(autoimmune) diabetes mellitus - Wiley Online Library

Immunology and Cell Biology (2009) 87, 315–323 & 2009 Australasian Society for Immunology Inc. All rights reserved 0818-9641/09 $32.00 www.nature.com/icb

REVIEW

Clinical application of NKT cell biology in type I (autoimmune) diabetes mellitus Marie T Fletcher1 and Alan G Baxter2 Type 1 natural killer T (NKT) cells are a population of CD1d-restricted, regulatory T cells that exhibit various NK cell characteristics and rapidly produce cytokines on stimulation with glycolipid antigen. In type I diabetes (TID), NKT cells are thought to have a tolerogenic function, evidenced by NKT cell numerical and functional deficiencies in the nonobese diabetic (NOD) mouse, which when corrected, can ameliorate disease. The mechanisms by which NKT cells can mediate their immunosuppressive effects in NOD mice are still poorly understood, which makes successful clinical translation of NKTcell-based therapies challenging. However, new insights into the genetic control of NKT cell deficiencies have provided some understanding of the genes that may control NKT cell number and function, potentially offering a new avenue for assessing TID risk in humans. Here, we review the mechanisms by which NKT cells are thought to prevent TID, discuss the evidence for involvement of NKT cells in the regulation of human TID and examine the genetic control of NKT cell number and function. A greater understanding of these areas will increase the chances of successful clinical manipulation of NKT cells to prevent or treat TID. Immunology and Cell Biology (2009) 87, 315–323; doi:10.1038/icb.2009.5; published online 17 February 2009 Keywords: immunoregulation; autoimmunity; mouse models; tolerance

Type I (autoimmune) diabetes (TID) is an organ-specific autoimmune disease in which the insulin-producing b cells of the pancreas are selectively destroyed, resulting in loss of glycemic control.1 The pathogenesis of TID is complex, resulting from multiple combined genetic, stochastic and environmental factors that affect various tolerance pathways.2 Animal models of TID such as the nonobese diabetic (NOD) mouse strain have provided valuable insights into human TID genetics and pathogenesis. NOD mice spontaneously develop TID between 15 and 30 weeks of age; disease progression is characterized by advancing infiltration of pancreatic islets by immune cells (insulitis) leading to specific b-cell destruction and loss of glucose homeostasis.3 Studies in NOD mice have demonstrated a key function for both CD4+ and CD8+ autoreactive T cells in the autoimmune process, and a number of b-cell autoantigens have been identified, including insulin, insulinoma-associated protein 2, islet-specific glucose-6-phosphatase catalytic subunit-related protein and glutamic acid decarboxylase.4–7 Multiple genetic loci are associated with TID risk in humans and mice, some of which appear to encode defects in tolerance mechanisms.8 NOD mice have a variety of immunological abnormalities that lead to inefficient central and peripheral tolerance, including the employment of a relatively rare major histocompatibility complex (MHC) class II variant linked to altered thymic selection and aberrant

peptide presentation9–11 and deficiencies in important regulatory cell types, such as CD25+ Treg12 and natural killer T (NKT) cells.13–15 Type 1 NKT cells are a population of T cells with unique immunoregulatory properties, that can affect the outcomes of a broad range of immune responses, including autoimmunity.16 Unlike conventional T cells, NKT cells can exhibit various NK cell characteristics, including expression of CD161c (NK1.1 in mice), and express a semi-invariant T-cell receptor (TCR) consisting of an invariant Va24-Ja18 (Va14-Ja18 in mice) chain coupled to Vb11 (Vb2, 8.2 or 7 in mice).17–19 The NKT TCR recognizes glycolipid, rather than peptide antigen, presented by the MHC class-I-like molecule CD1d.20 On activation, NKT cells rapidly respond with vigorous cytokine production, thus modulating the behavior of other immune cells.21 Through the production of a diverse range of cytokines, including interferon-g (IFNg), tumor necrosis factor-a (TNF-a), interleukin (IL)-4, IL-13, IL-10 and IL-17,21–25 NKT cells can paradoxically both suppress and promote cell-mediated immunity. The outcome of NKT cell activation in different settings is still poorly understood and can vary according to the type of stimulation, the local microenvironment and the subset that is targeted.16 NKT CELLS AND TID NKT cells appear to have an immunosuppressive function in TID. The majority of data has come from the NOD mouse model, where a numerically and functionally deficient NKT cell compartment initially

1Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia and 2Comparative Genomics Centre, James Cook University, Townsville, Queensland, Australia Correspondence: Professor AG Baxter, Comparative Genomics Centre, Molecular Sciences Bldg 21, James Cook University, Townsville, 4811 Queensland, Australia. E-mail: [email protected] Received 9 November 2008; revised 22 December 2008; accepted 22 December 2008; published online 17 February 2009

NKT cells in diabetes MT Fletcher and AG Baxter 316

suggested they contribute to b-cell protection.13,14,26 The numerical deficiency is thought to develop before birth, and those NKT cells that do develop produce subnormal levels of IL-4 and IFNg when stimulated.26–28 A fundamental role for these NKT cell defects in the autoimmune profile of NOD mice was demonstrated by a number of studies that suppressed diabetes by partially restoring the NKT cell pool (Table 1). Adoptive transfer of thymic populations enriched for NKT cells, or transgenic Va14-Ja18 NKT cells from spleen, prevented TID.29,31 Transgenic overexpression of the Va14-Ja18 TCR in NOD mice increased NKT cell frequency and provided some protection against disease; the importance of NKT cell number was highlighted by the fact that the transgenic lines of NOD mice with the highest NKT cell numbers showed the most robust protection. Furthermore, TID was accelerated in CD1d / mice, which completely lack NKT cells.35,41,42 These studies suggest that a numerically complete NKT cell compartment is sufficient for the maintenance of b-cell tolerance in NOD mice. Addressing the NKT cell functional defects in NOD mice through specific NKT cell activation has also inhibited, albeit partially, diabetes

in NOD mice. Weekly administration of the NKT cell glycolipid agonist aGalCer, or the related glycolipids OCH and C20:2, from 3 to 4 weeks of age reduced the incidence of disease by between 30 and 70%, presumably by altering the amount or type of NKT-cell-derived cytokines.35–37,39,40 Upregulation of CD1d within the pancreas of NOD mice also prevented TID by enhancing NKT cell function.43 MECHANISMS OF NKT-CELL-MEDIATED PROTECTION Although there have been numerous successful NKT-cell-based therapies in NOD mice (summarized in Table 1), the mechanisms by which tolerance is achieved are still unclear. Various mechanisms have been proposed; they need not be mutually exclusive. It is possible that NKT cells regulate TID through a number of combined mechanisms and at multiple stages of diabetes pathogenesis. Altering the Th1–Th2 balance: influence of IL-4, IL-10 and IFNc An obvious candidate mechanism for the regulatory function of NKT cells in TID is their capacity to produce cytokines. NKT cells rapidly make large amounts of IL-4 and IFNg without de novo mRNA

Table 1 NKT cell-based treatments of TID in NOD mice Experimental protocol/NKT cell treatment

Diabetes model

Effect of treatment

Proposed mechanism(s)

Adoptive transfer of thymocyte populations enriched for NKT cells:

NOD—spontaneous model NOD—accelerated model (transfer

Significant protection

Promotion of Th2 responses, dependent on IL-4 and/or IL-10

13,29,30

Increase in NKT cell number

Decreased Th1 responses against islets

31–34

CD4 CD8 (DN) abTCR+DN

of diabetogenic splenocytes into irradiated recipients)

NK1.1+abTCR+DN Transgenic overexpression of Va24Ja18 NOD—spontaneous model NOD—accelerated model (transfer chain Adoptive transfer of Va24Ja18 NKT cell- of diabetogenic splenocytes into enriched splenocytes

References

NOD.scid recipients) NOD—cyclophosphamide induced

in autoreactive CD4+

Significantly reduced incidence of disease in lines with highest NKT

Induction of anergy effector T cells

cell number Prevention of cyclo-induced dia-

Dependent on cell–cell contact, independent of CD1d expression

betes Inability to transfer disease in accelerated model Repeated long-term administration NOD—spontaneous model of aGalCer (weekly from onset of insulitis)

Between 30 and 60% reduction in diabetes incidence

Increased IL-4/IL-10, decreased IFNg production from autoreactive T cells

35–38

Induction of tolerogenic DC subsets in PLN 50% reduction in disease incidence. Slightly improved outcome

Increased IL-4/IL-10, decreased IFNg production from autoreactive T cells,

compared to aGalCer

increase in IL-10 producing cells in islets

Repeated administration of C20:2 for NOD—spontaneous model 7 weeks (weekly from onset of insulitis)

30% reduction in disease incidence. Slightly improved outcome

Reduction and Th2 deviation of autoantigen-specific CD8+ T cells

Targeted deletion of CD1d locus

NOD—spontaneous model

compared to aGalCer Earlier onset, 10–20% increase in

Induction of tolerogenic DCs Increase in activated T cells in pancreas

Transgenic overexpression of CD1d

NOD—accelerated model NOD—spontaneous model

incidence in CD1d / mice Upregulation of CD1d within islets

Increased IL-4 and IL-10 in PLN

under control of insulin promoter Adoptive transfer of splenocyte

NOD—accelerated model Transfer of BDC2.5 NOD

40% reduction in disease incidence Inability to transfer diabetes IFNg-dependent alteration of host APCs

populations containing NKT cells Production of NOD mice congenic

T cells into NOD.scid NOD—spontaneous model

Increased NKT cell number but no

Correction of defective SLAM-SAP sig-


disease protection Increased NKT cell number and

naling Correction of NKT cell numerical defects

reduced disease incidence

Altered expression of peroxisomal membrane protein-encoding gene

Repeated long-term administration of OCH (weekly from onset of insulitis)

for C57BL/6 alleles at Nkt1 locus Production of NOD mice congenic for C57BL/6 alleles at Nkt2 locus Production of NOD mice congenic for NOR alleles at Idd13


Pxmp4

Abbreviations: APC, antigen-presenting cell; DC, dendritic cell; IFNg, interferon-g; NKT, natural killer T cell; NOD, nonobese diabetic; PLN, popliteal lymph node.

Immunology and Cell Biology

39

40

41,42

43

44

45,46

47,48


synthesis;49 however NOD NKT cells are functionally impaired, particularly with respect to Th2 cytokines such as IL-4.26,28 The association between Th2 cytokines and functional tolerance in diabetes has been established by studies showing disease prevention or amelioration following treatment with IL-4 and IL-10.50,51 NKTcell-derived Th2 cytokines could potentially inhibit b-cell destruction by inhibiting IFNg-induced proinflammatory antigen-presenting cell (APC) activation, suppressing the production of inflammatory cytokines such as IL-1, IL-6 and TNF-a,52 and downregulating the production of IFNg, which is important in effector differentiation of CD8+ T cells. Early studies examining this possibility focused on the production of IL-4 and IL-10, and an important role was ascribed to IL-4 and/or IL-10 in NKT-cell-mediated protection against both spontaneous and cyclophosphamide-induced diabetes.29,53 aGalCer treatment of NOD mice promoted a Th2 cytokineenriched environment in the spleens and pancreatic lymph nodes, and autoantigen-specific T cells from treated mice showed increased production of IL-4 and decreased IFNg production.36,37 This suggests that the activation of NKT cells can alter the Th1–Th2 balance in NOD mice. Similarly, treatment of NOD mice with the aGalCer analogues OCH and C20:2, which induce relatively more IL-4 and less IFNg than aGalCer, has slightly improved TID and insulitis outcomes, consistent with a role for Th2 cytokines in the mechanisms of NKTcell-mediated TID prevention.39,40 It is clear, however, that Th2 deviation of immune responses is not sufficient to prevent diabetes in NOD mice, as congenic lines expressing C57BL/6-derived alleles on distal chromosome 1 do not show the Th1-dominance characteristic of the strain, but have an unaltered susceptibility to diabetes.45,46 Furthermore, NOD mice expressing targeted gene-deficient mutant alleles for IFNg, or its receptor, are susceptible to diabetes.54,55 Consistent with these findings, TID prevention by NKT cells is sometimes not attributable to the production of IL-4 and/or IL-10. One study showed that diabetes induced by the adoptive transfer of diabetogenic autoreactive BDC2.5 T cells into NOD.scid mice could be prevented by co-transfer of splenocytes containing NKT cells by a mechanism dependent on IFNg.44 NKT cell influence on APCs: induction of tolerogenic DCs Professional APCs, such as dendritic cells (DCs), are central to tolerance induction and it is known that NKT cells can modulate DC differentiation and maturation status.56,57 The effect of aGalCer on DCs varies according to the administration protocol. Activation of NKT cells with a single dose of aGalCer can cause DC maturation, as defined by acquisition of co-stimulatory molecules, such as CD40, and production of cytokines, such as IL-12.58,59 As NOD mice have reported defects in DC maturation, including low expression of co-stimulatory molecules such as CD86, aGalCer-induced TID prevention may result from altered maturation of DCs.60,61 Indeed, one study found that activation of NKT cells with aGalCer induced migration and maturation of DCs that subsequently anergized effector T cells within the pancreatic lymph node (PLN).38 This finding is consistent with a requirement for maturation of DCs to induce tolerance in some settings.62 In contrast, it appears that repeated aGalCer treatment of C57BL/6 and NOD mice induced regulatory CD11chiCD8a DC: a subset that has previously been found to be tolerogenic in NOD mice.35,38,63,64 The interaction between NKT cells and DCs appears to be quite complex, as the two postulated mechanisms of action—DC maturation or induction of immature, tolerogenic DC subsets— appear to be at odds with each other, yet promote the same ultimate outcome.

Effects of NKT cells on other T cells NKT cells may also modulate TID by directly influencing the behavior of autoreactive effector T cells independently of Th2 polarization. aGalCer treatment of NOD mice prevented TID without an accompanying IL-4/IL-10 bias of the anti-islet response in one study.41 Strikingly, Beaudoin et al.32 found that NKT cells controlled the effector activity of diabetogenic BDC2.5 T cells by rendering them anergic, as demonstrated by lack of proliferation and cytokine production in response to re-stimulation with autoantigen. The NKT-cell-mediated control of effector T cells was subsequently found to be dependent on cell–cell contact rather than cytokines in vitro.33 Remarkably, this regulatory activity was also independent of peripheral CD1d expression, as NKT cells could suppress the activity of BDC2.5 T cells in vitro regardless of CD1d expression by APCs, and reconstitution of CD1d / mice with NKT cells still protected against disease.34 The exact nature of the interaction, and which surface molecules are involved, remains to be determined. One potential mechanism by which NKT cells could inhibit TID is by influencing the suppressive activity of CD4+CD25+ Tregs. In a mouse model of autoimmune myasthenia gravis, therapeutic aGalCer treatment induced expansion of Tregs; conversely, depletion of CD25+ cells reduced the disease-protective effects of aGalCer.65 Similarly, NKT cells caused Treg differentiation in a mouse model of oral tolerance,66 and promoted Treg proliferation in an in vitro model of human PBL activation.67 Although one group found that Tregs may be required for the protective activity of NKT cells following aGalCer treatment of NOD mice,68 the treatment does not appear to alter the frequency of Tregs.37,40,68 Furthermore, in experiments where the co-transfer of NKT cells prevented diabetes induced by autoreactive diabetogenic BDC2.5 T cells, the NKT cell populations transferred were either purified or lacked Tregs, suggesting that NKT cell control of effector T cells can occur independently of Treg activity.32,44 NKT cells and the Th17 response The field of autoimmunity has recently undergone a paradigm shift with the discovery of the Th17 lineage of effector T cells.69 The involvement of an effector T-cell subset other than Th1 cells in autoimmune pathogenesis was suggested by the seemingly paradoxical finding that mice lacking IFNg or the p35 subunit of IL-12 were highly susceptible to many autoimmune diseases.70–72 Th17 cells, which produce the proinflammatory cytokine IL-17, were subsequently found to be highly pathogenic and necessary for disease development in experimental autoimmune encephalomyelitis and collagen-induced arthritis.73–76 The involvement of Th17 cells in TID pathogenesis is still unclear, but some reports suggest IL-17 may be involved in TID onset in NOD mice, and the fact that NOD mice deficient in IFNg or its receptor are still susceptible to TID may indicate the pathogenic activity of non-Th1 cells.54,55,77,78 The majority of studies of NKT-cell-based therapies in NOD mice were conducted before the knowledge of the importance of IL-17 in autoimmunity, but a retrospective analysis of the literature, combined with recent reports of IL-17 production from NKT cells themselves, suggests that this is an area that warrants further investigation.22–25 There are numerous ways that NKT cells could influence a Th17 response. Th17 differentiation from naive T cells is potently inhibited by IFNg, IL-4 and IL-13, all cytokines that NKT cells produce in abundance.69 Inhibition of IL-17 secretion would be a mechanism consistent with the paradoxically protective role of NKT-cell-derived IFNg in some settings,44 and may go some way to explaining why poor cytokine production from NOD NKT cells contributes to Immunology and Cell Biology


autoimmunity and why altering NKT cell function by activation (thus increasing cytokine production) can inhibit TID. Conversely, NKT cells could support a Th17 response through the production of cytokines that stimulate IL-17 secretion. Importantly, NKT cells were recently found to produce IL-21,79 which in combination with transforming growth factor-b and IL-6, induces Th17 development in vitro and in vivo and can amplify a preexisting Th17 response.80–82 Furthermore, several studies have shown that the NK1.1 subset of NKT cells are themselves capable of producing IL-17,22,23,83 and a recent report has found that the rare CD4 NK1.1 subset are the primary producers of this cytokine.25 Studies using congenic NOD mice expressing NK1.1 found that, in addition to having fewer NKT cells than C57BL/6 mice, NOD mice have a lower proportion of NKT cells expressing NK1.1.30,84 CLINICAL RELEVANCE: EVIDENCE FOR NKT CELL INVOLVEMENT IN HUMAN TID The clinical relevance of the NKT cell defect of NOD mice is controversial. In a study of discordant twin pairs, Wilson et al.85 found that the frequency of NKT cells in peripheral blood of TID patients was lower than in their healthy identical twins, and in vitro derived NKT cell clones from TID patients showed selectively impaired IL-4 production.85,86 A subsequent study from the same group confirmed a numerical deficiency in NKT cells and poor production of both IL-4 and IFNg from NKT cells from the blood of TID patients and individuals at risk.87 These findings supported an important role for NKT cells in TID in humans, but they were contradicted by five studies reporting that TID patients did not have low peripheral blood NKT cell levels compared to normal subjects; some even had elevated NKT cell frequencies.88–92 One study found that although the frequency of NKT cells in peripheral blood mononuclear cell (PBMC) among patient groups did not differ, NKT cell clones from TID patients showed a Th1 bias and the CD4+ subset was reduced.92 The conflicting reports of human NKT cell frequencies and function in TID patients may be attributable to differences in the patient populations tested and in methods of NKT cell identification between studies. Diverse combinations of aGalCer-loaded CD1d tetramer, TCR-specific antibodies and surface markers were used, which do not necessarily identify the same populations.93 In the initial study by Wilson et al.,85 NKT cell frequency was deduced, rather than enumerated, by Va24-Ja18 sequence analysis of NKT cell clones derived in vitro from CD4 CD8 Va24+ cells, identified by flow cytometry. The use of the CD4 CD8 Va24+ phenotype is problematic, as it excludes the dominant CD4+ NKT cell subset. The exclusion of CD4+ NKT cells in the study may explain the selective impairment of IL-4 production observed, because this subset was shown to be the primary producers of Th2 cytokines in humans.94,95 Some other studies have used a combination of Va24 and Vb11 monoclonal antibodies (mAbs) to identify NKT cells.89,90,92,96 Although this phenotype includes both CD4+ and DN subsets, it may also include nonCD1d-restricted T cells. The inadvertent inclusion of conventional T cells in the analysis may explain the elevated frequencies of Va24+ Vb11+ cells observed in Japanese subjects by Oikawa et al.89,96 However, this could also be explained by genetic differences between Japanese and Caucasian populations, because the same technique did not find similarly elevated NKT cell levels in an Australian population.90 The combined use of a-GalCer-loaded CD1d tetramers and antiVa24 mAb appears to be the most specific and reliable method of NKT cell identification in humans, as 100% of CD1d-aGalCer+/ Immunology and Cell Biology

Va24+ cells co-express the characteristic TCR-b chain expressed by human NKT cells, Vb11.88 Using this method to enumerate directly NKT cells among PBMCs, Lee et al.88 found no difference in NKT cell frequency between TID patients, at-risk individuals and healthy controls. However, it is important to note that studies of human NKT cells are limited by the relative inaccessibility of the lymphoid organs. The problems stemming from an almost total reliance on peripheral blood assessment are illustrated by a study in various strains of mice. The numerical deficiency of NKT cells that characterizes the NOD strain was evident in all tissues with the notable exception of blood, a result that casts uncertainty over human studies that use blood sampling to predict systemic NKT cell levels.97 The use of NKT cell clones isolated from the lymphoid tissue of diabetic patients circumvents this problem to an extent. Kent et al.98 isolated and cloned NKT cells from the pancreatic draining lymph nodes, and found that NKT cell clones from TID patients produced lower levels of IL-4 than those from healthy controls, supporting an important role for NKT cell function in the control of TID.98 However, the use of clones as a measure of NKT cell function can also be problematic, as the amount and type of cytokines NKT cells produce may change over time and with repeated in vitro stimulation.99 GENETIC CONTROL OF NKT CELL NUMBER AND FUNCTION Although it seems likely that NKT cells are important in the control of human TID, using NKT cell frequency and function to predict TID risk in humans remains problematic, mainly due to technical difficulties associated with the accurate detection of this rare cell population in lymphoid tissues. Identification of genes controlling NKT cell numbers and function and correlating their expression with TID in humans may prove a more reliable, clinically translatable method of predicting TID risk attributable to NKT cell phenotypes. In mice, strain-specific NKT cell differences suggested a genetic basis for the control of NKT cell numbers.84 A genome-wide screen of a cross between NOD and C57BL/6 mice identified two loci with significant linkage to NKT cell numbers:100 Nkt1 on distal chromosome 1, which maps to the NOD lupus susceptibility gene Babs2/ Bana3101 and Nkt2 on chromosome 2, which maps to the NOD diabetes susceptibility region Idd13.102 Jordan et al.45 produced a NOD mouse line congenic for the C57BL/6 allele at the Nkt1 locus, and found that congenic NOD.Nkrp1bNkt1b mice had an increased NKT cell frequency and number in thymus and spleen compared to NOD.Nkrp1b parental strain controls (a congenic line that expresses the developmental marker NK1.1), particularly within the relatively immature NK1.1 subset. The function of the Nkt1 locus was confirmed by another study that reported restored NKT cell number and function in NOD mice congenic for C57BL/6 alleles in this region.46 Microarray gene expression analysis of thymi from congenic and parental mice by Jordan et al.45 identified Slamf1 and Slamf6 (which encode SLAM and ly108, respectively) as the most likely candidate genes for the control of NKT cell numbers by Nkt1, because both receptors signal via SLAM-associated protein (SAP), and SAP signaling is necessary for NKT cell development. The importance of SAP signaling in NKT cell ontogeny is evidenced by studies showing that mice deficient in SAP or the downstream Src kinase Fyn display severe NKT cell defects.103–107 Flow cytometry confirmed a significant increase in the expression level of the Slamf1-encoded Ig-like receptor SLAM on the cell surface of thymocytes from NOD.Nkrp1bNkt1b compared to NOD.Nkrp1b


mice, and this was attributed to aberrant expression of SLAM during the developmental program of thymocytes in the wild-type NOD mice. Although in C57BL/6 and NOD.Nkrp1bNkt1b mice, SLAM expression reached a peak on the double-positive (DP) thymocytes and was expressed at lower levels on the more mature single-positive (SP) cells, in NOD.Nkrp1b mice SLAM expression was at its highest on the SP thymocyte subset and was expressed at relatively low levels on DP cells.45 SLAM expression on splenocytes was similar between strains, consistent with normal expression on SP thymocytes in the NOD.Nkrp1b line. The SLAM expression pattern on thymocytes in C57BL/6 mice found by Jordan et al.45 was supported by a study showing that genes Slamf1 and Slamf6 similarly reached a peak of expression on DP thymocytes before declining on SP cells.108 Although the significance of allelic variation in expression levels of SLAM on DP thymocytes is yet to be formally demonstrated, SAP signaling is crucial for normal NKT cell ontogeny and reduced SLAM expression on DP thymocytes would result in decreased homotypic ligation of SLAM on developing NKT cells, with a subsequent reduction in SAP signaling. This may explain the numerical deficiency characteristic of the NOD strain. However, it is worth noting that congenic NOD.Nkrp1bNkt1b mice are not protected from TID, despite expression of C57BL/6 alleles at the Nkt1 locus and partial correction of NKT cell number.45,46 This may be due to the relative immaturity of the additional NKT cells found in the line. Defects in SLAM signaling may also contribute to NKT cell functional deficiencies in NOD mice. A selective impairment in Th2-type (IL-4/IL-10) cytokine production was found to result when SLAM–SLAM interactions between stimulating mDCs and NKT cell lines were inhibited by blocking peptide, suggesting that the subnormal production of these cytokines characteristic of NOD NKT cells may result from the relatively low expression of SLAM observed on NOD DC.109 Slamf6, which is tightly linked to Slamf1, is also a strong candidate gene for the effects of Nkt1.45 Further evidence that Slamf6 is also involved in NKT cell development was provided by a study that used mixed bone marrow (BM) and ‘double-mutant’ chimeras to investigate the effects of both Slamf1 and Slamf6 expression on NKT cell development.108 Reconstitution of irradiated, NKT-cell-deficient Ja18 / hosts with a 1:1 mixture of CD45-marked wild-type (WT) and Slamf6- or Slamf1-deficient BM found that Slamf1- and Slamf6deficient cells failed to give rise to the same number of NKT cells as WT. Phenotypic analysis of chimeric NKT cells demonstrated that lack of Slamf1 or Slamf6 disrupted normal NKT cell expansion following positive selection.108 The authors next analyzed the relative functions of Slamf1 and Slamf6 signaling in NKT cell development by creating ‘double-mutant’ mice in which neither Slamf1-encoded SLAM nor Slamf6-encoded ly108 could engage in homotypic interactions. Irradiated Ja18 / hosts were reconstituted with a 1:1 ratio of CD45-labeled Cd1d / Slamf6 / and Cd1d+/+Slamf1 / BM (Figure 1). This approach was necessary because Slamf1 and Slamf6 are tightly linked, thus doublemutant mice could not be produced by crossing single mutants. Owing to the requirement for CD1d signaling, the above method created both single- and double-mutant environments; the TCR of Slamf1 / NKT cell precursors could ligate CD1d expressed by other Slamf1 / cells, which expressed Slamf6, whereas Slamf6 / cells could only respond to presentation by CD1d+/+Slamf1 / cells (Figure 1, lower panel), thus lacking co-stimulation by both SLAM family members. Analysis of the mice found that the single-mutant cells gave rise to up to 10 times more NKT cells compared to the double-mutant cells, suggesting that both Slamf1 and Slamf6

Figure 1 Double mutant SLAM/ly108 bone marrow chimeric mice. BM from CD1d / Slamf6 / and CD1d+/+Slamf1 / mice was mixed at a 1:1 ratio and injected into lethally irradiated J18 / hosts, thus preventing homotypic SLAM–SLAM and ly108–ly108 interactions (top). NKT cell precursors from CD1d / Slamf6 / (lacking ly108 expression but expressing SLAM) can only receive signals from CD1d-sufficient DP thymocytes, which express ly108 but do not express SLAM (bottom). In the single mutant, ly108expressing NKT precursors from CD1d+/+Slamf1 / mice can receive homotypic ly108 signals from other CD1d+/+Slamf1 / DP thymocytes.

contribute to NKT cell development but are partly redundant, and that both homotypic Slamf1–Slamf1 and Slamf6–Slamf6 interactions are important.108 The finding of an NKT cell numerical defect associated with defective expression of SLAM in NOD mice, combined with the evidence of redundancy in the effects of Slamf1 and Slamf6, suggests that allelic variation in Slamf6 may also contribute to the NOD NKT phenotype. The correction of NKT cell number in NOD mice by restoring normal SLAM signaling is therefore consistent with a redundant role for Slamf6-encoded ly108.45 The other genetic locus with significant linkage to NKT cell numbers was Nkt2 on chromosome 2,100 which mapped to the same region as the diabetes susceptibility gene Idd13. At least two loci are thought to contribute to Idd13, one of which is B2m, the gene that encodes b2-microglobulin.102,110 B2m was also proposed as a candidate for Nkt2, as it forms the light chain of the NKT-cellrestricting ligand CD1d. Fletcher et al.47 found that NOD.Nkrp1bNkt2b congenic mice had increased frequency and numbers of all thymic NKT cell subsets and Immunology and Cell Biology


decreased TID incidence compared to parental NOD.Nkrp1b strain controls. Microarray analysis of thymi from congenic and control mice identified the peroxisomal membrane protein-encoding gene Pxmp4 as a potential candidate gene for the control of NKT cell numbers, as peroxisomes are known to be important for glycolipid metabolism.111 Furthermore, Pxmp4 binds the chaperone/membrane transporter molecule Pex19,100,112 which is encoded within the Nkt1 linkage region and is highly differentially expressed in NOD mice congenic for the Nkt1 locus.45 Although NOD.Nkrp1bNkt2b congenic mice showed an increase in NKT cell number consistent with control of NKT cell numbers by a locus within the Nkt2b congenic region, the increase appeared to be only partial. NOD.Idd13NOR mice bearing the larger Idd13 congenic segment derived from the nonobese resistant (NOR) strain had an even greater increase in NKT cell numbers, suggesting that another locus within the Nkt2 but outside the Nkt2b region also affects NKT cell numbers.47 This was supported in a study by Chen et al.,48 which analyzed NKT cell numbers in NOR, NOD and NOD mice congenic for NOR-derived alleles at Idd13, and found evidence of at least two genes on chromosome 2 involved in the control of NKT cell numbers.48 B2m was excluded as a candidate, as there were no differences in NKT cell frequency or numbers between NOD.B2m / strains that transgenically expressed either the NOD-derived a or C57BL/6-derived b allele of B2m.45,102 The importance of NKT cell number in preventing TID was once again demonstrated by the fact that the incidence of diabetes and degree of insulitis was lowest in the NOD.Idd13NOR line, which had the highest number of NKT cells.47

NKT CELLS AND TID: CLINICAL APPLICATIONS The majority of NKT cell therapies for diabetes are prophylactic rather than curative in NOD mice, where the preexisting risk is known. To successfully translate our emerging understanding of NKT cell biology to the clinical management of TID, both diagnostic and therapeutic possibilities exist. Diagnostic possibilities (1) Correlation of genes controlling murine NKT cell number and function with human TID susceptibility alleles; (2) Assessment of NKT cell mass in vivo with low-dose aGalCerelicited serum cytokine response.

Therapeutic possibilities (1) Prevention of disease by glycolipid ligand-induced expansion of NKT cells in vivo in genetically susceptible individuals; (2) Isolation, in vitro expansion and reinfusion of NKT cells. The genetic information gleaned from the NOD model could be a useful way of determining susceptibility in humans. As discussed above, NKT cell number appears to be regulated by genes within the Nkt1 locus on chromosome 1,45,100 and Slamf1 and Slamf6 are two likely candidate genes, because SAP signaling is required for NKT cell development, and SLAM and ly108 both signal through SAP.103–105 Importantly, SAP signaling is also necessary for the development of human NKT cells; the development of NKT cells is impaired in patients with X-linked lymphoproliferative syndrome, where SAP is mutated.103 Furthermore, the human chromosomal segment syntenic Immunology and Cell Biology

to the mouse Nkt1 locus lies on distal chromosome 1q, which has been linked to TID susceptibility in humans.113,114 Once genetic risk has been determined, it is still unclear whether NKT-cell-based treatments such as adoptive transfer or glycolipid therapy could be successful in preventing TID in at-risk individuals, primarily because the mechanisms by which NKT cells regulate autoimmunity are poorly understood. Despite the success of NKT cell therapies in NOD mice, clinical translation may be stymied by the unpredictability surrounding the outcome of NKT cell manipulation. It is still unknown precisely how NKT cells can both suppress and promote cell-mediated immunity in different settings, or whether idiosyncratic differences would affect the outcome. aGalCer therapy for cancer has entered phase I clinical trials, establishing the relative safety of direct administration of the glycolipid alone and the re-administration of aGalCer-pulsed autologous DCs. Both treatments caused increases in serum cytokines such as IFNg and IL-12.115–117 The use of aGalCer for treatment or prevention of autoimmunity has not been tested, but it is worth remembering that prevention of TID in NOD mice using aGalCer requires prolonged treatment with weekly injections from around the time of the initiation of insulitis (3–4 weeks).35–37 The risks associated with long-term treatment with aGalCer in humans are unknown, despite the relative safety of the compound in the short term. Furthermore, anticancer studies of aGalCer treatment are designed to promote the proinflammatory or immune-potentiating function of NKT cells, which is not necessarily a desirable approach for the treatment or prevention of organ-specific autoimmune disease. The use of aGalCer analogues, which seem to preferentially promote production of IL-4 over IFNg, may be more useful. C20:2, a derivative of aGalCer containing an 11,14-cis-diunsaturated C20 fatty acid, slightly improved TID outcomes in NOD mice and successfully activated, and caused cytokine production from, human NKT cells.40 In contrast, although OCH has been shown to be effective for prevention of TID in NOD mice due to promotion of Th2 responses,39 this analogue was not able to bind to or activate human NKT cells, suggesting that it is unlikely to be suitable as an immunomodulator in humans.40 Replenishing NKT cell numbers by adoptive transfer has been a successful therapy in NOD mice.29,31 Clinical translation of this treatment requires research into methods enabling in vitro expansion and manipulation of NKT cells. Several studies have successfully expanded human NKT cells from PBMCs in vitro using aGalCer, DCs and cytokines such as IL-2 and IL-7.99,118,119 Transfer of autologous, in vitro expanded NKT cells harvested from the blood of at-risk individuals is a potential method for boosting NKT cell number and/or activity. Again, the use of Th2-favoring glycolipids to expand NKT cells, along with Th2-promoting reagents, such as IL4, may be a useful way of targeting their suppressive function.

CONCLUDING REMARKS NKT cells remain a tantalizing immunotherapeutic target for human TID, due to the many successful NKT-cell-based therapies in the NOD mouse model. However, the number and variety of mechanisms proposed to underlie the tolerogenic activity of NKT cells in TID pose a significant challenge to an uncomplicated clinical translation of the information obtained from the NOD model. Although NKT cells are thought to be important for regulating human TID, accurate assessment of NKT cell number and function in humans is difficult. It seems that a new approach, one which takes advantage of new techniques for assessing genetic control of NKT cell


number and function in mice, could be a useful and clinically translatable method for both assessing risk in humans and exploring new therapies.

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ACKNOWLEDGEMENTS This work was supported by the National Health and Medical Research Council (NHMRC) of Australia. MTF is a recipient of an Australian Postgraduate Award; AGB is a recipient of an NHMRC Senior Research Fellowship.

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