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39 Shapiro, V.S. et al. (1997) CD28 mediates transcriptional upregulation of the interleukin-2 (IL-2) promoter through a composite element containing the CD28RE and NF-IL-2B AP-1 sites. Mol. Cell. Biol. 17, 40514058 40 McGuire, K.L. and Iacobelli, M. (1997) Involvement of Rel, Fos, and Jun proteins in binding activity to the IL-2 promoter CD28 response element/AP-1 sequence in human T cells. J. Immunol. 159, 13191327 41 Coudronniere, N. et al. (2000) NF- B activation induced by CD28 costimulation is mediated by PKC . Proc. Natl. Acad. Sci. U. S. A. 97, 33943399 42 Lin, X. et al. (2000) Protein kinase C participates in NF- B/Rel activation induced by CD3/CD28 costimulation through selective activation of I B (IKK ). Mol. Cell. Biol. 20, 29332940 43 Downward J. et al. (1992) The regulation and function of p21ras in T cells. Immunol. Today 13, 8992 44 Ebinu, J.O. et al. (2000) RasGRP links T-cell receptor signaling to Ras. Blood 95, 31993203 45 Cantrell, D. (1996) T cell antigen receptor signal transduction pathways. Cancer Surv. 27, 165175 46 DAmbrosio, D. et al. (1994) Involvement of p21ras in T cell CD69 expression. Eur. J. Immunol. 24, 616620 47 Green, D.R. and Ware, C.F. (1997) Fas-ligand: privilege and peril. Proc. Natl. Acad. Sci U. S. A. 94, 59865990

48 Latinis, K.M. et al. (1997) Regulation of CD95 (Fas) ligand expression by TCR-mediated signaling events. J. Immunol. 158, 46024611 49 Rodriguez-Tarduchy, G. et al. (1996) Apoptosis but not other activation events is inhibited by a mutation in the transmembrane domain of T cell receptor chain that impairs CD3 association. J. Biol. Chem. 271, 3041730425 50 Villalba, M. et al. (1999) PKC is a necessary component, and cooperates with calcineurin, to induce FasL expression during activation-induced T cell death. J. Immunol. 163, 58135819 51 Villunger, A. et al. (1999) Synergistic action of protein kinase C and calcineurin is sufficient for Fas ligand expression and induction of a CrmA-sensitive apoptosis pathway in Jurkat T cells. Eur. J. Immunol. 29, 35493561 52 Rodriguez-Tarduchy, G. and Lopez-Rivas, A. (1989) Phorbol esters inhibit apoptosis in IL-2-dependent T lymphocytes. Biochem. Biophys. Res. Commun. 164, 10691075 53 Boise, L.H. et al. (1995) CD28 and apoptosis. Curr. Opin. Immunol. 7, 620625 54 Bertolotto, C. et al. PKC and promote T cell survival by a RSKdependent phosphorylation and inactivation of BAD. J. Biol. Chem. (in press) 55 Alcami, J. et al. (1995) Absolute dependence on B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes. EMBO J. 14, 15521560

NKT cells: facts, functions and fallacies

Dale I. Godfrey, Kirsten J.L. Hammond, Lynn D. Poulton, Mark J. Smyth and Alan G. Baxter
The proposed roles of NK1.1 T
on thymic TCR T cells that were CD4 KT cells are a population range from suppression of of T cells that share some and CD8 double negative ( DN) and autoimmunity to tumor rejection. characteristics with natuhighly biased toward V 8-2 expression. A ral killer (NK) cells (remajor subset of these cells was found to exHeterogeneity of these cells viewed in Refs 13). The key features charpress NK1.1, previously associated with NK contributes to the controversy acteristic of NKT cells include heavily biased cells, and to have the potential to secrete T-cell receptor (TCR) gene usage, CD1d rehigh levels of IL-4, IFN- and tumor necrosis surrounding their development and striction and high levels of cytokine producfactor (TNF). A population of NK1.1 CD4 function. This review aims to T cells was also identified with similar chartion, particularly interleukin 4 (IL-4) and inprovide an update on NKT cell acteristics13. Subsequent reports showed terferon (IFN- ) (Fig. 1). In mice, these cells TCR . that both populations predominantly express are commonly defined as NK1.1 biology and, whenever possible, to However, as cells bearing these markers are TCR V 14J 281 and that their development compare what is known about NKTphenotypically and functionally heterogenwas dependent on the MHC class I-like, 2cell subsets. microglobulin-associated molecule, CD1d. eous, it is appropriate to compare the differThese findings strongly suggested that ent subsets of cells encompassed within this DN and NK1.1 CD4 T cells were NK1.1 population. Most of the studies covered in part of the same lineage13. Thus, in recent years NKT cells are this review relate to NKT cells in mice, unless otherwise specified. usually identified by the coexpression of the TCR and NK1.1, as shown in Fig. 2. Subsets of NKT cells Several recent studies in mice have demonstrated the potential Although the term NKT was only recently applied, these cells were danger of oversimplifying our classification of NKT cells48. These first described in 1987. Most of the earlier studies in mice focussed studies have shown that CD4 , DN and CD8 NK1.1 T cells are
PII: S0167-5699(00)01735-7 0167-5699/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved.

(NKT) cells in immune responses

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NKT
Rapid/high IL-4 production CD1d restricted CD3/TCR+ Thymus-dependent TCR-dependent NK1.1+ Non-MHC restricted

Cytotoxic IFN- production

MHC-I/II restricted

Thymus-independent

NK
Immunology Today

Fig. 1. Mouse NKT cells share some characteristics with NK and T cells. This venn diagram incorporates some of the key features that are generally applied to NKT, T and NK cells. Characteristics that are unique to each lineage are shown in the non-overlapping sections. Characteristics that are common to any two of these lineages are indicated in the relevant overlapping areas (purple, orange or green). The central (white) area indicates characteristics that can be exhibited by all three lineages. Information derived from references (13). Abbreviations: MHC, major histocompatibility complex molecule; NK, natural killer; NKT, NK1.1 T cells; TCR, T-cell receptor.

(a)

Thymus

Spleen

Liver

NK1.1

0.6%

1.4%

24%

present in most tissues and are phenotypically and functionally distinct. Furthermore, peripheral NK1.1 T-cell subsets differ from their thymic counterparts (Fig. 3). Thymus and liver CD4 and DN NKT cells are generally alike, although those in the liver have greatly reduced expression of the MHC class-I ligands Ly49 A, C/I and G2 (Ref. 9). Spleen, lymph node and bone-marrow NKT cells are far more heterogeneous. For example, although splenic CD4 NKT cells are similar to thymic NKT cells, many splenic DN NKT cells are not CD1-dependent4,8, have a more heterogeneous TCR repertoire4,5,7, and produce lower levels of cytokines following short-term in vitro stimulation5. The likelihood that splenic DN NK1.1 T cells include two distinct subsets is supported by the bimodal expression of other cell-surface markers (Fig. 3) and by bimodal reactivity with CD1d- galactosylceramide ( GalCer) tetramers8. Splenic CD8 NK1.1 T cells are CD1d and thymusindependent, express heterogeneous TCR, and do not produce IL-4 rapidly. In addition, some NK1.1 CD4 T cells closely resemble cells of the NKT-cell family in their CD1dreactivity, TCR V 8-bias, and high IL-4production8,1012. One explanation for this might be that NK1.1 can be downregulated upon NKT-cell activation13.

Surrogate markers for NKT cells

TCR
Another difficulty with the use of NK1.1 to identify NKT cells is that this allelic marker is only expressed in a limited number of mouse strains, primarily C57BL/6 and related strains. A number of different surrogate phenotypes have been used, for example: DN (Ref. 14), DX5 CD3 (Ref. 15), and Ly49A CD122 CD3 (Ref. 16). A problem with these surrogate markers is that they do not accurately encompass NKT cells in C57BL/6 mice and their specificity for NKT cells in other strains is difficult to determine. In some cases, such as DN, they detect only a subset of NKT cells, and NK1.1 staining of these populations in C57BL/6 mice demonstrates the presence of other NK1.1 cells within these populations. Other surrogate phenotypes, such as DX5 CD3 , might exclude most NKT cells, particularly the CD1d-dependent subsets (CD4 and most DN), which are DX5

(b)
65 34 CD4 68 20 10 80 17 2

CD8
Immunology Today

Fig. 2. NK1.1 T cells and their CD4/CD8-defined subsets. Mononuclear cells from the thymus, spleen and liver were labelled with mAb specific for TCR, NK1.1, CD4 and CD8. (a) NK1.1 versus TCR on lymphoid gated cells. (b) CD4 versus CD8 expression by NKT cells gated as shown in (a). Data show representative 2-D dotplots derived from 57-week old female C57BL/6 mice, and acquired using a four-colour FACScalibur (Becton Dickinson, Mountain View, CA, USA). Abbreviations: mAb, monoclonal antibody, TCR, T cell receptor.

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(Refs 4,5) (Fig. 3). The use of NK1.1 congenic strains of mice will help with this problem but, ideally, V 14J 281 specific monoclonal antibody (mAb) or CD1d tetramers8,120 would most reliably identify these cells in all strains.

Thymus
Total
55

Peripherya
DN Total
57 42

CD4

+
54

CD4+
51

DN
31

CD8+
24

V8
76 81 69 96 99 89 97

Thy-1
74 69 84 64 80 40 27

NKT cells in humans

Humans also have NKT cells with very similar characteristics to mouse NKT cells (Table 1), including DN (Refs 17, 18) and CD4 subsets19. Both subsets react with human CD1d and produce high levels of IL-4 and IFN- when stimulated20. Significantly, human NKT cells express the homologous TCR gene products (V 11, the homologue of mouse V 8.2; and V 24J Q, the homologue of mouse V 14J 281). Furthermore, human NKT cells can recognize mouse CD1d and vice versa, indicating highly conserved specificity21.

CD69
98 97 99 97 97 99 100

CD45RB
7 6 12 13 4 35 43

DX5
70 69 76 54 48 70 86

Ly6C CD11a(LFA-1) CD24(HSA) CD25(IL2R) CD45R(B220) CD54(ICAM-1) CD62L CD127(IL7R) Ly49A Ly49C/I Ly49G2 CD1-dependent J281-dependent Thymus-dependent IL-4(U/ml)c IFN(ng/ml)c Low Low Neg 2% Pos 20% 51% 27% Y Pos Low Low Neg Pos 2% 15% Y Y Low Low Neg 2% 20% Y Y Y 600 8 Low Low Neg 60% 40% 10%b 11%b Y/N Y/N Y/N 300 0.2 Posb Low Low Neg Posb 50% 20% Y Y Y 200 0.3 Low Low Neg 60% 50% Y Low Low Neg 80% 45% N N N 2 0.8

Distribution of NKT cells

In mice, NKT cells can be detected wherever N Y conventional T cells are found, although the Y Y Y ratio of NKT to T cells varies widely in a 50 800 800 tissue-specific manner15. As a proportion of 0.1 6 5 mature T cells, NKT cells are most frequent in Immunology Today liver (3050%), bone marrow (2030%) and thymus (1020% of mature HSA T cells, although only 0.30.5% of total thymocytes). Fig. 3. Mouse NK1.1 T-cell subsets are phenotypically, developmentally and functionally distinct. They represent a smaller proportion of T cells Mouse NK1.1 T cells can be subdivided based on CD4/CD8 expression and on tissue of origin. This in other tissues including spleen (3%), lymph diagram shows representative histogram profiles or summarized data for a selection of features that node (0.3%), blood (4%) and lung (7%). Inter- reveal distinct subsets of NK1.1 T cells. Histogram profiles are representative of NK1.1 T-cell estingly, CD4 , DN and CD8 NK1.1 T cells subpopulations derived from 57-week old C57BL/6 mice. All NK1.1 T-cell subsets in the thymus are differentially distributed in a tissue-spe- and spleen are positive for CD122, Fas, CD44, CD1d, CD28, CD38 and CD45RC (not shown). Inforcific fashion46, supporting the concept that mation taken from Refs 16, 9. Abbreviations: DN, CD4 CD8 double negative; , not determined; these are functionally and/or developmen- IFN- , Interferon ; IL-4, interleukin 4; neg, negative; pos, positive; Y, yes; N, no; Y/N, partially a tally distinct subsets. Their intra-tissue local- dependent; J 281 dep., T-cell receptor V 14J 281-dependent development. Data relate to spleen b c ization remains unknown, partly because of NKT cells except where indicated. Data relate to liver NKT cells. IL-4/IFN- production measured 6 1 the relative scarcity of these cells and the lack by ELISA following 18 h culture on anti-CD3 coated plates, 10 cells ml . of a single, defining cell-surface marker. Although most reports on human NKT cells have been limited to that most NKT cells are thymus-dependent, some scientists argue for those in peripheral blood, V 24J Q NKT cells are clearly present in an extrathymic origin. This controversy has been discussed in detail human liver although apparently not as frequent as they are in mice in earlier reviews13, and will not be repeated here, except to say that (approximately 4% of hepatic T cells versus 50% in mice)22. The distri- the evidence for a thymic origin for most CD4 and DN NKT cells is bution of NKT cells in other human tissues remains to be determined. difficult to ignore. In mice, they develop in fetal thymus organ culture23, are present among recent thymic emigrants in both spleen and liver5 and fail to develop normally in nude24 or neonatally NKT cell development thymectomized mice25, or adult mice irradiated and thymectomized Thymus dependence prior to fetal liver-reconstitution26. By contrast, CD8 NK1.1 T cells A surprising amount of controversy surrounds studies of the origin are present in normal numbers in the periphery of neonatally of NKT cells. Although the overwhelming body of evidence indicates thymectomized mice5,25.

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Table 1. Features common to mouse and human NKT cells
Characteristic Major subsets Mouse CD4 , DN Human CD4 , DN Comment Proportions vary
NK1.1 following antigenic challenge in the periphery30. Although this would fit with the non-biased TCR repertoire of CD8 NK1.1 T T cell receptor cells, it is unlikely to completely explain the -chain V 14J 281 V 24J Q Homologous -chain V 8.2 V 11 Homologous presence of CD8 NK1.1 T cells in normal V 7, V 2 (Ref. 5). mice, many of which are CD8 Expression level Intermediate Intermediate Some studies have demonstrated and investigated the development of NK1.1 T Accessory molecules cells specific for conventional MHC plus NK associated NK1.1 NKR-P1 Homologous (CD161) peptide in TCR transgenic mice29,3133. CollecCD122 CD122 tively, these studies suggest that TCR-transLy49 genic, NK1.1-expressing, IL-4-secreting T Restriction element CD1d CD1d Homologous cells can be generated in the absence of endogenous TCR gene rearrangement. This Cognate antigen Glycolipid Glycolipid -GalCer stimulates implies that there is nothing inherently speCytokine production cial about the recognition of CD1d by the inIL-4 Rapid, high levels Rapid, high levels Following TCR ligation variant TCR V 14J 281, but rather, suggests IFNFollowing TCR ligation that NKT-cell development depends on the Frequency timing, avidity and type of selecting cell, and PBL ~1% ~0.10.5% More variable in humans that such selection might simply result more frequently through interaction with CD1d. aAbbreviations: GalCer, -galactosylceramide; DN, double negative; IFN- , interferon ; IL-4, interHowever, considering that NKT cells are leukin 4; NKR, NK-cell receptor; NKT, NK1.1 T cells; PBL, peripheral blood leukocytes; TCR, T-cell virtually absent in the thymus of CD1d / receptor. or TCR J 281 / mice, it seems that conventional TCRMHC interactions are, at best, a Appearance during ontogeny rare event in the development of NKT cells. Thus, although these Another debated question relates to the timing of NKT-cell develop- TCR transgenic approaches theoretically provide greater ability to ment. Most studies have indicated that NKT cells do not appear until manipulate the nature of the selecting environment (for example, relatively late in mouse ontogeny, well after conventional T cells13,25. non-selecting, positively selecting or negatively selecting), care However, one report suggested that NKT cells start to develop at E9.5 should be taken in extrapolating information from these studies to in the yolk sac and are one of the first T-cell populations to appear, non-transgenic NKT-cell development. arising in the fetal liver at E13.5 (Ref. 27). We (unpublished data) and others28 have been unable to verify these findings.

Factors influencing NKT-cell development NKT cell selection and branching

A largely unresolved problem relates to how NKT cells develop. Although this is known to involve a TCR-dependent interaction between NKT precursors and CD1d-expressing CD4 CD8 (DP) thymocytes13,24, many key questions remain. For example, the developmental relationship between the CD4 , DN and CD8 NK1.1 T-cell subsets is unknown. It is also unclear how recognition of CD1d cells results in commitment to this novel lineage, and whether high affinity recognition of self-antigen leads to negative selection of NKT cells. CD4 and MHC class II molecules appear to have, at best, limited roles in NKT cell development and function23. On the other hand, CD8 expression appears to strongly influence the development of NKT cells, such that high levels of CD8 transgene expression leads to their deletion in the thymus, whereas NKT cells have an altered TCR bias in CD8 / mice23. The development of CD8 NK1.1 T cells is even less well underNK1.1 T cells are stood. One study provided evidence that CD8 generated extrathymically following antigen encounter in the liver29. Another showed that mainstream CD8 T cells can upregulate In addition to CD1d, several other factors are known to influence NKT-cell development. Thymic stromal-cell-derived cytokines IL-15 and IL-7 are required for development of normal numbers and IL-4producing potential, respectively, of both CD4 and DN NKT cells34,35, and an intact thymic structure is also important36. In contrast to conventional T cells, NKT cell development requires an interaction with membrane lymphotoxin expressing cells37,38. Interestingly, lymphotoxin deficiency affected all three populations (CD4 , DN and CD8 ) of NK1.1 T cells, suggesting at least some commonality to the development of these subsets38. NKT-cell development is also absolutely dependent on pre-T signaling39 and at least partly dependent on granulocytemacrophage colony-stimulating factor (GM-CSF) signaling40. Fyn-deficient mice show a selective defect in the development of CD1d-dependent NKT cells, but not of conventional T cells, nor of CD8 NK1.1 T cells41,42. Analysis of NKT cells in common cytokine receptor -chain-deficient mice revealed at least two stages in NKT cell development43. Such mice generate thymocytes expressing normal amounts of V 14J 281 mRNA and develop IL-4 producing DN cells suggesting the presence of NKT cells yet these cells fail to express the NK receptors NK1.1 and Ly49, and are

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not exported to the periphery. Thus, intrathymic selection and development of IL-4-producing capacity seem to be earlier processes, whereas acquisition of the NK surface phenotype and emigration to the periphery are later, common -chain-dependent, events.

Maintenance of NKT cells

Similar to conventional T cells, the maintenance of peripheral NKT cell numbers is relatively thymus-independent. Following NKT cell depletion in the periphery, a wave of proliferation of NKT cells occurs in the bone marrow, quickly restoring the number of these cells, even in thymectomized mice44. Furthermore, whereas thymic CD4 NKT cells in young mice (less than 45 weeks old) turn over quite rapidly, almost no turnover is seen in the thymuses of older mice. By contrast, liver CD4 NKT cells continue to turn over in eight-week old mice, albeit at a low rate24. Liver NKT cells are also distinct in that the maintenance of normal numbers of these cells, but not NKT cells in other tissues, depends on LFA-1 expression by non-NKT cells45,46, possibly by NK cells47.

point, since GalCers show stronger immunostimulatory activities than GalCers (Ref. 56). It is therefore probably significant that the genes encoding the 31 galactosyl transferases, which are thought responsible for synthesizing such -linked carbohydrate moieties, are defective in humans and some other old-world primates57,58, suggesting at least on face value that GalCer cannot be a normal product of human cells. However, as this carbohydrate moiety also defines the human B blood group, this is clearly an area requiring more systematic study. If GalCer is not a product of human cells, it might either mimic a natural ligand, or displace CD1d-bound glycolipids through high-affinity interactions with CD1d.

Effector functions of NKT cells

The function most characteristic of CD4 and DN thymic NKT cells is the rapid production of high levels of immunoregulatory cytokines IL-4, IFN- and TNF following stimulation in vitro13,59. Although splenic CD4 NKT cells also produce these cytokines following short term in vitro stimulation, they produce less than do thymic NKT cells5. Furthermore, splenic DN and CD8 NK1.1 T cells secrete even lower levels of cytokines than do the splenic CD4 NKT cell subset5. Despite this, CD4 and DN NKT cells isolated from the spleen, within minutes of in vivo stimulation with anti-CD3, were a potent source of cytokines60,61. Curiously, this was not observed with splenic NKT cells in cell-suspension culture or in splenic fragments cultured with anti-CD3, suggesting that the potent cytokineproducing NKT cells might migrate rapidly into the spleen in response to in vivo activation61. Stimulation of mouse NKT cells by NK1.1 (NKRP1C/CD161) engagement, rather than by TCR cross-linking, led to increased IFNproduction in the absence of IL-4 (Ref. 62). Another member of the CD161 family (NKRP1B) inhibited mouse NKT activation when cross-linked63, and a similar effect was observed following CD161 cross-linking with human NKT cells64. Cytokines can also influence the response of NKT cells: IL-7 enhances IL-4 production by thymic and splenic NKT cells, whereas IL-12 enhances IFN- production by these cells11,34,65,66. Collectively, these results suggest that NKT cells have differential cytokine producing capacity depending on the microenvironment. In addition to cytokine production, NKT cells can exhibit potent lytic activity. They constitutively express Fas-L and can kill Fas target cells, including DP thymocytes67, and can also kill tumor targets in a perforin-dependent manner68.

NKT cell ligands

The expression of CD3/ TCR by NKT cells suggests that they require TCR-specific recognition to be activated. The biased TCR gene usage of mouse and human NKT cells probably reflects the fact that they are restricted to CD1d during their development and possibly their activation2,48. The TCR-bias and CD1d-dependence of mouse NK1.1 T cells varies between subsets and different tissues (Fig. 3). The target of the non-TCR-biased, CD1-independent NK1.1 T cells is unknown, but their more diverse TCR expression4,5 suggests it is a conventional MHCpeptide complex. Most NKT cells seem to recognize CD1d in conjunction with hydrophobic ligands (probably glycolipids)8,21, although the precise nature of these ligands is not yet clear. One candidate family of natural ligands might be the glycosylphosphatidylinositol (GPI)-anchors49,50 and phosphoinositol mannosides51, although this issue remains contentious52,53. The ability of NKT cells to respond to tumor-derived lipid extracts (including phospholipids) in the context of CD1d suggests that they might see a natural lipid ligand, possibly altered in tumor tissue54. There are obviously several possibilities that need not be mutually exclusive. Given that NKT cells appear to show tissue specificity in their recognition of CD1d (Ref. 48), it seems likely that these cells can recognize a diverse array of hydrophobic ligands in conjunction with CD1d, indicating the potential for antigen-specific activation, and perhaps even self-tolerance, of these cells.

-Galactosylceramide stimulation -Galactosylceramide

An GalCer, derived from marine sponges, binds CD1d and strongly stimulates both CD4 and DN NKT cells48,55. Although GalCers are a major constituent of some mammalian tissues, particularly in the central nervous system, at least the majority appear to be -linked galactosylceramides ( GalCers), rather than -linked GalCers. The nature of the galactosyl linkage might be a critical Many recent studies have focussed on in vitro and in vivo stimulation of NKT cells by GalCer presented by CD1d antigen presenting cells (APCs)21. As might be expected, such NKT cell stimulation can influence T-helper responses. However, the type of T-helper response induced by GalCer is contentious, suggesting complex consequences of treatment with this reagent. Although most studies showed that repeated exposure to GalCer in vivo leads to enhanced Th2 responses6971, one showed Th2 inhibition72. In line with the latter study, GalCer

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Table 2. NKT cells and disease
Disease Autoimmunity Type 1 diabetes NKT cells deficient in NOD mouse and BB rat models and in human diabetics Increased NKT cells protect from diabetes in NOD mice Lupus Decreased NKT cells associated with onset of disease (mice) DN transgenic CD1d-reactive T cell clone prevents lupus (mice) Experimental autoimmune gastritis Day 3 thymectomy selectively depletes NKT cells (mice) Systemic sclerosis NKT cells depleted in systemic sclerosis patients Multiple sclerosis NKT cells depleted in multiple sclerosis patients Infection Listeria DN NKT cells decreased in liver (mice) CD4 NKT cells increased in spleen (mice) Toxoplasma NKT cells help to generate CD8 effector cells (mice) Mycobacteria NKT cells required for granuloma formation in Mycobacterium tuberculosis infection (mice) BCG downregulates CD4 NKT cells and induces IFN- producing NKT cells (mice) Salmonella NKT cells exacerbate infection and are effectors of liver damage (mice) Plasmodium NKT cells directly inhibit parasite growth in liver (mice) CD1d-restricted NKT cells important in anti-parasite IgG formation (mice) No role for CD1d-restricted NKT cells in anti-parasite IgG formation (mice) Tumor NKT cells can be induced to mediate tumor rejection (mice) NKT cells play a natural role in tumor rejection (mice) Tolerance and immune deviation NKT cells required for ACAID (mice) NKT cells mediate suppression of acute GVHD (mice)
aData

Refs
mice48, protection against murine malaria80 and enhanced antigen-specific CTL activity74. However, a recent study demonstrated that GalCer treatment of mice rapidly led to massive, NKT cell-dependent liver damage, which was sufficient to kill older mice (presumably because of their higher liver NKT cell number)81. This might not be entirely surprising considering the widespread stimulation of potentially destructive lymphocytes such as NKT and NK cells and other bystander cells. Clearly, this result highlights the need for caution in applying the activities of GalCer observed in animal models to the clinic.

14, 16, 9498 14, 83, 96 92, 93 91 25 87 114

107 115 116 51 101, 105

Role of NKT cells in immune responses

The range of actions attributed to NKT cells is extremely diverse. Many studies have suggested that an important natural function for NKT cells might be to protect selftissues (particularly vital organs) from damaging inflammatory-type immune responses. There is also clear evidence that they can control immune responses to infection and some tumors (Table 2).

117, 118

102 50 52

Th2 induction
The strong capacity to produce IL-4 has led to speculation that NKT cells might drive the differentiation of Th2 responses13. Although demonstrated in a few studies, most investigations using NKT-deficient (CD1d / or 2M / ) mice do not support an essential role for these cells in such responses2,48. However, this does not exclude them as important players in some Th2-associated responses. For example, V 14 TCR transgenic mice, which have tenfold increased NKT-cell numbers, show elevated serum IgE and IL-4 (Ref. 82), and NKT-cell activation in vivo promotes Th2-associated immunity6971.

107109, 119 68 84, 85 86

from Refs 1721.

bAbbreviations: ACAID, anterior chamber-associated immune-deviation; DN, CD4

CD8 double negative; GVHD, graft versus host disease; IFN- , interferon- ; IL-4, interleukin 4; NK, natural killer; NKT, NK1.1 T cells; NOD, nonobese diabetic; TCR, T cell receptor.

induced CD40L expression by CD4 , but not CD4 , splenic NKT cells, which consequently promoted IL-12 production by CD40 APC (Ref. 73). GalCer stimulation of NKT cells induces proliferation by NK cells51 and promotes bystander activation of several other cell types including B, CD4 T, CD8 CTL and dendritic cells69,7477. These effects of GalCer are of potential clinical relevance: for example, NKT-cell stimulation through GalCer treatment leads to potent anti-tumor immunity78,79, prevention of type 1 diabetes in

Th1 inhibition
In some systems, NKT cells suppress Th1-associated cell-mediated immunity8386 through the production of IL-4, IL-10 and/or TGF- . NKT cells are essential for controlling anterior chamber-associated immune-deviation (ACAID), believed to prevent the eye from damage by inflammatory immune responses. This phenomenon was originally found to be thymus-dependent, and specifically regulated by thymic DN cells85, and more recently, it was associated with

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CD1-restricted NKT cells84. Curiously, the latter study also showed restoration of ACAID using adoptive transfer of NKT cells from spleen, in apparent contrast with the thymus-dependent population identified in the earlier study. Bone-marrow DN NKT cells were observed to be important in preventing graft-versus-host disease following allogeneic bone-marrow transplantation, in an IL-4dependent manner86, which might reflect a natural role for these cells in preventing inflammatory immune responses in bone marrow.

(iii) Type 1 diabetes

The situation in type 1 diabetes (a tissue-specific autoimmune disease) appears to be rather more straightforward. We and others found that non-obese diabetic (NOD) mice, which serve as a model of type 1 (autoimmune) diabetes, are numerically and functionally deficient in NKT cells in the thymus94,95 and, to a lesser extent, in the periphery14, well before the onset of diabetes. In adoptive transfer experiments, we have shown that diabetes in NOD mice can be prevented by the injection of 2 105 thymic DN (NKT) cells and that protection is mediated by an IL-4 and/or IL-10-dependent mechanism83. Lehuen and colleagues96 subsequently confirmed the protective role of NKT cells in NOD mice by increasing the numbers of these cells through the introduction of a V 14J 281 transgene. Adoptive transfer of splenic T cells, including approximately 8 105 DN and CD4 NKT cells, from these mice to non-transgenic syngeneic recipients also reduced diabetes development, although their protective effect was somewhat less efficient than the thymic NKT cells used in our study. Falcone and colleagues16 failed to prevent the onset of diabetes in NOD mice following adoptive transfer of 3 105 splenic Ly49A CD122 CD3 cells, thought to represent NKT cells. At least two factors contribute to this discrepancy: first, this dose was below the protective dose of splenic NKT cells described by Lehuen96; and second, cells with this phenotype did not produce detectable levels of IL-4, even in C57BL/6 mice16, indicating that this surrogate phenotype might not adequately encompass NKT cells. Collectively, these studies suggest that thymus-derived NKT cells afford more potent protection against diabetes in NOD mice compared with splenic NKT cells. This difference might be related to the varying functional and developmental characteristics of NKT-cell subsets in these organs. The association between a deficiency in NKT cells and autoimmune diabetes also holds true for other species. Diabetes-prone BB rats have approximately a tenth of the proportion of NKR-P1 TCR lymphocytes compared with diabetes-resistant BB rats97, and human diabetic patients were found to have lower frequencies of DN V 24J Q T cells in peripheral blood than their nondiabetic siblings98.

Prevention of autoimmune tissue destruction

Disturbances in numbers and function of NKT cells have been implicated in several autoimmune diseases, although in some of these studies it is unclear whether the changes reflect a cause or effect of disease.

(i) Scleroderma
Scleroderma (systemic sclerosis) is a systemic autoimmune disease. Following up on an earlier report that patients with scleroderma had increased numbers of circulating DN cells with limited TCRheterogeneity, Sumida and colleagues87 performed a detailed analysis of the TCR- chains of DN T cells from these patients. Although they found a fivefold increase in V 24 DN T cells, virtually none of these cells expressed the invariant NKT cell-associated V 24J Q TCR -chain, but instead carried alternate junctional regions. The authors concluded that scleroderma was associated with a deficiency of NKT cells and an oligoclonal expansion of other DN V 24 T cells, which they suggested were responsible for the ongoing tissue damage.

(ii) Systemic lupus erythematosus

The study of the role of NKT cells in human systemic lupus erythematosus (SLE) is less advanced, but shows striking parallels. Patients with SLE have a three- to tenfold increase in the proportions of DN T cells in the peripheral blood88,89 and an increased proportion of these cells produce IL-4 after polyclonal stimulation89. Significantly, these cells were capable of supporting the production of anti-DNA antibodies by syngeneic B-cells in vitro88, and might represent the SLE-associated counterpart of the pathogenic DN cells associated with scleroderma in which case, they are unlikely to be NKT cells. Similar results have been obtained in studies of the NZB/NZW mouse model of SLE (Refs 90, 91). SLE-prone lpr and gld strains have massive expansions of DN T cells. These differ from NKT cells by their expression of heterogeneous TCR, B220 expression, lack of NK1.1, and restriction to conventional MHC class I molecules3. By contrast, there is some suggestion that the onset of autoimmunity in lpr, gld and (NZBxNZW)F1 lupus-prone mice correlates with a spontaneous depletion of NK1.1 or V 14 NKT cells92,93, although the issue would benefit from being revisited. A significant problem with the latter study was the reliance on a V 14-specific mAb that does not recognize TCR with the NKT cell-associated J 281 junctional region2.
:

Control of infection
NKT cells appear to participate in immune responses to a range of different infectious agents. We have selected the three types of infections in which NKT cells have been more extensively studied.

(i) Mycobacteria
CD1-restricted T cells appear to play a major role in immune reand sponses to mycobacteria. Human clones and lines of CD8 DN T cells, that are restricted by CD1a, CD1b or CD1c and respond to mycobacterial lipid-containing antigens, express heterogeneous TCR (Ref. 99). This diversity is in marked contrast to NKT cells, which bind CD1d via their restricted selection of TCR chains. It is not known if CD1d-restricted T cells also play a role in human resistance to mycobacteria, and the results of studies in mouse models are inconsistent. For example, although CD1d-deficient mice did not differ significantly in susceptibility to Mycobacterium tuberculosis100, NKT

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cells predominate in the granulomatous reaction to M. tuberculosis cell-wall preparations, and such granulomas do not form in NKT celldeficient J 281 / mice51. Furthermore, NKT cells of normal mice respond to mycobacterial infection by decreasing IL-4 and increasing IFN- production101, changes likely to aid the host response to mycobacteria, since IFN- plays a critical role in pathogen clearance.

(ii) Plasmodium
In an experimental model of malaria, infection with Plasmodium yoelii sporozoites induced a decrease in CD4 NKT cells in the liver, but a corresponding increase in DN NKT cells, which directly inhibited growth of the pathogen in hepatocytes in vitro via an IFN- -dependent mechanism102. CD1-restricted presentation of Plasmodium berghei sporozoite-derived GPI anchor has been shown to stimulate NKT-cell-mediated B-cell help and specific antibody production50. Moreover, this response was severely impaired in CD1d / mice, suggesting a key role for NKT cells50. However, these data are in conflict with a more recent study which demonstrated that the IgG response to P. berghei occurred normally in CD1d / mice and was MHC class-II restricted52. The basis of this discrepancy is unresolved.

rejection, which has been suggested to be NKT cell-mediated66,107109. However, other studies indicate that the dependence on NKT cells in IL-12 mediated tumor rejection might be model and dose-dependent110,111. GalCer-treatment of mice also induces CD1d-restricted, NKT-dependent tumor rejection48,78,79. Although in vitro experiments support a role for NKT cells as direct anti-tumor effectors, they do not exclude the possibility that the main function of NKT cells is to activate other effector cell types, such as NK cells76,77. These studies show that mouse NKT cells can be induced to participate in antitumor responses, but do not indicate whether this is a natural function of these cells. At least two findings support such a role. First, IL-4-producing DN NKT cells accumulated at the site of an embryonal carcinoma and appeared to be necessary for tumor rejection112. Second, we recently demonstrated that NKT cells might be important in protecting against some, but not all, types of tumors. For example, NKT-deficient J 281 / mice had impaired protection against spontaneous sarcomas initiated by the chemical carcinogen methylcolanthrene, but were resistant to other tumor types controlled by NK cells68. Human V 24 NKT cells (including CD4 and DN subsets), stimulated in vitro with GalCer-pulsed dendritic cells, mediated strong perforin-dependent cytotoxic activity against the U937 tumor line20.

(iii) Listeria
Although DN T cells accumulate in the peritoneal cavity following intraperitoneal infection of mice with Listeria monocytogenes103, the investigators were unable to demonstrate proliferation of these cells in response to Listeria lysates or Listeria-infected macrophages in vitro104. Immediately ex vivo, the cells did contain mRNA for GMCSF, Eta-1 and MCP-1 (but not IL-4), and secreted IFN- following in vitro stimulation with Listeria-infected macrophages104. These results collectively suggest that NKT cells are capable of responding to L. monocytogenes antigens. Emoto and colleagues105 reported an IL-12-dependent decrease in numbers of IL-4-producing CD4 NKT cells in livers of mice infected intravenously with L. monocytogenes. In this model, the effect on NKT cells was transient and correlated with the period during which viable bacteria were identified in the liver (that is, the first week post-infection). The changes in DN and CD4 NKT cell numbers during L. monocytogenes infection were consistent with those identified in Plasmodium infection, as both were associated with a decrease in IL-4-producing CD4 NKT cells and a concomitant increase in IFN- -secreting DN NKT cells. Flesch and colleagues106 described an increase in IL-4 production by splenic CD4 NKT cells in response to intravenous L. monocytogenes infection, so it is possible that the immune response to this organism involves recruitment of IFN- -producing DN NKT cells to the site of infection, and deployment of IL-4-producing CD4 NKT cells to the spleen for support of antibody production.

Concluding remarks
This is clearly a controversial field. Many of these disagreements would be resolved by increasing the precision with which the identification of NKT cells is reported. The proportions of CD4 , DN and CD8 NK1.1 T cells in mice vary between tissues, and represent functionally discrete populations. As the literature currently stands, TCR cells virtually it is probably safe to say that of NK1.1 all CD4 and at least half of the DN (tissue-dependent) subset are bona fide, CD1d-restricted NKT cells. However, some CD4 NKT cells might not express NK1.1, and some DN and most TCR cells are not NKT cells. Improved underCD8 NK1.1 standing is likely to come from further dissection of NKT-cell subsets and the relationships between them. It will be important to determine what phenotypic changes occur in their development and following activation. Certainly, further improvement would be attained if a reliable antibody specific for the canonical V 14J 281 TCR was available for identifying these cells in mice, and CD1d- GalCer tetramers8,120 represent an important tool for identifying these cells in both mice and humans. Both V 24 and V 11 mAbs are available for identification of NKT cells in humans, and the combination of reagents appears to be highly specific113. The continued use of antibodies to the surrogate markers CD56 and CD57 to identify human NKT cells is therefore difficult to justify. Little is known about what determines the functions attributed to NKT cells. The influence of costimulation and other NK-associated receptors is unclear. It will be necessary to develop a better understanding of the signal transduction machinery and functional outcomes of ligating the TCR and NK/NKT-cell receptors on these cells. Although it is clear that NKT cells respond to CD1d molecules via their TCR, the identity of the natural ligands presented to NKT cells

Tumor rejection
In some models, NKT cells mediate cytotoxicity against tumor cell lines in vitro2,68, suggesting an important role for these cells in tumor rejection. In vivo treatment of mice with IL-12 induces potent tumor

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remain uncertain, as do the roles they play in NKT-cell differentiation and function. NKT cells might also respond to pathogen-specific ligands, and play a role in determining the direction of immunedeviation early in infections. Some of these uncertainties should be resolved by extending the catalogue of known CD1d-binding NKT cell ligands and other NKT-cell stimulatory factors. Tetramer technology will be an important tool for measuring NKT cell recognition of different CD1d-ligand complexes. Finally, it will be necessary to understand the roles of NKT cells in health and disease. Although much is known about the involvement of CD1 presentation in responses to mycobacteria, it is still not known if CD1d contributes to immune responses to mycobacteria. Much less is known about the changes in NKT-cell numbers and functions in other disease states, especially autoimmune diseases and cancer. Careful clinical identification of NKT cells by the application of TCR-chain-specific reagents is needed. The normal range of NKT-cell numbers in human peripheral blood is not known, and the association of NKT numbers and activity with defined disease states remains to be determined. It will be very interesting to see whether NKT-cell numbers can be used to estimate the risk of disease or predict clinical outcomes.

This work was funded by the National Health and Medical Research Council of Australia (NHMRC) and the Juvenile Diabetes Foundation. DIG is a recipient of an ADCORP/Diabetes Australia Fellowship. AGB is a recipient of an R. Douglas Wright Fellowship from the NHMRC. LDP and KJLH are recipients of Australian Postgraduate Research Awards. MJS is an NHMRC Principal Research Fellow.

Dale I. Godfrey (dale.godfrey@med.monash.edu.au) and Kirsten J.L. Hammond are at the Dept of Pathology and Immunology, Monash University Medical School, Commercial Road, Prahran, VIC. 3181, Australia; Lynn D. Poulton and Alan G. Baxter (a.baxter@centenary.usyd.edu.au) are at the Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag #6, Newtown, NSW 2042, Australia; Mark J. Smyth (m.smyth@pmci.unimelb.edu.au) is at the Peter MacCallum Cancer Institute, Locked Bag 1, A Beckett St, VIC. 8006, Australia. References
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