promote CD1d expression and suppress iNKT-cell development. Our results indicate that genetically controlled physiological variation of CD1d expression levels modulates iNKT-cell development.

Genetic control of murine invariant natural killer T-cell development dynamically differs dependent on the examined tissue type.

Previous studies using gene-targeted mutant mice revealed several molecules important for the development or function of invariant natural killer T (iNKT) cells. However, these gene knockout mice represent cases that are rare in humans. Thus, it remains unclear how naturally occurring allelic variants of these genes or others regulate the numerical and functional diversity of iNKT cells in both mice and humans. Studies in humans are mostly limited to iNKT cells in peripheral blood (PB). It is not known if the relative distribution of iNKT cells between PB and other lymphoid organs is correlated or under common genetic control. To initially address these questions, we analyzed iNKT cells in the spleen, thymus and PB of 38 inbred mouse strains. Percentages of iNKT cells in these three anatomical sites varied significantly in a strain-dependent manner. The correlation between PB and spleen was moderate, and none was observed between PB and thymus. Similarly, proportions of the CD4-expressing subset of iNKT cells differed significantly among inbred strains. The percentages of CD4-positive iNKT cells displayed a strong correlation between PB and spleen, although it remained poor between PB and thymus. Genome-wide association studies across strains identified only partially overlapping loci associated with variability of iNKT cell frequencies within and between differing anatomical sites.

Comparative therapeutic effects of orally administered 1,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(3) on type-1 diabetes in non-obese diabetic mice fed a normal-calcaemic diet.

Frequent injections of the hormonal form of vitamin D(3), 1,25 dihydroxyvitamin D(3) (1,25D3) reportedly inhibits autoimmune type 1 diabetes (T1D) in non-obese diabetic (NOD) mice by correcting some of the abnormalities in antigen-presenting cells which contribute the development of pathogenic T cell responses. This route of administration greatly elevates the levels of these compounds in the bloodstream for hours after treatment, which requires mice to be fed diets formulated to contain much reduced levels of Ca to avoid the toxic effects of hypercalcaemia. In the current work, we demonstrate that feeding 1,25D3 or its synthetic precursor, 1alpha(OH) vitamin D(3) (1alphaD3), as part of a T1D supportive chow diet containing normal levels of Ca, is an effective means of reducing the incidence of disease in NOD mice, but the doses required for protection elicited hypercalcaemia. However, T1D protection elicited by D3 analogue feeding appears, at least partially, to have an immunological basis, as splenic T cells from treated mice had a decreased capacity to adoptively transfer disease. Protection is associated with an increased proportion of T cells with CD4+ forkhead box P3+ regulatory phenotype within the islet infiltrate of treated animals. The 1alphaD3 precursor is converted rapidly to the active 1,25D3 isoform in vivo. However, feeding the 1alphaD3 analogue elicited stronger T1D protection than the 1,25D3 compound, but also induced more severe hypercalcaemia. In future, the dietary supplementation of novel low-calcaemic D3 analogues may enable their continuous delivery at levels that inhibit T1D development in susceptible humans consuming normal levels of Ca.

Unexpected functional consequences of xenogeneic transgene expression in beta-cells of NOD mice.

We describe unexpected alterations in the non-obese diabetic (NOD/Lt) mouse model of type 1 diabetes (T1D) following forced beta-cell expression of non-mammalian genes ligated to an insulin promoter sequence. These include the jellyfish green fluorescent protein (GFP), useful for beta-cell identification, and the bacteriophage P1 Cre recombinase, necessary for beta cell-specific ablation of a gene using a Cre-loxP system. Homozygous expression of GFP, driven by the mouse insulin 1 gene promoter (MIP-GFP) in a single transgenic line of NOD mice, produced T1D in postnatal mice that was not associated with insulitis, but rather beta cell-depleted islets. Hemizygous transgene expression suppressed spontaneous autoimmune T1D in females, and produced a male glucose intolerance syndrome associated with age-dependent declines in plasma insulin content. Among lines of transgenic NOD/Lt mice expressing Cre recombinase driven by the rat insulin 2 promoter (RIP-Cre), high, non-mosaic expression correlated with suppressed T1D development. These findings emphasize the need for careful characterization of genetically manipulated NOD mouse stocks to insure that model characteristics have not been compromised.

Human clonal CD8 autoreactivity to an IGRP islet epitope shared between mice and men.

Type 1 diabetes (T1D) is a multifactorial disease characterized by the infiltration and subsequent destruction of the pancreatic insulin-producing beta cells by autoreactive T cells. CD8(+) T cells play an essential role in this beta cell destruction. However, little is known about the target antigens of CD8(+) T cells in human T1D patients. The aim of this study was to assess whether an epitope derived from the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), IGRP(265-273,) which has recently been identified as a target in non-obese diabetic (NOD) mice and is fully homologous to the human epitope, is a target of human diabetogenic CD8(+) T cells. We isolated a human CD8 T cell clone against this epitope, which confirms that this IGRP epitope is shared across species.

Conditioning the genome identifies additional diabetes resistance loci in Type I diabetes resistant NOR/Lt mice.

While sharing the H2g7 MHC and many other important Type I diabetes susceptibility (Idd) genes with NOD mice, the NOR strain remains disease free due to resistance alleles within the approximately 12% portion of their genome that is of C57BLKS/J origin. Previous F2 segregation analyses indicated multiple genes within the 'Idd13' locus on Chromosome 2 provide the primary component of NOR diabetes resistance. However, it was clear other genes also contribute to NOR diabetes resistance, but were difficult to detect in the original segregation analyses because they were relatively weak compared to the strong Idd13 protection component. To identify these further genetic components of diabetes resistance, we performed a new F2 segregation analyses in which NOD mice were outcrossed to a 'genome-conditioned' NOR stock in which a large component of Idd13-mediated resistance was replaced with NOD alleles. These F2 segregation studies combined with subsequent congenic analyses confirmed the presence of additional NOR resistance genes on Chr. 1 and Chr. 4, and also potentially on Chr. 11. These findings emphasize the value for diabetes gene discovery of stratifying not only MHC loci conferring the highest relative risk but also as many as possible of the non-MHC loci presumed to contribute significantly.

Transgenic rescue implicates beta2-microglobulin as a diabetes susceptibility gene in nonobese diabetic (NOD) mice.

Type 1 diabetes in both humans and nonobese diabetic (NOD) mice results from T-cell-mediated autoimmune destruction of insulin-producing pancreatic beta cells. Linkage studies have shown that type 1 diabetes in NOD mice is a polygenic disease involving more than 15 chromosomal susceptibility regions. Despite extensive investigation, the identification of individual susceptibility genes either within or outside the major histocompatibility complex region has proven problematic because of the limitations of linkage analysis. In this paper, we provide evidence implicating a single diabetes susceptibility gene, which lies outside the major histocompatibility complex region. Using allelic reconstitution by transgenic rescue, we show that NOD mice expressing the beta(2) microglobulin (beta(2)M)(a) allele develop diabetes, whereas NOD mice expressing a murine beta(2)M(b) or human allele are protected. The murine beta(2)M(a) allele differs from the beta(2)M(b) allele only at a single amino acid. Mechanistic studies indicate that the absence of the NOD beta(2)M(a) isoform on nonhematopoietic cells inhibits the development or activation of diabetogenic T cells.

Autoreactive diabetogenic T-cells in NOD mice can efficiently expand from a greatly reduced precursor pool.

A broad repertoire of pancreatic beta-cell autoreactive T-cells normally contributes to the development of type 1 diabetes in NOD mice. However, it has been unknown if a large reduction in the precursor pool from which autoreactive T-cells are drawn would inhibit the development of type 1 diabetes. To address this issue, we reduced the precursor frequency of autoreactive T-cells in NOD mice through allelic exclusion induced by transgenic expression of an H2-Db class I-restricted T-cell receptor (TCR) specific for a pathologically irrelevant lymphocytic choriomeningitis virus (LCMV) peptide. TCR allelic exclusion greatly reduced the pool of T-cells from which diabetogenic effectors could be derived in these NODxLCMV TCR Tg mice. Surprisingly, this did not impair their type 1 diabetes susceptibility. Furthermore, a diabetogenic CD8 T-cell population that is prevalent in standard NOD mice was present at essentially equivalent levels in pancreatic islets of NODxLCMV TCR Tg mice. Other data indicated that the antigenic specificity of these CD8 T-cells is primarily the function of a shared TCR-alpha chain. Although the percentage of TCR transgenic T-cells decreased in NOD versus B6,D2 control mice, much higher total numbers of both the TCR transgenic and the nontransgenic T-cells accumulated in the NOD strain. This transgenic T-cell accumulation in the absence of the cognate peptide indicated that the NOD genetic background preferentially promotes a highly efficient antigen-independent T-cell expansion. This might allow diabetogenic T-cells in NOD mice to undergo an efficient expansion before encountering antigen, which would represent an important and previously unconsidered aspect of pathogenesis.

Inhibition of autoimmune diabetes in nonobese diabetic mice by transgenic restoration of H2-E MHC class II expression: additive, but unequal, involvement of multiple APC subtypes.

Transgenic restoration of normally absent H2-E MHC class II molecules on APC dominantly inhibits T cell-mediated autoimmune diabetes (IDDM) in nonobese diabetic (NOD) mice. We analyzed the minimal requirements for transgenic H2-E expression on APC subtypes (B lymphocytes vs macrophages/dendritic cells (DC)) to inhibit IDDM. This issue was addressed through the use of NOD stocks transgenically expressing high levels of H2-E and/or made genetically deficient in B lymphocytes in a series of genetic intercross and bone marrow/lymphocyte chimera experiments. Standard (H2-E(null)) NOD B lymphocytes exert a pathogenic function(s) necessary for IDDM. However, IDDM was inhibited in mixed chimeras where H2-E was solely expressed on all B lymphocytes. Interestingly, this resistance was abrogated when even a minority of standard NOD H2-E(null) B lymphocytes were also present. In contrast, in NOD chimeras where H2-E expression was solely limited to approximately half the macrophages/DC, an active immunoregulatory process was induced that inhibited IDDM. Introduction of a disrupted IL-4 gene into the NOD-H2-E transgenic stock demonstrated that induction of this Th2 cytokine does not represent the IDDM protective immunoregulatory process mediated by H2-E expression. In conclusion, high numbers of multiple subtypes of APC must express H2-E MHC class II molecules to additively inhibit IDDM in NOD mice. This raises a high threshold for success in future intervention protocols designed to inhibit IDDM by introduction of putatively protective MHC molecules into hemopoietic precursors of APC.

A glutamic acid decarboxylase 65-specific Th2 cell clone immunoregulates autoimmune diabetes in nonobese diabetic mice.

Several studies have provided indirect evidence in support of a role for beta cell-specific Th2 cells in regulating insulin-dependent diabetes (IDDM). Whether a homogeneous population of Th2 cells having a defined beta cell Ag specificity can prevent or suppress autoimmune diabetes is still unclear. In fact, recent studies have demonstrated that beta cell-specific Th2 cell clones can induce IDDM. In this study we have established Th cell clones specific for glutamic acid decarboxylase 65 (GAD65), a known beta cell autoantigen, from young unimmunized nonobese diabetic (NOD) mice. Adoptive transfer of a GAD65-specific Th2 cell clone (characterized by the secretion of IL-4, IL-5, and IL-10, but not IFN-gamma or TGF-beta) into 2- or 12-wk-old NOD female recipients prevented the progression of insulitis and subsequent development of overt IDDM. This prevention was marked by the establishment of a Th2-like cytokine profile in response to a panel of beta cell autoantigens in cultures established from the spleen and pancreatic lymph nodes of recipient mice. The immunoregulatory function of a given Th cell clone was dependent on the relative levels of IFN-gamma vs IL-4 and IL-10 secreted. These results provide direct evidence that beta cell-specific Th2 cells can indeed prevent and suppress autoimmune diabetes in NOD mice.

Development and function of diabetogenic T-cells in B-cell-deficient nonobese diabetic mice.

Insulin-dependent diabetes (type 1 diabetes) in the NOD mouse is a T-cell-mediated autoimmune disease. However, B-cells may also play a critical role in disease pathogenesis, as genetically B-cell-deficient NOD mice (NOD.microMT) have been shown to be protected from type 1 diabetes and to display reduced responses to certain islet autoantigens. To examine the requirements for B-cells in the development of type 1 diabetes, we generated a B-cell-naive T-cell repertoire by transplantation of NOD fetal thymuses (FTs) into NOD.scid recipients. Surprisingly, these FT-derived NOD T-cells were diabetogenic in 36% of NOD.scid recipients, despite the absence of B-cells. In addition, T-cells isolated from NOD.microMT mice were diabetogenic in 22% of NOD.scid recipients. Together, these results indicate that B-cells are not an absolute requirement for the generation or effector function of an islet-reactive T-cell repertoire in NOD mice. We suggest that conditions favoring rapid lymphocyte expansion can reveal autoreactive T-cell activity and precipitate disease in genetically susceptible individuals.

Antigen-specific mediated suppression of beta cell autoimmunity by plasmid DNA vaccination.

In this study, we have investigated the use of plasmid DNA (pDNA) vaccination to elicit Th2 effector cell function in an Ag-specific manner and in turn prevent insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. pDNA recombinants were engineered encoding a secreted fusion protein consisting of a fragment of glutamic acid decarboxylase 65 (GAD65) linked to IgGFc, and IL-4. Intramuscular injection of pDNA encoding GAD65-IgGFc and IL-4 effectively prevented diabetes in NOD mice treated at early or late preclinical stages of IDDM. This protection was GAD65-specific since NOD mice immunized with pDNA encoding hen egg lysozyme-IgGFc and IL-4 continued to develop diabetes. Furthermore, disease prevention correlated with suppression of insulitis and induction of GAD65-specific regulatory Th2 cells. Importantly, GAD65-specific immune deviation was dependent on pDNA-encoded IL-4. In fact, GAD65-specific Th1 cell reactivity was significantly enhanced in animals immunized with pDNA encoding only GAD65-IgGFc. Finally, NOD.IL4(null) mice treated with pDNA encoding GAD65-IgGFc and IL-4 continued to develop diabetes, indicating that endogenous IL-4 was also required for disease prevention. These results demonstrate that pDNA vaccination is an effective strategy to elicit beta cell-specific Th2 regulatory cell function for the purpose of preventing IDDM even at a late stage of disease development.

Th1 to Th2 cytokine shifts in nonobese diabetic mice: sometimes an outcome, rather than the cause, of diabetes resistance elicited by immunostimulation.

Numerous immunostimulatory protocols inhibit the development of T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse model. Many of these protocols, including treatment with the nonspecific immunostimulatory agents CFA or bacillus Calmette-Guérin (BCG) vaccine, have been reported to mediate protection by skewing the pattern of cytokines produced by pancreatic beta-cell autoreactive T cells from a Th1 (IFN-gamma) to a Th2 (IL-4 and IL-10) profile. However, most of these studies have documented associations between such cytokine shifts and disease protection rather than a cause/effect relationship. To partially address this issue we produced NOD mice genetically deficient in IFN-gamma, IL-4, or IL-10. Elimination of any of these cytokines did not significantly alter the rate of spontaneous IDDM development. Additional experiments using these mice confirmed that CFA- or BCG-elicited diabetes protection is associated with a decreased IFN-gamma to IL-4 mRNA ratio within T cell-infiltrated pancreatic islets, but this is a secondary consequence rather than the cause of disease resistance. Unexpectedly, we also found that the ability of BCG and, to a lesser extent, CFA to inhibit IDDM development in standard NOD mice is actually dependent upon the presence of the Th1 cytokine, IFN-gamma. Collectively, our studies demonstrate that while Th1 and Th2 cytokine shifts may occur among beta-cell autoreactive T cells of NOD mice protected from overt IDDM by various immunomodulatory therapies, it cannot automatically be assumed that this is the cause of their disease resistance.

Unusual resistance of ALR/Lt mouse beta cells to autoimmune destruction: role for beta cell-expressed resistance determinants.

Genetic analysis of autoimmune insulin-dependent diabetes mellitus (IDDM) has focused on genes controlling immune functions, with little investigation of innate susceptibility determinants expressed at the level of target beta cells. The Alloxan (AL) Resistant (R) Leiter (Lt) mouse strain, closely related to the IDDM-prone nonobese diabetic (NOD)/Lt strain, demonstrates the importance of such determinants. ALR mice are unusual in their high constitutive expression of molecules associated with dissipation of free-radical stress systemically and at the beta-cell level. ALR islets were found to be remarkably resistant to two different combinations of beta-cytotoxic cytokines (IL-1beta, tumor necrosis factor alpha, and IFN-gamma) that destroyed islets from the related NOD and alloxan-susceptible strains. The close MHC relatedness between the NOD and ALR strains (H2-Kd and H2-Ag7 identical) allowed us to examine whether ALR islet cells could survive autoimmune destruction by NOD-derived Kd-restricted diabetogenic cytotoxic T lymphocyte clones (AI4 and the insulin-reactive G9C8 clones). Both clones killed islet cells from all Kd-expressing strains except ALR. ALR resistance to diabetogenic immune systems was determined in vivo by means of adoptive transfer of the G9C8 clone or by chimerizing lethally irradiated ALR or reciprocal (ALR x NOD)F1 recipients with NOD bone marrow. In all in vivo systems, ALR and F1 female recipients of NOD marrow remained IDDM free; in contrast, all of the NOD recipients became diabetic. In conclusion, the ALR mouse presents a unique opportunity to identify dominant IDDM resistance determinants expressed at the beta cell level.

Interferon-gamma receptor signaling is dispensable in the development of autoimmune type 1 diabetes in NOD mice.

There have been two previous conflicting reports that the development of T-cell-mediated autoimmune diabetes (type 1 diabetes) was respectively unaffected or inhibited in NOD mice genetically deficient in the T-helper (Th) 1 cytokine interferon (IFN)-gamma or the alpha-chain subunit of its receptor. Our goal was to resolve this conundrum by congenically transferring, from a 129 donor strain to the NOD background, a functionally inactivated gene for the beta-chain signaling (located on chromosome 16) rather than the alpha-chain ligand binding domain (located on chromosome 10) of the IFN-gamma receptor. These NOD.IFNgammaRBnull mice were characterized by normal patterns of leukocyte development and T-cells that produced greatly enhanced levels of the putatively type 1 diabetes-protective Th2 cytokine interleukin (IL)-4. However, despite being unable to respond to the primary Thl cytokine IFN-gamma and having T-cells that produce greatly enhanced levels of IL-4, NOD.IFNgammaRBnull mice remained highly susceptible to type 1 diabetes. This result indicated that the previously reported inhibition of type 1 diabetes in NOD mice carrying a functionally inactivated IFN-gamma receptor alpha-chain gene may have been due to a closely linked and previously unidentified diabetes resistance allele. Furthermore, our results indicate that the pathogenicity of diabetogenic T-cells in NOD mice is not dampened by an inability to respond to IFN-gamma and a concurrent shift to greatly enhanced Th2 cytokine production. This finding calls into question whether clinical protocols designed to shift beta-cell autoreactive T-cells from a Thl to Th2 cytokine production profile will truly be safe and efficacious in blocking the development of type 1 diabetes in humans.

Acceleration of type 1 diabetes by a coxsackievirus infection requires a preexisting critical mass of autoreactive T-cells in pancreatic islets.

Coxsackievirus infections have been proposed as an environmental trigger for the development of T-cell-mediated autoimmune (type 1) diabetes by either providing a molecular mimic of the candidate pancreatic beta-cell autoantigen GAD or inducing bystander inflammation in the pancreas. In this study in the NOD mouse model, we found that infection with a pancreatrophic coxsackievirus isolate can accelerate type 1 diabetes development through the induction of a bystander activation effect, but only after a critical threshold level of insulitic beta-cell-autoreactive T-cells has accumulated. Thus, coxsackievirus infections do not appear to initiate beta-cell autoreactive immunity but can accelerate the process once it is underway. These findings indicate that the timing of a coxsackievirus infection, rather than its simple presence or absence, may have important etiological implications for the development of T-cell-mediated autoimmune type 1 diabetes in humans.

Identification of a CD8 T cell that can independently mediate autoimmune diabetes development in the complete absence of CD4 T cell helper functions.

Previous work has indicated that an important component for the initiation of autoimmune insulin-dependent diabetes mellitus (IDDM) in the NOD mouse model entails MHC class I-restricted CD8 T cell responses against pancreatic beta cell Ags. However, unless previously activated in vitro, such CD8 T cells have previously been thought to require helper functions provided by MHC class II-restricted CD4 T cells to exert their full diabetogenic effects. In this study, we show that IDDM development is greatly accelerated in a stock of NOD mice expressing TCR transgenes derived from a MHC class I-restricted CD8 T cell clone (designated AI4) previously found to contribute to the earliest preclinical stages of pancreatic beta cell destruction. Importantly, these TCR transgenic NOD mice (designated NOD.AI4alphabeta Tg) continued to develop IDDM at a greatly accelerated rate when residual CD4 helper T cells were eliminated by introduction of the scid mutation or a functionally inactivated CD4 allele. In a previously described stock of NOD mice expressing TCR transgenes derived from another MHC class I-restricted beta cell autoreactive T cell clone, IDDM development was retarded by elimination of residual CD4 T cells. Hence, there is variability in the helper dependence of CD8 T cells contributing to the development of autoimmune IDDM. The AI4 clonotype represents the first CD8 T cell with a demonstrated ability to progress from a naive to functionally activated state and rapidly mediate autoimmune IDDM development in the complete absence of CD4 T cell helper functions.

Reevaluation of the major histocompatibility complex genes of the NOD-progenitor CTS/Shi strain.

The common Kd and/or Db alleles of NOD mice contribute to the development of autoimmune diabetes, but their respective contributions are unresolved. The major histocompatibility complex (MHC) of the CTS/Shi mouse, originally designated as H2ct, shares MHC class II region identity with the H2g7 haplotype of NOD mice. However, CTS mice were reported to express distinct but undefined MHC class I gene products. Because diabetes frequency was reduced 56% in females of a NOD stock congenic for H2ct, this partial resistance may have derived from the MHC class I allelic differences. In the present report, we use a combination of serologic analysis and sequencing of MHC class I cDNAs to establish that NOD/Lt and CTS/Shi share a common H2-Kd allele but differ at the H2-D end of the MHC complex. The H2-D allele of CTS/Shi was identified as the rare H2-Ddx recently described in ALR/Lt, another NOD-related strain. These results in mouse model systems show that multiple MHC genes confer diabetes resistance and suggest that at least one of the protective MHC or MHC-linked genes in CTS mice may be at the H2-D end of the complex.

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.