Introduction

Type 1 diabetes (T1D) is a T-cell-mediatedautoimmune disease that results in thedestruction of insulin-producing β-cells of theislets of Langerhans of the pancreas (1,2).T1D arises in two distinct stages. First, a het-erogeneous population of leukocytes com-

prising T-cells, B-cells, macrophages (Mϕs),and dendritic cells (DCs) begins to invade theperivascular region of the islets (periinsulitis),and an invasive and destructive insulitisslowly progresses (3–5). Second, T1D devel-ops as a result of islet β cell destruction byislet-infiltrated immune cells and is mani-fested by hyperglycemia (2,6). The period of

AbstractInvariant CD1d-restricted natural killer T (iNKT) cells functionduring innate and adaptive immune responses. A functional andnumerical deficiency of iNKT cells is well documented in bothnonobese diabetic (NOD) mice and humans with autoimmune type1 diabetes (T1D). Restoring the numerical and/or functional defi-ciency of iNKT cells in NOD mice by either treatment with α-galac-tosylceramide, transgenic induction of Vα14-Jα18 expression, ortransgenic expression of CD1d in NOD islets under the control ofthe human insulin promoter confers protection from T1D in thesemice. Recently, considerable progress has been made in under-standing the developmental and functional activities of iNKT cells.In this review, we discuss the role of iNKT cell deficiency anddefective development in the onset of T1D in NOD mice and the dif-ferent protective mechanisms known to restore these defects.

Key WordsInvariant CD1d-restricted

natural killer T cellsType 1 diabetesNonobese diabetic miceRegulatory T cellsImmunoregulation

Dr. Terry L. DelovitchDirector, Autoimmunity/Diabetes GroupRobarts Research Institute1400 Western RoadLondon, Ontario N6G 2V4, CanadaE-mail: del@robarts.ca

177© 2005Humana Press Inc.0257–277X/05/31/3:177–188/$30.00

Role of Regulatory Invariant CD1d-Restricted Natural Killer T-Cellsin Protection Against Type 1 Diabetes

Immunologic Research 2005;31/3:177–188

Shabbir Hussain1

Melany Wagner2

Dalam Ly2

Terry L. Delovitch1,2

1Autoimmunity/Diabetes Group,Robarts Research Institute,London, Ontario, Canada2Department of Microbiology &Immunology,University of Western Ontario,London, Ontario, Canada

insulitis can vary among individuals (years inhumans, months in rodents) before it finallyprogresses to overt T1D. Both CD4+ andCD8+ T-cells are required for islet β celldestruction (2,7). CD8+ T-cells function aseffector cells but require help from CD4+ T-cells (7–9). The presence of T-cells, B-cells,Mϕs, and DCs in islets suggests that interac-tions between these cells and their secretedproducts may lead to the destruction of islet βcells and onset of T1D.

The nonobese diabetic (NOD) mouse is anextensively studied animal model of T1D (10).NOD mice spontaneously develop T1D as aresult of immune dysregulation with animmunopathologic profile very similar to that

of the human disease (2). Immune dysregula-tion in NOD mice is mediated by defectiveantigen presentation, impaired deletion and/orsuppression of autoreactive T-cells (2), anddeficiencies in subsets of regulatory T (Treg)cells including CD4+CD25+ T-cells (11,12)and invariant CD1d-restricted natural killer T(iNKT) cells (13–21). Although iNKT cellsrepresent a small T-cell subset, their potentimmunoregulatory activity is sufficient to pro-tect NOD mice from T1D (19,20,22–29).Figure 1 shows that interactions among variousimmune cells lead to immune dysregulation inNOD mice and that iNKT cell activation cor-rects this dysregulation and protects these micefrom T1D.

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Fig. 1. Immune dysregulation in NOD mice initiates the onset of T1D. iNKT cell activation corrects thisdysregulation and protects NOD mice from diabetes. (A) Defective antigen presentation together with sub-optimal costimulation by antigen-presenting cell (APC) costimulatory molecules causes the generation andaccumulation of islet β cell autoreactive T-cells. If the activity of these autoreactive T-cells is not controlledby Treg cells, such as iNKT cells and CD4+CD25+ Treg cells, islet β cell destruction and onset of T1D willresult. (B) On activation by α-galactosylceramide (α-GalCer), iNKT cells secrete low levels of Th1 cytokine(interferon-γ [IFN-γ]) and increased levels of Th2 cytokines (interleukin-4, [IL-4], IL-10), which polarizesthe immune response toward a Th2 phenotype. (C) iNKT cell activation by α-GalCer may correct the defi-ciency in CD4+CD25+ Treg cells via cell:cell contact and/or cytokine secretion. (D) Various mechanisms maybe involved in iNKT cell–mediated protection from T1D.

iNKT cells are defined by their T-cell recep-tor (TCR) α gene rearrangements and CD1ddependency. iNKT cells express a specificinvariant TCR α gene rearrangement: Vα14-Jα18 and Vβ8.2 in mice, and Vα24-Jα15 andVβ11 in humans (30). iNKT cells recognizeglycolipid antigen presented in the context ofthe major histocompatibility complex classI–like protein, CD1d (31,32). iNKT cells arereactive to endogenous ligands (31), and astrong stimulator of iNKT cell activity hasbeen identified to be a glycolipid, α-GalCer,derived from a marine sponge (33,34). Whenpresented in the context of CD1d, α-GalCercan potently stimulate iNKT cells to rapidlysecrete IL-4, IL-10, and IFN-γ (19,20,35,36),suggesting that iNKT cells are capable ofimmune regulation. Protection of NOD micefrom T1D by increasing iNKT cell number byeither α-GalCer treatment (19,20,22), trans-genic induction of Vα14-Jα281 expression(27,28), or transgenic expression of CD1d inNOD islets (25) also supports the immunoreg-ulatory function of iNKT cells. Altogether,these data suggest that a functional andnumerical deficiency of iNKT cells in NODmice may be one of the major contributingfactors responsible for T1D development. Inthis review we focus on the role of iNKT cellsin protection against T1D with an emphasison the defects in iNKT cell development andthe mechanisms that may be involved in iNKTcell activation-dependent protection againstT1D.

Role of iNKT Cell Deficiency in Development of T1D

NOD mice are deficient in the number andfunction of iNKT cells, and correction of thisdeficiency protects these mice from T1D(13,19,20,25,27,28). The most severe defi-ciency in iNKT cell numbers in NOD micecompared with nondiabetic C57BL/6, Balb/c,

and AKR mice occurs in the thymus andspleen (13,21,37). iNKT cells from NODmice also show decreased IL-4 secretion fol-lowing TCR crosslinking and α-GalCer treat-ment (19). Thus, in addition to the low IL-4secretion by NOD T-cells (38), decreasednumbers of IL-4-producing iNKT cells (19)may be an important factor that precipitatesthe onset of T1D. The deficiency in iNKT cellnumber and function in NOD mice occursbefore the onset of insulitis and is restored by12–15 wk of age in the spleen but not thethymus (37). In humans with T1D, the issueof whether an iNKT cell deficiency occurs hasbeen controversial, because both an increaseand a decrease in the number of circulatingiNKT cells has been reported in patients withT1D. The initial result by Wilson et al. (39)suggested that humans with T1D have areduced frequency of iNKT cells as well asimpaired cytokine secretion following stimu-lation with α-GalCer. It was found that iNKTcell clones from nondiabetic siblings wereable to produce both IL-4 and IFN-γ, whereasthose from diabetic siblings only producedIFN-γ, not IL-4, indicating a Th2 abnormalityin the diabetic siblings (39). Other investiga-tors (40,41) have not found an iNKT defi-ciency in patients with T1D. The reason(s) forthe discrepancy between these studies is notknown and may result from the differentreagents and cell populations used in the stud-ies. Nonetheless, an increased frequency ofiNKT cells in the peripheral blood of patientswith T1D is associated with recent onset ofT1D rather than established disease (42). Thediscrepancy regarding iNKT cell numbers inpatients with T1D has cast some doubt oniNKT cell–based clinical application. How-ever, a recent study (43) found normal or evenelevated iNKT cell numbers in the peripheralblood of NOD mice, implying that circulatingiNKT frequencies may not reflect lymphoidtissue deficiencies in iNKT cells. Although

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the iNKT cell frequency in peripheral bloodmay not be a reliable tool for the diagnosis ofT1D, the immunoregulatory properties ofiNKT cells may still offer much potential forfuture therapies.

Development of Defective iNKT Cells May Cause iNKT Cell Deficiency

Based on the expression of the CD4 andCD8 coreceptors, CD4+CD8– and CD4–CD8–

(double-negative), are two well-characterizedsubsets of iNKT cells present in the thymus,the main site of T-cell development (44).Generally, iNKT cells and conventional αβ T-cells follow the same developmental path-ways, with the exception that a few studiessuggest that an alternative pathway for thedevelopment of iNKT cells may exist. Thelatter studies are based mainly on findings thata dominant-negative ras transgene inhibits thepositive selection of conventional αβ T-cellsbut not iNKT cells (45). By contrast, theLy49D, IL-15, RelB, and Fyn cell signalingmolecules are required for the development ofiNKT cells and have no detectable effect onthe development of conventional αβ T-cells(46,47). Therefore, it is evident that develop-ing conventional αβ T-cells and iNKT cellsrespond differently to the aforementioned sig-naling molecules. Accordingly, signals fromthese molecules might be required for thematuration of iNKT cells but not conventionalT-cells after positive selection. Alternatively,these molecules may provide signals thatassist in the positive selection of iNKT cellsover conventional T-cells by cross talk withsignals from the invariant TCR and/or otheriNKT cell–specific receptors.

Strong evidence suggests that iNKT cellsfollow the conventional αβ T-cell develop-mental pathway. For instance, the pre-TCRplays a key role in the expansion of conven-tional T-cells and is absolutely required for

the development of iNKT cells (48,49). CD4+

and double-negative iNKT cells, like conven-tional αβ T-cells, also pass through thedouble-positive stage and the immatureCD44low T-cell stage in the thymus (50,51).Furthermore, iNKT cell selection followsboth positive and negative selection. Deathowing to neglect or lack of positive selectionpresumably accounts for the lack of matureCD4+ and double-negative iNKT cells in thethymus of C57BL/6.CD1d–/– mice (50). iNKTcells also undergo negative selection in astrain-dependent manner on administration ofexcess amounts of α-GalCer in vitro and invivo (52,53). Although studies of the devel-opment of iNKT cells have identified simi-larities and differences between thedevelopment of conventional αβ T-cells andiNKT cells the defect(s) in central tolerancethat gives rise to the iNKT cell deficiency andthe generation of islet β cell autoreactive T-cells in NOD mice remains to be determined.Precursors common to conventional αβ T-cells and iNKT cells, such as CD44low iNKTor double-positive thymocytes, should beexamined for the expression of the invariantVα14Jα18 TCR to determine the stage atwhich the iNKT cell deficiency occurs in theNOD thymus (50,51).

iNKT cells are preferentially selected invivo over conventional αβ T-cells, because theinvariant TCR rearrangement of iNKT cells isdetectable by α-GalCer-loaded CD1d tetramerstaining in the thymus of both NOD andautoimmune-resistant mice at approx 105 timeshigher frequency than would be expected byrandom rearrangement (32). Clones of iNKTcells also express other VαJα rearrangements,suggesting that the invariant TCR is not pref-erentially rearranged, but must be preferen-tially selected when random rearrangementresults in a Vα14Jα18/Vβ8/7/2 pairing (54).Indeed, expression of the invariant TCR ofiNKT cells directly affects the number of iNKT

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cells. Transgenic expression of Vα14Jα18enhances iNKT cells in NOD mice, and doubletransgenic Vα14/Vβ8.Rag1–/– mice develop ahomogeneous NK1.1+ αβ T-cell population,whereas Jα18–/– mice lack NK1.1+ T-cells(32,44). Selection of conventional αβ T-cellsoccurs via TCR interactions during positiveand negative selection at the double-positivestage of development. Given that iNKT cellspass through the double-positive stage of con-ventional αβ T-cell development and undergopositive and negative selection, positive/nega-tive selection of all αβ T-cells at the double-positive stage would likely preferentiallyenhance the iNKT cell population (50). Thus,a survival advantage for iNKT cells over con-ventional αβ T-cells may be provided by theinvariant TCR under normal conditions, but thestrength of the invariant signal may be toostrong and selectively deplete iNKT cells overconventional αβ T-cells on manipulation ofother signaling molecules.

Early blockade of the development of iNKTcells by thymectomy at 3 d of age severelyreduces the number of peripheral NKT cellsand causes gastritis in (Balb/c x C57BL6) F1mice (55). This implies that significant devel-opment of NKT cells takes place early in lifeto prevent autoimmune disease. In NOD mice,NKT cell deficiency has been reported asearly as 3 wk of age (37). Interestingly, ourreal-time polymerase chain reaction analysesof Vα14Jα18 expression indicate that theiNKT cell deficiency may be present even ear-lier in NOD mice, by 1 wk of age (unpub-lished data), a time point considered to be theimmunologic equivalent of a newborn human(56). This result raises the possibility that aniNKT cell deficiency may be present at birthand may predispose to the development ofT1D. NOD mice possess a defect in centraltolerance; thymic epithelia from NOD miceengrafted into athymic C57BL/6 mice is suf-ficient to induce insulitis in these diabetes-

resistant mice (57). Besides thymic epithelia,other unknown factor(s) may also contributeto the iNKT cell deficiency in NOD mice,since intrathymic cotransfer of thymic pre-cursors (1:1) from diabetes-susceptible NODand diabetes-resistant ACK mice into NODmice restored the frequency of iNKT cells inan NOD thymus (18). The factor(s) responsi-ble for defects in central tolerance and aniNKT cell deficiency in the NOD thymusremains to be defined.

Activation of iNKT Cells Protects Against T1D

iNKT cells are implicated in the regula-tion of susceptibility to several autoimmunediseases, including systemic lupus eryth-matosus (17), rheumatoid arthritis (58), mul-tiple sclerosis (59), and T1D in humans (39)and in animal models (13,19,20,25,60).Recent progress in understanding CD1d-dependent iNKT cell activation-induced pro-tection from T1D has identified severalpossible mechanisms of protection. Theseinclude a shift in the immune responsetoward a Th2 phenotype, iNKT cell/APCinteraction-dependent tolerance induction,regulation of chemokine/chemokine receptorexpression and interaction, and induction ofimmune homeostasis. These mechanisms aresummarized in Table 1 and are discussed inlight of current findings.

iNKT Cell Activation Shifts Immune ResponseToward a Th2-Like Phenotype

NOD CD4+ T-cells are believed to be biasedtoward Th1 cytokine (IFN-γ) production,while showing defects in Th2 cytokine (IL-4,IL-10, IL-13, and transforming growth factor-β) polarization (2). We previously reportedthat NOD T-cells are deficient in IL-4 produc-tion and are hyporesponsive to TCR-inducedproliferation (61). On restoration of IL-4

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expression to normal levels by either admin-istration of recombinant IL-4, IL-4 gene ther-apy, or anti-CD28 costimulation, T-cellresponsiveness to TCR stimulation is restoredand NOD mice are protected from destructiveinsulitis and T1D (38,61–63).

In NOD mice, iNKT cell activation byadministration of α-GalCer preferentiallyenhances IL-4 and IL-10 production by spleencells and the upregulation of IL-10 receptor(IL-10R) expression (19,20). The significanceof elevated IL-10R expression is unclear, butit may indicate enhanced utilization of IL-10produced in response to α-GalCer treatment.To clarify the relative importance of IL-4 andIL-10 in iNKT cell–mediated protectionagainst T1D, we treated IL-4–/– and IL-10–/–

NOD mice with α-GalCer and monitored their

incidence of spontaneous and cyclophos-phamide-induced T1D. Interestingly, wefound that NOD.IL-10–/– but not NOD.IL-4–/–

mice were protected against T1D (64), demon-strating that IL-4 but not IL-10 is required forα-GalCer-mediated protection against T1D.These results highlight the relative contribu-tions of IL-4 and IL-10 in the protectionagainst T1D in NOD mice.

iNKT Cell–APC Interaction Induces ToleranceNKT cells recognize lipid antigen when

presented in the context of CD1d (31,34).CD1d is expressed on APCs such as DCs,Mϕs, and B-cells (65,66). In the anteriorchamber–associated immune deviation modelof NKT cell–dependent tolerance, CD1d+

spleen marginal zone (MZ) B-cells promote

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Table 1. iNKT Cell–Mediated Protection from T1D in NOD Mice

Restoration of iNKT Significant findings with proposed cell deficiency mechanism(s) of protectiona References

α-GalCer treatment ↑ iNKT cell numbers in spleen, PLN, and islets; ↑ IL-2, IL-4, 19,20,22,23,26IL-10, and CCL4 expression; ↓ IL-16 expression; ↓ insulitis;↓ Th1 Ab isotypes (IgG1, IgG2a) in response to GAD65; ↑ IgE recruitment of DCs to PLN; polarization to Th2-like antibody and cytokine profiles

Transfer of double-negative Double-negative αβ T-cells rich in iNKT cells, thus causing 29αβ T-cells (thymocytes) systemic ↑ number of iNKT cells; ↓ insulitis; protection from

T1D abrogated after neutralization of IL-4 and/or IL-10 with Abs

Transgenic expression ↑ number of iNKT cells in thymus, spleen, PLN, and islets; 27,28of Vα14-Jα281 ↓ IL-4 and ↓ IFN-γ and IL-2; ↓ Th1 Ab isotype (IgG2c) and

↑ Th2 Ab isotype (IgG1) in response to GAD65; shift of Ab and cytokine profiles from Th1 to Th2

NOD.Vα14Cα–/– ↑ iNKT cell numbers in PLN harbored by NOD.Vα14Cα–/– 24mice; on transfer of diabetogenic T-cells, show ↓ insulitis;↓ IFN-γ, TNF-α, and IL-2 production; ↓ activity of T-cells reactive to pancreatic β-cells

Transgenic expression of ↑ iNKT cell numbers in PLN; ↓ T-cell proliferation in response 25CD1d under control of to GAD65; ↓ insulitis; ↓ IFN-γ ↑ IL-4 and IL-10; shift in insulin promoter in islets cytokine profile induced by GAD65 from Th1 to Th2 phenotype

a ↑, increase in; ↓, decrease in; PLN, pancreatic draining lymph node; Ab, antibody; TNF-α, tumor necrosis factor-α.

tolerance by enhancing the development ofantigen-specific T-regulatory (Treg) cells (66).The exact mechanism of NKT cell–dependentincrease in Treg development is not known butmay be owing to an increase in IL-10 secre-tion by activated NKT cells (67). Alterna-tively, MZ B-cell–NKT cell interaction mayactivate MZ B-cells to secrete IL-10 to gen-erate Treg cells. The latter possibility is sup-ported by the notion that activated B-cells arean excellent source of IL-10 (68), and ivtransfusion of B-cell receptor–stimulated B-cells into NOD mice protects these mice fromT1D by increasing the number of CD4+CD25+

Treg cells in islets (unpublished data).DCs represent another important APC

subset. CD1d is preferentially expressed by amouse lymphoid DC subset (CD11c+ CD8α+)relative to myeloid DCs (CD11c+CD8α–)(65). Lymphoid DCs are an excellent sourceof IL-12 and promote a Th1 immune responseafter activation (69), whereas myeloid DCsfunction as “tolerogenic DCs” and protectNOD mice from T1D (70). This notion is sup-ported by the inability of DCs isolated frompancreatic draining lymph node of α-GalCer-treated NOD mice to secrete IL-12 inresponse to in vitro LPS stimulation (23). Fur-thermore, these pancreatic draining lymphnode–derived DCs are of myeloid phenotype(CD11c+CD8α–) and protect NOD mice fromT1D when transferred into naive prediabeticmice. Thus, iNKT cell activation appears toinduce the accumulation of CD11c+CD8α–

tolerogenic DCs in the pancreatic draininglymph node of α-GalCer-treated NOD mice.

iNKT Cell Activation RegulatesCytokine/Cytokine Receptor andChemokine/Chemokine Receptor Expression and Interaction

Chemokines and their receptors play a keyrole in the pathogenesis of T1D owing to theirability to recruit leukocytes to islets. An ele-

vated ratio of macrophage inflammatory pro-tein-1α (MIP-1α):MIP-1β in the pancreas ofNOD mice relative to diabetes-resistant NORmice correlates with destructive insulitis andprogression to diabetes (71). MIP-1α is nowtermed CCL3, and MIP-1β is termed CCL4.Systemic IL-4 therapy of NOD mice lowersthe CCL3:CCL4 ratio and protects these micefrom T1D (71). iNKT cell activation by α-GalCer also preferentially enhances IL-4 pro-duction (19,20) and increases CCL4 mRNAexpression in the spleen of NOD mice (26).Interestingly, antibody neutralization ofCCL4 abrogates IL-4-mediated protectionand identifies a requirement for CCL4 activ-ity in IL-4-mediated protection from T1D(26). CCL4 has been shown to recruitCD4+CD25+ Treg cells in vitro (72). Whetherprotection from T1D in α-GalCer- and IL-4-treated NOD mice is associated with the pref-erential recruitment and/or generation ofCD4+CD25+ Treg cells requires further inves-tigation.

IL-16 is a cytokine with chemoattractantactivity that when neutralized in NOD miceby a mouse antihuman IL-16 antibodyreduces the incidence of T1D (26). Interest-ingly, this protection is abrogated on coinjec-tion of anti-IL-16 and anti-CCL4 antibodies(26). IL-16 is proinflammatory in nature andis produced by several cell types, includingCD4+ and CD8+ T-cells, B-cells, and DCs(73,74). In contrast to CCL4 gene expression,we found a decrease in the IL-16 mRNAexpression in the spleen of NOD mice treatedwith α-GalCer (26). IL-16 binds to its CD4receptor and preferentially recruits CD4+ Th1cells (75). Thus, IL-16 neutralization mayblock the migration of CD4+ Th1 cells toislets and thereby block the onset of T1D.Alternatively, because the binding of IL-16 toCD4 desensitizes the ability of CCL4 tosignal through its receptor CCR5 (76), neu-tralization of IL-16 by an anti-IL-16 antibody

iNKT Cells and Type 1 Diabetes 183

may block this desensitization and enableCCL4 to signal normally to CCR5. Thisnotion is further supported by the increasedincidence of T1D in NOD mice coinjectedwith anti-IL-16- and anti-CCL4-neutralizingantibodies (26).

Stromal cell–derived factor 1 (SDF-1)mRNA transcripts are reduced in the spleensof α-GalCer-treated NOD mice (26). SDF-1recruits both T- and B-cells (77). Neutraliza-tion of SDF-1 in NOD mice by administrationof an antimouse SDF-1 polyclonal antibodyprotects these mice from T1D by decreasingthe number of B-cells that migrate to thespleen (78). The expression levels ofchemokine 6ckine (CCL21) and chemokinereceptors CCR2, CCR3, and CCR4 are alsoreduced in the spleen of α-GalCer-treatedNOD mice (26). The expression of the CCL4as well as the eotaxin (CCL24), IFN-γ-inducible protein 10 (CXCL10), and monokineinduced by IFN-γ (CXCL9) chemokine genesis increased in the spleens of α-GalCer-treatedNOD mice (26). Together, these findings sug-gest that iNKT cell activation regulateschemokines and chemokine receptor geneexpression. Currently, the mechanism of reg-ulation of chemokines and chemokine recep-tors genes following α-GalCer treatment is notknown but once identified may open newavenues for therapeutic intervention.

iNKT Cell Activation Induces ImmuneHomeostasis

NOD mice not only harbor a numerical andfunctional deficit of iNKT cells but are alsodeficient in T-cells (79) and B-cells (79,80).Numerical and functional abnormalities werealso reported in NOD bone marrow–derivedDCs and Mϕs (81–85). Recently, it wasreported that correction of this T-cell deficiencyin NOD mice either by administration of com-plete Freund’s adjuvant or by iv transfusion ofprediabetic syngeneic T-cells into naive NOD

mice induces protection from T1D (79). Inter-estingly, treatment of NOD mice with α-GalCerresults in a 2.5-fold increase in spleen cells rel-ative to vehicle-treated control mice (unpub-lished data). Whether the increase in totalspleen cells represents a preferential increase inT- and B-cells or all spleen cells are increasedproportionally remains to be determined. Sinceα-GalCer-treated NOD mice are protected fromcyclophosphamide-induced T1D (19,26,64)and cyclophosphamide elicits a lymphopenia,activation-induced lymphocyte homeostasismay be one of the iNKT cell–mediated mech-anisms of protection from T1D.

Conclusion

The precise mechanism whereby iNKTcell activation by α-GalCer protects NODmice from developing T1D is still not com-pletely understood. Studies to date indicatethat polarization of an immune responsetoward a Th2 phenotype may be involved;however, tolerogenic DCs that are recruited tolymph nodes on α-GalCer treatment may alsodrive this polarization. Although variouscytokines, chemokines, and their respectivereceptors have been shown to regulate theactivation and migration of iNKT cells, Th2cells, DCs, and other interacting types ofcells, further study is required to understandbetter the mechanism of regulation involved.Whereas an iNKT cell deficiency has beenreported to occur in NOD mice as early as 3wk after birth, our studies indicate that thisdeficiency may occur during fetal develop-ment and be present at birth. Precursors thathave been identified in the iNKT cell path-way, such as CD44low iNKT or double-posi-tive thymocytes, need to be examined todetermine the earliest stage at which theiNKT cell deficiency occurs in the thymus.Emphasis should be given to the identificationof the subset of APCs that presents glycolipid

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antigen and stimulates the production of Th1(IL-12, IFN-γ) or Th2 (IL-4, IL-10, IL-13)cytokines. What are the natural self-ligandsthat regulate iNKT cell activity in various tis-sues? Do iNKT cells protect NOD mice fromT1D by correcting lymphopenia? Do iNKTcells also provide protection against diabetesin other induced models of T1D? If so, whatare the mechanisms of protection? DoesiNKT cell activation maintain homeostasis ofthe immune system by enhancing the numer-ical and functional activity of CD4+CD25+

Treg cells? Answers to these questions mayopen up new avenues for the design and exe-cution of new strategies to treat T1D.

Acknowledgments

This work was supported by grants fromthe Canadian Institutes of Health Research;the Juvenile Diabetes Research Foundation;the Canadian Diabetes Association, in honorof the late Olive I. Moore; and the OntarioResearch and Development Challenge Fund.

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