The NOD Mouse Beyond Autoimmune Diabetes.

Autoimmune diabetes arises spontaneously in Non-Obese Diabetic (NOD) mice, and the pathophysiology of this disease shares many similarities with human type 1 diabetes. Since its generation in 1980, the NOD mouse, derived from the Cataract Shinogi strain, has represented the gold standard of spontaneous disease models, allowing to investigate autoimmune diabetes disease progression and susceptibility traits, as well as to test a wide array of potential treatments and therapies. Beyond autoimmune diabetes, NOD mice also exhibit polyautoimmunity, presenting with a low incidence of autoimmune thyroiditis and Sjögren's syndrome. Genetic manipulation of the NOD strain has led to the generation of new mouse models facilitating the study of these and other autoimmune pathologies. For instance, following deletion of specific genes or via insertion of resistance alleles at genetic loci, NOD mice can become fully resistant to autoimmune diabetes; yet the newly generated diabetes-resistant NOD strains often show a high incidence of other autoimmune diseases. This suggests that the NOD genetic background is highly autoimmune-prone and that genetic manipulations can shift the autoimmune response from the pancreas to other organs. Overall, multiple NOD variant strains have become invaluable tools for understanding the pathophysiology of and for dissecting the genetic susceptibility of organ-specific autoimmune diseases. An interesting commonality to all autoimmune diseases developing in variant strains of the NOD mice is the presence of autoantibodies. This review will present the NOD mouse as a model for studying autoimmune diseases beyond autoimmune diabetes.

The Idd2 Locus Confers Prominent Resistance to Autoimmune Diabetes.

Type 1 diabetes is an autoimmune disease characterized by pancreatic ß cell destruction. It is a complex genetic trait driven by >30 genetic loci with parallels between humans and mice. The NOD mouse spontaneously develops autoimmune diabetes and is widely used to identify insulin-dependent diabetes (Idd) genetic loci linked to diabetes susceptibility. Although many Idd loci have been extensively studied, the impact of the Idd2 locus on autoimmune diabetes susceptibility remains to be defined. To address this, we generated a NOD congenic mouse bearing B10 resistance alleles on chromosome 9 in a locus coinciding with part of the Idd2 locus and found that NOD.B10-Idd2 congenic mice are highly resistant to diabetes. Bone marrow chimera and adoptive transfer experiments showed that the B10 protective alleles provide resistance in an immune cell-intrinsic manner. Although no T cell-intrinsic differences between NOD and NOD.B10-Idd2 mice were observed, we found that the Idd2 resistance alleles limit the formation of spontaneous and induced germinal centers. Comparison of B cell and dendritic cell transcriptome profiles from NOD and NOD.B10-Idd2 mice reveal that resistance alleles at the Idd2 locus affect the expression of specific MHC molecules, a result confirmed by flow cytometry. Altogether, these data demonstrate that resistance alleles at the Idd2 locus impair germinal center formation and influence MHC expression, both of which likely contribute to reduced diabetes incidence.

Evidence of genetic epistasis in autoimmune diabetes susceptibility revealed by mouse congenic sublines.

Susceptibility to autoimmune diabetes is a complex genetic trait. Linkage analyses exploiting the NOD mouse, which spontaneously develops autoimmune diabetes, have proved to be a useful tool for the characterization of some of these traits. In a linkage analysis using 3A9 TCR transgenic mice on both B10.BR and NOD.H2k backgrounds, we previously determined that both the Idd2 and Idd13 loci were linked to the proportion of immunoregulatory CD4-CD8- double negative (DN) T cells. In addition to Idd2 and Idd13, five other loci showed weak linkage to the proportion of DN T cells. Of interest, in an interim analysis, a locus on chromosome 12 is linked to DN T cell proportion in both the spleen and the lymph nodes. To determine the impact of this locus on DN T cells, we generated two congenic sublines, which we named Chr12P and Chr12D for proximal and distal, respectively. While 3A9 TCR:insHEL NOD.H2k-Chr12D mice were protected from diabetes, 3A9 TCR:insHEL NOD.H2k-Chr12P showed an increase in diabetes incidence. Yet, the proportion of DN T cells was similar to the parental 3A9 TCR NOD.H2k strain for both of these congenic sublines. A genome-wide two dimensional LOD score analysis reveals genetic epistasis between chromosome 12 and the Idd13 locus. Altogether, this study identified further complex genetic interactions in defining the proportion of DN T cells, along with evidence of genetic epistasis within a locus on chromosome 12 influencing autoimmune susceptibility.

MHC-Independent Thymic Selection of CD4 and CD8 Coreceptor Negative αß T Cells.

It is becoming increasingly clear that unconventional T cell subsets, such as NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells, each play distinct roles in the immune response. Subsets of these cell types can lack both CD4 and CD8 coreceptor expression. Beyond these known subsets, we identify CD4-CD8-TCRαß+, double-negative (DN) T cells, in mouse secondary lymphoid organs. DN T cells are a unique unconventional thymic-derived T cell subset. In contrast to CD5high DN thymocytes that preferentially yield TCRαß+ CD8αα intestinal lymphocytes, we find that mature CD5low DN thymocytes are precursors to peripheral DN T cells. Using reporter mouse strains, we show that DN T cells transit through the immature CD4+CD8+ (double-positive) thymocyte stage. Moreover, we provide evidence that DN T cells can differentiate in MHC-deficient mice. Our study demonstrates that MHC-independent thymic selection can yield DN T cells that are distinct from NKT, γδ T, mucosal-associated invariant T, and CD8αα T cells.

Common Heritable Immunological Variations Revealed in Genetically Diverse Inbred Mouse Strains of the Collaborative Cross.

Variations in the proportion and number of specific immune cell types among healthy individuals are influenced by both heritable and nonheritable factors. Mouse models, subjected to fewer nonheritable factors than humans, allow the identification of genetic factors that shape the immune system. We characterized immunological trait variability in the Collaborative Cross (CC), a powerful genetic resource of recombinant inbred mouse strains derived from eight diverse founder strains. Of the 18 immunological traits studied in more than 60 CC strains, eight showed genome-wide significant linkage, revealing new genetic loci linked to specific immune traits. We also found that these traits were highly subject to heritable influences. As for humans, mouse immunological traits varied as a continuum rather than as discrete immunophenotypes. The CC thus represents a useful resource to identify factors that determine immunological variations, as well as defining other immune traits likely to be heritable in humans.

Genetic interaction between two insulin-dependent diabetes susceptibility loci, Idd2 and Idd13, in determining immunoregulatory DN T cell proportion.

Several immune regulatory cell types participate in the protection against autoimmune diseases such as autoimmune diabetes. Of these immunoregulatory cells, we and others have shown that peripheral CD4-CD8- double negative (DN) T cells can induce antigen-specific immune tolerance. Particularly, we have described that diabetes-prone mice exhibit a lower number of peripheral DN T cells compared to diabetes-resistant mice. Identifying the molecular pathways that influence the size of the DN T cell pool in peripheral lymphoid organs may thus be of interest for maintaining antigen-specific immune tolerance. Hence, through immunogenetic approaches, we found that two genetic loci linked to autoimmune diabetes susceptibility, namely Idd2 and Idd13, independently contribute to the partial restoration of DN T cell proportion in secondary lymphoid organs. We now extend these findings to show an interaction between the Idd2 and Idd13 loci in determining the number of DN T cells in secondary lymphoid organs. Using bioinformatics tools, we link potential biological pathways arising from interactions of genes encoded within the two loci. By focusing on cell cycle, we validate that both the Idd2 and Idd13 loci influence RAD51 expression as well as DN T cell progression through the cell cycle. Altogether, we find that genetic interactions between Idd2 and Idd13 loci modulate cell cycle progression, which contributes, at least in part, to defining the proportion of DN T cells in secondary lymphoid organs.

Abnormal differentiation of B cells and megakaryocytes in patients with Roifman syndrome.

BACKGROUND: Roifman syndrome is a rare inherited disorder characterized by spondyloepiphyseal dysplasia, growth retardation, cognitive delay, hypogammaglobulinemia, and, in some patients, thrombocytopenia. Compound heterozygous variants in the small nuclear RNA gene RNU4ATAC, which is necessary for U12-type intron splicing, were identified recently as driving Roifman syndrome. OBJECTIVE: We studied 3 patients from 2 unrelated kindreds harboring compound heterozygous or homozygous stem II variants in RNU4ATAC to gain insight into the mechanisms behind this disorder. METHODS: We systematically profiled the immunologic and hematologic compartments of the 3 patients with Roifman syndrome and performed RNA sequencing to unravel important splicing defects in both cell lineages. RESULTS: The patients exhibited a dramatic reduction in B-cell numbers, with differentiation halted at the transitional B-cell stage. Despite abundant B-cell activating factor availability, development past this B-cell activating factor-dependent stage was crippled, with disturbed minor splicing of the critical mitogen-activated protein kinase 1 signaling component. In the hematologic compartment patients with Roifman syndrome demonstrated defects in megakaryocyte differentiation, with inadequate generation of proplatelets. Platelets from patients with Roifman syndrome were rounder, with increased tubulin and actin levels, and contained increased α-granule and dense granule markers. Significant minor intron retention in 354 megakaryocyte genes was observed, including DIAPH1 and HPS1, genes known to regulate platelet and dense granule formation, respectively. CONCLUSION: Together, our results provide novel molecular and cellular data toward understanding the immunologic and hematologic features of Roifman syndrome.

An Unbiased Linkage Approach Reveals That the p53 Pathway Is Coupled to NK Cell Maturation.

Natural killer cells constitute potent innate lymphoid cells that play a major role in both tumor immunosurveillance and viral clearance via their effector functions. A four-stage model of NK cell functional maturation has been established according to the expression of CD11b and CD27, separating mature NK (mNK) cells into distinct populations that exhibit specific phenotypic and functional properties. To identify genetic factors involved in the regulation of NK cell functional maturation, we performed a linkage analysis on F2 (B6.Rag1-/- × NOD.Rag1-/- intercross) mice. We identified six loci on chromosomes 2, 4, 7, 10, 11, and 18 that were linked to one or more mNK cell subsets. Subsequently, we performed an in silico analysis exploiting mNK cell subset microarray data, highlighting various genes and microRNAs as potential regulators of the functional maturation of NK cells. Together, the combination of our unbiased genetic linkage study and the in silico analysis positions genes known to affect NK cell biology along the specific stages of NK cell functional maturation. Moreover, this approach allowed us to uncover a novel candidate gene in the regulation of NK cell maturation, namely Trp53 Using mice deficient for Trp53, we confirm that this tumor suppressor regulates NK cell functional maturation. Additional candidate genes revealed in this study may eventually serve as targets for the modulation of NK cell functional maturation to potentiate both tumor immunosurveillance and viral clearance.

Genes Outside the Major Histocompatibility Complex Locus Are Linked to the Development of Thyroid Autoantibodies and Thyroiditis in NOD.H2h4 Mice.

Thyroiditis and autoantibodies to thyroglobulin (TgAb) and thyroid peroxidase (TPOAb) develop spontaneously in NOD.H2h4 mice, a phenotype enhanced by dietary iodine. NOD.H2h4 mice were derived by introducing the major histocompatibility class (MHC) molecule I-Ak from B10.A(4R) mice to nonobese diabetic (NOD) mice. Apart from I-Ak, the genes responsible for the NOD.H2h4 phenotype are unknown. Extending serendipitous observations from crossing BALB/c to NOD.H2h4 mice, thyroid autoimmunity was investigated in both genders of the F1, F2, and the second-generation backcross of F1 to NOD.H2h4 (N2). Medium-density linkage analysis was performed on thyroid autoimmunity traits in F2 and N2 progeny. TgAb develop before TPOAb and were measured after 8 and 16 weeks of iodide exposure; TPOAb and thyroiditis were studied at 16 weeks. TgAb, TPOAb, and thyroiditis, absent in BALB/c and F1 mice, developed in most NOD.H2h4 and in more N2 than F2 progeny. No linkages were observed in F2 progeny, probably because of the small number of autoantibody-positive mice. In N2 progeny (equal numbers of males and females), a chromosome 17 locus is linked to thyroiditis and TgAb and is suggestively linked to TPOAb. This locus includes MHC region genes from B10.A(4R) mice (such as I-Ak and Tnf, the latter involved in thyrocyte apoptosis) and genes from NOD mice such as Satb1, which most likely plays a role in immune tolerance. In conclusion, MHC and non-MHC genes, encoded within the chromosome 17 locus from both B10.A(4R) and NOD strains, are most likely responsible for the Hashimoto disease-like phenotype of NOD.H2h4 mice.

TCR transgenic mice reveal the impact of type 1 diabetes loci on early and late disease checkpoints.

Linkage analysis studies for autoimmune diabetes have revealed multiple non-major histocompatibility complex (MHC) chromosomal regions linked to disease susceptibility. To date, more than 20 insulin-dependent diabetes (Idd) loci linked to diabetes susceptibility have been identified in NOD mice and validated via congenic breeding. Importantly, evidence suggests that Idd loci may regulate at least two pathological steps during autoimmune diabetes development, namely the onset of insulitis and the transition from insulitis to overt diabetes. Here we assess the role of various non-MHC Idd diabetes-resistance loci, which have been validated in the non-transgenic setting, on autoimmune diabetes progression in the transgenic setting. Specifically, we generated multiple Idd congenic strains in the 3A9-TCR:insHEL NOD.H2(k) transgenic model and monitored their diabetes incidence. We show that 3A9-TCR:insHEL NOD.H2(k) mice congenic for Idd3 or Idd5 display a reduction in diabetes development, whereas mice congenic for Idd9 or Idd13 exhibit an increase, in comparison with 3A9-TCR:insHEL NOD.H2(k) mice. These results suggest that the presence of the 3A9-TCR and hen egg lysosyme transgenes can offset the regulatory function of certain diabetes-resistance genetic variants contained within the Idd loci, including Idd9 and Idd13. We propose the antigen-specific 3A9-TCR:insHEL transgenic model as a useful tool for the study of the genetics of autoimmune diabetes development.

Autoimmunity and antibody affinity maturation are modulated by genetic variants on mouse chromosome 12.

Autoimmune diseases result from a break in immune tolerance leading to an attack on self-antigens. Autoantibody levels serve as a predictive tool for the early diagnosis of many autoimmune diseases, including type 1 diabetes. We find that a genetic locus on mouse chromosome 12 influences the affinity maturation of antibodies as well as autoantibody production. Thus, we generated a NOD.H2(k) congenic strain bearing B10 alleles at the locus comprised within the D12Mit184 and D12Mit12 markers, which we named NOD.H2(k)-Chr12. We determined the biological relevance of the Chr12 locus on the autoimmune process using an antigen-specific TCR transgenic autoimmune mouse model. Specifically, the 3A9 TCR transgene, which recognizes a peptide from hen egg lysozyme (HEL) in the context of I-A(k), and the HEL transgene, which is expressed under the rat-insulin promoter (iHEL), were bred into the NOD.H2(k)-Chr12 congenic strain. In the resulting 3A9 TCR:iHEL NOD.H2(k)-Chr12 mice, we observed a significant decrease in diabetes incidence as well as a decrease in both the quantity and affinity of HEL-specific IgG autoantibodies relative to 3A9 TCR:iHEL NOD.H2(k) mice. Notably, the decrease in autoantibodies due to the Chr12 locus was not restricted to the TCR transgenic model, as it was also observed in the non-transgenic NOD.H2(k) setting. Of importance, antibody affinity maturation upon immunization and re-challenge was also impeded in NOD.H2(k)-Chr12 congenic mice relative to NOD.H2(k) mice. Together, these results demonstrate that a genetic variant(s) present within the Chr12 locus plays a global role in modulating antibody affinity maturation.

The mouse idd2 locus is linked to the proportion of immunoregulatory double-negative T cells, a trait associated with autoimmune diabetes resistance.

Autoimmune diseases result from a break in immune tolerance. Various mechanisms of peripheral tolerance can protect against autoimmunity, including immunoregulatory CD4(-)CD8(-) double-negative (DN) T cells. Indeed, we have previously shown that diabetes-prone mouse strains exhibit a low proportion of DN T cells relative to that of diabetes-resistant mice, and that a single autologous transfer of DN T cells can impede autoimmune diabetes development, at least in the 3A9 TCR transgenic setting. In this study, we aim to understand the genetic basis for the difference in DN T cell proportion between diabetes-resistant and diabetes-prone mice. We thus perform an unbiased linkage analysis in 3A9 TCR F2 (NOD.H2(k) × B10.BR) mice and reveal that a locus on chromosome 9, which coincides with Idd2, is linked to the proportion of DN T cells in the lymph nodes. We generate two NOD.H2(k).B10-Chr9 congenic mouse strains and validate the role of this genetic interval in defining the proportion of DN T cells. Moreover, we find that the increased proportion of DN T cells in lymphoid organs is associated with a decrease in both diabetes incidence and serum IgG Ab levels. Together, the data suggest that Idd2 is linked to DN T cell proportion and that a physiological increase in DN T cell number may be sufficient to confer resistance to autoimmune diabetes. Altogether, these findings could help identify new candidate genes for the development of therapeutic avenues aimed at modulating DN T cell number for the prevention of autoimmune diseases.

Nearby Construction Impedes the Progression to Overt Autoimmune Diabetes in NOD Mice.

Construction nearby animal houses has sporadically been reported to affect various aspects of animal health. Most of the reports have focussed on the impact on stress hormone levels and the hypersensitivity of animals relative to humans. There has also been an anecdotal report on the impact of construction on autoimmune diabetes in NOD mice. Here, we describe that nearby construction significantly impedes the progression to overt diabetes in female NOD mice offspring. We demonstrate that this was not due to a genetic drift or to particularities associated with our specific mouse colony. Interestingly, although the glycemia levels remained low in mice born from mothers subject to construction stress during gestation, we detected an active autoimmune reaction towards pancreatic islet cells, as measured by both the degree of insulitis and the presence of insulin autoantibody levels in the serum. These results suggest that the external stress imposed during embryonic development does not prevent but significantly delays the autoimmune process. Together, our findings emphasize the impact of surrounding factors during in vivo studies and are in agreement with the hypothesis that both environmental and genetic cues contribute to autoimmune diabetes development.