Reviews in Endocrine & Metabolic Disorders 2003;4:255–261C© 2003 Kluwer Academic Publishers. Manufactured in The Netherlands.

Genetic Separation of the Transplantation Tolerance andAutoimmune Phenotypes in NOD Mice

Todd Pearson1, Thomas G. Markees2, David V.Serreze1,2,4, Melissa A. Pierce4, Linda S. Wicker5,Laurence B. Peterson6, Leonard D. Shultz1,2,4,John P. Mordes2, Aldo A. Rossini1–3 andDale L. Greiner1,2

1Program in Immunology and Virology, 2Department of Medicine,and 3Program in Molecular Medicine at The University ofMassachusetts Medical School, 55 Lake Avenue North,Worcester, MA 01655; 4The Jackson Laboratory, 600 Main Street,Bar Harbor, ME 04609; 5Juvenile Diabetes ResearchFoundation/Wellcome Trust Diabetes and InflammationLaboratory, University of Cambridge, Cambridge, UK;6Department of Pharmacology, Merck Research Laboratories,Rahway, NJ 07065

Key Words. NOD mice, transplantation tolerance, costimulationblockade, CD154, type 1 diabetes

Introduction

Type 1 diabetes is caused by autoimmune destructionof the insulin-producing beta cells within the pancreaticislets of Langerhans, and affected persons require ex-ogenous insulin to survive [1]. Insulin injections con-trol hyperglycemia, but only imperfectly, and they donot prevent the secondary complications of diabetes [2].Islet (or pancreas) transplantation can be curative, butcurrently requires lifelong immunosuppression [3]. In-ducing islet transplantation tolerance would be prefer-able, but transplanting islets into persons with type 1diabetes requires both induction of allotransplantationtolerance and the prevention of recurrent autoimmunity[4]. Autoimmune recurrence was elegantly demonstratedwhen patients with type 1 diabetes were transplantedwith pancreata from discordant monozygotic siblings.In the absence of immunosuppression, the beta cellswithin the grafts were destroyed [5]. Because the graftswere syngeneic, ordinary allograft rejection could nothave occurred, and are indicative of autoimmune dis-ease recurrence. To better understand the interrelation-ship between autoimmune recurrence and allograft re-jection, researchers using animal models have attemptedto define the mechanisms that control each process[4,6,7].

Costimulation Blockade Inductionof Transplantation Tolerance

Several costimulation blockade-based transplantation tol-erance protocols have been developed [4]. Our laboratoryuses a protocol that combines a single donor-specific trans-fusion (DST) of spleen cells and injection of a anti-CD154mAb which blocks co-stimulation to abortively activateand then tolerize alloreactive T cells [4]. This protocolinduces permanent islet and prolonged skin allograft sur-vival in non-autoimmune mouse strains. Allograft survivalrequires depletion of most alloreactive CD8+ T cells and isdependent on CTLA-4, CD4+ T cells, and IFNγ [8,9]. Toinvestigate if costimulation blockade protocols will pro-long allograft survival in autoimmune hosts, investigatorshave focused principally on the NOD mouse [10,11]. Thesequencing of the mouse genome and genetically modifiedstocks make the NOD mouse a powerful system for inves-tigating mechanisms underlying self- and transplantationtolerance in the context of autoimmunity.

Pathogenesis and Genetics of Type 1 Diabetesin NOD Mice

NOD mouse diabetes is characterized by sponta-neous T cell-mediated beta cell destruction [6,10,11].

Corresponding author: Dale L. Greiner.E-mail: dale.greiner@umassmed.edu

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Table 1. Cell subsets important in the pathogenesis of diabetes in NODmice and in transplantation tolerance

Cell subset Defects in NOD mice

Role intransplantationtolerance

Effector CD4+and CD8+ Tcells

Abnormal deletion ofactivated T cells

Deletion required fortransplantationtolerance

CD4+(CD25+)regulatoryT cells

Abnormal function Required fortransplantationtolerance

NKT cells Defective cytokinesecretion andability to maturedendritic cells

Suggested to beimportant intransplantationtolerance

Dendritic cells Abnormal maturation Tolerogenic dendriticcells important intolerance induction,required forinduction ofregulatory T cells

NK cells Defective, notrequired forexpression ofdiabetes

Role in transplantationtolerance unknown

Macrophages Abnormal maturation Role in transplantationtolerance unknown

Immune cells hypothesized to be important in the pathogenesis of diabetes and in

the induction of transplantation tolerance.

Mononuclear cells infiltrate into islets at 4 to 5 weeks ofage and diabetes follows soon after. When maintained inspecific pathogen free environments the prevalence of dia-betes in NOD/Lt mice 20 weeks of age is >90% in femalesand ∼60% in males. The development of autoimmune dia-betes appears to involve failures of both central and periph-eral T cell tolerance mechanisms [6,10,11]. NOD mice donot delete autoreactive thymocytes cells during develop-ment in the thymus, and defects in peripheral CD4+ andCD8+ T cells have been observed [6,10,11]. T cell self-tolerance defects are likely to be both intrinsic [12], andextrinsic, involving defects in other immunological com-partments including antigen presenting cells (APC) [10].In addition to pathogenic autoreactive T cells, NOD miceexhibit other immunological defects, some of which mayhave counterparts in human type 1 diabetes pathogene-sis [6,10,11]. Many cell populations associated with thepathogenesis of diabetes in NOD mice are also involvedin the induction of transplantation tolerance (Table 1).

Preventing and Curing Type 1 Diabetesin NOD Mice

More than 100 interventions prevent diabetes in NODmice [6,10,11]. These interventions range from non-

specific (e.g. elevated temperature, overcrowding) tohighly specific (e.g. treatment with exogenous cytokinesor islet antigens). Obviously, many protocols that preventdiabetes in NOD mice are unlikely to yield effective ther-apy for humans [7]. In most cases, the mechanisms bywhich these treatments prevent diabetes in NOD miceare unknown, but various hypotheses have been proposed.These include skewing of cytokine responses from a Th1to a Th2 profile [13], the induction of regulatory T cells(Treg) [14] and the maturation of APC improving theirtolerogenic capacity [10].

Autoimmune Recurrence in NOD Mice

Although many interventions readily prevent its sponta-neous diabetes, remarkably few prevent recurrent autoim-munity in NOD mice. These include immunosuppressivedrugs [15,16], anti-CD3 antibody [17], and the genera-tion of hematopoietic chimerism [18]. The data suggestthat transplanting islets into persons with type 1 diabetesmay require induction of central tolerance, induction of arobust form of peripheral tolerance, or both.

Hematopoietic chimerism establishes central tolerance,prevents autoimmunity, and leads to transplantationtolerance in NOD miceThe establishment of hematopoietic chimerism can pre-vent autoimmune diabetes and induce tolerance to tis-sue allografts of the same donor type as the stem cellgraft [4]. The mechanism is thought to involve dele-tion of autoreactive and alloreactive T cells during in-trathymic development [19]. Elimination of autoreactiveT cells and protection from diabetes are mediated by ex-pression of a protective major histocompatibility complex(MHC) haplotype on bone marrow-derived antigen pre-senting cells intrathymically [10]. Establishment of allo-geneic hematopoietic chimerism in combination with anallogeneic islet graft of the same donor strain can perma-nently reverse type 1 diabetes in both normal mice withchemically-induced diabetes, and NOD mice with autoim-mune diabetes [18–21]. To date, the establishment of al-logeneic hematopoietic chimerism is the most effectivemethod known for generating transplantation tolerance inNOD mice.

Peripheral transplantation tolerance in NOD miceSeveral protocols can establish peripheral transplantationtolerance in normal, non-autoimmune mice [4], but mostwork poorly in the NOD mouse. Based on studies we per-formed using donor-specific transfusion and anti-CD154mAb [22,23], we initially hypothesized that this outcomewas due to a generalized defect in the response of NODmice to tolerance induction. When treated with protocols

Autoimmunity and Transplantation Tolerance 257

that induce prolonged or even permanent survival of al-lografts in non-autoimmune strains, NOD mice rapidlyreject not only islet but also skin allografts [22,24].

Genetic and Cellular Basis of Resistanceto Transplantation Tolerance vs. Expressionof Autoimmunity in NOD Mice

The cellular basis for the resistance of NOD mice to trans-plantation tolerance induction was thought to result fromtheir autoimmunity. This concept seemed attractive, asthere are multiple immune defects that could be involvedin both the autoimmune phenotype and resistance to trans-plantation tolerance phenotype (Table 1). These two NODphenotypes have usually been thought to be: (1) controlledby the same genes (2) mediated by similar cellular abnor-malities, and (3) result from the ongoing autoimmunity(Fig. 1A).

An alternative hypothesis is that these two phenotypesare: (1) controlled by different or only partially overlap-ping sets of genes, (2) mediated by different sets of cellu-lar abnormalities, and (3) not due to a common underlyingpredisposition to autoimmunity (Fig. 1B). These compet-ing hypotheses have in the past been difficult to evaluateexperimentally, but newer analytic tools are now available.We can now begin to identify the genetic and cellular sim-ilarities and differences associated with autoimmune dia-

Fig. 1. Schematic diagram depicting the contribution of genetic andcellular defects to autoimmunity and resistance to transplantationtolerance in NOD mice. Panel A depicts the conventional view that theIdd loci are responsible for a set of cellular immunological defects thatcause both autoimmune diabetes and resistance to transplantationtolerance. These cellular abnormalities with a single genetic origincould cause transplantation tolerance resistance either directly or as asecondary consequence of the diabetes. Panel B offers an alternativeview based on new published and preliminary data. This view proposesthat different sets of genes contribute independently to the twophenotypes and that the ongoing autoimmune process does not controltransplantation tolerance. The model does not exclude the possibility,as indicated by question marks, that the gene sets and the cellularabnormalities they cause overlap.

betes and resistance to transplantation tolerance inductionin NOD mice. New data suggest that the two phenotypescan be genetically separated.

Cellular mechanisms of autoimmunityand transplantation tolerance

Effector CD4+ and CD8+ T cells in autoimmunediabetes and transplantation tolerance. AutoreactiveCD4+ and CD8+ T cell clones that can adoptively transferdiabetes to naıve recipients have been derived from NODmice [6,10,25]. Interventions that impair the developmentof these autoreactive T lymphocytes or lead to their dele-tion in vivo decrease the incidence of diabetes [6,10].

In transplantation tolerance induction, deletion of al-loreactive CD8+ T cells is required to prolong allograftsurvival [8,26]. In addition, we have recently observed thatmost alloreactive CD4+ T cells are also deleted by cos-timulation blockade (TGM, unpublished observations). InNOD mice, mechanisms important for deletion of autore-active and alloreactive T cells may be similar and mayreflect an underlying genetic defect in T cell apoptosis.

Regulatory CD4+ T cells in autoimmune diabetes andtransplantation tolerance. Regulatory T cells that ex-press the CD4+CD25+ phenotype are believed to playa critical role in the prevention of autoimmune diabetes[27,28]. These cells are absent from both CD28 knockoutand B7-1/B7-2 knockout mice, and spontaneous diabetesis exacerbated in both B7-1/B7-2-deficient and CD28-deficient NOD mice. Transfer of this regulatory T cellsubset from control NOD animals into CD28-deficientanimals can delay or prevent diabetes [28]. The resultssuggest that the CD28/B7 costimulatory pathway is es-sential for the development and homeostasis of regulatoryT cells that control spontaneous autoimmune diseases. Abrief course of anti-CD3 mAb also reverses hyperglycemiain NOD mice permanently, presumptively due to the in-duction of regulatory T cells [17]. Interestingly, data froma clinical trial of a humanized anti-CD3 mAb in new on-set Type 1 diabetes suggest that this treatment prolongsthe “honeymoon period” [29]. Although based on cor-relations, these studies suggest that regulatory CD4+ Tcells, particularly CD4+CD25+ T cells, are important inautoimmune diabetes.

CD4+ T cells are also important in transplantation tol-erance. In mice tolerized with a donor-specific transfu-sion (DST) plus anti-CD154 mAb, depletion of CD4+

T cells leads to rapid rejection of skin allografts [9,30].Mice genetically deficient in CD4+ T cells are also re-sistant to costimulation blockade-based transplantationtolerance [9]. In vitro studies have demonstrated thatalloantigen tolerance induced by costimulatory blockade

258 Pearson et al.

is maintained by CD4+CD25+ T cells [31]. In another re-cent report [32], peripheral transplantation tolerance wasshown to depend on a population of CD4+CD25+ T cellsthat regulate the activity of CD154-independent CD8+ Tcells. The relationship of the CD4+ T cells that regulateautoimmune diabetes and those that regulate transplanta-tion tolerance is not known.

NKT cells in autoimmune diabetes and transplantationtolerance. NKT cells are hypothesized to be importantin the pathogenesis of type 1 diabetes in both humans [33]and NOD mice [34]. Their importance in NOD mice hasbeen demonstrated by genetic disruption of CD1, whichresults in the absence of CD1-restricted invariant NKTcells [35]. In these mice, diabetes occurs at an earlier ageand with increased frequency in association with an in-creased number of activated and memory autoreactive Tcells. Congenic introgression of a resistance variant ofthe Idd6 locus, which includes the NKRP-1 complex, im-proves NKT cell function and protects some animals fromdiabetes [36]. Administration of the NKT agonist α galac-tosyl ceramide (αGalCer) dramatically reduces the fre-quency of diabetes in NOD mice [34]. αGalCer also pre-vents autoimmune recurrence in spontaneously diabeticmice transplanted with syngeneic islet grafts. Proposedmechanisms of action include 1) induction of regulatorycytokines [34] and 2) NKT cell induction of tolerogenicdendritic cells in draining pancreatic lymph nodes [37].

NKT cells may also be important in transplantationtolerance. NKT cell deficient mice rapidly reject cornealallografts [38]. Mice deficient in NKT cells are also resis-tant to a transplantation tolerance induction protocol basedon anti-LFA-1 and anti-B7.1/2 antibodies [39]. WhetherNKT cell defects in NOD mice are responsible for theirunderlying resistance to transplantation tolerance is notknown.

The role of T cell apoptosis in autoimmune diabetesand transplantation tolerance. The CD4+ T cells ofNOD mice appear to respond abnormally to apoptoticsignals, whether in the form of death-by-neglect or ac-tivation induced cell death. IL-2 deprivation, for example,fails to induce apoptosis and is associated with upregu-lation of Bcl-x [40]. In another study, maximally stim-ulated NOD CD4 T cells failed to undergo activation-induced death, a critical mechanism for the regulation ofperipheral T cell tolerance [41]. Interventions based onovercoming this resistance and inducing apoptosis in Tcells can prevent spontaneous and recurrent autoimmu-nity in these animals [15,42]. Combination pro-apoptotictherapy using sirolimus and IL-2 that is effective in theNOD mouse [15] is under consideration for use in hu-

mans (www.immunetolerance.org/). Apoptosis of acti-vated CD8+ T cells also is important for the inductionof costimulation blockade-based transplantation toler-ance, and interventions that prevent CD8+ T cell deletionabrogate allograft survival [8,26,30].

Dendritic cells in autoimmune diabetes and trans-plantation tolerance. Dendritic cells regulate both self-tolerance and immunity. Abnormalities of NOD mousedendritic cells include impaired maturation [43] and ahyper-stimulatory capacity of the minority that do ma-ture [44,45]. NOD dendritic cells also express low levelsof CD86, which could lead to impaired self-tolerance dueto inability to upregulate CTLA-4 on CD4+ T cells [46].Adoptive transfer of either freshly isolated or in vitro cul-tured syngeneic bone marrow-derived dendritic cells pre-vents diabetes in NOD mice [10,37,47].

In transplantation studies, adoptive transfer of imma-ture donor dendritic cells can prolong allograft survival[48]. Dendritic cells that ingest apoptotic cells are tolero-genic, produce low levels of IL-12 upon stimulation, andhave a reduced capacity to stimulate T cell proliferation[49,50]. The genetic basis for the dendritic cell abnormal-ities in NOD mice is unknown, as is their precise role inautoimmunity and resistance to transplantation tolerance.

Genetic mechanisms of transplantation toleranceand autoimmunity

Genetic strain background and transplantation toler-ance. Responses to costimulation blockade-based trans-plantation tolerance vary widely among inbred mousestrains [51], and studies in recombinant inbred strainssuggest that multiple loci control susceptibility to trans-plantation tolerance [51]. We are using a genome widescanning approach to identify chromosomal regions thatassociate with the resistance of NOD mice to transplan-tation tolerance. This approach, similar to that used toidentify diabetes-associated loci in NOD mice [52], willreveal whether the genes that control autoimmune diabetesin NOD mice are the same, partially overlapping, or dif-ferent from those that control resistance to transplantationtolerance (Fig. 1).

Idd loci and autoimmunity. Investigators have analyzedin detail the genetic loci (designated Idd loci) associ-ated with diabetes susceptibility and resistance in NODmice [6,10]. The analyses reveal at least 27 loci on15 different chromosomes that associate with diabetesor insulitis (the inflammatory lesion of islets that pre-cedes and accompanies diabetes onset). The majority ofdiabetes susceptibility alleles are of NOD origin, but

Autoimmunity and Transplantation Tolerance 259

Table 2. Idd loci and candidate genes potentially involved inautoimmunity and transplantation tolerance

Locus Candidate genes

Candidate genes intransplantationtolerance

Idd1 MHC class II and class I genes Unknown; may conferresistance

Idd3 Il2, Fgf2, Il21 Il2Idd5.1 Cd152 (CTLA-4), Cd28 Cd152, Cd28Idd5.2 Nramp1, Cmkar2 UnknownIdd6 Nkrp1 UnknownIdd9.1 Lck LckIdd9.2 Tnfr2, Cd30 Tnfr2Idd9.3 Cd137 (4-1BB) Cd137Idd10 Csfm, Cd53, Kcna2, Nras,

Rap1aUnknown

Idd13 B2m (β2 microglobulin) UnknownIdd16 Proximal to MHC class I K

geneUnknown

Idd17 Unknown UnknownIdd18 Csfm, Cd53, Kcna3, Nras,

Rap1aUnknown

Idd loci, candidate genes within the Idd loci, and their relationship, if known, in

transplantation tolerance.

strains free of autoimmunity can harbor susceptibilityalleles, and the NOD mouse harbors some diabetes re-sistance loci [6]. Many NOD Idd loci have human or-thologues, including the most powerful susceptibility lo-cus, Idd1, which maps to the MHC [6,52]. Other non-MHC Idd susceptibility loci have been mapped in detailand include candidate genes important in immune func-tion [52]. Identifying the pathogenic genes with Idd locihas proven difficult; to date β2 microglobulin (β2M) isthe only non-MHC gene documented to be an Idd gene[53]. Table 2 lists some of the many other candidategenes.

Idd congenic mice, autoimmune diabetes, and transplan-tation tolerance. We have recently shown that a numberof non-MHC Idd resistance loci that profoundly attenuatethe expression of diabetes do not improve the responseof NOD mice to transplantation tolerance induction [23].This finding suggests that, even if the mechanisms of self-tolerance and transplantation tolerance involve the samecell types, the underlying basis for these cellular abnor-malities may be different. In genetic terms, we hypothesizethat the genes controlling the autoimmune phenotype inNOD mice are not completely overlapping, and may bedifferent from those controlling the phenotype of resis-tance to transplantation tolerance.

(NOD × C57BL/6)F1 mice, autoimmune diabetes, andtransplantation tolerance. As one approach to test the

above hypothesis, we assessed the response of dia-betes resistant, (NOD × C57BL/6)F1 mice to costimula-tion blockade-based transplantation tolerance induction.These animals are completely free of both diabetes andinsulitis and are resistant to cyclophosphamide-induceddiabetes. (NOD × C57BL/6)F1 mice are heterozygous atall Idd alleles, and the great majority of susceptibility al-leles are recessive [52]. Surprisingly, we observed that,following tolerance induction with DST and anti-CD154mAb, skin allograft survival on (NOD × C57BL/6)F1mice is only slightly longer that on tolerized NOD miceand much shorter than on tolerized C57BL/6 mice [54].We have also found that both dendritic cell maturation andthe response of CD4+ and CD8+ T cells to costimulationblockade are abnormal in (NOD × C57BL/6)F1 mice. Incontrast, defects in macrophage maturation and NK cellactivity are normal in (NOD × C57BL/6)F1 mice [54].At the cellular level, these promising data imply that atleast two immune defects (in macrophages and NK cells)potentially associated with diabetes susceptibility havebeen dissected away from the resistance of NOD miceto transplantation tolerance. At the genetic level, theseobservations have at least partially separated the autoim-mune phenotype from the transplantation tolerance phe-notype in NOD mice. The data further imply that autoim-mune diabetes in (NOD × C57BL/6)F1 mice is a reces-sive trait whereas resistance to transplantation tolerance isdominant.

Summary

Published and preliminary data generated in NOD, NODIdd congenic, and (NOD × C57BL/6)F1 mice suggest thatan autoimmune diabetes phenotype and a resistance totransplantation tolerance phenotype may be co-expressedbut are not under identical genetic control. We cannotas yet determine if (1) separate genes or (2) differingcombinations of the same genes control these two phe-notypes. It is clear, however, that if Idd loci also controlresistance to transplantation tolerance, that the “thresh-old” for prevention of autoimmunity is much lower thanthe threshold for induction of transplantation tolerance.The available data suggest that at least some genes maydiffer between autoimmunity and transplantation toler-ance (Fig. 1B) and that many of the cell types involved inboth processes are likely to be same (Table 1). If autoim-mune diabetes and transplantation tolerance induction areshown to have different genetic bases, it would suggestthe strategies required for treating autoimmune diabetesand preventing recurrent autoimmunity may need to bedifferent from those required for inducing transplantationtolerance.

260 Pearson et al.

Acknowledgments

Supported in part by grants AR35506 and AI42669 andan institutional Diabetes Endocrinology Research Center(DERC) grant DK52530 from the National Institutes ofHealth and by grant DK53006 jointly funded by the Na-tional Institutes of Health and the Juvenile Diabetes Re-search Foundation (JDRF). LSW is supported by a jointgrant from the JDRF and the Wellcome Trust. DVS issupported by grants DK46266 and DK51090 from theNational Institutes of Health and by a grant from theJDRF. The availability of NOD congenic mice throughthe Taconic Farms Emerging Models Program has beensupported by grants from the Merck Genome ResearchInstitute, NIAID and the JDRF. The contents of this pub-lication are solely the responsibility of the authors and donot necessarily represent the official views of the NationalInstitutes of Health.

References

1. Gabir MM, Hanson RL, Dabelea D, Imperatore G, Roumain J,Bennett PH, Knowler WC. The 1997 American Diabetes Asso-ciation and 1999 World Health Organization criteria for hyper-glycemia in the diagnosis and prediction of diabetes. Diab Care2000;23:1108–1112.

2. American Diabetes Association: Implications of the Diabetes Con-trol and Complications Trial. Diab Care 2002;25(suppl. 1):S27.

3. Shapiro AMJ, Lakey JRT, Ryan EA, Korbutt GS, Toth E, WarnockGL, Kneteman NM, Rajotte RV. Islet transplantation in seven pa-tients with type 1 diabetes mellitus using a glucocorticoid-free im-munosuppressive regimen. N Engl J Med 2000;343:230–238.

4. Rossini AA, Greiner DL, Mordes JP. Induction of immunologicaltolerance for transplantation. Physiol Rev 1999;79:99–141.

5. Sutherland DE, Sibley R, Xu XZ, Michael A, Srikanta AM, TaubF, Najarian J, Goetz FC. Twin-to-twin pancreas transplantation: Re-versal and reenactment of the pathogenesis of type I diabetes. TransAssoc Am Physicians 1984;97:80–87.

6. Mordes JP, Serreze DV, Greiner DL, Rossini AA. Animal modelsof autoimmune diabetes mellitus. Diabetes mellitus. A fundamentaland clinical text 2003, in press.

7. Greiner DL, Rossini AA, Mordes JP. Translating data from ani-mal models into methods for preventing human autoimmune dia-betes mellitus: caveat emptor and primum non nocere. Clin Immunol2001;100:134–143.

8. Iwakoshi NN, Mordes JP, Markees TG, Phillips NE, Greiner DL,Rossini AA. Treatment of allograft recipients with donor specifictransfusion and anti-CD154 antibody leads to deletion of alloreactiveCD8+ T cells and prolonged graft survival in a CTLA4-dependentmanner. J Immunol 2000;164:512–521.

9. Markees TG, Phillips NE, Gordon EJ, Noelle RJ, Shultz LD, MordesJP, Greiner DL, Rossini AA. Long-term survival of skin allo-grafts induced by donor splenocytes and anti-CD154 antibody inthymectomized mice requires CD4+ T cells, interferon-gamma, andCTLA4. J Clin Invest 1998;101:2446–2455.

10. Serreze DV, Leiter EH. Genes and pathways underlying autoimmunediabetes in NOD mice. In: Molecular Pathology of Insulin Depen-dent Diabetes Mellitus. Von Herrath MG, ed. New York: KargerPress, 2001:31–67.

11. Atkinson M, Leiter EH. The NOD mouse model of insulin dependentdiabetes: As good as it gets? Nature Med 1999;5:601–604.

12. Kishimoto H, Sprent J. A defect in central tolerance in NOD mice.Nat Immunol 2001;2:1025–1031.

13. Delovitch TL, Singh B. The nonobese diabetic mouse as a model ofautoimmune diabetes: Immune dysregulation gets the NOD. Immu-nity 1997;7:727–738.

14. Bach JF. Immunotherapy of insulin-dependent diabetes mellitus.Curr Opinion Immunol 2001;13:601–605.

15. Rabinovitch A, Suarez-Pinzon WL, Shapiro AMJ, Rajotte RV,Power R. Combination therapy with sirolimus and interleukin-2 pre-vents spontaneous and recurrent autoimmune diabetes in NOD mice.Diabetes 2002;51:638–645.

16. Shapiro AMJ, Suarez-Pinzon WL, Power R, Rabinovitch A. Combi-nation therapy with low dose sirolimus and tacrolimus is synergisticin preventing spontaneous and recurrent autoimmune diabetes innon-obese diabetic mice. Diabetologia 2002;45:224–230.

17. Chatenoud L, Primo J, Bach JF. CD3 antibody-induced domi-nant self tolerance in overtly diabetic NOD mice. J Immunol1997;158:2947–2954.

18. Seung E, Iwakoshi N, Woda BA, Markees TG, Mordes JP, RossiniAA, Greiner DL. Allogeneic hematopoietic chimerism in micetreated with sublethal myeloablation and anti-CD154 antibody: Ab-sence of graft-versus-host disease, induction of skin allograft toler-ance, and prevention of recurrent autoimmunity in islet-allograftedNOD/Lt mice. Blood 2000;95:2175–2182.

19. Sykes M. Mixed chimerism and transplant tolerance. Immunity2001;14:417–424.

20. Leykin I, Nikolic B, Sykes M. Mixed bone marrow chimerism asa treatment for autoimmune diabetes. Transplant Proc 2001;33:120.

21. Li H, Kaufman CL, Ildstad ST. Allogeneic chimerism induces donor-specific tolerance to simultaneous islet allografts in nonobese dia-betic mice. Surgery 1995;118:192–198.

22. Markees TG, Serreze DV, Phillips NE, Sorli CH, Noelle RJ, WodaBA, Greiner DL, Mordes JP, Rossini AA. NOD mice have a general-ized defect in their response to transplantation tolerance induction.Diabetes 1999;48:967–974.

23. Pearson T, Markees TG, Wicker LS, Serreze DV, Peterson LB,Mordes JP, Rossini AA, Greiner DL. NOD congenic mice geneti-cally protected from autoimmune diabetes remain resistant to trans-plantation tolerance induction. Diabetes 2003;52:321–326.

24. Molano RD, Berney T, Li H, Cattan P, Pileggi A, Vizzardelli C,Kenyon NS, Ricordi C, Burkly LC, Inverardi L. Prolonged islet graftsurvival in NOD mice by blockade of the CD40-CD154 pathway ofT-cell costimulation. Diabetes 2001;50:270–276.

25. Haskins K, Wegmann D. Diabetogenic T-cell clones. Diabetes1996;45:1299–1305.

26. Li XC, Wells AD, Strom TB, Turka LA. The role of T cell apoptosisin transplantation tolerance. Curr Opinion Immunol 2000;12:522–527.

27. Green EA, Choi Y, Flavell RA. Pancreatic lymph node-derivedCD4(+)CD25(+) Treg cells: Highly potent regulators of diabetesthat require TRANCE-RANK signals. Immunity 2002;16:183–191.

28. Salomon B, Lenschow DJ, Rhee L, Ashourian N, Singh B, SharpeA, Bluestone JA. B7/CD28 costimulation is essential for the home-ostasis of the CD4+CD25+ immunoregulatory T cells that controlautoimmune diabetes. Immunity 2000;12:431–440.

29. Herold KC, Hagopian W, Auger JA, Poumian-Ruiz E, Taylor L,Donaldson D, Gitelman SE, Harlan DM, Xu DL, Zivin RA,Bluestone JA. Anti-CD3 monoclonal antibody in new-onset type1 diabetes mellitus. N Engl J Med 2002;346:1692–1698.

30. Iwakoshi NN, Markees TG, Turgeon NA, Thornley T, Cuthbert A,Leif JH, Phillips NE, Mordes JP, Greiner DL, Rossini AA. Skin

Autoimmunity and Transplantation Tolerance 261

allograft maintenance in a new synchimeric model system of toler-ance. J Immunol 2001;167:6623–6630.

31. Taylor PA, Noelle RJ, Blazar BR. CD4+CD25+ immune regu-latory cells are required for induction of tolerance to alloanti-gen via costimulatory blockade. J Exp Med 2001;193:1311–1317.

32. van Maurik A, Herber M, Wood KJ, Jones ND. Cutting edge:CD4+CD25+ alloantigen-specific immunoregulatory cells thatcan prevent CD8+ T cell-mediated graft rejection: Implica-tions for anti-CD154 immunotherapy. J Immunol 2002;169:5401–5404.

33. Wilson SB, Kent SC, Horton HF, Hill AA, Bollyky PL, Hafler DA,Strominger JL, Byrne MC. Multiple differences in gene expressionin regulatory Vα24JαQ T cells from identical twins discordant fortype 1 diabetes. Proc Natl Acad Sci USA 2000;97:7411–7416.

34. Sharif S, Arreaza GA, Zucker P, Mi QS, Delovitch TL. Regula-tion of autoimmune disease by natural killer T cells. J Mol Med2002;80:290–300.

35. Shi FD, Flodstrom M, Balasa B, Kim SH, Van Gunst K, StromingerJL, Wilson SB, Sarvetnick N. Germ line deletion of the CD1 locusexacerbates diabetes in the NOD mouse. Proc Natl Acad Sci USA2001;98:6777–6782.

36. Carnaud C, Gombert JM, Donnars O, Garchon HJ, Herbelin A.Protection against diabetes and improved NW/NKT cell perfor-mance in NOD.NK1.1 mice congenic at the NK complex. J Immunol2001;166:2404–2411.

37. Naumov YN, Bahjat KS, Gausling R, Abraham R, Exley MA,Koezuka Y, Balk SB, Strominger JL, Clare-Salzler M, Wilson SB.Activation of CD1d-restricted T cells protects NOD mice from de-veloping diabetes by regulating dendritic cell subsets. Proc NatlAcad Sci USA 2001;98:13838–13843.

38. Sonoda KH, Faunce DE, Taniguchi M, Exley M, Balk S, Stein-Streilein J. NK T cell-derived IL-10 is essential for the differenti-ation of antigen-specific T regulatory cells in systemic tolerance.J Immunol 2001;166:42–50.

39. Seino KI, Fukao K, Muramoto K, Yanagisawa K, Takada Y, KakutaS, Iwakura Y, Van Kaer L, Takeda K, Nakayama T, Taniguchi M,Bashuda H, Yagita H, Okumura K. Requirement for natural killer T(NKT) cells in the induction of allograft tolerance. Proc Natl AcadSci USA 2001;98:2577–2581.

40. Lamhamedi-Cherradi SE, Luan JJ, Eloy L, Fluteau G, Bach JF,Garchon HJ. Resistance of T-cells to apoptosis in autoimmunediabetic (NOD) mice is increased early in life and is associ-ated with dysregulated expression of Bcl-x. Diabetologia 1998;41:178–184.

41. Noorchashm H, Moore DJ, Noto LE, Noorchashm N, Reed AJ,Reed AL, Song HK, Mozaffari R, Jevnikar AM, Barker CF, NajiA. Impaired CD4 T cell activation due to reliance upon B cell-

mediated costimulation in nonobese diabetic (NOD) mice. J Im-munol 2000;165:4685–4696.

42. Zheng XX, Steele AW, Hancock WW, Kawamoto K, Li XC,Nickerson PW, Li YS, Tian Y, Strom TB. IL-2 receptor-targetedcytolytic IL-2/Fc fusion protein treatment blocks diabetogenic au-toimmunity in nonobese diabetic mice. J Immunol 1999;163:4041–4048.

43. Prasad SJ, Goodnow CC. Cell-intrinsic effects of non-MHC NODgenes on dendritic cell generation in vivo. Int Immunol 2002;14:677–684.

44. Poligone B, Weaver DJ Jr, Sen P, Baldwin AS Jr, Tisch R. ElevatedNF-kappaB activation in nonobese diabetic mouse dendritic cellsresults in enhanced APC function. J Immunol 2002;168:188–196.

45. Steptoe RJ, Ritchie JM, Harrison LC. Increased generation ofdendritic cells from myeloid progenitors in autoimmune-pronenonobese diabetic mice. J Immunol 2002;168:5032–5041.

46. Dahlen E, Hedlund G, Dawe K. Low CD86 expression in thenonobese diabetic mouse results in the impairment of both T cellactivation and CTLA-4 up-regulation. J Immunol 2000;164:2444–2456.

47. Feili-Hariri M, Dong X, Alber SM, Watkins SC, Salter RD, MorelPA. Immunotherapy of NOD mice with bone marrow-derived den-dritic cells. Diabetes 1999;48:2300–2308.

48. Thomson AW, Lu L. Are dendritic cells the key to liver transplanttolerance? Immunol Today 1999;20:27–32.

49. Lutz M, Schuler G. Immature, semi-mature and fully mature den-dritic cells: Which signals induce tolerance or immunity? TrendsImmunol 2002;23:445.

50. Steinman RM, Turley S, Mellman I, Inaba K. The induction of tol-erance by dendritic cells that have captured apoptotic cells. J ExpMed 2000;191:411–416.

51. Williams MA, Trambley J, Ha J, Adams AB, Durham MM, ReesP, Cowan SR, Pearson TC, Larsen CP. Genetic characterization ofstrain differences in the ability to mediate CD40/CD28-independentrejection of skin allografts. J Immunol 2000;165:6849–6857.

52. Todd JA, Wicker LS. Genetic protection from the inflammatorydisease type 1 diabetes in humans and animal models. Immunity2001;15:387–395.

53. Hamilton-Williams EE, Serreze DV, Charlton B, Johnson EA,Marron MP, Mullbacher A, Slattery RM. Transgenic rescue impli-cates β2-microglobulin as a diabetes susceptibility gene in nonobesediabetic (NOD) mice. Proc Natl Acad Sci USA 2001;98:11533–11538.

54. Pearson T, Markees TG, Serreze DV, Pierce MA, Marron MP, WickerLS, Peterson LB, Shultz LD, Mordes MP, Rossini AA, Greiner DL.Genetic disassociation of autoimmunity and resistance to costimu-lation blockade-induced transplantation tolerance in nonobese dia-betic mice. J Immunol 2003;171:in press.