T1D (Type 1 diabetes) is an autoimmune disease caused by the immune-mediated destruction of pancreatic β-cells. Studies in T1D patients have been limited by the availability of pancreatic samples, a protracted pre-diabetic phase and limitations in markers that reflect β-cell mass and function. The NOD (non-obese diabetic) mouse is currently the best available animal model of T1D, since it develops disease spontaneously and shares many genetic and immunopathogenic features with human T1D. Consequently, the NOD mouse has been extensively studied and has made a tremendous contribution to our understanding of human T1D. The present review summarizes the key lessons from NOD mouse studies concerning the genetic susceptibility, aetiology and immunopathogenic mechanisms that contribute to autoimmune destruction of β-cells. Finally, we summarize the potential and limitations of immunotherapeutic strategies, successful in NOD mice, now being trialled in T1D patients and individuals at risk of developing T1D.

INTRODUCTION

Experiments using NOD (non-obese diabetic) mice have paved the way for significant advances in the current understanding of T1D (Type 1 diabetes). T1D is an autoimmune disorder in which the insulin-producing β-cells of the pancreas are selectively destroyed. In this condition, inflammatory infiltrates, mainly comprising DCs (dendritic cells), macrophages and B- and T-lymphocytes, invade the islets. The progressive loss of β-cells ultimately causes insulin deficiency and consequent hyperglycaemia. Despite exogenous insulin administration, many patients develop debilitating microvascular and macrovascular complications, leading to increased morbidity and mortality.

Investigation into the genetics and immunopathological mechanisms that lead to initial progression and β-cell destruction in individuals at risk of developing T1D is difficult due to a protracted pre-diabetic phase, inaccessibility of islet tissue and limitations in β-cell markers that reflect cell mass and functionality. Although GWAS (genome-wide association studies) have identified more than 40 distinct susceptibility regions linked to T1D that may assist in the screening for those at risk of developing T1D, a comprehensive understanding of the immunopathological mechanisms underlying the development of T1D is also required. The knowledge gained from such studies will help develop immunomodulatory strategies to prevent the onset of disease in genetically susceptible individuals. Accordingly, our understanding of the genetics, aetiology and pathogenesis of T1D has heavily depended on studies using the NOD mouse strain that spontaneously develops T1D. The present review will discuss the lessons learned from NOD mice in determining the mechanisms underlying T1D susceptibility and pathogenesis, as well as the value and limitations of this model.

THE NOD MOUSE MODEL

Genetics

The inbred NOD mouse strain originated as a hyperglycaemic sub-strain of the CTS (cataract-prone mouse) at the Shionogi Laboratories, Fukushima-ku, Japan [1]. At the time of weaning, NOD mice develop around pancreatic islets a mononuclear cell infiltrate (insulitis) that progresses at approximately 100 days of age to invasive insulitis and complete β-cell destruction [2]. Although NOD mice have an increased genetic susceptibility to T1D, the penetrance of disease can be modulated by various environmental factors. Hence not all NOD mice in a colony will develop T1D. Importantly, a number of T1D-susceptibility genes identified in NOD mice (designated Idd) have been found to contribute to T1D susceptibility in humans (designated IDDM). To date >40 Idd regions have been identified [3,4], but only a small number of these regions have been localized to candidate genes, and these will be discussed below.

The MHC is the strongest susceptibility region and was the first identified in both humans (IDDM1) and in mice (Idd1). The mouse MHC region is located on chromosome 17 and contains a number of genes known to contribute to disease. These genes make up a haplotype that contains both MHC class I- and class II-susceptibility genes. In mice, MHC class I genes comprise Kd and Db alleles and MHC class II genes comprise I-Ag7 and I-Eb alleles [5,6]. Although NOD mice express the I-A heterodimer that is composed of I-Aα and I-Aβ chains, the I-E heterodimer is not expressed due to a deletion in the promoter region of the I-Eα gene. Furthermore, replacement of histidine and serine residues with proline and aspartic acid residues at positions 56 and 57 respectively within the I-Aβ chain prevents T1D in NOD mice [79].

The Idd3 locus encodes the cytokines IL (interleukin)-2 and IL-21, and these are strong candidate genes for T1D susceptibility [1012]. In NOD mice, IL-2 expression levels are abnormally low, with low-dose IL-2 treatment reducing the severity of insulitis and inhibiting T1D onset [13,14]. Further reduction in IL-2 levels in NOD mice heterozygous for a deletion of the IL-2 gene accelerated T1D development [15]. In contrast, IL-21 is highly expressed in NOD mice and NOD mice deficient in the IL-21 receptor are protected from T1D [12,16].

The Idd5.1 locus includes two candidate susceptibility genes encoding CTLA-4 (cytotoxic T-lymphocyte-associated antigen-4) and ICOS (inducible T-cell co-stimulator). CTLA-4, a receptor involved in inhibiting T-cell activation, is present in four distinct isoforms in NOD mice, one of which is the ligand-independent CTLA-4 isoform (liCTLA-4). It contains a SNP (single nucleotide polymorphism) in exon 2 that causes liCTLA-4 to be expressed at reduced levels, decreasing the activation threshold of T-cells and consequently increasing disease susceptibility. Similarly, expression levels of the sCTLA-4 (soluble CTLA-4) isoform are reduced in T1D patients [17]. CTLA-4 therefore represents an attractive target for immunotherapeutic intervention. The ICOS gene in NOD mice has a SNP encoding a non-conservative amino acid change in the leader sequence of exon 1. This change causes higher expression of ICOS, which heightens T-cell co-stimulation. Consistent with the idea that increased expression of ICOS contributes to susceptibility, ICOS−/− NOD mice are protected from T1D [18,19].

The Idd7 locus contains a gene, or several linked genes, thought to influence allelic exclusion of TCR (T-cell receptor) genes during T-cell development [20]. These studies were performed in TCR transgenic mice and may not represent the normal path of TCR rearrangement found in human T-cells. However, if such defects are present, this could lead to dual TCR expression and autoimmunity [21].

The Idd9 locus contains three regions: Idd9.1, Idd9.2 and Idd9.3 [22]. Although the genes localized at Idd9.1 are unknown, they are associated with increased B-cell pathogenic activity [23], low numbers of induced iNKT cells [invariant NKT cells (natural killer T-cells)] and reduced Treg cell (regulatory T-cell) development and activity in NOD mice [24,25]. Candidate susceptibility genes at Idd9.2 and Idd9.3 encode CD30, TNFR2 (tumour necrosis factor receptor 2) and CD137 respectively [22].

The Idd13 locus contains multiple susceptibility genes, including the candidate genes β2-microglobulin (B2m), Cd93, Nkt2 and Bim [26,27,3032]. In inbred mouse populations, there are three allelic variants of B2m that encode isoforms differing by a single amino acid at residue 85 [28,29]. Wild-type NOD mice express the B2ma isoform, whereas NOD mice congenic for the NOR (non-obese-resistant) Idd13 region express the B2mb isoform and are protected from T1D. B2m was confirmed as a diabetes-susceptibility gene in reconstitution experiments in which NOD mice lacking endogenous B2m and transgenic for B2mb were protected from the development of T1D compared with mice transgenic for B2ma [30]. The mechanism of protection conferred by different B2m isoforms has not been elucidated, but is proposed to relate to the expression level of MHC class I (R.M. Slattery, unpublished work). As such, although humans are non-polymorphic at the B2m loci, there may be related changes in the expression level of MHC class I that influence antigen presentation, thereby modulating thymic selection and/or peripheral activation of CD8+ T-cells [26]. In NOD mice, the Cd93 gene has a SNP that results in a conformational change in the CD93 protein [27]. Although the function of this protein is not yet well defined, its absence in C57Bl/6 (B6) CD93−/− mice results in a reduced number of iNKT cells, which may promote T1D in NOD mice [27]. A third gene within the Idd13 region that controls NKT cell numbers has been mapped to the Nkt2 gene. NOD mice congenic for the B6 Nkt2 region had increased NKT cell numbers and a reduced incidence of T1D [31]. The fourth candidate susceptibility gene to be localized within the Idd13 locus is Bim, which encodes the pro-apoptotic protein BIM. The failure to induce BIM in thymocytes confers resistance to thymic deletion in NOD mice [32].

Of the >40 susceptibility loci identified in NOD mice only a small number of these regions contain genes that have orthologues associated with human T1D. Nevertheless, a number of the non-orthologous candidate susceptibility genes in NOD mice have led to studies that have provided valuable insights into the immunopathogenic mechanisms of T1D relevant to humans, and these are discussed later in the review.

Environment

Geography

Various environmental parameters have been associated with T1D susceptibility in humans. They include geographical location, dietary components and infectious agents. Worldwide incidence data on T1D suggests that there is an inverse correlation between disease incidence and proximity to the equator that could be explained by a number of variables. Regions furthest from the equator have reduced exposure to sunlight with a concomitant reduction in both UV radiation and temperature. In human T1D it is difficult to segregate the roles of sunlight variables from the confounding co-variables of genetics and culture. However, genetically controlled studies utilizing inbred NOD mice have allowed the independent contribution of UV radiation and temperature on T1D to be assessed.

NOD mice maintained at a temperature of 23.7°C compared with 21°C had a lower incidence of T1D. This suggests that the inverse correlation between equatorial distance and incidence of T1D may in part be explained by temperature [33]. Since UV radiation is essential for epidermal vitamin D synthesis, and the active form of vitamin D 1,25 D3 (1,25-dihydroxyvitamin D3) influences the development of Treg cells, the effect of vitamin D on T1D has been studied in NOD mice. These studies revealed that early deficiency of vitamin D in NOD mice resulted in accelerated T1D development [34], where NOD mice administered supplementary vitamin D were protected from developing T1D, and this was correlated with an increased frequency of Treg cells within the pLN [pancreatic LN (lymph node)] [35,36]. It is thought that vitamin D also modulates the immune response through inhibition of the NF-κB (nuclear factor κB) pathway in DCs and macrophages. This results in decreased production of the pro-inflammatory cytokines IL-12 and IFNγ (interferon γ) that leads to decreased MHC class II expression on APCs (antigen-presenting cells) and MHC class I expression on β-cells [37]. Vitamin D supplementation at birth has been shown to significantly protect against T1D in humans [38]. However, supplementation of vitamin D in patients with recent-onset T1D failed to reduce the loss of β-cell function [39].

Diet

The earliest evidence for dietary influences in T1D susceptibility came from studies in NOD mice. A positive association was found between a high-fat diet and T1D incidence in these mice [40]. An increase in T1D progression in NOD mice has also been correlated with wheat- or corn-enriched diets and this has been attributed to the wheat protein gluten [41]. It has been speculated that a mechanism by which dietary antigens could influence susceptibility is by modulation of the mucosal immune system via the release of tolerogenic, allergenic or pro-inflammatory cytokines [42]. NOD mice fed on cereal/wheat-based diets expressed significantly higher levels of IFNγ, TNFα (tumour necrosis factor α) and iNOS (inducible NO synthase) that are known to drive the immune response towards T1D [42].

Conversely, EFAs (essential fatty acids) have been shown to protect from T1D. The offspring of mice fed on a low omega-6/omega-3 (n−6/n−3) EFA ratio diet showed a decrease in T1D incidence [43]. Similarly, omega-3 reduced the incidence of T1D in genetically susceptible children, indicating its benefit as a supplement to assist in the prevention of human T1D [44]. The protective effect of omega-3 may partly explain why T1D incidence is lower in Japan, where there is a high consumption of fish compared with countries with westernised diets [45]. One mechanism by which omega-3 may protect from T1D is through direct binding to GPR120 (G-protein-coupled receptor 120) on macrophages, which exerts a wide range of potent anti-inflammatory effects [46]. Additionally, omega-3 may act indirectly through the anti-inflammatory influence of bioactive products resulting from its breakdown and enzymatic conversion, such as resolvins [47].

Diet and the use of antibiotics can also modulate the gut microbial diversity, which has recently been shown to regulate the immune system. NOD mice housed under ‘specific germ-free’ (SGF) conditions have a higher incidence of T1D than those housed in a non-germ-free environment. Furthermore, NOD mice treated with probiotics have elevated IL-10 production and reduced T1D development [48]. The bacteria responsible for this protection are members of the Bacteroidetes phyla, which are capable of producing SCFAs (short-chain fatty acids). SCFAs can bind GPRs on immune cells and thereby mediate an anti-inflammatory response that protects from T1D [49]. Whether probiotics, or SCFA treatment, will prevent T1D in those at risk remains to be determined, although probiotics are currently being trialled in at-risk children [50]. In contrast, some bacterial strains promote inflammation and thereby exacerbate the development of T1D. Gluten-fed NOD mice contain greater quantities of aerobic, micro-aerophilic and caecal bacteria and are more likely to develop T1D than those fed on gluten-free diets [51]. Furthermore, a positive correlation was observed between the numbers of Gram-positive aerophilic and anaerobic bacteria found in the gut of NOD mice and the incidence of T1D, regardless of diet [51]. It is thought that such bacterial strains promote a pro-inflammatory cytokine environment that drives T1D by inducing mucosal DCs to secrete Th1-type cytokines.

Viruses

A number of viruses have been positively associated with T1D onset and several mechanisms have been proposed to explain this association [52]. Viral infection of the gastrointestinal mucosa with rotavirus increases the intestinal permeability, creating a ‘leaky gut’. Opportunistic gut bacteria that migrate through the leaky gut can induce inappropriate sub-mucosal immune responses that signal through TLRs (Toll-like receptors) and drive inflammation [53]. Other viruses, such as CVB (Coxsackie virus B), can directly infect islet tissue or neighbouring neuro-endocrine cells, promoting inflammation in both islets and associated ECs (endothelial cells) [54]. At the time of insulitis development, NOD mouse islet ECs develop an activated phenotype with up-regulation of adhesion molecules, such as CD54 [ICAM-1 (intercellular adhesion molecule-1)], CD106 [VCAM-1 (vascular cell adhesion molecule-1)] and of MHC class I and II molecules [54,55,55a]. Islet ECs from human histological samples taken during the peri-onset stage of T1D show a similar phenotype, displaying increased expression of adhesion and MHC molecules [56].

Another potential mechanism of virally induced T1D is molecular mimicry, whereby T-cells that are activated by specific viral antigens cross-react with AAgs (autoantigens) that share structural similarities. This model has been investigated for its capacity to trigger T1D in transgenic mice expressing the LCMV (lymphocytic choriomeningitis virus) antigen NP (nucleoprotein) or GP (glycoprotein). Mice expressing these antigens on pancreatic β-cells remained tolerant and free from T1D in the absence of viral infection [57]. However, when challenged with LCMV, NP- and GP-specific T-cells were activated and β-cells were killed, causing T1D [57]. Blocking or depletion of pro-inflammatory cytokines has been found to reduce T1D incidence in NOD mice following viral infection [58].

IMMUNOPATHOLOGY

Disease initiation

The inaccessibility of pAPCs (professional APCs) has made it challenging to measure their role in human T1D. Researchers rely heavily on studies in NOD mice to understand how pAPCs may promote T1D and, importantly, how their central role in tolerance may be exploited in immunotherapeutic approaches (Figure 1). pAPCs are the first cells to accumulate marginally around the islets, thereby initiating a cascade of immunopathological events that culminate in β-cell destruction and T1D [59,60]. The events triggering the accumulation of pAPCs in the islet milieu remain to be fully elucidated. It has been proposed that a wave of β-cell death occurs in NOD mice as part of tissue remodelling. This could occur either in response to viral infection or other metabolic changes around the time of weaning [61,62]. The migration of CCR7 (CC chemokine receptor 7)-positive monocyte/macrophages and DCs to the islets is promoted by the elevated expression of lymphoid-tissue-related chemokines, such as CCL19 (CC chemokine ligand 19), found in NOD mice [63]. Macrophages produce the cytokines TNFα and IL-1β that are directly β-cell toxic and over-produce IL-12, driving the further recruitment of DCs to the accumulated β-cell debris. This exacerbates the inflammatory response and leads to the inappropriate activation of autoreactive T-cells in NOD mice [64]. Furthermore, due to increased prostaglandin (PGE2) production, macrophages from NOD mice are impaired in their phagocytic ability and their ability to present self-antigens for the induction of tolerogenic immune responses [6567]. Therefore NOD mouse macrophages have an important role not only in the initiation of insulitis, but also in driving its progression towards β-cell destruction and T1D. On the basis of the important roles of IL-1β and TNFα in β-cell damage, children recently diagnosed with T1D have been treated with IL-1RAs (IL-1 receptor antagonists) and TNF antagonists in a number of different trials. Both treatments promoted small increases in insulin production within 4–5 months of treatment [68].

Key immunopathogenic features of β-cell destruction in NOD mice

Figure 1
Key immunopathogenic features of β-cell destruction in NOD mice

(1) The initial trigger that causes the release of β-cell antigens is unknown, but it has been proposed that viral infection and/or a wave of β-cell death may promote the initial recruitment of NK cells, DCs and macrophages to the islet milieu. NOD mouse macrophages have a reduced phagocytic ability and secrete increased levels of ROS, TNFα and IL-1β that promote β-cell death, as well as increased levels of the pro-inflammatory cytokines IFNγ and IL-12 that increase local MHC expression and promote DC activation. (2) Activated DCs migrate to the pLN, where they deliver β-cell antigen to resident APCs and T-cells. NOD mouse DCs have a reduced tolerogenic capacity due to decreased expression of PD-L1 and IDO, as well as increased expression of NF-κB, CD80/CD86, TNFα and IL-12, leading to activation of autoreactive CD4 and CD8 T-cells. Activated T-cells are expanded further by encounter with activated B-cells presenting β-cell antigen. (3) Activated lymphocytes migrate across a chemokine gradient from the inflamed islet associated vascular endothelium, expressing up-regulated HA, ICOS, VCAM-1, ICAM-1, MADCAM-1, MHC class I and MHC class II, into the islet tissue. Local activated APCs in the islet milieu continue to take up, process and present antigen, promoting the retention and expansion of CD8 and CD4 T-cells that kill β-cells via perforin/granzyme and Fas/FasL mechanisms respectively. RAE1, retinoic acid early transcript 1.

Figure 1
Key immunopathogenic features of β-cell destruction in NOD mice

(1) The initial trigger that causes the release of β-cell antigens is unknown, but it has been proposed that viral infection and/or a wave of β-cell death may promote the initial recruitment of NK cells, DCs and macrophages to the islet milieu. NOD mouse macrophages have a reduced phagocytic ability and secrete increased levels of ROS, TNFα and IL-1β that promote β-cell death, as well as increased levels of the pro-inflammatory cytokines IFNγ and IL-12 that increase local MHC expression and promote DC activation. (2) Activated DCs migrate to the pLN, where they deliver β-cell antigen to resident APCs and T-cells. NOD mouse DCs have a reduced tolerogenic capacity due to decreased expression of PD-L1 and IDO, as well as increased expression of NF-κB, CD80/CD86, TNFα and IL-12, leading to activation of autoreactive CD4 and CD8 T-cells. Activated T-cells are expanded further by encounter with activated B-cells presenting β-cell antigen. (3) Activated lymphocytes migrate across a chemokine gradient from the inflamed islet associated vascular endothelium, expressing up-regulated HA, ICOS, VCAM-1, ICAM-1, MADCAM-1, MHC class I and MHC class II, into the islet tissue. Local activated APCs in the islet milieu continue to take up, process and present antigen, promoting the retention and expansion of CD8 and CD4 T-cells that kill β-cells via perforin/granzyme and Fas/FasL mechanisms respectively. RAE1, retinoic acid early transcript 1.

In NOD mice multiple DC subsets are present including mDCs (myeloid DCs), lymphoid DCs and pDCs (plasmacytoid DCs). mDCs have an important role in antigen processing and presentation to autoreactive T-cells leading to their activation. In NOD mice there is a 5-fold increase in the number of immature mDCs and fewer mature mDCs. The immature DCs underproduce IL-10, have deficient responses to GM-CSF (granulocyte/macrophage colony-stimulating factor), decreased intracellular and surface expression of MHC class II, reduced co-stimulatory molecule expression and lowered expression of the CD40 signalling molecule [69]. However, the mature mDCs in NOD mice express elevated NF-κB in response to antigen, and this leads to increased secretion of IL-12p70 and TNFα, and increased expression of co-stimulatory molecules. Increased IL-12 leads to autocrine activation of DCs, as well as increased activation of antigen-specific CD8+ T-cells [70]. This intrinsic defect of the mature mDC population therefore tips the balance towards a Th1 immune response that drives β-cell destruction and T1D. Likewise, mDCs from T1D patients display elevated NF-κB activation and IL-12 secretion [71]. In addition to defects in the myeloid population there are also defects in the lymphoid DC population in NOD mice. Lymphoid DCs in NOD mice show reduced IDO (indoleamine 2,3-dioxygenase)-mediated catabolism of tryptophan in response to IFNγ, resulting in the increased proliferation of effector T-cells [72]. pDCs have an immature phenotype, are poor antigen presenters and are therefore known to be important for the induction of tolerogenic immune responses. In NOD mice depletion of pDCs was associated with a reduction in IDO in the pancreas and this correlated directly with the development and severity of insulitis [73]. The importance of DCs in T1D has been exploited in on-going clinical trials. Autologous DCs modified ex vivo to have reduced co-stimulatory ability have been used in T1D patients and at risk individuals to drive tolerogenic immune responses [179]. Although safety has been established, the efficacy of this approach is not yet known.

NK (natural killer) cells

NK cells are normally associated with defence against viruses, intracellular pathogen infected cells, malignant cells and foreign or transplanted cells. It is thought that NK cells may have an important early role in the immunopathogenesis of T1D since they are found to infiltrate pancreatic islets of NOD mice and have also been detected in the pancreatic islets of cadaveric T1D patients [74,75]. Furthermore, NK cells within the insulitis lesion display an activated phenotype, expressing higher levels of KIRG1 (killer cell lectin-like receptor group G1), PD-1 (programmed cell death-1), IL-2R (IL-2 receptor; CD25) and CD69 than NK cells from the pLN or spleen, and this correlates with increased β-cell destruction. The activated NK cells found in the insulitis lesion may directly induce β-cell apoptosis through perforin- and granzyme-mediated cytotoxic damage, as they express CD107a which is a marker of granule exocytosis [76]. NK cells are able to recognize NOD β-cells as these express RAE1 (retinoic acid early transcript 1) and NKp46 ligand, which are the ligands for NK cell receptors NKG2D (natural killer group 2D) and NKp46 respectively. Although it has been shown that blockade of NKG2D and NKp46 reduces T1D incidence, it may be that the protection is due to inhibition of interactions other than with NK cells, such as with cytotoxic T-cells that also express NKG2D [77]. Alternatively, they may indirectly damage β-cells since they also express high levels of pro-inflammatory cytokines such as IFNγ [78]. Although NK cells undoubtedly have a role in the early insulitis lesion it is unlikely that they are essential in the immunopathogenic process that drives complete β-cell destruction as NOD β-cells lacking MHC class I/B2m yet retaining NK receptor ligands are not killed by NK cells [79].

B-lymphocytes

Although macrophages and DCs are the primary pAPCs involved in the insulitis initiation, B-lymphocytes also have a pivotal early role in the pathogenesis of T1D development. B-cells have been identified in the insulitic lesions of biopsies from human T1D patients, as well as in the pancreatic biopsies of diabetes-prone mice and rats [80]. The significance of B-cells as important contributors to disease became clear when diabetes-prone NOD mice lacking B-cells were generated. Although B-cell-deficient NOD mice developed mild insulitis, suggesting that B-cells are not required for the initiation of disease, they were significantly protected from the development of diabetes, confirming their role in disease progression [8184]. Similar results were obtained following antibody-mediated depletion of B-cells [85].

Two main roles have been proposed for B-cells in the pathogenesis of T1D. (i) B-cells may contribute to disease via the production of AAbs (autoantibodies), known to correlate with early pathogenesis. A role for AAbs in promoting T1D development has also been supported by maternal AAb studies whereby inhibition of AAb transfer from mothers to NOD offspring in several models was found to protect against T1D development in the offspring [86,87]. Additionally, there is indirect evidence that suggests that AAbs enhance the effector functions of DCs and NK cells, as FcRγ (Fc receptor γ)−/− NOD mice are protected from disease [88]. These findings have led to the proposal that AAbs may augment β-cell destruction through binding AAgs and promoting FcRγ-mediated antigen uptake by APCs or by promoting antibody-dependent cellular cytotoxicity of β-cells. However, AAbs are not requisite for T1D development as NOD transgenic mice expressing only membrane-bound BCRs (B-cell receptors) and without the capacity to secrete antibody developed an increased incidence of insulitis and T1D compared with NOD B-cell-deficient mice [89]. (ii) B-cells may contribute to disease via the recognition, uptake and processing of AAgs and presentation to diabetogenic T-cells. This antigen-presenting role of B-cells must occur after the activation of T-cells involved in the initial immune phase of T1D, as many B-cell-deficient mice develop non-invasive insulitis [81]. The progression from non-invasive to invasive insulitis is well documented, but a mechanistic understanding of this crucial switch is not well understood [90,91]. It is likely that the highly proliferative nature of B-cells allows them to efficiently capture β-cell antigen for processing and presentation to activated diabetogenic CD4+ T-cells and CD8+ T-cells, resulting in the rapid expansion of cells invading and killing the islet β-cells [92,93].

Because of their known role in antigen presentation, and because they are expanded in NOD mice, the MZ (marginal zone) B-cell population has been implicated in the pathogenesis of T1D [94]. However, it is unlikely that the increase in MZ numbers observed in NOD mice is a primary defect promoting T1D, since loss of MZ B-cells following complete splenectomy failed to protect NOD mice from T1D, whereas the removal of follicular (FO), and not MZ, B-cells in anti-CD20-treated NOD mice did protect from T1D [95].

Treatment of recently diagnosed T1D patients with anti-CD20 mAb (monoclonal antibody) transiently depleted B-cells and resulted in transient preservation of β-cell mass. Although B-cells clearly contribute to T1D pathogenesis in NOD mice and humans, it is also clear that T1D can develop via alternative pathways in both species. T-cells from B-cell-deficient NOD mice were able to transfer T1D to NODscid recipients, albeit at a reduced frequency compared with T-cells from B-cell-sufficient donors [82]. In humans, T1D has likewise been reported in B-cell-deficient patients [96]. Therefore, although B-cells present as an attractive therapeutic target in those at risk of developing T1D, it is unlikely that their blockade will provide protection in all patients.

T-lymphocytes

There is a large body of evidence implicating T-cells in the development and progression of T1D in humans and NOD mice. Both CD4+ and CD8+ T-lymphocytes are pivotal during the early and late stages of disease in mice. Whole splenocytes or purified populations of both CD4+ and CD8+ T-cells can transfer T1D to young NOD and non-diabetes-prone F1 mouse strains, whereas neither CD4+ nor CD8+ T-cells alone can transfer disease [97,98]. Likewise, T-cell-depletion of susceptible NOD mice inhibited disease progression and T1D, reinforcing the central role of T-cells in immunopathogenesis [99]. The early discovery that T-cells are essential in the immunopathogenesis of T1D has led to a major focus on these cells, from thymic development to understanding the key mediators of cytotoxic versus regulatory subset development and β-cell killing.

Development

The architecture of the NOD mouse thymus is abnormal, comprising unusually large perivascular spaces and disorganized thymic medulla. Additionally, NOD mouse thymocytes have reduced expression of the integrin-type fibronectin receptors α4β1 (VLA-4) and α5β1 (VLA-5) that cause defects in cell migration. Since the thymocytes trapped within the giant perivascular spaces of the NOD mouse thymus are consistently VLA-5-negative, their accumulation may be due to an impairment of normal thymocyte migration [100,100a]. Although the overall number of T-cells emigrating from the NOD mouse thymus is normal, there may be an increase in the proportion of T-cells that bear TCRs with autoreactive specificity. The unique MHC class II haplotype present in NOD mice, and in many Caucasoid T1D patients, influences the ability to bind to self-peptide and mediate negative selection. This deficiency was tested in transgenic NOD mice expressing non-autoimmune-associated MHC class II haplotypes. These mice were protected from developing T1D, a protection that involved thymic deletion of autoreactive CD4+ T-cells in TCR transgenic 4.1NOD mice expressing the MHC class II molecule I-E [101].

It has been proposed that NOD mice fail to express self-antigens efficiently. This could be due to the lack of the MHC class II molecule I-E or, alternatively, due to the inability of AIREs (autoimmune regulators) to induce expression of self-antigens in mTECs (medullary thymic epithelial cells). NOD mice have reduced thymic expression of the AAg ICA69 (islet cell autoantigen of 69 kDa), and the gene encoding this protein carries a SNP in the promoter region important for AIRE binding [102]. This could explain the reduced thymic expression of this self-antigen and the potential for reduced deletion of thymocytes with specificity for it. Although there is no direct evidence that the expression of proinsulin is similarly reduced in the NOD thymus, enhanced expression of this important AAg in transgenic NOD mice protects from T1D [103]. In humans elevated thymic expression of proinsulin is also associated with protection from T1D [104].

NOD mice have a defect that limits allelic exclusion, which is identified by the increased heterogeneity of TCRα genes expressed on T-cells from transgenic NOD AI4 mice [105]. As a consequence of inefficient allelic exclusion, two different TCRs can be expressed on the surface of developing thymocytes and this has been shown to allow escape from negative selection and development of autoimmunity in other models [21].

Once the process of thymic selection is complete, thymocytes up-regulate receptors on their surface, such as CCR7, allowing them to respond to chemokines and emigrate from the thymus to the peripheral lymphoid organs. Interestingly, CCR7 is elevated on NOD mouse T-cells and CCR7-deficient NOD mice are protected from T1D [106,107]. However, there is to date no evidence that this polymorphism contributes to altered thymic emigration.

Activation

Since autoreactive T-cells are found in normal healthy people and mice, the escape of such cells from the NOD thymus cannot alone account for the development of T1D. Therefore there must be defects in the peripheral regulation of autoreactive T-cells. Following selection within the thymus, naive CD4+ and CD8+ T-cells travel to LNs, where they await activation upon MHC presentation of their complementary antigens by pAPCs and co-stimulatory signals. The activation of islet-reactive T-cells occurs within the pLN as their early removal in NOD mice was found to prevent development of T1D, whereas early removal of spleen had no impact on T1D [108].

The unique MHC class II of NOD mice contributes not only to the loss of thymic tolerance, but also to the loss of peripheral tolerance. Autoreactive CD4+ T-cell activation is associated with the unique I-Ag7 molecule. The lack of an acidic residue at position 57 of the β chain prevents formation of a salt bridge with Arg76 in the α chain [109111]. As a consequence, I-Ag7 is able to form salt bridges with bound peptides, enhancing peptide–MHC class II binding. A similar binding property has been found in the human MHC class II genotype HLA DQA1*0301, DQB1*0302 associated with T1D [112], suggesting that the homologous human HLA haplotype may lead to T1D development through the same mechanism.

The maintenance of tolerance to islet cell antigen requires their expression by stromal cells in the pLN, and this is regulated by the Deaf1 (deformed epidermal autoregulatory factor 1) transcription factor. Deaf1 is negatively regulated by its binding to a Deaf1 isoform called Deaf1-VAR, which prevents localization of Deaf1 to the nucleus. In the absence of Deaf1 in the nucleus, the transcription of TSAs (tissue-specific antigens) is reduced. In NOD mice undergoing destructive insulitis, Deaf1-VAR is in excess and the expression of TSAs are consequently down-regulated [113]. Reduced expression of Deaf1 was also found in T1D patients. However, in NOD mice, islet-reactive T-cells are activated as early as 3 weeks of age, well before any changes in Deaf1 expression are observed, and as such this cannot be the primary peripheral tolerance defect that leads to the development of insulitis.

The activation of islet-reactive T-cells requires signalling through co-stimulatory molecules expressed by pAPCs, including CD40, CD80 and CD86. CD40 binds CD40L (CD40 ligand) on T-cells, leading to an up-regulation of CD40 and TNFRs on APCs. Early inhibition of CD40L in NOD mice caused a significant reduction in the development of insulitis and T1D, suggesting that autoreactive T-cells are dependent on this interaction for activation [114]. In addition to the importance of the CD40/CD40L interaction for activation of T-cells, this interaction may also be important for the expansion of T-cells in the insulitis lesion. CD40 has been identified on the surface of islet-invasive T-cells, raising the possibility that pathogenic T-cells may cross-stimulate via CD40/CD40L interaction [115]. CD80/CD86 on APCs can ligate CD28 on T-cells and lead to their activation. This activation can be negatively regulated by the inhibitory molecule CTLA-4 on T-cells, which also binds CD80 and CD86. CTLA-4 negative regulation promotes the maturation of DCs that express IDO and regulate effector T-cells. NOD mice express reduced levels of liCTLA-4 with a concomitant reduction in the usual negative regulation of T-cell activation [17]. Consistent with this, deletion or blocking of CTLA-4 in NOD mice resulted in exacerbation of T1D [116]. Furthermore, NOD mice with induced overexpression of liCTLA-4 had a reduction in the incidence of T1D [117]. Although liCTLA-4 is not found in humans, sCTLA-4 is reduced in T1D patients [17]. On the basis of these studies, patients with recent-onset T1D were treated with CTLA-4Ig, a fusion protein composed of the Fc region of IgG1 and extracellular domain of CTLA-4, which inhibits the co-stimulation of T cells. Although C-peptide levels were initially higher after CTLA-4Ig treatment, there was no significant preservation of β-cell mass after 2 years [118].

Another negative regulatory molecule belonging to the CD28 family and involved in T1D development in NOD mice is PD-1. PD-1 is expressed on activated T-cells and its ligands, PD-L1 and PD-L2, are expressed on DCs. T-cell proliferation is inhibited when PD-1 binds to its ligands on DCs. Interestingly PD-L1 is also expressed on islet cells and blockade of PD-L1 in NOD mice, using mAbs, accelerated the rate of insulitis and T1D progression, suggesting that the PD-1/PD-L1 pathway negatively regulates autoreactive T-cells [119]. Consistent with this, T1D patients have reduced expression of this important negative regulator of T-cell proliferation [120].

In addition, within the CD28 family is ICOS, a co-stimulatory molecule up-regulated on activated T-cells and important in T1D. NOD mice have a higher expression level of ICOS than non-autoimmune strains. Both ICOS- and ICOSL (ICOS ligand)-deficient NOD mice are protected from T1D and this is caused by the failure to activate β-cell-reactive T-cells [18]. ICOS may therefore have an important role in maintaining the balance between the activation of effector and regulatory T-cells that control the development of T1D in NOD mice.

Homing to the islet

Activated islet-specific T-cells in NOD mice show normal up-regulation of CD44 and down-regulation of CD62L (CD62 ligand), allowing them to migrate out of the LNs via the efferent lymphatics. They then re-enter the circulation via the thoracic duct and migrate along the ECs associated with islet tissue. In pre-diabetic NOD mice, the β-cells, ECs and mononuclear cells infiltrating the islets produce multiple chemokine proteins that facilitate the tissue-specific homing of effector cells. The expression pattern of these chemokines is complex and it is unlikely that targeting of single chemokines will be of significant therapeutic benefit. However, β-cell expression of a chemokine decoy receptor that binds an array of chemokines completely prevented T1D in NOD mice [121].

The islet ECs normally protect β-cells from inappropriate infiltration by immune cells travelling in the blood. However, NOD mouse islet ECs take on an activated phenotype at the time of insulitis development, expressing HA (hyaluronan), MADCAM-1 (mucosal vascular addressin cell adhesion molecule-1), and ICAM-1 and VCAM-1, which are important for adhesion of activated monocytes and T-cells [122]. T-cell expression of MT1-MMP (membrane type-1 matrix metalloproteinase) facilitates their transmigration into the islets by cleaving the CD44/HA interaction [123]. Activated ECs also up-regulate MHC class I and II expression important for the presentation of antigens to CD8+ and CD4+ T-cells respectively. The reasons for this up-regulation are not known, but many viruses thought to be associated with T1D are known to infect microvasculature ECs and persistent infection of these cells has been associated with increased expression of adhesion molecules and MHC molecules [5456,124]. The importance of ICAM-1 in the development of T1D has been demonstrated in anti-ICAM-1-treated NOD mice that were found to be protected from the development of T1D [125,126]. Others have reported that an increase in MHC class I expression on islet ECs was associated with T1D in NOD mice [55], and human histological samples taken during the peri-onset stage of T1D show up-regulation of MHC class I and II, as well as adhesion molecules, on the islet ECs [56]. A novel therapeutic approach has been tested in NOD mice using a mAb that blocks MT1-MMP cleavage of CD44/HA and thereby prevents the transmigration of activated T-cells into the islet, resulting in protection from T1D [123].

Insulitis

Despite the many studies showing that both CD4+ and CD8+ T-cells are required for progression of disease to complete β-cell destruction and T1D, the precise role of CD4+ and CD8+ T-cells in the initiation phase of disease has been controversial. β2M−/− NOD mice, which lack MHC class I expression and CD8+ T-cells, do not develop insulitis [127]. This work was interpreted to mean that CD8+ T-cells are critical for the initiation of insulitis. However, an alternative explanation for the complete lack of insulitis is that protection was conferred by other B6-derived Idd13-linked protective genes back-crossed to the NOD with the β2M deficiency [127130]. NOD mice lacking β2M/MHC class I on APCs only developed a mild peri-islet infiltrate consisting of CD4+ T-cells that did not progress to invasive insulitis in the absence of CD8+ T-cells [131]. This indicates that CD4+ T-cells initiate peri-insulitis independently of CD8+ T-cells and subsequently provide help to CD8+ T-cells that can then respond to the earliest β-cell antigens.

It is thought that the earliest autoantigenic target of T-cells is insulin because a high frequency of both CD4+ and CD8+ T-cell clones isolated from the insulitic lesion of NOD mice react to peptides from insulin. In particular, early insulitic CD4+ T-cells predominantly respond to insulin peptide B-(9–23) [132], and CD8+ T-cells respond to peptide B-(15–23) [133]. Replacement of the NOD mouse insulin gene with a mutated insulin gene, encoding Al16, protected from T1D, but periductal insulitis persisted around some islets [134]. This suggests that, although insulin is an important early AAg recognized by T-cells, there may be upstream AAgs that precede insulin. Whatever the upstream antigens are, clearly the induction of tolerance to insulin is important and blocks the progression of insulitis. NOD mice treated with oral porcine insulin or peptides were protected from T1D [135], and those administered with human insulin developed Treg cells that were also able to transfer this protection [136]. However, unfortunately, in none of the clinical trials in which insulin was delivered systemically or orally was there a delay in onset of T1D compared with untreated control subjects at risk of developing T1D [137,138]. However, the problem with these trials may have been the route of administration as a pilot study using a single intramuscular injection of insulin with IFA (Incomplete Freund's Adjuvant) in recent-onset T1D patients showed an increase in insulin-specific Treg cells 2 years after treatment [139].

Once initiated by T-cells specific for insulin, islet cell damage ensues with concomitant release of AAgs that results in the activation of an increasingly heterogeneous autoreactive T-cell repertoire. A large number of these AAgs have been defined in NOD mice and have been reviewed extensively elsewhere [140]. It is more difficult to study the expanding heterogeneity of human T-cell responses in T1D due to the low T-cell frequency in peripheral blood and the inaccessibility of islets. However, if we are to design therapeutic intervention strategies that induce tolerance in T-cells of diabetic patients, it is imperative that we identify the human antigenic targets. Currently, there are only three targets recognized by both CD4+ and CD8+ T-cells that have been identified in the NOD mouse and that are also found in human T1D patients. These include two β-cell antigens, insulin and IGRP (islet-specific glucose-6-phosphatase catalytic subunit-related protein), and a neuroendocrine antigen, GAD (glutamic acid decarboxylase) [141]. GAD-65 alum has been used to treat new-onset T1D patients, but failed to show significant improvement in C-peptide levels. Combination therapy with GAD-65 alum and vitamin D/ibuprofen are currently in Phase II clinical trials [142].

Balance of immunopathogenesis and regulation

The insulitic lesion of NOD mice includes a number of CD4+ effector T-cell subsets that have been categorized based on their cytokine production as Th1, Th2, Th17, Th40, Treg cells and NKT cells. On the basis of these subsets, T1D has been considered a Th1-mediated disease, because increased levels of IFNγ and lower levels of IL-4 were correlated with β-cell destructive insulitis [143]. Furthermore, induction of a Th2 bias by administration of IL-4, and infection with various helminths, confers protection from T1D in NOD mice [144,145]. However, this simple categorization of T1D as a Th1-mediated disease was brought into question when IFNγ- and IFNγR (IFNγ receptor)-deficient NOD mice remained susceptible [146,147]. It is now recognized that cytokines are involved in the cross-talk between a greater range of T-cell subsets that control the balance between effector and regulatory immune responses. Th17-cells were identified as a subset of effector T-helper cells that differentiate in response to TGF-β (transforming growth factor-β) and IL-6, and require IL-23 for population expansion. They produce mainly IL-17A, IL-17F and IL-22, which have broad actions on the immune system due to the distribution of the IL-17 and IL-22 receptors [148]. Th17-cells have been shown to be important for a number of autoimmune diseases, such as asthma and psoriasis, but their role in T1D has only been reported in a few studies and remains controversial. Serum IL-17 is elevated in NOD mice and there are reports of increased numbers of Th17-cells in NOD mouse islets [149]. Consistent with the proposed role for Th17 cells in disease, the blockade of IL-17 in NOD mice with a mAb resulted in reduced insulitis [150]. Conversely, IL-17-deficient NOD mice were not protected from T1D [151]. Furthermore, when Th1- and Th17-cells from NOD BDC2.5 mice were independently transferred to NODscid recipients, those receiving Th1-cells developed more severe disease than those receiving Th17-cells. Interestingly, recipients of Th17-cells were found to have converted into a Th1 phenotype as measured by their production of IFNγ, suggesting that Th17-cells in NOD mice are not the primary cell type driving pathogenesis [152]. These studies also highlight the plasticity of the different CD4+ T-cell effector populations involved in a dynamic immune response.

A highly pathogenic subset of CD4+ effector T-cells, termed Th40-cells, has been identified in the insulitis lesion of NOD mice [153]. In addition to CD40L expressed on all activated T-cells, this subset expresses the CD40 protein itself and produces the pro-inflammatory cytokines IFNγ, TNFα, IL-6 and, sometimes, IL-17. The percentage of Th40-cells in NOD mice correlates with increased insulitis, and this population is essential for the transfer of disease to NODscid recipients [154]. Further adding to the complexity of the dynamic interactions between the different CD4+ effector T-cell populations, the production of IL-6 by Th40-cells can convert Treg cells into Th17-cells in mice [154a]. Importantly, Th40-cells have been identified in increased numbers in T1D patients and were found to be reactive to known islet autoantigens [155].

Treg cells are another important subset of effector T-helper cells involved in resolution of immune responses and in preventing autoimmunity. Although NOD mice have normal numbers of Treg cells, their ability to regulate pathogenic T-cells is limited [156]. IL-2, encoded within the Idd3 susceptibility loci, is expressed at reduced levels in NOD mice compared with non-autoimmune-prone strains, and this is correlated with the development of T1D. Since IL-2 has an important role in the induction and maintenance of FoxP3 (forkhead box P3)-expressing Treg cells, it has been proposed that reduced IL-2 promotes T1D by causing an imbalance between Treg cells and pathogenic T-cells [157]. Indeed, it has been shown that islet-infiltrating Treg cells in NOD mice have reduced levels of IL-2R (CD25), FoxP3 and Bcl2 as a consequence of reduced IL-2 signalling [157a]. Consistent with this, administration of low-dose IL-2 to susceptible NOD mice restored CD25 expression on islet-infiltrating Treg cells and protected from T1D [158]. It was also possible to correct the functional defect in Treg cells of NOD mice by co-culturing them with cord blood stem cells [159]. This enhancement of Treg cell function using stem cells has recently been tested in T1D patients and showed promising therapeutic potential [160].

NKT cells are a rare but an important effector T-helper cell subset, expressing an invariant TCRα chain Vα14-Jα18 in mice and Vα24-Jα18 in humans, and are also important in regulating effector T-cells. In NOD mice, NKT cells are severely reduced in number and are functionally deficient with an impaired ability to produce cytokines upon stimulation [161,162]. Although NKT cells can usually produce large quantities of both IL-4 and IFNγ, NKT cells in NOD mice produce decreased levels of Th2 cytokines, particularly IL-4, which may lead to a Th1 bias [163]. The reduced number of NKT cells correlates with exacerbation of T1D, and reconstitution of NOD mice with normal numbers of NKT cells prevents T1D [164]. Furthermore, it has been found that the impact of NKT cells on T1D in NOD mice is not always attributable to cytokine production [163]. NKT cells may also regulate T1D development through cell–cell contact with conventional T-cells, since this has been found to inhibit differentiation and induce anergy of islet-reactive T-cells in NOD mice [165]. These findings demonstrate that NKT cells may influence T1D via multiple non-mutually exclusive mechanisms. However, the significance of NKT cells in human T1D is yet to be definitively confirmed.

Killing of β-cells

As the infiltration of macrophages within the inflamed islet precedes that of T-cells, it is thought that initial β-cell death may not be antigen-specific, but instead be mediated by cytokines produced by macrophages. These cytokines include TNFα, IFNγ and IL-1β that bind their respective receptors on β-cells and induce apoptosis of β-cells via STAT1 (signal transducer and activator of transcription 1) and NF-κB pathways [166]. They can also induce the expression of ROS (reactive oxygen species) such as NO, which initiates DNA strand breakage and activation of PARP [poly(ADP ribose) polymerase], causing β-cell apoptosis. Although macrophages are important for initial β-cell damage, they do not kill sufficient numbers of β-cells to cause T1D, since NODscid mice with functional macrophages, but lacking lymphocytes, do not develop T1D.

The events that cause the non-invasive insulitis lesion to become invasive are not understood. However, the mechanisms by which β-cells are killed once this switch occurs have been well defined using NOD mice deficient in β-cell MHC class I, Fas or FasL (Fas ligand), perforin or granzyme. After a critical threshold of β-cell antigen has been released by non-specific killing and presented to islet-specific T-cells, they become activated, and are recruited to and retained within the islet tissue where they proliferate and contribute to β-cell death [167]. Activated T-cells can kill β-cells through a Fas/FasL pathway. NOD mice deficient for either Fas or FasL are protected from T1D and transfer of wild-type NOD splenocytes into Fas-deficient NODscid mice resulted in delayed T1D [168].

Unlike CD4+ T-cells, which cannot kill β-cells in an antigen-specific manner, CD8+ T-cells recognize up-regulated MHC class I on β-cells and can mediate antigen-specific β-cell killing [169]. NOD mice lacking β-cell MHC class I expression are largely protected from the development of T1D. This demonstrates that a direct interaction between CD8+ T-cells and β-cells is the primary mechanism for antigen-specific β-cell killing [79,170]. Perforin-deficient NOD mice have a similar reduction in T1D incidence, suggesting that CD8+ T-cells use the perforin/granzyme cytotoxicity pathway to kill β-cells [171]. Recently, the role of CD8+ T-cells in human T1D was also demonstrated. Islet autoreactive CD8+ T-cells were identified in focal islet regions from cadaveric T1D donors and this was associated with β-cell destruction [172]. Furthermore, in humans, it has been shown that the structural basis of CD8+-mediated killing of human β-cells is different from other TCR–MHC class I interactions [173]. The TCR of a human autoreactive CD8+ T-cell was highly peptide-centric in its recognition of MHC class I bearing proinsulin peptide, thus forming a very weak interaction with the MHC molecule itself. This interaction may explain why such autoreactive T-cells escape thymic selection. On the basis of these important studies implicating T-cells in the development of T1D, there have been a number of trials using anti-CD3. Early studies using humanized anti-CD3 were promising, with treated patients showing a reduced dependence on insulin over 4 years. However a more recent and large trial using anti-CD3 (otelixizumab) showed no improvement in C peptide, insulin dependence or HbA1c (glycated haemoglobin) levels [174].

SUMMARY

Studies in NOD mice over the past few decades have contributed to numerous immunomodulatory therapies and many of them have been tested in humans. Despite the successful protection from T1D seen in NOD mice, there has been limited success with therapeutic interventions in individuals at risk of T1D and patients. In NOD mice, non-antigen-specific therapies, including those that target the T-cells using anti-CD3 mAbs, B-cells using anti-CD20 mAbs, and APCs using a sCTLA4Ig, have shown some protection from T1D. Unfortunately, however, similar therapies showed limited efficacy in humans. Treatment of recently diagnosed T1D patients with rituximab, a humanized anti-CD20 mAb, resulted in transient preservation of β-cell mass. Although improvement was transient, this therapy holds promise for those at risk of T1D if administered earlier in the disease process. Genetic screening and earlier diagnosis will be important for opening an earlier therapeutic window of intervention [175,176].

Antigen-specific immune modulation trials in NOD mice, such as those involving insulin, were successful. However, none of the human trials in at-risk children have demonstrated significant efficacy. Explanations that have been proposed for the failure of these trials include the possibility that the route of administration rendered the insulin ineffective, the dose was inadequate to induce tolerance or, alternatively, such an approach may work only in those at-risk individuals that entered the trial prior to the development of activated T-cells and insulin AAbs [137,138].

Combinational therapies that suppress T-cell activation and enhance tolerance have also been successful in NOD mice. A recent pilot study [177] using combinational therapy in T1D patients involved the use of rapamycin to supress effector T-cell proliferation in combination with IL-2 to induce the formation of Treg cells. Although this approach did promote an increase in Treg cells, unfortunately none of the treated patients showed an increase in preserved β-cell mass [177].

Another innovative therapeutic approach that has been successfully tested in NOD mice is the use of immature DCs to induce tolerance [178]. This approach involves the ex vivo engineering of NOD DCs with AS-ODNs (antisense oligodeoxyribonucleotides) to inhibit the expression of CD80/CD86. After transfer back into NOD mice, these immature DCs migrated to the pLN and induced tolerance to β-cell antigens. Clinical trials using engineered DCs have now been initiated and support the on-going investigation of this approach for treatment of at-risk individuals in further trials [179]. One of the most recent and exciting therapeutic approaches, currently in Phase I clinical trials, is known as SCE (stem cell educator) therapy. This approach originated from NOD mouse studies in which splenocytes were cultured with human cord blood stem cells. Treg cells cultured in this way showed an increase in the CD4+CD62L+ sub-population and were able to suppress T1D in NOD recipients [180].

In summary, the NOD mouse has been studied extensively and has informed much of the current understanding of the immunopathogenesis of T1D. There are many similarities in the genetics and immunopathogenic mechanisms that lead to T1D in NOD mice and humans. Understanding these pathways has given us insight into a number of potential therapeutic avenues that are currently being trialled (Table 1).

Table 1
Summary of clinical outcomes for T1D patients based on NOD mouse studies
Lessons from NOD mice Potential relevance to human T1D Clinical significance 
Role of environment   
 Vitamin D protects from T1D in NOD mice [35,36Reduced incidence of T1D in equatorial regions of high sunlightUV-B is essential for the synthesis of vitamin D Vitamin D supplementation: at birth protected from T1D; recent-onset T1D had no significant protection [38,39
 Vitamin D has anti-inflammatory effects  
 Omega-3 fatty acids protect from T1D in NOD mice [43Reduced incidence of T1D in ethnic groups with a culture of increased fish consumptionOmega 3 fatty acids are anti-inflammatory Omega-3 supplementation: in genetically susceptible children, T1D incidence was reduced; in pregnant mothers and their HLA higher T1D risk babies efficacy not yet known [44]; trials is on-going 
 Probiotics protect from T1D in NOD mice [48Higher T1D incidence associated with higher standard of living, hygiene and antibiotic use Probiotic supplementation administered to at-risk children (PRODIA study) established safety; efficacy is not yet known [50
Role of cytokine/cytokine receptors   
 Macrophage production of TNFα and IL-1β are directly β-cell toxic in NOD mice [64Macrophages present in pancreatic samples of cadaveric T1D patients [181Recombinant IL-1RA (anakinra/kineret): in recent-onset T1D children, no change in pro-inflammatory cytokine gene expression, insulin secretory capacity or HbA1c levels was observed; lower insulin requirements were reported up to 4 months post-treatment [68
  TNF antagonist (etanercept, infliximab and adalimumab): in recent-onset T1D children, HbA1c was reduced and insulin production was increased at 24 weeks post-treatment [68
Role of DCs   
 Defective DC maturation in NOD mice [69DCs control tolerance compared with activation of T-cells Autologous DCs, manipulated to reduce co-stimulatory ability and promote tolerance, were given to T1D patients and at-risk individuals; safety was established, but efficacy is not yet known [179]; trial is on-going 
 Increased NF-κB activation in myeloid DCs of NOD mice [70Increased NF-κB activation in myeloid DCs of T1D patients [71 
 Increased IL-12 production by NOD mice DCs [70  
Role of co-stimulation   
 Reduced expression of co-stimulation inhibitory molecule liCTLA-4 in NOD mice [17Reduced expression of soluble CTLA-4 in human T1D patients [17CTLA-4Ig fusion protein co-stimulation blockade (abatacept): in recent-onset T1D patients, treatment resulted in an initial improvement in C-peptide levels; no preservation of β-cell mass was observed after 2 years [118
 Overexpression of liCTLA-4 reduced T1D incidence in NOD mice [117  
 Early treatment with CTLA-4Ig protected from T1D in NOD mice [116  
 Reduced expression of inhibitory molecule PD-L1 [119Reduced expression of PD-1 on T-cells in human T1D patients [120 
 Higher expression of ICOS [182  
Role of β-cell antigens   
 Proinsulin   
  Transgenic NOD mice overexpressing proinsulin in the thymus were protected from T1D, suggesting the role of thymic expression of insulin in maintaining tolerance [103Polymorphism in human insulin promoter is associated with lower thymic proinsulin expression, loss of tolerance to insulin and T1D [104Induction of tolerance to insulin was trialled with:(i) Intra-nasal delivery: autoantibody-positive individuals had some increase in antibody and decrease in T-cell responses to insulin [184
  Transgenic NOD mice expressing mutated proinsulin (lacking immunogenic peptide) were protected from T1D [134T-cell responses against insulin in human T1D patients [183]Insulin autoantibodies present in human T1D patients [183(ii) Oral delivery: recent-onset T1D patients showed no improvement in C-peptide secretion or IgG insulin antibodies; accelerated β-cell loss in some treated patients was detected [185,186
  Insulin autoantibodies precede T1D in NOD mice [183 (iii) Intramuscular delivery with IFA: recent-onset T1D patients showed some increased insulin-specific Treg cells at 2 years post-treatment [139
  T-cell responses against insulin present in NOD mice [183  
 GAD65  Induction of tolerance to GAD65/alum was trialled with: 
  GAD65 autoantibodies present in NOD mice [183GAD65 autoantibodies present in human T1D patients [183a(i) Subcutaneous delivery: recent-onset T1D patients had no significant improvement in C-peptide levels [187]; combination therapy with vitamin D and ibuprofen is currently in Phase II trials 
Role of T-cells   
   
 Autoreactive T-cells present in insulitis lesion of NOD mice [80Autoreactive T-cells present in T1D patients [187aAnti-CD3 mAb in recent-onset T1D patients and at risk individuals reduced the dependence on insulin over 4 years [190]; however, a recent large trial (otelixizumab) showed no efficacy in terms of C-peptide, insulin-dependence and HbA1c [191]; trials are on-going 
 Autoreactive T-cells transfer disease to NODscid mice [82CD8 T-cells present in islets of cadaveric T1D patients [181 
 CD8 T-cells are the primary mediators of β-cell killing in NOD mice [129  
 Intravenous treatment of anti-CD3 protected from T1D in NOD mice [99  
 Oral anti-CD3 reversed T1D in NOD mice [188  
 Combined treatment of anti-CD3 with IL-1RA caused synergistic reversal of T1D in NOD mice [189  
Role of B-cells   
 B-cells present in insulitic lesion of NOD mice [80B-cells present in pancreatic samples of cadaveric T1D patients [193Anti-CD20 (rituximab): recent-onset T1D patients had improved HbA1c and insulin-dependence at 1 year; however, depressed IgM levels indicated B-cell immunosuppression [175
 Antibodies against β-cell antigens present in NOD mice [183Antibodies against β-cell antigens present in human T1D patients [195 
 B-cell-deficient NOD mice were protected from T1D [85  
 Anti-CD20 depletion of B-cells protected from T1D in NOD mice [192  
Role of Treg cells   
 Lower levels of IL-2 in NOD mice [194Polymorphism in IL-2RA causing diminished IL-2 response in Treg cells from human T1D patients [196Stem cell educator (SCE) to promote Treg cell development; autologous lymphocytes co-cultured with cord blood stem cells (CB-SC) given to T1D patients improved C-peptide, HbA1c and insulin-dependence at 40 weeks post-treatment [180
 Treg cells have reduced levels of IL-2R and FoxP3 expression in NOD mice   
Lessons from NOD mice Potential relevance to human T1D Clinical significance 
Role of environment   
 Vitamin D protects from T1D in NOD mice [35,36Reduced incidence of T1D in equatorial regions of high sunlightUV-B is essential for the synthesis of vitamin D Vitamin D supplementation: at birth protected from T1D; recent-onset T1D had no significant protection [38,39
 Vitamin D has anti-inflammatory effects  
 Omega-3 fatty acids protect from T1D in NOD mice [43Reduced incidence of T1D in ethnic groups with a culture of increased fish consumptionOmega 3 fatty acids are anti-inflammatory Omega-3 supplementation: in genetically susceptible children, T1D incidence was reduced; in pregnant mothers and their HLA higher T1D risk babies efficacy not yet known [44]; trials is on-going 
 Probiotics protect from T1D in NOD mice [48Higher T1D incidence associated with higher standard of living, hygiene and antibiotic use Probiotic supplementation administered to at-risk children (PRODIA study) established safety; efficacy is not yet known [50
Role of cytokine/cytokine receptors   
 Macrophage production of TNFα and IL-1β are directly β-cell toxic in NOD mice [64Macrophages present in pancreatic samples of cadaveric T1D patients [181Recombinant IL-1RA (anakinra/kineret): in recent-onset T1D children, no change in pro-inflammatory cytokine gene expression, insulin secretory capacity or HbA1c levels was observed; lower insulin requirements were reported up to 4 months post-treatment [68
  TNF antagonist (etanercept, infliximab and adalimumab): in recent-onset T1D children, HbA1c was reduced and insulin production was increased at 24 weeks post-treatment [68
Role of DCs   
 Defective DC maturation in NOD mice [69DCs control tolerance compared with activation of T-cells Autologous DCs, manipulated to reduce co-stimulatory ability and promote tolerance, were given to T1D patients and at-risk individuals; safety was established, but efficacy is not yet known [179]; trial is on-going 
 Increased NF-κB activation in myeloid DCs of NOD mice [70Increased NF-κB activation in myeloid DCs of T1D patients [71 
 Increased IL-12 production by NOD mice DCs [70  
Role of co-stimulation   
 Reduced expression of co-stimulation inhibitory molecule liCTLA-4 in NOD mice [17Reduced expression of soluble CTLA-4 in human T1D patients [17CTLA-4Ig fusion protein co-stimulation blockade (abatacept): in recent-onset T1D patients, treatment resulted in an initial improvement in C-peptide levels; no preservation of β-cell mass was observed after 2 years [118
 Overexpression of liCTLA-4 reduced T1D incidence in NOD mice [117  
 Early treatment with CTLA-4Ig protected from T1D in NOD mice [116  
 Reduced expression of inhibitory molecule PD-L1 [119Reduced expression of PD-1 on T-cells in human T1D patients [120 
 Higher expression of ICOS [182  
Role of β-cell antigens   
 Proinsulin   
  Transgenic NOD mice overexpressing proinsulin in the thymus were protected from T1D, suggesting the role of thymic expression of insulin in maintaining tolerance [103Polymorphism in human insulin promoter is associated with lower thymic proinsulin expression, loss of tolerance to insulin and T1D [104Induction of tolerance to insulin was trialled with:(i) Intra-nasal delivery: autoantibody-positive individuals had some increase in antibody and decrease in T-cell responses to insulin [184
  Transgenic NOD mice expressing mutated proinsulin (lacking immunogenic peptide) were protected from T1D [134T-cell responses against insulin in human T1D patients [183]Insulin autoantibodies present in human T1D patients [183(ii) Oral delivery: recent-onset T1D patients showed no improvement in C-peptide secretion or IgG insulin antibodies; accelerated β-cell loss in some treated patients was detected [185,186
  Insulin autoantibodies precede T1D in NOD mice [183 (iii) Intramuscular delivery with IFA: recent-onset T1D patients showed some increased insulin-specific Treg cells at 2 years post-treatment [139
  T-cell responses against insulin present in NOD mice [183  
 GAD65  Induction of tolerance to GAD65/alum was trialled with: 
  GAD65 autoantibodies present in NOD mice [183GAD65 autoantibodies present in human T1D patients [183a(i) Subcutaneous delivery: recent-onset T1D patients had no significant improvement in C-peptide levels [187]; combination therapy with vitamin D and ibuprofen is currently in Phase II trials 
Role of T-cells   
   
 Autoreactive T-cells present in insulitis lesion of NOD mice [80Autoreactive T-cells present in T1D patients [187aAnti-CD3 mAb in recent-onset T1D patients and at risk individuals reduced the dependence on insulin over 4 years [190]; however, a recent large trial (otelixizumab) showed no efficacy in terms of C-peptide, insulin-dependence and HbA1c [191]; trials are on-going 
 Autoreactive T-cells transfer disease to NODscid mice [82CD8 T-cells present in islets of cadaveric T1D patients [181 
 CD8 T-cells are the primary mediators of β-cell killing in NOD mice [129  
 Intravenous treatment of anti-CD3 protected from T1D in NOD mice [99  
 Oral anti-CD3 reversed T1D in NOD mice [188  
 Combined treatment of anti-CD3 with IL-1RA caused synergistic reversal of T1D in NOD mice [189  
Role of B-cells   
 B-cells present in insulitic lesion of NOD mice [80B-cells present in pancreatic samples of cadaveric T1D patients [193Anti-CD20 (rituximab): recent-onset T1D patients had improved HbA1c and insulin-dependence at 1 year; however, depressed IgM levels indicated B-cell immunosuppression [175
 Antibodies against β-cell antigens present in NOD mice [183Antibodies against β-cell antigens present in human T1D patients [195 
 B-cell-deficient NOD mice were protected from T1D [85  
 Anti-CD20 depletion of B-cells protected from T1D in NOD mice [192  
Role of Treg cells   
 Lower levels of IL-2 in NOD mice [194Polymorphism in IL-2RA causing diminished IL-2 response in Treg cells from human T1D patients [196Stem cell educator (SCE) to promote Treg cell development; autologous lymphocytes co-cultured with cord blood stem cells (CB-SC) given to T1D patients improved C-peptide, HbA1c and insulin-dependence at 40 weeks post-treatment [180
 Treg cells have reduced levels of IL-2R and FoxP3 expression in NOD mice   

We thank Jacques F. Miller and Pablo A. Silveira for critical reading of the paper before submission.

Abbreviations

     
  • AAb

    autoantibody

  •  
  • AAg

    autoantigen

  •  
  • AIRE

    autoimmune regulator

  •  
  • APC

    antigen-presenting cell

  •  
  • β2m

    (B2m), β2-microglobulin

  •  
  • CCR

    CC chemokine receptor

  •  
  • CD40L

    CD40 ligand

  •  
  • CD62L

    CD62 ligand

  •  
  • CTLA-4

    cytotoxic T-lymphocyte-associated antigen-4

  •  
  • DC

    dendritic cell

  •  
  • Deaf1

    deformed epidermal autoregulatory factor 1

  •  
  • EC

    endothelial cell

  •  
  • EFA

    essential fatty acid

  •  
  • FasL

    Fas ligand

  •  
  • FcRγ

    Fc receptor γ

  •  
  • FoxP3

    forkhead box P3

  •  
  • GAD

    glutamic acid decarboxylase

  •  
  • GP

    glycoprotein

  •  
  • GPR

    G-protein-coupled receptor

  •  
  • HA

    hyaluronan

  •  
  • HbA1c

    glycated haemoglobin

  •  
  • ICAM-1

    intercellular adhesion molecule-1

  •  
  • ICOS

    inducible T-cell co-stimulator

  •  
  • IDO

    indoleamine 2,3-dioxygenase

  •  
  • IFA

    Incomplete Freund's Adjuvant

  •  
  • IFNγ

    interferon γ

  •  
  • IL

    interleukin

  •  
  • IL-1RA

    IL-1 receptor antagonist

  •  
  • iNKT cell

    invariant natural killer T-cell

  •  
  • iNOS

    inducible NO synthase

  •  
  • liCTLA-4

    ligand-independent CTLA-4

  •  
  • LCMV

    lymphocytic choriomeningitis virus

  •  
  • LN

    lymph node

  •  
  • mAb

    monoclonal antibody

  •  
  • mDC

    myeloid DC

  •  
  • MT-1-MTP

    membrane type-1 matrix metalloproteinase

  •  
  • MZ

    marginal zone

  •  
  • NF-κB

    nuclear factor κB

  •  
  • NK

    natural killer

  •  
  • NKG2D

    natural killer group 2D

  •  
  • NKT cell

    natural killer T-cell

  •  
  • NOD

    non-obese diabetic

  •  
  • NP

    nucleoprotein

  •  
  • pAPC

    professional APC

  •  
  • PD-1

    programmed cell death 1

  •  
  • pDC

    plasmacytoid DC

  •  
  • PD-L1

    PD-1 ligand

  •  
  • pLN

    pancreatic lymph node

  •  
  • ROS

    reactive oxygen species

  •  
  • SCFA

    short-chain fatty acid

  •  
  • sCTLA-4

    soluble CTLA-4

  •  
  • SNP

    single nucleotide polymorphism

  •  
  • T1D

    Type 1 diabetes

  •  
  • TCR

    T-cell receptor

  •  
  • TNF

    tumour necrosis factor

  •  
  • TNFR2

    TNF receptor 2

  •  
  • Treg cell

    regulatory T-cell

  •  
  • TSA

    tissue-specific antigen

  •  
  • VCAM-1

    vascular cell adhesion molecule-1

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Author notes

All authors contributed equally to this review.