NOD mice and autoimmunity

Autoimmunity Reviews 4 (2005) 373 – 379 www.elsevier.com/locate/autrev

NOD mice and autoimmunity Christopher A. Aokia, Andrea T. Borchersa, William M. Ridgwayb, Carl L. Keenc, Aftab A. Ansarid, M. Eric Gershwina,T a

Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, 451 E. Health Sciences Drive, Suite 6510, Davis, CA 95616, United States b Division of Rheumatology, University of Pittsburgh, Pittsburgh, PA 15213, United States c Department of Nutrition, University of California, Davis, CA 95616, United States d Department of Pathology, Emory University School of Medicine, Atlanta GA 30322, United States Received 14 January 2005; accepted 3 February 2005 Available online 29 March 2005

Abstract The NOD mouse has been an important model of type 1 diabetes and autoimmune diseases for over 20 years. Experimental and genetic manipulations of the NOD mouse have demonstrated a broad susceptibility to multiple autoimmune syndromes. This predisposition to autoimmunity is due to defects in both central and peripheral tolerance. The defect of central tolerance is likely secondary to improper negative selection mediated by the unique MHC Class II molecule, I-Ag7 as well as intrinsic T cell signaling defects. The genetic basis for impaired peripheral tolerance is controlled by over 20 susceptibility loci termed insulin-dependent diabetes (idd) loci. The maintenance of peripheral tolerance is impaired by alterations in T cell signaling and apoptosis. In addition, insufficient co-stimulation from accessory cells, and defective regulatory T cells, may promote the production of autoreactive T cells. D 2005 Elsevier B.V. All rights reserved. Keywords: Tolerance; Thymic education; Apoptosis; T cell signaling

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . Genetic susceptibility to autoimmune disease Impaired thymic selection . . . . . . . . . . T cells and autoimmunity . . . . . . . . . . . Impaired co-stimulation and autoimmunity . .

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T Corresponding author. Tel.: +1 530 752 2884; fax: +1 530 752 4669. E-mail address: [email protected] (M.E. Gershwin). 1568-9972/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2005.02.002

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6. Regulatory T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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immune responses in a variety of tissues, including salivary, lacrimal, thyroid, parathyroid, adrenal, testis, large bowel and red blood cells [1]. NOD mice are also susceptible to the experimental induction of a variety of autoimmune diseases, including experimental autoimmune thyroiditis, colitis-like wasting disease, encephalomyelitis, and manifestations of systemic lupus erythematosus (SLE). Genetic alter-

1. Introduction Non-obese diabetic (NOD) mice are an inbred strain originally developed from the cataract-prone strain of outbred Jcl:ICR mice. Inbreeding of the founders established the NOD strain as a model of spontaneous type one diabetes (T1D). In addition to diabetes, NOD mice spontaneously develop autoTable 1 NOD mouse manipulations resulting in autoimmune diseases Model Treatment

Genetic manipulation

Insulitis

Heat-killed BCG

Diabetes

Other autoimmune manifestations

Ref.

0

SLE-like disease with immune complex deposition in the kidney, hemolytic anemia, and ANA (including anti-dsDNA, anti-Sm, and anti-nRNP) SLE-like disease with leukopenia, thrombocytopenia, proteinuria, immune complex deposition in the kidney Autoimmune thyroiditis Autoimmune cardiomyopathy strongly resembling human idiopathic dilated cardiomyopathy; characterized by mononuclear cell infiltration of the heart and the outermost muscle layer surrounding the pulmonary veins, anti-cardiac autoAb, progressive atrioventricular block Autoimmune peripheral polyneuropathy Fatal paralytic autoimmune myositis CD8 T cell mediated

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Idiotypic induction of SLE via immunization with a human IgM Ab carrying the 16/6 idiotype (MIV-7) NOD.H2h4 DQ8+/+, IA! / NOD mice, i.e., NOD mice expressing the human HLA-DQ8 transgene in the absence of mouse IA!

Significantly reduced

Decreased incidence (25% vs. 90%)

0

0 0

NOD mice with B7-2 deficiency CD2 promoter-driven transgene encoding the beta chain of the IFN-g receptor (IFN-gRB) in all T cells in order to eliminate production of IFN-g; intended as a model of deficient Th1 cytokine production NOD.c3c4 congenic mice

Peri-insulitis in some animals

0 Not prevented, sialadenitis also not prevented

0, Reduced sialadenitis

Fatal autoimmune polycystic biliary tract disease characterized by massive hepatomegaly, lymphocytic peribiliary infiltrates, and anti-ssDNA, anti-dsDNA and anti-Sm Ab

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ations imposed onto the NOD background have also led to the development of a variety of unique autoimmune conditions. Therefore either genetic or therapeutic manipulations of NOD can result in the abrogation of diabetes and the development of different autoimmune disease (Table 1). There are also reports of manipulations that do not protect NOD mice from diabetes, and instead induce additional autoimmune disorders (Table 1). These results suggest that NOD mice have an underlying susceptibility to autoimmune disease, and have marked variation in the target tissue of their autoimmune illness.

2. Genetic susceptibility to autoimmune disease Nod mice are homozygous for the H-2g7 haplotype (K , I-Ag7, I-Enull, Db), that encodes an I-Aad/I-Ahg7 heterodimer. The MHC Class II molecule, I-Ag7, is essential to the development of T1DM in the NOD mouse and is the strongest genetic contributor to the disease [2]. I-Ag7 has proline and serine at positions 56 and 57, instead of the histidine and aspartic acid found in most murine I-Ah chains. This unique structure may facilitate the binding and presentation of peptides that are not usually presented by other MHC haplotypes. In addition, I-Ag7 binds with low affinity to many peptides, and may also be a bpromiscuousQ peptide binder [3–5]. The resulting complexes are unstable and characterized by poor T cell stimulatory ability compared to other I-A molecules [4]. Although the low affinity binding between I-Ag7 and a self peptide does not necessarily result in failure to induce tolerance in NOD mice, it seems likely that it contributes to the emergence of a large number and a diverse repertoire of autoreactive T cells [6,7]. Over 20 non-MHC Idd loci have been associated with diabetes pathogenesis as identified in genome scanning and congenic mapping experiments [8]. Fine mapping of the Idd3 locus using congenic strains and an ancestral haplotype analysis has revealed the Il2 gene as the strongest candidate within this region [9]. No polymorphisms were detected within regulatory elements of the promoter region, and steady-state IL-2 mRNA expression was similar among a variety of diabetes-susceptible and resistant mouse strains analyzed. Only variations in the coding sequence of IL-2

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allotypes have been found to correlate with the extent of glycosylation and disease susceptibility [9]. A noncoding polymorphism in the CTLA-4 gene in the NOD mouse was found to correlate with alternative splicing of a ligand independent form of CTLA-4 [10–12]. This ligand independent form of CTLA-4 has been shown to inhibit T cell response and is highly expressed in memory and regulatory T cells from diabetes resistant-NOD congenic mice compared to wild type NOD mice [11]. Although this may suggest a direct inhibitory role, this has not been demonstrated in vivo. A striking feature of these and other Idd loci is that no single locus, even MHC class II, is both necessary and sufficient to mediate autoimmune diabetes, showing that a complex and multi-step genetic pathway mediates the development of autoimmune diseases in the NOD mouse.

d

3. Impaired thymic selection There is significant evidence that impaired thymic selection contributes to the development of autoreactive NOD T cells. As mentioned above, the NOD IAg7 structure has been shown to be intrinsically unstable resulting in poor self-peptide binding, which may alter the thresholds for thymic selection. Thymic selection may also fail due to altered sensitivity of NOD thymocytes to undergo negative selection. This has been demonstrated using an anti-CD3 mAb or a superantigen in both in vitro and in vivo assays of NOD thymocytes [13]. Non-MHC NOD genes have been shown to be responsible for a primary defect in thymic selection, causing high affinity autoreactive T cells to be resistant to clonal deletion at the doublepositive and CD4+CD24high single-positive stages [14]. Most recently, resistance to thymic deletion in NOD was shown to result from failure to induce Bim, a proapoptotic protein, during in vivo encounter with a high avidity antigen [15].

4. T cells and autoimmunity A variety of defects in NOD T cell activation and function have been described. One example is the increased IFN-g and decreased IL-4 in NOD activated CD4+ T cells [16,17]. These cytokine defects can be

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partially corrected by the addition of IL-2 and completely restored by the addition of IL-4 or CD28 stimulation [17,18]. NOD lymphocytes are also resistant to induction of apoptosis by a variety of stimuli [19]. Although this is not a consistent finding, the resistance to activation-induced cell death seems to arise from the defective proliferative response of NOD T cells and can be overcome by IL-4 [19]. These are just 2 examples from a large literature implicating abnormal NOD T cell function.

5. Impaired co-stimulation and autoimmunity In addition to intrinsic defects, NOD T cells receive insufficient co-stimulation from accessory cells during negative selection. Tolerance induction requires a high level of co-stimulation from accessory cells and when impaired results in defective negative selection. Splenic CD8a+ DC and macrophages from NOD mice were found to express lower basal levels of CD86 (B7-2) compared to C57BL/6 and BALB/c mice. This was associated with reduced expression of CD86 and CTLA-4 on NOD T cells following activation with anti-CD3 [20]. Diabetes is much worsened in NOD B7-1/2 and CD-28 knockout mice; disruption of the CD28/B7 pathway is believed to result in a Th1 immune response to autoantigens and a reduction in number and function of CD4+CD25+ regulatory T cells [21,22]. CTLA-4 is another member of the co-stimulatory pathway that appears to be dysregulated in the NOD mouse. When expressed by activated T cells; CTLA-4 inhibits T cell proliferation, IL-2 production and cell cycle proliferation. In humans, CTLA-4 is a candidate gene for susceptibility to a number of autoimmune diseases. In NOD lymphocytes, CTLA-4 surface expression may be decreased compared to other murine strains [23]; as mentioned above NOD lymphocytes show decreased expression of the ligand independent form of CTLA4 [11]. In the BDC2.5 TCR transgenic NOD mice model, treatment with anti-CTLA-4 mAb before the development of insulitis, results in a more aggressive infiltrate and an increase in diabetes compared to animals treated with control mAb [24]. Experiments involving the adoptive transfer of naive BDC2.5 T cells into T cell deficient NOD recipients indicate that CTLA-4 is not essential

during the initial priming of the transferred T cells. Instead it acts when primed T cells encounter their antigen at the target organ [24]. Other costimulatory molecules in the CD28 family that appear to be dysregulated in NOD include ICOS and PD-1. ICOS promotes IL-10 production and regulates the TH2 pathway. BDC 2.5TCR transgenic mice treated with anti-ICOS have decreased regulatory T cells and acceleration of diabetes [25]. PD-1 is believed to act as a negative regulator of the T cell response and NOD mice deficient in PD-1 have an accelerated development of diabetes [26]. The costimulatory molecules expressed by antigen presenting cells may also be defective in the NOD mouse. In NOD mice less than 3 weeks of age, anti-CD40L mAb blocked insulitis and diabetes supporting the hypothesis that the CD40/CD154 interaction is important for the initial priming of the disease [27]. Furthermore mice deficient in CD154 have reduced incidence of diabetes [28]. In general, the importance of costimulatory signals in pathogenesis of T1D in NOD mice highlights the likely importance of antigen presenting cell (APC) biology. A variety of other APC defects have been reported on NOD, for example APC produced IL-12 may be important, since IL-12 blockade inhibits the development of diabetes in NOD mice while exogenous IL-12 can accelerate it [29].

6. Regulatory T cells Recent years have seen an explosion of reports implicating a role for regulatory T cells in NOD diabetes pathogenesis. The acceleration of diabetes after thymectomy at weaning is thought to be due to the inability to generate protective regulatory T cells. In addition, CD4+ T cells from young non-diabetic mice can prevent the transfer of diabetes by coinoculation with splenocytes from diabetic mice. Various tolerizing regimens via administration of autoantigens have been reported to involve the generation of T cells capable of actively suppressing autoreactive T cell responses [30]. It has been reported that prediabetic NOD mice also have a lower number of CD4+CD25+ T cells when compared to mouse strains not susceptible to autoimmune diseases [21]. Finally, several treatments associated with the ameli-

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Regulatory T cells: Reduced number and function Natural Killer T cells: Reduced number and function Autoreactive T cells

Impaired negative selection in thymus

Defective apoptosis and signaling

Antigen Presenting Cells: Impaired co-stimulation and increased IL-12 production

Fig. 1. The pathophysiology of autoimmunity in NOD mice.

oration of diabetes in NOD mice, such as administration of TNF-a to neonates have been associated with an increase in CD4+CD25+ T cells [31] (Fig. 1). Regulatory CD4+ T cell subsets have different effects on the autoimmune manifestations of NOD. scid mice reconstituted with prediabetic NOD splenocytes. These diseases include diabetes, sialitis, moderate colitis, and mild gastritis [1]. The transfer of splenocytes depleted of CD25 + , CD62 + or CD45RBlow cells indicate that CD25+CD62L T cells provide protection from gastritis, CD25 CD62L+ T cells selectively inhibit diabetes, and CD45RBlow cells are essential for the control of colitis. In addition, 30% of the recipients of CD25+-depleted T cells also exhibit thyroiditis. Interestingly, most of the regulatory T cell subsets appear to afford some protection from sialitis, with CD25+ T cells being the most effective. Besides impairment in the CD4+CD25+ T cell, NOD mice have 2- to 4-fold reduction in the number of NK-T cells in the thymus, spleen and liver compared to non-autoimmune mouse strains. The reduction in NK-

T cells is probably due to an intrinsic defect of NOD T cell precursors [32]. Numerous functional deficiencies of NOD NKT cells have been described, including defective proliferation in response to stimulation with IL-12. Both thymic and peripheral NKT cells from NOD mice have significantly reduced in vitro production of IL-4 and/or IFN-g in response to TCR cross linking. The stimulation of NK-T cells by a-GalCer is also reduced in NOD mice [33]. Take-home messages ! Experimental and genetic manipulations of the NOD mouse produce a wide variety of models of human autoimmune diseases suggesting a broad underlying predisposition to autoimmunity. ! Multiple candidate genes, including the MHC Class II, CTLA-4, and IL-2, have been associated with the development of diabetes. ! Defective central tolerance leading to autoimmune disease may be mediated by both structurally

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abnormal MHC molecules and defective T cell signaling. ! T cells (activated, regulatory and natural killer) have defective signaling and function in the NOD mouse contributing to the autoimmune disease process. ! Antigen presenting cell (APC) mediated costimulation of T cells at various developmental checkpoints is a critical regulator of autoimmunity. References [1] Alyanakian MA, You S, Damotte D, Gouarin C, Esling A, Garcia C, et al. Diversity of regulatory CD4+ T cells controlling distinct organ-specific autoimmune diseases. Proc Natl Acad Sci U S A 2003 Dec. 23;100(26):15806 – 11. Epub 2003 Dec. 12. Erratum in: Proc Natl Acad Sci U S A. 2004 Mar. 23;101(12):4331. [2] Tisch R, McDevitt H. Insulin-dependent diabetes mellitus. Cell 1996;85:291 – 7. [3] Carrasco-Marin E, Shimizu J, Kanagawa O, Unanue ER. The class II MHC I-Ag7 molecules from non-obese diabetic mice are poor peptide binders. Immunology 1996 (Jan. 15);156(2): 450 – 8. [4] Hausmann DH, Yu B, Hausmann S, Wucherpfennig KW. pHdependent peptide binding properties of the type I diabetesassociated I-Ag7 molecule: rapid release of CLIP at an endosomal pH. J Exp Med 1999;189:1723 – 34. [5] Stratmann T, Apostolopoulos V, Mallet-Designe V, Corper AL, Scott CA, Wilson IA, et al. The I-Ag7 MHC class II molecule linked to murine diabetes is a promiscuous peptide binder. J Immunol 2000 (Sep. 15);165(6):3214 – 25. [6] Kanagawa O, Martin SM, Vaupel BA, Carrasco-Marin E, Unanue ER. Autoreactivity of T cells from nonobese diabetic mice: an I-Ag7-dependent reaction. Proc Natl Acad Sci U S A 1998;95:1721 – 4. [7] Ridgway WM, Ito H, Fasso M, Yu C, Fathman CG. Analysis of the role of variation of major histocompatibility complex class II expression on nonobese diabetic (NOD) peripheral T cell response. J Exp Med 1998 (Dec. 21);188(12):2267 – 75. [8] Wicker LS, Todd JA, Peterson LB. Genetic control of autoimmune diabetes in the NOD mouse. Annu Rev Immunol 1995;13:179 – 200. [9] Podolin PL, Wilusz MB, Cubbon RM, Pajvani U, Lord CJ, Todd JA, et al. Differential glycosylation of interleukin 2, the molecular basis for the NOD Idd3 type 1 diabetes gene? Cytokine 2000 May;12(5):477 – 82. [10] Hill NJ, Lyons PA, Armitage N, Todd JA, Wicker LS, Peterson LB. NOD Idd5 locus controls insulitis and diabetes and overlaps the orthologous CTLA4/IDDM12 and NRAMP1 loci in humans. Diabetes 2000;49:1744 – 7. [11] Vijayakrishnan L, Slavik JM, Illes Z, Greenwald RJ, Rainbow D, Greve B, et al. An autoimmune disease-

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T-cell molecular mimicry in Chagas disease: identification and partial structural analysis of multiple crossreactive epitopes between Trypanosoma cruzi B13 and cardiac myosin heavy chain. Chagas disease cardiomyopathy (CCC) is one of the few examples of post-infectious autoimmunity, where infectious episodes with an established pathogen, such as Trypanosoma cruzi, clearly triggers molecular mimicry-related target organ immune damage. CD4+ T-cell clones infiltrating hearts from CCC patients cross-reactively recognize human cardiac myosin and the immunodominant B13 protein from T.cruzi. In order to identify cross-reactive epitopes between B13 protein and human cardiac myosin, Iwai LK. et al. (J Autoimmunity 2005;24:111-117) used B13 peptide S15, 4, preferentially recognized by CCC patients, to establish a T-cell clone from an HLA-DQ7 individual. The B13 S15, 4 peptide-specific CD4+ T-cell clone 3E5 was tested in proliferation assays against S15,4-derived peptides for TCR/HLA contact analysis. Clone ˆ -cardiac myosin heavy chain bearing the central HLA3E5 was also tested against peptides from human O DQ7 binding motif. Clone 3E5 recognized 13 peptides from cardiac myosin. The alignment of cross-reactive peptides in cardiac myosin showed very limited sharing of residues with similar structural features at aligned positions, indicative of a very degenerate TCR recognition pattern. The existence of degenerate intramolecular recognition, with multiple low-homology, cross-reactive epitopes in a single autoantigenic protein may have implications in increasing the magnitude of the autoimmune response in CCC and other autoimmune diseases.