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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, 4051–4058 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, 1319–1327 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, 3394–3399 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, 2933–2940 43 Downward J. et al. (1992) The regulation and function of p21ras in T cells. Immunol. Today 13, 89–92 44 Ebinu, J.O. et al. (2000) RasGRP links T-cell receptor signaling to Ras. Blood 95, 3199–3203 45 Cantrell, D. (1996) T cell antigen receptor signal transduction pathways. Cancer Surv. 27, 165–175 46 D’Ambrosio, D. et al. (1994) Involvement of p21ras in T cell CD69 expression. Eur. J. Immunol. 24, 616–620 47 Green, D.R. and Ware, C.F. (1997) Fas-ligand: privilege and peril. Proc. Natl. Acad. Sci U. S. A. 94, 5986–5990

48 Latinis, K.M. et al. (1997) Regulation of CD95 (Fas) ligand expression by TCR-mediated signaling events. J. Immunol. 158, 4602–4611 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, 30417–30425 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, 5813–5819 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, 3549–3561 52 Rodriguez-Tarduchy, G. and Lopez-Rivas, A. (1989) Phorbol esters inhibit apoptosis in IL-2-dependent T lymphocytes. Biochem. Biophys. Res. Commun. 164, 1069–1075 53 Boise, L.H. et al. (1995) CD28 and apoptosis. Curr. Opin. Immunol. 7, 620–625 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, 1552–1560

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 1–3). 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 acteristics1–3. 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 lineage1–3. 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 cells4–8. 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.

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2. and produce lower levels of cytokines following short-term in vitro stimulation5. orange or green). Furthermore. spleen and liver were labelled with mAb specific for TCR. TCR.1+ Non-MHC restricted Cytotoxic IFN-γ production MHC-I/II restricted Thymus-independent T NK Immunology Today Fig.5.galactosylceramide ( GalCer) tetramers8. and high IL-4production8. have a more heterogeneous TCR repertoire4. A number of different surrogate phenotypes have been used.REVIEW I M M U N O L O G Y T O D AY NKT Rapid/high IL-4 production CD1d restricted CD3/αβTCR+ Thymus-dependent TCR-dependent NK1. Surrogate markers for NKT cells αβTCR Another difficulty with the use of NK1. 2 1 2 0 0 0 No.1 T cells are CD1d and thymusindependent. major histocompatibility complex molecule. NK1. 15). (b) CD4 versus CD8 expression by NKT cells gated as shown in (a). for example: DN (Ref. Mountain View.1 T cells.1.1 can be downregulated upon NKT-cell activation13. might exclude most NKT cells. C/I and G2 (Ref. 16). which are DX5– (b) 65 34 CD4 68 20 10 80 17 2 CD8 Immunology Today Fig.8. Data show representative 2-D dotplots derived from 5–7-week old female C57BL/6 mice. particularly the CD1d-dependent subsets (CD4 and most DN). Other surrogate phenotypes. Abbreviations: mAb. 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.1 cells within these populations.1 T-cell subsets differ from their thymic counterparts (Fig. NKT. Characteristics that are unique to each lineage are shown in the non-overlapping sections. Mononuclear cells from the thymus. T cell receptor.4% 24% present in most tissues and are phenotypically and functionally distinct. T and NK cells. N O V E M B E R 574 Vo l . primarily C57BL/6 and related strains. Splenic CD8 NK1. In some cases.1 staining of these populations in C57BL/6 mice demonstrates the presence of other NK1.1 T cells and their CD4/CD8-defined subsets. although those in the liver have greatly reduced expression of the MHC class-I ligands Ly49 A. The likelihood that splenic DN NK1. such as DX5 CD3 . Thymus and liver CD4 and DN NKT cells are generally alike. CD4 and CD8. NK. (a) Thymus Spleen Liver NK1. TCR.1 0. USA). they detect only a subset of NKT cells. and NK1. monoclonal antibody. although splenic CD4 NKT cells are similar to thymic NKT cells.1 T cells include two distinct subsets is supported by the bimodal expression of other cell-surface markers (Fig. Mouse NKT cells share some characteristics with NK and T cells. One explanation for this might be that NK1.1 to identify NKT cells is that this allelic marker is only expressed in a limited number of mouse strains. DX5 CD3 (Ref.6% 1. CA. and do not produce IL-4 rapidly. (a) NK1. Characteristics that are common to any two of these lineages are indicated in the relevant overlapping areas (purple.1 CD4 T cells closely resemble cells of the NKT-cell family in their CD1dreactivity. NK1. 14). such as DN. Abbreviations: MHC. 3). Information derived from references (1–3).11 . lymph node and bone-marrow NKT cells are far more heterogeneous. NK1. and acquired using a four-colour FACScalibur (Becton Dickinson. The central (white) area indicates characteristics that can be exhibited by all three lineages. TCR V 8-bias. Spleen. This venn diagram incorporates some of the key features that are generally applied to NKT. In addition.7.1 versus TCR on lymphoid gated cells. some NK1. 9). natural killer. T-cell receptor. many splenic DN NKT cells are not CD1-dependent4. For example. and Ly49A CD122 CD3 (Ref.10–12. 3) and by bimodal reactivity with CD1d. express heterogeneous TCR. peripheral NK1. 1.

although only 0.production measured 6 1 the relative scarcity of these cells and the lack by ELISA following 18 h culture on anti-CD3 coated plates.5% of total thymocytes).. they develop in fetal thymus organ culture23. 10 cells ml . no. DN and CD8 NK1. Fas.1 T cells. the homologue of mouse V 8.120 would most reliably identify these cells in all strains. Abbreviations: DN.REVIEW I M M U N O L O G Y TO D AY (Refs 4. 3). By contrast. N. and V 24J Q.5) (Fig. CD4 CD8 double negative. neg. interleukin 4. of a single.2 Posb Low Low Neg Posb 50% – 20% – – Y Y Y 200 0. The distri.8 Distribution of NKT cells In mice.11 2 0 0 0 575 . CD4 .when stimulated20. N O V E M B E R Vo l . indicating highly conserved specificity21. Y/N. Inter. Fig. Although most reports on human NKT cells have been limited to that most NKT cells are thymus-dependent. They represent a smaller proportion of T cells Mouse NK1.1 T-cell estingly. J 281 dep. some scientists argue for those in peripheral blood. IL-4/IFN. 9.IFN. not determined. the homologue of mouse V 14J 281). Inforcific fashion4–6. defining cell-surface marker. T-cell receptor V 14J 281-dependent development. Their intra-tissue local.1 6 5 mature T cells. positive. As a proportion of 0. Interferon .3 – Low Low Neg – 60% – 50% – – Y – Low Low Neg – 80% – 45% – – N N N 2 0. NKT cells can be detected wherever N Y conventional T cells are found.25. blood (4%) and lung (7%). This in other tissues including spleen (3%).. or adult mice irradiated and thymectomized Thymus dependence prior to fetal liver-reconstitution26. V 14J 281 specific monoclonal antibody (mAb) or CD1d tetramers8. CD44.reveal distinct subsets of NK1. human NKT cells express the homologous TCR gene products (V 11. difficult to ignore. CD28.1 congenic strains of mice will help with this problem but. CD38 and CD45RC (not shown). IL-4. including DN (Refs 17. Significantly. CD1d.and spleen are positive for CD122. 18) and CD4 subsets19. Although the overwhelming body of evidence indicates thymectomized mice5. lymph diagram shows representative histogram profiles or summarized data for a selection of features that node (0.1 T cells subpopulations derived from 5–7-week old C57BL/6 mice. partly because of NKT cells except where indicated.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. Data relate to spleen b c ization remains unknown.1 T cells can be subdivided based on CD4/CD8 expression and on tissue of origin.3%). developmentally and functionally distinct. partially a tally distinct subsets. these are functionally and/or developmen.2. although the Y Y Y ratio of NKT to T cells varies widely in a 50 800 800 tissue-specific manner1–5. and will not be repeated here. ideally.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. –. 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. 2 1 No. In mice. NKT cells are most frequent in Immunology Today liver (30–50%). The use of NK1. Both subsets react with human CD1d and produce high levels of IL-4 and IFN. All NK1. 3. V 24J Q NKT cells are clearly present in an extrathymic origin.1 T-cell subsets in the thymus are differentially distributed in a tissue-spe. except to say that (approximately 4% of hepatic T cells versus 50% in mice)22. yes. Mouse NK1. pos. This controversy has been discussed in detail human liver although apparently not as frequent as they are in mice in earlier reviews1–3. Thymus Total 55 Peripherya DN Total 57 42 CD4 + 54 CD4+ 51 DN 31 CD8+ 24 Vβ8 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). Furthermore.dependent. negative. Data relate to liver NKT cells. Histogram profiles are representative of NK1. supporting the concept that mation taken from Refs 1–6.3–0. 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-1α) CD24(HSA) CD25(IL2Rα) CD45R(B220) CD54(ICAM-1) CD62L CD127(IL7Rα) Ly49A Ly49C/I Ly49G2 CD1-dependent Jα281-dependent Thymus-dependent IL-4(U/ml)c IFNγ(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. human NKT cells can recognize mouse CD1d and vice versa.1 T-cell subsets are phenotypically. CD8 NK1. Y. bone marrow (20–30%) and thymus (10–20% of mature HSA– T cells.

One study provided evidence that CD8 generated extrathymically following antigen encounter in the liver29. IFN. NKR. We (unpublished data) and others28 have been unable to verify these findings. IL-4-secreting T Restriction element CD1d CD1d Homologous cells can be generated in the absence of endogenous TCR gene rearrangement.2 V 11 Homologous presence of CD8 NK1. It is also unclear how recognition of CD1d cells results in commitment to this novel lineage. although these Another debated question relates to the timing of NKT-cell develop. T-cell virtually absent in the thymus of CD1d / receptor.11 . limited roles in NKT cell development and function23. one report suggested that NKT cells start to develop at E9. CollecCD122 CD122 tively. aAbbreviations: GalCer. TCR. -galactosylceramide. relatively late in mouse ontogeny. Interestingly. and are N O V E M B E R 576 Vo l .35.5 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.25.31–33. a Appearance during ontogeny rare event in the development of NKT cells. NK-cell receptor. non-transgenic NKT-cell development.TCR transgenic approaches theoretically provide greater ability to ment. Another showed that mainstream CD8 T cells can upregulate In addition to CD1d. at best. it seems that conventional TCR–MHC interactions are. 2 1 2 0 0 0 No. Analysis of NKT cells in common cytokine receptor -chain-deficient mice revealed at least two stages in NKT cell development43.1-expressing. of both CD4 and DN NKT cells34. high levels Rapid. DN Comment Proportions vary NK1. peripheral blood leukocytes.1 T cells. but not of conventional T cells. 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 T cells41. several other factors are known to influence NKT-cell development. NKT-cell development is also absolutely dependent on pre-T signaling39 and at least partly dependent on granulocyte–macrophage colony-stimulating factor (GM-CSF) signaling40.1 T T cell receptor cells. NK1.1 T cells is even less well underNK1. Although this is known to involve a TCR-dependent interaction between NKT precursors and CD1d-expressing CD4 CD8 (DP) thymocytes1–3. whereas NKT cells have an altered TCR bias in CD8 / mice23.1 T-cell subsets is unknown. V 2 (Ref. these studies suggest that TCR-transLy49 genic. CD4 and MHC class II molecules appear to have. it is unlikely to completely explain the -chain V 14J 281 V 24J Q Homologous -chain V 8. On the other hand. 5). avidity and type of selecting cell.38. suggesting at least some commonality to the development of these subsets38. Most studies have indicated that NKT cells do not appear until manipulate the nature of the selecting environment (for example. high levels Following TCR ligation variant TCR V 14J 281.1 following antigenic challenge in the periphery30. arising in the fetal liver at E13.24. Fyn-deficient mice show a selective defect in the development of CD1d-dependent NKT cells. lymphotoxin deficiency affected all three populations (CD4 . NKT. double negative. 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. and whether high affinity recognition of self-antigen leads to negative selection of NKT cells.5% More variable in humans that such selection might simply result more frequently through interaction with CD1d. many key questions remain.1 T cells are stood. positively selecting or negatively selecting). the developmental relationship between the CD4 .1 and Ly49. DN and CD8 ) of NK1. at best. suggests IFNFollowing TCR ligation that NKT-cell development depends on the Frequency timing..1–0. mice.1 T cells in normal V 7. many of which are CD8 Expression level Intermediate Intermediate Some studies have demonstrated and investigated the development of NK1. Thymic stromal-cell-derived cytokines IL-15 and IL-7 are required for development of normal numbers and IL-4producing potential. considering that NKT cells are leukin 4. Factors influencing NKT-cell development NKT cell selection and branching A largely unresolved problem relates to how NKT cells develop.1 T cells. The development of CD8 NK1.1 NKR-P1 Homologous (CD161) peptide in TCR transgenic mice29.1 T Accessory molecules cells specific for conventional MHC plus NK associated NK1. PBL. respectively. well after conventional T cells1–3. and PBL ~1% ~0. DN Human CD4 . CD8 expression appears to strongly influence the development of NKT cells. IL-4. Features common to mouse and human NKT cells Characteristic Major subsets Mouse CD4 . DN and CD8 NK1.5 (Ref. but rather. For example.42. nor of CD8 NK1. interHowever. interferon . non-selecting. such that high levels of CD8 transgene expression leads to their deletion in the thymus. In contrast to conventional T cells. NK1. or TCR J 281 / mice. Thus. NKT cell development requires an interaction with membrane lymphotoxin expressing cells37.REVIEW I M M U N O L O G Y T O D AY Table 1. and an intact thymic structure is also important36. Although this would fit with the non-biased TCR repertoire of CD8 NK1. DN. care However. 27).

particularly in the central nervous system. There are obviously several possibilities that need not be mutually exclusive. within minutes of in vivo stimulation with anti-CD3. Although GalCers are a major constituent of some mammalian tissues.66. 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. 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. intrathymic selection and development of IL-4-producing capacity seem to be earlier processes. this is clearly an area requiring more systematic study. suggesting – at least on face value – that GalCer cannot be a normal product of human cells. Furthermore. which are thought responsible for synthesizing such -linked carbohydrate moieties. or displace CD1d-bound glycolipids through high-affinity interactions with CD1d.production by these cells11. Stimulation of mouse NKT cells by NK1. a wave of proliferation of NKT cells occurs in the bone marrow. Liver NKT cells are also distinct in that the maintenance of normal numbers of these cells. suggesting complex consequences of treatment with this reagent. although this issue remains contentious52. Cytokines can also influence the response of NKT cells: IL-7 enhances IL-4 production by thymic and splenic NKT cells. the type of T-helper response induced by GalCer is contentious. Given that NKT cells appear to show tissue specificity in their recognition of CD1d (Ref. They constitutively express Fas-L and can kill Fas target cells. almost no turnover is seen in the thymuses of older mice. possibly by NK cells47.and TNF following stimulation in vitro1–3. point. By contrast. It is therefore probably significant that the genes encoding the 3–1 galactosyl transferases.1 T cells varies between subsets and different tissues (Fig. Maintenance of NKT cells Similar to conventional T cells. events. rather than by TCR cross-linking. In line with the latter study.1 T cells is unknown. even in thymectomized mice44. were a potent source of cytokines60. Curiously. CD1-independent NK1.59. rather than -linked GalCers. but their more diverse TCR expression4. 62). these results suggest that NKT cells have differential cytokine producing capacity depending on the microenvironment.1 (NKRP1C/CD161) engagement.58. liver CD4 NKT cells continue to turn over in eight-week old mice. derived from marine sponges. are defective in humans and some other old-world primates57. and can also kill tumor targets in a perforin-dependent manner68. Although most studies showed that repeated exposure to GalCer in vivo leads to enhanced Th2 responses69–71. If GalCer is not a product of human cells.1 T cells secrete even lower levels of cytokines than do the splenic CD4 NKT cell subset5.21. 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. they produce less than do thymic NKT cells5. suggesting that the potent cytokineproducing NKT cells might migrate rapidly into the spleen in response to in vivo activation61. since GalCers show stronger immunostimulatory activities than GalCers (Ref. Furthermore. 3). One candidate family of natural ligands might be the glycosylphosphatidylinositol (GPI)-anchors49. Most NKT cells seem to recognize CD1d in conjunction with hydrophobic ligands (probably glycolipids)8. and a similar effect was observed following CD161 cross-linking with human NKT cells64. As might be expected. it might either mimic a natural ligand. The target of the non-TCR-biased. but not NKT cells in other tissues. CD4 and DN NKT cells isolated from the spleen. 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)21.48.11 2 0 0 0 577 . as this carbohydrate moiety also defines the human B blood group. splenic DN and CD8 NK1. whereas IL-12 enhances IFN. 2 1 No. Following NKT cell depletion in the periphery. of these cells. binds CD1d and strongly stimulates both CD4 and DN NKT cells48. The TCR-bias and CD1d-dependence of mouse NK1. this was not observed with splenic NKT cells in cell-suspension culture or in splenic fragments cultured with anti-CD3.5 suggests it is a conventional MHC–peptide complex. at least the majority appear to be -linked galactosylceramides ( GalCers). -Galactosylceramide stimulation -Galactosylceramide An GalCer. NKT cell ligands The expression of CD3/ TCR by NKT cells suggests that they require TCR-specific recognition to be activated.46.REVIEW I M M U N O L O G Y TO D AY not exported to the periphery. depends on LFA-1 expression by non-NKT cells45. one showed Th2 inhibition72. such NKT cell stimulation can influence T-helper responses. and perhaps even self-tolerance. IFN. albeit at a low rate24. GalCer N O V E M B E R Vo l . Although splenic CD4 NKT cells also produce these cytokines following short term in vitro stimulation.65.55. including DP thymocytes67. NKT cells can exhibit potent lytic activity. the maintenance of peripheral NKT cell numbers is relatively thymus-independent. indicating the potential for antigen-specific activation. However. Despite this. possibly altered in tumor tissue54. 48). quickly restoring the number of these cells. whereas thymic CD4 NKT cells in young mice (less than 4–5 weeks old) turn over quite rapidly. although the precise nature of these ligands is not yet clear. Another member of the CD161 family (NKRP1B) inhibited mouse NKT activation when cross-linked63. In addition to cytokine production. led to increased IFNproduction in the absence of IL-4 (Ref. common -chain-dependent. However. Thus.50 and phosphoinositol mannosides51.61. it seems likely that these cells can recognize a diverse array of hydrophobic ligands in conjunction with CD1d. Collectively. 56). whereas acquisition of the NK surface phenotype and emigration to the periphery are later.53.34.

85 86 from Refs 17–21. For example.REVIEW I M M U N O L O G Y T O D AY Table 2. 83. which was sufficient to kill older mice (presumably because of their higher liver NKT cell number)81. nonobese diabetic. IL-4. splenic NKT cells. CD8 CTL and dendritic cells69. and more recently. but not CD4 . NK. this result highlights the need for caution in applying the activities of GalCer observed in animal models to the clinic. TCR. prevention of type 1 diabetes in Th1 inhibition In some systems. DN.. NK1. 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. this does not exclude them as important players in some Th2-associated responses. 93 91 25 87 114 107 115 116 51 101. which have tenfold increased NKT-cell numbers. a recent study demonstrated that GalCer treatment of mice rapidly led to massive. NKT cells are essential for controlling anterior chamber-associated immune-deviation (ACAID). interleukin 4. This might not be entirely surprising considering the widespread stimulation of potentially destructive lymphocytes such as NKT and NK cells and other bystander cells. 107–109. CD4 T. 16. V 14 TCR transgenic mice. 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. However. and specifically regulated by thymic DN cells85. IL-10 and/or TGF. These effects of GalCer are of potential clinical relevance: for example. CD4 CD8 double negative. anterior chamber-associated immune-deviation. 105 Role of NKT cells in immune responses The range of actions attributed to NKT cells is extremely diverse. Clearly. it was associated with N O V E M B E R 578 Vo l .. T cell receptor. 96 92. believed to prevent the eye from damage by inflammatory immune responses. show elevated serum IgE and IL-4 (Ref. 14. 94–98 14. NKT cells suppress Th1-associated cell-mediated immunity83–86 through the production of IL-4. GalCer stimulation of NKT cells induces proliferation by NK cells51 and promotes bystander activation of several other cell types including B. protection against murine malaria80 and enhanced antigen-specific CTL activity74. NKT-cell stimulation through GalCer treatment leads to potent anti-tumor immunity78. NKT. induced CD40L expression by CD4 . most investigations using NKT-deficient (CD1d / or 2M / ) mice do not support an essential role for these cells in such responses2. NKT cell-dependent liver damage..74–77. which consequently promoted IL-12 production by CD40 APC (Ref. 2 1 2 0 0 0 No.11 . 82). This phenomenon was originally found to be thymus-dependent. Although demonstrated in a few studies. NOD. IFN. natural killer. 119 68 84. 117. graft versus host disease. 73).1 T cells. GVHD. interferon.79.48. 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 responses1–3. However. bAbbreviations: ACAID. and NKT-cell activation in vivo promotes Th2-associated immunity69–71. There is also clear evidence that they can control immune responses to infection and some tumors (Table 2).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.

We and others found that non-obese diabetic (NOD) mice. which serve as a model of type 1 (autoimmune) diabetes. and restriction to conventional MHC class I molecules3.REVIEW I M M U N O L O G Y TO D AY CD1-restricted NKT cells84. and the results of studies in mouse models are inconsistent. from these mice to non-transgenic syngeneic recipients also reduced diabetes development. they are unlikely to be NKT cells. NKT N O V E M B E R Vo l . Curiously. including approximately 8 105 DN and CD4 NKT cells. (i) Scleroderma Scleroderma (systemic sclerosis) is a systemic autoimmune disease. 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. Bone-marrow DN NKT cells were observed to be important in preventing graft-versus-host disease following allogeneic bone-marrow transplantation. even in C57BL/6 mice16. indicating that this surrogate phenotype might not adequately encompass NKT cells. The authors concluded that scleroderma was associated with a deficiency of NKT cells and an oligoclonal expansion of other DN V 24 T cells.1 or V 14 NKT cells92. cells with this phenotype did not produce detectable levels of IL-4. although CD1d-deficient mice did not differ significantly in susceptibility to Mycobacterium tuberculosis100. Prevention of autoimmune tissue destruction Disturbances in numbers and function of NKT cells have been implicated in several autoimmune diseases. which bind CD1d via their restricted selection of TCR chains. 91). there is some suggestion that the onset of autoimmunity in lpr. (iii) Type 1 diabetes The situation in type 1 diabetes (a tissue-specific autoimmune disease) appears to be rather more straightforward. virtually none of these cells expressed the invariant NKT cell-associated V 24J Q TCR -chain. well before the onset of diabetes. 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. Significantly. which might reflect a natural role for these cells in preventing inflammatory immune responses in bone marrow. and human diabetic patients were found to have lower frequencies of DN V 24J Q T cells in peripheral blood than their nondiabetic siblings98. At least two factors contribute to this discrepancy: first. are numerically and functionally deficient in NKT cells in the thymus94.chains of DN T cells from these patients. that are restricted by CD1a.89 and an increased proportion of these cells produce IL-4 after polyclonal stimulation89.to tenfold increase in the proportions of DN T cells in the peripheral blood88. and might represent the SLE-associated counterpart of the pathogenic DN cells associated with scleroderma – in which case. although in some of these studies it is unclear whether the changes reflect a cause or effect of disease. lack of NK1. This difference might be related to the varying functional and developmental characteristics of NKT-cell subsets in these organs. this dose was below the protective dose of splenic NKT cells described by Lehuen96. Human clones and lines of CD8 DN T cells. gld and (NZBxNZW)F1 lupus-prone mice correlates with a spontaneous depletion of NK1. although their protective effect was somewhat less efficient than the thymic NKT cells used in our study. Adoptive transfer of splenic T cells. These differ from NKT cells by their expression of heterogeneous TCR. SLE-prone lpr and gld strains have massive expansions of DN T cells. although the issue would benefit from being revisited. in the periphery14. which they suggested were responsible for the ongoing tissue damage. CD1b or CD1c and respond to mycobacterial lipid-containing antigens. Patients with SLE have a three. in an IL-4dependent manner86. 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.95 and. 2 1 No. Sumida and colleagues87 performed a detailed analysis of the TCR. Although they found a fivefold increase in V 24 DN T cells. : Control of infection NKT cells appear to participate in immune responses to a range of different infectious agents. but shows striking parallels. Following up on an earlier report that patients with scleroderma had increased numbers of circulating DN cells with limited TCRheterogeneity.93. (i) Mycobacteria CD1-restricted T cells appear to play a major role in immune reand sponses to mycobacteria. Similar results have been obtained in studies of the NZB/NZW mouse model of SLE (Refs 90. to a lesser extent. We have selected the three types of infections in which NKT cells have been more extensively studied. thought to represent NKT cells. Collectively. these cells were capable of supporting the production of anti-DNA antibodies by syngeneic B-cells in vitro88. This diversity is in marked contrast to NKT cells. 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. B220 expression.11 2 0 0 0 579 . but instead carried alternate junctional regions. Falcone and colleagues16 failed to prevent the onset of diabetes in NOD mice following adoptive transfer of 3 105 splenic Ly49A CD122 CD3 cells. (ii) Systemic lupus erythematosus The study of the role of NKT cells in human systemic lupus erythematosus (SLE) is less advanced. and second. express heterogeneous TCR (Ref. By contrast. For example. 99). in apparent contrast with the thymus-dependent population identified in the earlier study. In adoptive transfer experiments. the latter study also showed restoration of ACAID using adoptive transfer of NKT cells from spleen.1. It is not known if CD1d-restricted T cells also play a role in human resistance to mycobacteria. these studies suggest that thymus-derived NKT cells afford more potent protection against diabetes in NOD mice compared with splenic NKT cells.

NKT cells of normal mice respond to mycobacterial infection by decreasing IL-4 and increasing IFN. (ii) Plasmodium In an experimental model of malaria. Flesch and colleagues106 described an increase in IL-4 production by splenic CD4 NKT cells in response to intravenous L. First. other studies indicate that the dependence on NKT cells in IL-12 mediated tumor rejection might be model and dose-dependent110. and secreted IFN. (iii) Listeria Although DN T cells accumulate in the peritoneal cavity following intraperitoneal infection of mice with Listeria monocytogenes103.1 T cells in mice vary between tissues. The changes in DN and CD4 NKT cell numbers during L. suggesting a key role for NKT cells50. this response was severely impaired in CD1d / mice. At least two findings support such a role. as both were associated with a decrease in IL-4-producing CD4 NKT cells and a concomitant increase in IFN. monocytogenes infection were consistent with those identified in Plasmodium infection.77. 2 1 2 0 0 0 No.111. but not all. In this model. since IFN. the cells did contain mRNA for GMCSF. infection with Plasmodium yoelii sporozoites induced a decrease in CD4 NKT cells in the liver.1. Little is known about what determines the functions attributed to NKT cells.78. the first week post-infection). NKT-deficient J 281 / mice had impaired protection against spontaneous sarcomas initiated by the chemical carcinogen methylcolanthrene. berghei occurred normally in CD1d / mice and was MHC class-II restricted52.plays a critical role in pathogen clearance. The basis of this discrepancy is unresolved.107–109. such as NK cells76. stimulated in vitro with GalCer-pulsed dendritic cells. tuberculosis cell-wall preparations. but do not indicate whether this is a natural function of these cells.REVIEW I M M U N O L O G Y T O D AY cells predominate in the granulomatous reaction to M.68. The continued use of antibodies to the surrogate markers CD56 and CD57 to identify human NKT cells is therefore difficult to justify. they do not exclude the possibility that the main function of NKT cells is to activate other effector cell types. rejection. Many of these disagreements would be resolved by increasing the precision with which the identification of NKT cells is reported. NKT-dependent tumor rejection48. IL-4-producing DN NKT cells accumulated at the site of an embryonal carcinoma and appeared to be necessary for tumor rejection112. and the combination of reagents appears to be highly specific113. monocytogenes infection. the identity of the natural ligands presented to NKT cells Tumor rejection In some models. types of tumors.1 standing is likely to come from further dissection of NKT-cell subsets and the relationships between them.GalCer tetramers8. Concluding remarks This is clearly a controversial field. Although in vitro experiments support a role for NKT cells as direct anti-tumor effectors. TCR cells – virtually it is probably safe to say that – of NK1. the investigators were unable to demonstrate proliferation of these cells in response to Listeria lysates or Listeria-infected macrophages in vitro104.-dependent mechanism102. However. 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. Eta-1 and MCP-1 (but not IL-4). Moreover. As the literature currently stands. and represent functionally discrete populations. For example. Improved underCD8 NK1. but were resistant to other tumor types controlled by NK cells68. 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. monocytogenes. The influence of costimulation and other NK-associated receptors is unclear. Furthermore.1 all CD4 and at least half of the DN (tissue-dependent) subset are bona fide. mediated strong perforin-dependent cytotoxic activity against the U937 tumor line20. but a corresponding increase in DN NKT cells. which directly inhibited growth of the pathogen in hepatocytes in vitro via an IFN. some CD4 ‘NKT’ cells might not express NK1.-producing DN NKT cells to the site of infection. It will be important to determine what phenotypic changes occur in their development and following activation.11 . Although it is clear that NKT cells respond to CD1d molecules via their TCR. NKT cells mediate cytotoxicity against tumor cell lines in vitro2. CD1d-restricted NKT cells. Both V 24 and V 11 mAbs are available for identification of NKT cells in humans. In vivo treatment of mice with IL-12 induces potent tumor N O V E M B E R 580 Vo l . DN and CD8 NK1. Immediately ex vivo. Certainly. the effect on NKT cells was transient and correlated with the period during which viable bacteria were identified in the liver (that is. suggesting an important role for these cells in tumor rejection. monocytogenes antigens. and some DN and most TCR cells are not NKT cells. These studies show that mouse NKT cells can be induced to participate in antitumor responses. and such granulomas do not form in NKT celldeficient J 281 / mice51. GalCer-treatment of mice also induces CD1d-restricted.79. so it is possible that the immune response to this organism involves recruitment of IFN.following in vitro stimulation with Listeria-infected macrophages104. changes likely to aid the host response to mycobacteria. these data are in conflict with a more recent study which demonstrated that the IgG response to P. However.120 represent an important tool for identifying these cells in both mice and humans. The proportions of CD4 . Human V 24 NKT cells (including CD4 and DN subsets). which has been suggested to be NKT cell-mediated66. and deployment of IL-4-producing CD4 NKT cells to the spleen for support of antibody production.-secreting DN NKT cells. However. 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. we recently demonstrated that NKT cells might be important in protecting against some. and CD1d. 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. These results collectively suggest that NKT cells are capable of responding to L. Second.production101.

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