The diabetes susceptibility locus Idd5.1 on mouse chromosome 1 regulates ICOS expression and modulates murine experimental autoimmune encephalomyelitis

Linkage analysis and congenic mapping in NOD mice have identified a susceptibility locus for type 1 diabetes, Idd5.1 on mouse chromosome 1, which includes the Ctla4 and Icos genes. Besides type 1 diabetes, numerous autoimmune diseases have been mapped to a syntenic region on human chromosome 2q33. In this study we determined how the costimulatory molecules encoded by these genes contribute to the immunopathogenesis of experimental autoimmune encephalomyelitis (EAE). When we compared levels of expression of costimulatory molecules on T cells, we found higher ICOS and lower full-length CTLA-4 expression on activated NOD T cells compared with C57BL/6 (B6) and C57BL/10 (B10) T cells. Using NOD.B10 Idd5 congenic strains, we determined that a 2.1-Mb region controls the observed expression differences of ICOS. Although Idd5.1 congenic mice are resistant to diabetes, we found them more susceptible to myelin oligodendrocyte glycoprotein 35-55-induced EAE compared with NOD mice. Our data demonstrate that higher ICOS expression correlates with more IL-10 production by NOD-derived T cells, and this may be responsible for the less severe EAE in NOD mice compared with Idd5.1 congenic mice. Paradoxically, alleles at the Idd5.1 locus have opposite effects on two autoimmune diseases, diabetes and EAE. This may reflect differential roles for costimulatory pathways in inducing autoimmune responses depending upon the origin (tissue) of the target Ag.     

Congenic mice reveal genetic epistasis and overlapping disease loci for autoimmune diabetes and listeriosis

The nonobese diabetic (NOD) mouse strain serves as a genomic standard for assessing how allelic variation for insulin-dependent diabetes (Idd) loci affects the development of autoimmune diabetes. We previously demonstrated that C57BL/6 (B6) mice harbor a more diabetogenic allele than NOD mice for the Idd14 locus when introduced onto the NOD genetic background. New congenic NOD mouse strains, harboring smaller B6-derived intervals on chromosome 13, now localize Idd14 to an ~18-Mb interval and reveal a new locus, Idd31. Notably, the B6 allele for Idd31 confers protection against diabetes, but only in the absence of the diabetogenic B6 allele for Idd14, indicating genetic epistasis between these two loci. Moreover, congenic mice that are more susceptible to diabetes are more resistant to Listeria monocytogenes infection. This result co-localizes Idd14 and Listr2, a resistance locus for listeriosis, to the same genomic interval and indicates that congenic NOD mice may also be useful for localizing resistance loci for infectious disease.     

Molecular genetic analysis of the Idd4 locus implicates the IFN response in type 1 diabetes susceptibility in nonobese diabetic mice

High-resolution mapping and identification of the genes responsible for type 1 diabetes (T1D) has proved difficult because of the multigenic etiology and low penetrance of the disease phenotype in linkage studies. Mouse congenic strains have been useful in refining Idd susceptibility loci in the NOD mouse model and providing a framework for identification of genes underlying complex autoimmune syndromes. Previously, we used NOD and a nonobese diabetes-resistant strain to map the susceptibility to T1D to the Idd4 locus on chromosome 11. Here, we report high-resolution mapping of this locus to 1.4 megabases. The NOD Idd4 locus was fully sequenced, permitting a detailed comparison with C57BL/6 and DBA/2J strains, the progenitors of T1D resistance alleles found in the nonobese diabetes-resistant strain. Gene expression arrays and quantitative real-time PCR were used to prioritize Idd4 candidate genes by comparing macrophages/dendritic cells from congenic strains where allelic variation was confined to the Idd4 interval. The differentially expressed genes either were mapped to Idd4 or were components of the IFN response pathway regulated in trans by Idd4. Reflecting central roles of Idd4 genes in Ag presentation, arachidonic acid metabolism and inflammation, phagocytosis, and lymphocyte trafficking, our combined analyses identified Alox15, Alox12e, Psmb6, Pld2, and Cxcl16 as excellent candidate genes for the effects of the Idd4 locus.     

Evidence for Cd101 but not Fcgr1 as candidate for type 1 diabetes locus, Idd10

Among polygenes conferring susceptibility to type 1 diabetes in the NOD mouse, Idd10 on distal chromosome 3 has been shown to be important for disease susceptibility. In this study, we investigated the candidacy of Fcgr1 and Cd101 for Idd10, by congenic mapping and candidate gene sequencing. Among seven NOD-related strains studied, the IIS mouse was found to possess a recombinant Idd10 interval with the same sequence at Fcgr1 as the NOD mouse, but a different sequence at Cd101 from that in the NOD mouse with 10 amino acid substitutions. The frequency of type 1 diabetes in NOD mice congenic for IIS Idd10 (NOD.IISIdd10) was significantly reduced as compared to that in the NOD mouse, despite the presence of the identical Fcgr1 sequence. These data indicate that IIS mice possess a resistant allele at Idd10, and suggest that Cd101, but not Fcgr1, is responsible for the Idd10 effect.     

Impact of protective IL-2 allelic variants on CD4+ Foxp3+ regulatory T cell function in situ and resistance to autoimmune diabetes in NOD mice

Type I diabetes (T1D) susceptibility is inherited through multiple insulin-dependent diabetes (Idd) genes. NOD.B6 Idd3 congenic mice, introgressed with an Idd3 allele from T1D-resistant C57BL/6 mice (Idd3(B6)), show a marked resistance to T1D compared with control NOD mice. The protective function of the Idd3 locus is confined to the Il2 gene, whose expression is critical for naturally occurring CD4(+)Foxp3(+) regulatory T (nT(reg)) cell development and function. In this study, we asked whether Idd3(B6) protective alleles in the NOD mouse model confer T1D resistance by promoting the cellular frequency, function, or homeostasis of nT(reg) cells in vivo. We show that resistance to T1D in NOD.B6 Idd3 congenic mice correlates with increased levels of IL-2 mRNA and protein production in Ag-activated diabetogenic CD4(+) T cells. We also observe that protective IL2 allelic variants (Idd3(B6) resistance allele) also favor the expansion and suppressive functions of CD4(+)Foxp3(+) nT(reg) cells in vitro, as well as restrain the proliferation, IL-17 production, and pathogenicity of diabetogenic CD4(+) T cells in vivo more efficiently than control do nT(reg) cells. Lastly, the resistance to T1D in Idd3 congenic mice does not correlate with an augmented systemic frequency of CD4(+)Foxp3(+) nT(reg) cells but more so with the ability of protective IL2 allelic variants to promote the expansion of CD4(+)Foxp3(+) nT(reg) cells directly in the target organ undergoing autoimmune attack. Thus, protective, IL2 allelic variants impinge the development of organ-specific autoimmunity by bolstering the IL-2 producing capacity of self-reactive CD4(+) T cells and, in turn, favor the function and homeostasis of CD4(+)Foxp3(+) nT(reg) cells in vivo.     

Genome-wide microarray expression analysis of CD4+ T Cells from nonobese diabetic congenic mice identifies Cd55 (Daf1) and Acadl as candidate genes for type 1 diabetes

NOD.Idd3/5 congenic mice have insulin-dependent diabetes (Idd) regions on chromosomes 1 (Idd5) and 3 (Idd3) derived from the nondiabetic strains B10 and B6, respectively. NOD.Idd3/5 mice are almost completely protected from type 1 diabetes (T1D) but the genes within Idd3 and Idd5 responsible for the disease-altering phenotype have been only partially characterized. To test the hypothesis that candidate Idd genes can be identified by differential gene expression between activated CD4+ T cells from the diabetes-susceptible NOD strain and the diabetes-resistant NOD.Idd3/5 congenic strain, genome-wide microarray expression analysis was performed using an empirical Bayes method. Remarkably, 16 of the 20 most differentially expressed genes were located in the introgressed regions on chromosomes 1 and 3, validating our initial hypothesis. The two genes with the greatest differential RNA expression on chromosome 1 were those encoding decay-accelerating factor (DAF, also known as CD55) and acyl-coenzyme A dehydrogenase, long chain, which are located in the Idd5.4 and Idd5.3 regions, respectively. Neither gene has been implicated previously in the pathogenesis of T1D. In the case of DAF, differential expression of mRNA was extended to the protein level; NOD CD4+ T cells expressed higher levels of cell surface DAF compared with NOD.Idd3/5 CD4+ T cells following activation with anti-CD3 and -CD28. DAF up-regulation was IL-4 dependent and blocked under Th1 conditions. These results validate the approach of using congenic mice together with genome-wide analysis of tissue-specific gene expression to identify novel candidate genes in T1D.     

Localization of Idd11 using NOD congenic mouse strains: elimination of Slc9a1 as a candidate gene

 Type 1 diabetes is a multigenic autoimmune disease, the genetic basis for which is perhaps best characterized in the nonobese diabetic (NOD) mouse model. We previously located a NOD diabetes susceptibility locus, designated Idd11, on mouse Chromosome (Chr) 4 by analyzing diabetic backcross mice produced after crossing NOD/Lt with the nondiabetic resistant strain C57BL/6 (B6) strain. In order to confirm Idd11 and further refine its location, three NOD congenic mouse strains with different B6 derived intervals within Chr 4 were generated. Two of the congenic strains had a significant decrease in the cumulative incidence of diabetes compared with NOD/Lt control mice. The third NOD congenic strain, containing a B6 interval surrounding the Slc9a1 locus, was not protected against diabetes. These results define a new distal boundary for Idd11 and eliminate the Slc9a1 gene as a candidate. The Idd11 locus has now been definitively mapped to a 13cM interval on mouse Chr 4. Received: 15 May 1999 / Revised: 25 September 1999     

Increased Autoimmune Diabetes in pIgR-Deficient NOD Mice Is Due to a "Hitchhiking" Interval that Refines the Genetic Effect of Idd5.4

Selective breeding to introduce a gene mutation from one mouse strain onto the genetic background of another strain invariably produces “hitchhiking” (i.e. flanking) genomic intervals, which may independently affect a disease trait of interest. To investigate a role for the polymeric Ig receptor in autoimmune diabetes, a congenic nonobese diabetic (NOD) mouse strain was generated that harbors a Pigr null allele derived from C57BL/6 (B6) mice. These pIgR-deficient NOD mice exhibited increased serum IgA along with an increased diabetes incidence. However, the Pigr null allele was encompassed by a relatively large “hitchhiking” genomic interval that was derived from B6 mice and overlaps Idd5.4, a susceptibility locus for autoimmune diabetes. Additional congenic NOD mouse strains, harboring smaller B6-derived intervals, confirmed Idd5.4 independently of the other three known susceptibility loci on chromosome 1, and further localized Idd5.4 to an interval proximal to Pigr. Moreover, these congenic NOD mice showed that B6 mice harbor a more diabetogenic allele than NOD mice for this locus. The smallest B6-derived interval encompassing the Pigr null allele may, however, confer a small degree of protection against diabetes, but this protection appears to be dependent on the absence of the diabetogenic B6 allele for Idd5.4. This study provides another example of the potential hidden effects of “hitchhiking" genomic intervals and how such intervals can be used to localize disease susceptibility loci.     

Influence of a non-NK complex region of chromosome 6 on CD4+ invariant NK T cell homeostasis

The number and function of immunoregulatory invariant NKT (iNKT) cells are genetically controlled. A defect of iNKT cell ontogeny and function has been implicated as one causal factor of NOD mouse susceptibility to type 1 diabetes. Other factors of diabetes susceptibility, such as a decrease of regulatory T cell function or an increase in TLR1 expression, are corrected in diabetes-resistant Idd6 NOD.C3H 6.VIII congenic mice. Thus, we surmised that the iNKT cell defects found in NOD mice may also be rescued in congenic mice. Unexpectedly, we found, in both the thymus and the periphery, a 50% reduction in iNKT cell number in NOD.C3H 6.VIII mice as compared with NOD mice. This reduction only affected CD4(+) iNKT cells, and left the double negative iNKT cells unchanged. In parallel, the production of IL-4 and IFN-gamma following alpha-GalCer stimulation was proportionally reduced. Using three subcongenic strains, we have narrowed down the region controlling iNKT development within Idd6 (5.8 Mb) to Idd6.2 region (2.5 Mb). Idd6 region had no effect on NK cell number and in vivo cytotoxic activity. These results indicate that the role of iNKT cells in diabetes development is equivocal and more complex than initially considered. In addition, they bring strong evidence that the regulation of CD4(+) iNKT cell production is independent from that of DN iNKT cells, and involves genes of the Idd6 locus.     

Genetic dissection of V alpha 14J alpha 18 natural T cell number and function in autoimmune-prone mice

Nonobese diabetic (NOD) mice, a model for type I diabetes (TID), have reduced numbers of invariant V alpha 14J alpha 18 TCR alpha-chain-positive natural T (iNKT) cells that do not release IL-4 in response to in vivo activation through their Ag receptor. The deficit in iNKT cell number and function is implicated in immune dysregulation and the etiology of TID. Therefore, we reasoned that the genetic determinant(s) that controls iNKT cell number and function might lie within Idd (insulin-dependent diabetes susceptibility locus) regions, which are known to contain TID resistance or susceptibility genes. A systematic analysis of iNKT cell number and function in Idd congenic mice revealed that neither iNKT cell number nor their inability to rapidly secrete IL-4 in response to acute in vivo activation by Ag underlies the mechanism of protection from diabetes in Idd congenic mice. Moreover, the regulation of iNKT cell number and function appears to be under the control of several genes. The most notable of these map to the Idd4, Idd5, Idd9.1, and Idd13 regions of the mouse genome. Together these findings provide a clue to the genetic mechanism(s) underlying iNKT cell deficiency in NOD mice.     

Three loci on mouse chromosome 6 influence onset and final incidence of type I diabetes in NOD.C3H congenic strains

The development of insulin-dependent diabetes mellitus in both human and mouse is dependent on the interaction between genetic and environmental factors. The analysis of newly created NOD.C3H congenic strains for spontaneous and cyclophosphamide-induced diabetes has allowed the definition of three controlling genetic loci on mouse chromosome 6. A NOD-derived susceptibility allele at the Idd6 locus strongly influences the onset of diabetes in spontaneous diabetes. A NOD-derived resistance allele at the Idd19 locus affects the final diabetes incidence observed in both models, while a novel locus, provisionally termed Idd20, appears to control Idd19 in an epistatic manner. Decreased diabetes incidence is observed in CY-induced diabetes when Idd20 is homozygous for the C3H allele, while heterozygosity is associated with an increase in diabetes incidence. The Idd20, Idd19, and Idd6 candidate regions fall respectively within genetically defined intervals of 4, 7, and 4.5 cM on mouse chromosome 6. From our YAC contig, Idd6 would appear to localize within a ca. 1.5-Mb region on distal chromosome 6.     

Genetic control of anti-Sm autoantibody production in NOD congenic mice narrowed to the Idd9.3 region

Anti-Smith (anti-Sm) autoantibodies are directed to proteins in the small-nuclear ribonucleoprotein (snRNP) family and are considered specific for systemic lupus erythematosus (SLE) in both humans and mice. We previously established that NOD.c3c4 mice, carrying B6 and B10 congenic segments from chromosomes 3 to 4 on an nonobese diabetic (NOD) background, and NOD.Idd9R28 mice, carrying a B10 segment on c4 alone, developed significant penetrance of anti-Sm antibody production. Here we determine autoantibody incidence in additional NOD.Idd9 congenic strains and use a congenic mapping approach to narrow the interval necessary for enhanced autoantibody production to a ∼5.6-Mb region containing insulin-dependent diabetes (Idd)9.3. The Idd9.3 interval contains the candidate molecule cluster of differentiation (CD)137, which is a member of the tumor necrosis factor (TNF) receptor superfamily, functions as an inducible costimulator of T cells, and controls T–B interactions. The NOD and B10 CD137 alleles have sequence polymorphisms and different functional effects on T cells; the NOD CD137 allele mediates weaker T cell proliferative responses and decreased interleukin (IL)-2 production after CD137-mediated costimulation. Our work establishes CD137 as a candidate gene for control of autoantibody production in NOD.Idd9.3 congenic mice.     

Fine mapping, gene content, comparative sequencing, and expression analyses support Ctla4 and Nramp1 as candidates for Idd5.1 and Idd5.2 in the nonobese diabetic mouse

At least two loci that determine susceptibility to type 1 diabetes in the NOD mouse have been mapped to chromosome 1, Idd5.1 (insulin-dependent diabetes 5.1) and Idd5.2. In this study, using a series of novel NOD.B10 congenic strains, Idd5.1 has been defined to a 2.1-Mb region containing only four genes, Ctla4, Icos, Als2cr19, and Nrp2 (neuropilin-2), thereby excluding a major candidate gene, Cd28. Genomic sequence comparison of the two functional candidate genes, Ctla4 and Icos, from the B6 (resistant at Idd5.1) and the NOD (susceptible at Idd5.1) strains revealed 62 single nucleotide polymorphisms (SNPs), only two of which were in coding regions. One of these coding SNPs, base 77 of Ctla4 exon 2, is a synonymous SNP and has been correlated previously with type 1 diabetes susceptibility and differential expression of a CTLA-4 isoform. Additional expression studies in this work support the hypothesis that this SNP in exon 2 is the genetic variation causing the biological effects of Idd5.1. Analysis of additional congenic strains has also localized Idd5.2 to a small region (1.52 Mb) of chromosome 1, but in contrast to the Idd5.1 interval, Idd5.2 contains at least 45 genes. Notably, the Idd5.2 region still includes the functionally polymorphic Nramp1 gene. Future experiments to test the identity of Idd5.1 and Idd5.2 as Ctla4 and Nramp1, respectively, can now be justified using approaches to specifically alter or mimic the candidate causative SNPs.     

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.     

Subcongenic analysis of genetic basis for impaired development of invariant NKT cells in NOD mice

Reduced numbers and function of invariant NKT (iNKT) cells partially contribute to type 1 diabetes (T1D) development in NOD mice. Previous linkage analysis identified a genetic locus on chromosome 2 controlling numbers of thymic iNKT cells. Interestingly, this locus resides within the Idd13 region that distinguishes NOD mice from the closely genetically related, but strongly T1D-resistant NOR strain. Thus, we tested if a genetic variant that confers T1D resistance in NOR mice may do so by enhancing iNKT cell numbers. iNKT cells were enumerated by an α-GalCer analog loaded CD1d tetramer in NOD and NOR mice as well as in NOD stocks carrying NOR-derived congenic regions on chromosome 1, 2, or 4. Significantly, more thymic and splenic iNKT cells were present in NOR than NOD mice. The NOR-derived Idd13 region on chromosome 2 contributed the most significant effect on increasing iNKT cell numbers. Subcongenic analyses indicated that at least two genes within the Idd13 region regulate iNKT cell numbers. These results further define the genetic basis for numerical iNKT cell defects contributing to T1D development in NOD mice.     

Protection against diabetes and improved NK/NKT cell performance in NOD.NK1.1 mice congenic at the NK complex

The NK1.1 cell surface receptor, which belongs to the NKR-P1 gene cluster, has been bred onto nonobese diabetic (NOD) mice for two purposes. The first was to tag NK and NKT cells for easier experimental identification of those subsets and better analysis of their implication in type 1 diabetes. The second was to produce a congenic strain carrying Idd6, a susceptibility locus that has been repeatedly mapped in the vicinity of the NKR-P1 gene cluster and the NK complex, to explore the impact of this locus upon autoimmune diabetes. NOD.NK1.1 mice express the NK1.1 marker selectively on the surface of their NK and NKT cell subsets. In addition, the mice manifest reduced disease incidence and improved NK and NKT cell performance, as compared with wild-type NOD mice. The association of those two features in the same congenic strain constitutes a strong argument in favor of Idd6 being associated to the NK complex. This could explain at the same time the multiple alterations of innate immunity reported in NOD mice and the fact that disease onset can be readily modified by boosting the innate immune system of the mouse.     

Sex-specific effect of insulin-dependent diabetes 4 on regulation of diabetes pathogenesis in the nonobese diabetic mouse

Many human autoimmune diseases are more frequent in females than males, and their clinical severity is affected by sex hormone levels. A strong female bias is also observed in the NOD mouse model of type I diabetes (T1D). In both NOD mice and humans, T1D displays complex polygenic inheritance and T cell-mediated autoimmune pathogenesis. The identities of many of the insulin-dependent diabetes (Idd) loci, their influence on specific stages of autoimmune pathogenesis, and sex-specific effects of Idd loci in the NOD model are not well understood. To address these questions, we analyzed cyclophosphamide-accelerated T1D (CY-T1D) that causes disease with high and similar frequencies in male and female NOD mice, but not in diabetes-resistant animals, including the nonobese diabetes-resistant (NOR) strain. In this study we show by genetic linkage analysis of (NOD x NOR) x NOD backcross mice that progression to severe islet inflammation after CY treatment was controlled by the Idd4 and Idd9 loci. Congenic strains on both the NOD and NOR backgrounds confirmed the roles of Idd4 and Idd9 in CY-T1D susceptibility and revealed the contribution of a third locus, Idd5. Importantly, we show that the three loci acted at distinct stages of islet inflammation and disease progression. Among these three loci, Idd4 alleles alone displayed striking sex-specific behavior in CY-accelerated disease. Additional studies will be required to address the question of whether a sex-specific effect of Idd4, observed in this study, is also present in the spontaneous model of the disease with striking female bias.     

Inhibition of type 1 diabetes by upregulation of the circadian rhythm-related aryl hydrocarbon receptor nuclear translocator-like 2

The genetic locus Idd6 is involved in type 1 diabetes development in the non-obese diabetic (NOD) mouse through its effect on the immune system and in particular, on T cell activities. Analysis of congenic strains for Idd6 has established the Aryl hydrocarbon receptor nuclear translocator-like 2 (Arntl2) as a likely candidate gene. In this study we investigate the role of Arntl2 in the autoimmune disease and T cell activation. An Arntl2 expressing plasmid was transfected into CD4⁺ T cells by nucleofection. Expression levels of cytokines and CD4⁺ T cell activation markers, cell death, apoptosis, and cell proliferation rates were characterized in ex vivo experiments whilst in vivo the transfected cells were transferred into NOD.SCID mice to monitor diabetes development. The results demonstrate that Arntl2 overexpression leads to inhibition of CD4⁺ T cell proliferation and decreases in their diabetogenic activity without influence on the expression levels of cytokines, CD4⁺ T cell activation markers, cell death, and apoptosis. Our findings suggest that Arntl2 at the Idd6 locus may act via the inhibition of CD4⁺ T cell proliferation and the reduction in the diabetogenic activity of CD4⁺ T cells to protect against autoimmune type 1 diabetes in the NOD mice.