natural killer cells 2

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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,

4051405840 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 IB (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 transductionpathways. 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 Tcell 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 RSK-dependent 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

KT cells are a population

of T cells that share some

characteristics with natu-

ral killer (NK) cells (re-

viewed in Refs 13). The key features char-

acteristic of NKT cells include heavily biased

T-cell receptor (TCR) gene usage, CD1d re-striction and high levels of cytokine produc-

tion, particularly interleukin 4 (IL-4) and in-

terferon (IFN-) (Fig. 1). In mice, these cells

are commonly defined as NK1.1TCR.

However, as cells bearing these markers are

phenotypically and functionally heterogen-

eous, it is appropriate to compare the differ-

ent subsets of cells encompassed within this

population. Most of the studies covered in

this review relate to NKT cells in mice, unless otherwise specified.

Subsets of NKT cells

Although the term NKT was only recently applied, these cells were

first described in 1987. Most of the earlier studies in mice focussed

on thymic TCR T cells that were CD4

and CD8 double negative (DN) and

highly biased toward V8-2 expression. A

major subset of these cells was found to ex-

press NK1.1, previously associated with NK

cells, and to have the potential to secrete

high levels of IL-4, IFN- and tumor necrosisfactor (TNF). A population of NK1.1 CD4

T cells was also identified with similar char-

acteristics13. Subsequent reports showed

that both populations predominantly express

TCR V14J281 and that their development

was dependent on the MHC class I-like, 2-

microglobulin-associated molecule, CD1d.

These findings strongly suggested that

NK1.1DN and NK1.1CD4 T cells were

part of the same lineage13. Thus, in recent years NKT cells are

usually identified by the coexpression of the TCR and NK1.1, as

shown in Fig. 2.

Several recent studies in mice have demonstrated the potential

danger of oversimplifying our classification of NKT cells48. These

studies have shown that CD4, DN and CD8 NK1.1 T cells are

R E V I E WI M M U N O L O G Y T O D A Y

V o l . 2 1 N o . 1 1 5 7 3

0167-5699/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved.PII: S0167-5699(00)01735-7

N O V E M B E R 2 0 0 0

NKT cells: facts, functions and fallacies

Dale I. Godfrey, Kirsten J.L. Hammond, Lynn D. Poulton, Mark J. Smythand Alan G. Baxter

The proposed roles of NK1.1 T

(NKT) cells in immune responses

range from suppression of

autoimmunity to tumor rejection.

Heterogeneity of these cells

contributes to the controversy

surrounding their development and

function. This review aims to

provide an update on NKT cell

biology and, whenever possible, to

compare what is known about NKT-

cell subsets.

N


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present in most tissues and are phenotypi-

cally and functionally distinct. Furthermore,

peripheral NK1.1

T-cell subsets differ fromtheir thymic counterparts (Fig. 3). Thymus

and liver CD4 and DN NKT cells are gen-

erally 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-depen-

dent4,8, have a more heterogeneous TCR

repertoire4,5,7

, and produce lower levels ofcytokines 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- galactosyl-

ceramide (GalCer) tetramers8. Splenic

CD8 NK1.1 T cells are CD1d and thymus-

independent, express heterogeneous TCR,

and do not produce IL-4 rapidly. In addition,

some NK1.1CD4 T cells closely resemble

cells of the NKT-cell family in their CD1d-reactivity, TCR V8-bias, and high IL-4-

production8,1012. One explanation for this

might be that NK1.1 can be downregulated

upon NKT-cell activation13.

Surrogate markers for NKT cells

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 phe-notypes have been used, for example: DN

(Ref. 14), DX5CD3 (Ref. 15), and Ly49A

CD122CD3 (Ref. 16). A problem with these

surrogate markers is that they do not accu-

rately 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 popu-

lations in C57BL/6 mice demonstrates the

presence of other NK1.1 cells within these

populations. Other surrogate phenotypes,

such as DX5CD3, might exclude most NKT

cells, particularly the CD1d-dependent sub-

sets (CD4 and most DN), which are DX5

R E V I E WI M M U N O L O G Y T O D AY

5 7 4 V o l . 2 1 N o . 1 1

N O V E M B E R 2 0 0 0

Fig. 1.Mouse NKT cells share some characteristics with NK and T cells. This venn diagram incor-

porates 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.

Immunology Today

NKT

NKT

Thymus-independentMHC-I/II restricted

CD3/TCR+

Thymus-dependentTCR-dependent Cytotoxic

IFN-production

NK1.1+

Non-MHCrestricted

Rapid/high IL-4 productionCD1d restricted

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 ver-

sus 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.

Immunology Today

80

17 2

65

34

0.6%

68

20 10

1.4% 24%

TCR

CD8

(a)

(b)

Thymus Spleen Liver

NK1.1

CD4


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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, V14J281 specific monoclonalantibody (mAb) or CD1d tetramers8,120 would

most reliably identify these cells in all strains.

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. Signifi-

cantly, human NKT cells express the homol-ogous TCR gene products (V11, the homo-

logue of mouse V8.2; and V24JQ, the

homologue of mouse V14J281). Further-

more, human NKT cells can recognize

mouse CD1d and vice versa, indicating

highly conserved specificity21.

Distribution of NKT cells

In mice, NKT cells can be detected wherever

conventional T cells are found, although the

ratio of NKT to T cells varies widely in atissue-specific manner15. As a proportion of

mature T cells, NKT cells are most frequent in

liver (3050%), bone marrow (2030%) and

thymus (1020% of mature HSA T cells, al-

though only 0.30.5% of total thymocytes).

They represent a smaller proportion of T cells

in other tissues including spleen (3%), lymph

node (0.3%), blood (4%) and lung (7%). Inter-

estingly, CD4, DN and CD8 NK1.1 T cells

are differentially distributed in a tissue-spe-

cific fashion46, supporting the concept that

these are functionally and/or developmen-tally distinct subsets. Their intra-tissue local-

ization remains unknown, partly because of

the relative scarcity of these cells and the lack

of a single, defining cell-surface marker.

Although most reports on human NKT cells have been limited to

those in peripheral blood, V24JQ NKT cells are clearly present in

human liver although apparently not as frequent as they are in mice

(approximately 4% of hepatic T cells versus 50% in mice)22. The distri-

bution of NKT cells in other human tissues remains to be determined.

NKT cell developmentThymus dependence

A surprising amount of controversy surrounds studies of the origin

of NKT cells. Although the overwhelming body of evidence indicates

that most NKT cells are thymus-dependent, some scientists argue for

an extrathymic origin. This controversy has been discussed in detail

in earlier reviews13, and will not be repeated here, except to say that

the evidence for a thymic origin for most CD4 and DN NKT cells is

difficult to ignore. In mice, they develop in fetal thymus organ cul-

ture23, are present among recent thymic emigrants in both spleen

and liver5 and fail to develop normally in nude24 or neonatally

thymectomized mice25, or adult mice irradiated and thymectomized

prior to fetal liver-reconstitution26. By contrast, CD8NK1.1 T cells

are present in normal numbers in the periphery of neonatally

thymectomized mice5,25.


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N O V E M B E R 2 0 0 0

Fig. 3.Mouse NK1.1 T-cell subsets are phenotypically, developmentally and functionally distinct.

Mouse NK1.1 T cells can be subdivided based on CD4/CD8 expression and on tissue of origin. This

diagram shows representative histogram profiles or summarized data for a selection of features that

reveal distinct subsets of NK1.1 T cells. Histogram profiles are representative of NK1.1 T-cell

subpopulations derived from 57-week old C57BL/6 mice. All NK1.1 T-cell subsets in the thymus

and spleen are positive for CD122, Fas, CD44, CD1d, CD28, CD38 and CD45RC (not shown). Infor-

mation taken from Refs 16, 9. Abbreviations: DN, CD4CD8 double negative; , not determined;

IFN-, Interferon ; IL-4, interleukin 4; neg, negative; pos, positive; Y, yes; N, no; Y/N, partiallydependent; J281 dep., T-cell receptor V14J281-dependent development. aData relate to spleen

NKT cells except where indicated. bData relate to liver NKT cells. cIL-4/IFN-production measured

by ELISA following 18 h culture on anti-CD3 coated plates, 106 cells ml1.

Immunology Today

24315142

9996 9789

64 274080

97 99 10097

433513 4

8654 48 70

55 5754

698176

74 69 84

98 97 99

7 6 12

69 7670

Thymus Peripherya

Total CD4+ DN Total CD4+ CD8+DN

V8

Thy-1

CD69

CD45RB

DX5

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

Y

Y

800

5

Pos

Low

Low

Neg

Pos

2%

15%

Y

Y

Y

800

6

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

N

Y

50

0.1

Low

Low

Neg

80%

45%

N

N

N

2

0.8


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Appearance during ontogeny

Another debated question relates to the timing of NKT-cell develop-ment. Most studies have indicated that NKT cells do not appear until

relatively late in mouse ontogeny, well after conventional T cells13,25.

However, one report suggested that NKT cells start to develop at E9.5

in the yolk sac and are one of the first T-cell populations to appear,

arising in the fetal liver at E13.5 (Ref. 27). We (unpublished data) and

others28 have been unable to verify these findings.

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 CD4CD8 (DP)thymocytes13,24, many key questions remain. For example, the de-

velopmental 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 CD8NK1.1 T cells is even less well under-

stood. One study provided evidence that CD8NK1.1 T cells are

generated extrathymically following antigen encounter in the liver29.

Another showed that mainstream CD8 T cells can upregulate

NK1.1 following antigenic challenge in the

periphery30. Although this would fit with the

non-biased TCR repertoire of CD8

NK1.1

Tcells, it is unlikely to completely explain the

presence of CD8 NK1.1 T cells in normal

mice, many of which are CD8 (Ref. 5).

Some studies have demonstrated and

investigated the development of NK1.1 T

cells specific for conventional MHC plus

peptide in TCR transgenic mice29,3133. Collec-

tively, these studies suggest that TCR-trans-

genic, NK1.1-expressing, IL-4-secreting T

cells can be generated in the absence of

endogenous TCR gene rearrangement. This

implies that there is nothing inherently spe-cial about the recognition of CD1d by the in-

variant TCR V14J281, but rather, suggests

that NKT-cell development depends on the

timing, avidity and type of selecting cell, and

that such selection might simply result more

frequently through interaction with CD1d.

However, considering that NKT cells are

virtually absent in the thymus of CD1d/

or TCR J281/ mice, it seems that conven-

tional TCRMHC interactions are, at best, a

rare event in the development of NKT cells. Thus, although these

TCR transgenic approaches theoretically provide greater ability tomanipulate the nature of the selecting environment (for example,

non-selecting, positively selecting or negatively selecting), care

should be taken in extrapolating information from these studies to

non-transgenic NKT-cell development.

Factors influencing NKT-cell development

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-4-

producing potential, respectively, of both CD4 and DN NKT

cells34,35, and an intact thymic structure is also important 36. In con-trast to conventional T cells, NKT cell development requires an in-

teraction with membrane lymphotoxin expressing cells37,38. Interest-

ingly, lymphotoxin deficiency affected all three populations (CD4,

DN and CD8) of NK1.1 T cells, suggesting at least some common-

ality 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 conven-

tional T cells, nor of CD8NK1.1 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 thymo-

cytes expressing normal amounts of V14J281 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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N O V E M B E R 2 0 0 0

Table 1. Features common to mouse and human NKT cells

Characteristic Mouse Human Comment

Major subsets CD4, DN CD4, DN Proportions vary

T cell receptor-chain V14J281 V24JQ Homologous

-chain V8.2 V11 Homologous

V7, V2

Expression level Intermediate Intermediate

Accessory molecules

NK associated NK1.1 NKR-P1 Homologous (CD161)

CD122 CD122

Ly49

Restriction element CD1d CD1d Homologous

Cognate antigen Glycolipid Glycolipid -GalCer stimulates

Cytokine production

IL-4 Rapid, high levels Rapid, high levels Following TCR ligation

IFN- Following TCR ligation

Frequency

PBL ~1% ~0.10.5% More variable in humans

aAbbreviations: GalCer, -galactosylceramide; DN, double negative; IFN-, interferon ; IL-4, inter-leukin 4; NKR, NK-cell receptor; NKT, NK1.1 T cells; PBL, peripheral blood leukocytes; TCR, T-cellreceptor.


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not exported to the periphery. Thus, intrathymic selection and de-

velopment of IL-4-producing capacity seem to be earlier processes,

whereas acquisition of the NK surface phenotype and emigration tothe 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 oc-

curs 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-weekold 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.

NKT cell ligands

The expression of CD3/TCR by NKT cells suggests that they re-

quire 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 mouseNK1.1 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 hy-

drophobic ligands (probably glycolipids)8,21, although the precise na-

ture 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 con-

tentious52,53. The ability of NKT cells to respond to tumor-derived

lipid extracts (including phospholipids) in the context of CD1d sug-

gests that they might see a natural lipid ligand, possibly altered intumor 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

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, partic-

ularly 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

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 thoughtresponsible for synthesizing such -linked carbohydrate moieties,

are defective in humans and some other old-world primates57,58, sug-

gesting 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. IfGalCer 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 cellsis the rapid production of high levels of immunoregulatory cyto-

kines 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 ofin 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 frag-

ments cultured with anti-CD3, suggesting that the potent cytokine-

producing NKT cells might migrate rapidly into the spleen in responseto in vivo activation61.

Stimulation of mouse NKT cells by NK1.1 (NKRP1C/CD161) en-

gagement, rather than by TCR cross-linking, led to increased IFN-

production 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 tar-

get cells, including DP thymocytes67, and can also kill tumor targets

in a perforin-dependent manner68.

-Galactosylceramide stimulation

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 re-

peated exposure toGalCer in vivo leads to enhanced Th2 responses6971,

one showed Th2 inhibition72. In line with the latter study, GalCer


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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 prolifera-

tion 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

mice48, protection against murine malaria80

and enhanced antigen-specific CTL activ-

ity74

. However, a recent study demonstratedthat GalCer treatment of mice rapidly led

to massive, NKT cell-dependent liver dam-

age, which was sufficient to kill older mice

(presumably because of their higher liver

NKT cell number)81. This might not be en-

tirely surprising considering the widespread

stimulation of potentially destructive lym-

phocytes such as NKT and NK cells and

other bystander cells. Clearly, this result

highlights the need for caution in applying

the activities ofGalCer observed in animal

models to the clinic.

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 func-

tion for NKT cells might be to protect self-

tissues (particularly vital organs) from dam-

aging inflammatory-type immune responses.

There is also clear evidence that they can

control immune responses to infection andsome tumors (Table 2).

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 investi-

gations 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 im-portant players in some Th2-associated re-

sponses. For example, V14 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.

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 dam-

age 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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Table 2. NKT cells and disease

Disease Refs

Autoimmunity

Type 1 diabetes

NKT cells deficient in NOD mouse and BB rat models and in 14, 16, 9498

human diabetics

Increased NKT cells protect from diabetes in NOD mice 14, 83, 96

Lupus

Decreased NKT cells associated with onset of disease (mice) 92, 93

DN transgenic CD1d-reactive T cell clone prevents lupus (mice) 91

Experimental autoimmune gastritis

Day 3 thymectomy selectively depletes NKT cells (mice) 25

Systemic sclerosis

NKT cells depleted in systemic sclerosis patients 87

Multiple sclerosis

NKT cells depleted in multiple sclerosis patients 114

Infection

Listeria

DN NKT cells decreased in liver (mice) 107

CD4NKT cells increased in spleen (mice) 115

Toxoplasma

NKT cells help to generate CD8 effector cells (mice) 116

Mycobacteria

NKT cells required for granuloma formation inMycobacterium 51

tuberculosis infection (mice)

BCG downregulates CD4NKT cells and induces IFN-- 101, 105

producing NKT cells (mice)

Salmonella

NKT cells exacerbate infection and are effectors of liver 117, 118damage (mice)

Plasmodium

NKT cells directly inhibit parasite growth in liver (mice) 102

CD1d-restricted NKT cells important in anti-parasite IgG 50

formation (mice)

No role for CD1d-restricted NKT cells in anti-parasite IgG 52

formation (mice)

Tumor

NKT cells can be induced to mediate tumor rejection (mice) 107109, 119

NKT cells play a natural role in tumor rejection (mice) 68

Tolerance and immune deviationNKT cells required for ACAID (mice) 84, 85

NKT cells mediate suppression of acute GVHD (mice) 86

aData from Refs 1721.bAbbreviations: ACAID, anterior chamber-associated immune-deviation; DN, CD4CD8 double nega-tive; 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.


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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 populationidentified 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-4-

dependent manner86, which might reflect a natural role for these cells

in preventing inflammatory immune responses in bone marrow.

Prevention of autoimmune tissue destruction

Disturbances in numbers and function of NKT cells have been im-

plicated 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 TCR-

heterogeneity, Sumida and colleagues87 performed a detailed analy-

sis of the TCR- chains of DN T cells from these patients. Al-

though they found a fivefold increase in V24 DN T cells, virtually

none of these cells expressed the invariant NKT cell-associated

V24JQ TCR -chain, but instead carried alternate junctional re-

gions. The authors concluded that scleroderma was associated witha deficiency of NKT cells and an oligoclonal expansion of other DN

V24 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 eryth-

ematosus (SLE) is less advanced, but shows striking parallels. Pa-

tients with SLE have a three- to tenfold increase in the proportions of

DN T cells in the peripheral blood88,89 and an increased propor-

tion of these cells produce IL-4 after polyclonal stimulation89. Sig-

nificantly, these cells were capable of supporting the production ofanti-DNA antibodies by syngeneic B-cells in vitro88, and might rep-

resent 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 andgld strains have massive expansions of DN T

cells. These differ from NKT cells by their expression of hetero-

geneous 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 de-

pletion of NK1.1 or V14 NKT cells92,93, although the issue would

benefit from being revisited. A significant problem with the latter

study was the reliance on a V14-specific mAb that does not recog-

nize TCR with the NKT cell-associated J281 junctional region2.

(iii) Type 1 diabetes

The situation in type 1 diabetes (a tissue-specific autoimmune dis-

ease) appears to be rather more straightforward. We and othersfound 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 pre-

vented by the injection of 2 105 thymic DN (NKT) cells and that

protection is mediated by an IL-4 and/or IL-10-dependent mecha-

nism83. Lehuen and colleagues96 subsequently confirmed the protec-

tive role of NKT cells in NOD mice by increasing the numbers of

these cells through the introduction of a V14J281 transgene.

Adoptive transfer of splenic T cells, including approximately 8 105

DN and CD4

NKT cells, from these mice to non-transgenic syn-geneic 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 3105

splenic Ly49ACD122CD3 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 lev-

els 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 po-

tent protection against diabetes in NOD mice compared with splenicNKT 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 auto-

immune 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

V24JQ T cells in peripheral blood than their nondiabetic siblings98.

Control of infection

NKT cells appear to participate in immune responses to a range ofdifferent 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 re-

sponses to mycobacteria. Human clones and lines of CD8 and

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 dif-

fer significantly in susceptibility toMycobacterium 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 cell-

deficient J281/

mice51

. Furthermore, NKT cells of normal mice re-spond 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 inhib-

ited growth of the pathogen in hepatocytes in vitro via an IFN--de-

pendent mechanism102. CD1-restricted presentation of Plasmodium

berghei sporozoite-derived GPI anchor has been shown to stimulateNKT-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.

(iii) Listeria

Although DN T cells accumulate in the peritoneal cavity follow-

ing intraperitoneal infection of mice with Listeria monocytogenes103,

the investigators were unable to demonstrate proliferation of thesecells in response to Listeria lysates or Listeria-infected macrophages

in vitro104. Immediately ex vivo, the cells did contain mRNA for GM-

CSF, Eta-1 and MCP-1 (but not IL-4), and secreted IFN- following

in vitro stimulation with Listeria-infected macrophages104. These re-

sults 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 con-sistent 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 infec-

tion, so it is possible that the immune response to this organism in-

volves 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.

Tumor rejectionIn 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

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-depen-dent110,111. 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 anti-

tumor responses, but do not indicate whether this is a natural func-

tion of these cells. At least two findings support such a role. First,

IL-4-producing DN NKT cells accumulated at the site of an embry-

onal carcinoma and appeared to be necessary for tumor rejection112.

Second, we recently demonstrated that NKT cells might be impor-

tant in protecting against some, but not all, types of tumors. Forexample, NKT-deficient J281/ mice had impaired protection

against spontaneous sarcomas initiated by the chemical carcinogen

methylcolanthrene, but were resistant to other tumor types con-

trolled by NK cells68. Human V24 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.

Concluding remarks

This is clearly a controversial field. Many of these disagreements

would be resolved by increasing the precision with which the iden-tification 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,

it is probably safe to say that of NK1.1TCR cells virtually

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

CD8NK1.1TCR cells are not NKT cells. Improved under-

standing is likely to come from further dissection of NKT-cell subsets

and the relationships between them. It will be important to deter-

mine what phenotypic changes occur in their development and fol-

lowing activation. Certainly, further improvement would be attainedif a reliable antibody specific for the canonical V14J281 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 V24 and V11 mAbs are available

for identification of NKT cells in humans, and the combination of

reagents appears to be highly specific113. The continued use of anti-

bodies 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 under-

standing of the signal transduction machinery and functional out-

comes 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


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remain uncertain, as do the roles they play in NKT-cell differentia-

tion and function. NKT cells might also respond to pathogen-specific

ligands, and play a role in determining the direction of immune-deviation 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 tech-

nology 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 appli-cation 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 recipi-

ent of an ADCORP/Diabetes Australia Fellowship. AGB is a recipient of an

R. Douglas Wright Fellowship from the NHMRC. LDP and KJLH are recipientsof Australian Postgraduate Research Awards. MJS is an NHMRC Principal

Research Fellow.

Dale I. Godfrey ([email protected]) and Kirsten J.L.

Hammond are at the Dept of Pathology and Immunology, Monash Uni-

versity Medical School, Commercial Road, Prahran, VIC. 3181, Australia;

Lynn D. Poulton andAlan G. Baxter([email protected])

are at the Centenary Institute of Cancer Medicine and Cell Biology,

Locked Bag #6, Newtown, NSW 2042, Australia; Mark J. Smyth

([email protected]) is at the Peter MacCallum Cancer Institute,

Locked Bag 1, A Beckett St, VIC. 8006, Australia.

References

1 Bendelac, A. (1995) Mouse NK1 T cells. Curr. Opin. Immunol. 7, 367

2 Bendelac, A. et al. (1997) Mouse CD1-specific NK1 T cells

development, specificity, and function.Ann. Rev. Immunol. 15, 535562

3 Macdonald, H.R. (1995) NK1.1 T cell receptor- cells new clues

to their origin, specificity, and function.J. Exp. Med. 182, 633638

4 Eberl, G. et al. (1999) Tissue-specific segregation of CD1d-dependent

and CD1d-independent NK T cells.J. Immunol. 162, 64106419

5 Hammond, K.J.L. et al. (1999) NKT cells are phenotypically and

functionally diverse. Eur. J. Immunol. 29, 37683781

6 Zeng, D.F. et al. (1999) Heterogeneity of NK1.1 T cells in the bone

marrow: Divergence from the thymus.J. Immunol. 163, 53385345

7 Apostolou, I. et al. (2000) Evidence for two subgroups of CD4CD8

NKT cells with distinct TCRalphabeta repertoires and differentialdistribution in lymphoid tissues.J. Immunol. 165, 24812490

8 Benlagha, K. et al. (2000) In vivo identification of glycolipid antigen-

specific T cells using fluorescent CD1d tetramers.J. Exp. Med. 191,

18951903

9 MacDonald, H.R. et al. (1998) Developmentally regulated extinction of

Ly-49 receptor expression permits maturation and selection of NK1.1

T cells.J. Exp. Med. 187, 21092114

10 Chen, H.J. and Paul, W.E. (1998) A population of CD62Llow NK1.1

CD4 T cells that resembles NK1.1 CD4 T cells. Eur. J. Immunol. 28,

31723182

11 Hameg, A. et al. (1999) IL-7 up-regulates IL-4 production by splenic

NK1.1 and NK1.1 MHC class I-like/CD1-dependent CD4 T cells.

J. Immunol. 162, 70677074

12 Moodycliffe, A.M. et al. (1999) Splenic NK1.1-negative, TCR

intermediate CD4 T cells exist in naive NK1.1 allelic positive and

negative mice, with the capacity to rapidly secrete large amounts of

IL-4 and IFN-gamma upon primary TCR stimulation.J. Immunol. 162,

51565163

13 Chen, H.J. et al. (1997) NK1.1CD4 T cells lose NK1.1 expression

upon in vitro activation.J. Immunol. 158, 51125119

14 Baxter, A.G. et al. (1997) Association between TCRCD4CD8 T cell

deficiency and IDDM in NOD/Lt mice. Diabetes 46, 572582

15 Ortaldo, J.R. et al. (1998) The Ly-49 family: regulation of cytotoxicity

and cytokine production in murine CD3 cells.J. Immunol. 160,

11581165

16 Falcone, M. et al. (1999) A defect in interleukin 12-induced activation

and interferon secretion of peripheral natural killer T cells in

nonobese diabetic mice suggests new pathogenic mechanisms for

insulin-dependent diabetes mellitus.J. Exp. Med. 190, 963972

17 Exley, M. et al. (1997) Requirements for CD1d recognition by human

invariant V-alpha-24 CD4CD8 T cells.J. Exp. Med. 186, 109120

18 Dellabona, P. et al. (1994) An invariant V24-JQ/V11 T cell receptor

is expressed in all individuals by clonally expanded CD48 T cells.

J. Exp. Med. 180, 11711176

19 Davodeau, F. et al. (1997) Close phenotypic and functional similarities

between human and murine T cells expressing invariant TCR -chains.J. Immunol. 158, 56035611

20 Takahashi, T. et al. (2000) Analysis of human V 24 CD4 NKT cells

activated by -glycosylceramide-pulsed monocyte-derived dendritic

cells.J. Immunol. 164, 44584464

21 Burdin, N. and Kronenberg, M. (1999) CD1-mediated immune

responses to glycolipids. Curr. Opin. Immunol. 11, 326331

22 Doherty, D.G. et al. (1999) The human liver contains multiple

populations of NK cells, T cells, and CD3CD56 natural T cells with

distinct cytotoxic activities and Th1, Th2, and Th0 cytokine secretion

patterns.J. Immunol. 163, 23142321

23 Bendelac, A. et al. (1994) A subset of CD4 thymocytes selected by

MHC class I molecules. Science 263, 17741778

24 Coles, M.C. and Raulet, D.H. (2000) NK1.1 T cells in the liver arise in

the thymus and are selected by interactions with class I molecules onCD4CD8 cells.J. Immunol. 164, 24122418

25 Hammond, K.J.L. et al. (1998) Three day neonatal thymectomy

selectively depletes NK1.1 T cells.Int. Immunol. 10, 14911499

26 Tilloy, F. et al. (1999) Thymic dependence of invariant V 14 natural

killer-T cell development. Eur. J. Immunol. 29, 33133318

27 Makino, Y. et al. (1996) Development of V14 NKT cells in the early

stages of embryogenesis. Proc. Natl. Acad. Sci. U. S. A. 93, 65166520

28 Shimamura, M. et al. (1997) Thymus-independent generation of NK1

T cells in vitro from fetal liver precursors.J. Immunol. 158, 36823689

29 Legendre, V. et al. (1999) Selection of phenotypically distinct NK1.1

T cells upon antigen expression in the thymus or in the liver. Eur. J.

Immunol. 29, 23302343

30 Slifka, M.K. et al. (2000) NK markers are expressed on a high

percentage of virus-specific CD8

and CD4

T cells.J. Immunol. 164,20092015

31 Schulz, R.J. et al. (1996) Development of CD4CD8 TCRNK1.1

T lymphocytes: thymic selection by self antigen.J. Immunol. 157,

43794389


V o l . 2 1 N o . 1 1 5 8 1

N O V E M B E R 2 0 0 0


10/11

32 Iwabuchi, C. et al. (1998) Intrathymic selection of NK1.1 T cell

antigen receptor TCR cells in transgenic mice bearing TCR specific

for chicken ovalbumin and restricted to I-A(d). Proc. Natl. Acad. Sci.

U. S. A. 95, 8199820433 Curnow, S.J. et al. (1995) TCR-associated -FcRI- heterodimers on

CD4CD8 NK1.1 T cells selected by specific class I MHC antigen.

Immunity 3, 427438

34 Vicari, A.P. et al. (1996) NK1.1 T cells from IL-7-deficient mice have a

normal distribution and selection but exhibit impaired cytokine

production.Int. Immunol. 8, 17591766

35 Ohteki, T. et al. (1997) Role for IL-15/IL-15 receptor -chain in NK1.1

T cell receptor- cell development.J. Immunol. 159, 59315935

36 Nakagawa, K. et al. (1997) Generation of NK1.1 T cell antigen

receptor thymocytes associated with intact thymic structure. Proc.

Natl. Acad. Sci. U. S. A. 94, 24722477

37 Iizuka, K. et al. (1999) Requirement for membrane lymphotoxin in

natural killer cell development. Proc. Natl. Acad. Sci. U. S. A. 96,

6336634038 Elewaut, D. et al. (2000) Membrane lymphotoxin is required for the

development of different subpopulations of NK T cells.J. Immunol.

165, 671679

39 Eberl, G. et al. (1999) Absolute requirement for the pre-T cell receptor

chain during NK1.1 TCR cell development. Eur. J. Immunol. 29,

19661971

40 Sato, H. et al. (1999) Induction of differentiation of pre-NKT cells to

mature V 14 NKT cells by granulocyte macrophage colony-

stimulating factor. Proc. Natl. Acad. Sci. U. S. A. 96, 74397444

41 Eberl, G. et al. (1999) Cutting edge: NKT cell development is

selectively impaired in Fyn-deficient mice.J. Immunol. 163, 40914094

42 Gadue, P. et al. (1999) The Src family tyrosine kinase Fyn regulates

natural killer T cell development.J. Exp. Med. 190, 11891196

43 Lantz, O. et al. (1997) Lineage relationships and differentiation ofnatural killer (NK) T cells intrathymic selection and interleukin

(IL)-4 production in the absence of NKR-P1 and Ly49 molecules.

J. Exp. Med. 185, 13951401

44 Eberl, G. and MacDonald, H.R. (1998) Rapid death and regeneration of

NKT cells in anti-CD3-- or IL-12-treated mice a major role for bone

marrow in NKT cell homeostasis.Immunity 9, 345353

45 Emoto, M. et al. (1999) Critical role of leukocyte function-associated

antigen-1 in liver accumulation of CD4 NKT cells.J. Immunol. 162,

50945098

46 Ohteki, T. et al. (1999) Cutting edge: LFA-1 is required for liver

NK1.1TCR cell development: evidence that liver NK1.1TCR

cells originate from multiple pathways.J. Immunol. 162, 37533756

47 Miyamoto, M. et al. (2000) Cutting edge: contribution of NK cells to the

homing of thymic CD4NKT cells to the liver.J. Immunol. 165, 17291732

48 Hong, S.M. et al. (1999) Lipid antigen presentation in the immune system:

lessons learned from CD1d knockout mice.Immunol. Rev. 169, 3144

49 Joyce, S. et al. (1998) Natural ligand of mouse CD1d1 cellular

glycosylphosphatidylinositol.Science 279, 15411544

50 Schofield, L. et al. (1999) CD1d-restricted immunoglobulin G formation

to GPI-anchored antigens mediated by NKT cells. Science 283, 225229

51 Apostolou, I. et al. (1999) Murine natural killer T (NKT) cells

contribute to the granulomatous reaction caused by mycobacterial cell

walls. Proc. Natl. Acad. Sci. U. S. A. 96, 51415146

52 Molano, A. et al. (2000) Cutting edge: the IgG response to the

circumsporozoite protein is MHC class II-dependent and CD1d-

independent: exploring the role of GPIs in NK T cell activation and

antimalarial responses.J. Immunol. 164, 50055009

53 Burdin, N. et al. (1998) Selective ability of mouse CD1 to present

glycolipids -galactosylceramide specifically stimulates V--14

NKT lymphocytes.J. Immunol. 161, 32713281

54 Gumperz, J.E. et al. (2000) Murine CD1d-restricted T cell recognition of

cellular lipids.Immunity 12, 211221

55 Kawano, T. et al. (1997) CD1d-restricted and TCR-mediated activation

of V()14 NKT cells by glycosylceramides. Science 278, 16261629

56 Motoki, K. et al. (1995) Immunostimulatory and antitumor activities of

monoglycosylceramides having various sugar moieties. Biol. Pharm.Bull. 18, 14871491

57 Galili, U. and Swanson, K. (1991) Gene sequences suggest inactivation

of -1,3-galactosyltransferase in catarrhines after the divergence of

apes from monkeys. Proc. Natl. Acad. Sci. U. S. A. 88, 74017404

58 Larsen, R.D. et al. (1990) Frameshift and nonsense mutations in a

human genomic sequence homologous to a murine UDP-Gal:-D-

Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA.J. Biol.

Chem. 265, 70557061

59 Zlotnik, A. et al. (1992) Cytokine production by mature and immature

CD4-CD8- T cells. -T cell receptor CD4CD8 T cells produce

IL-4.J. Immunol. 149, 12111215

60 Leite de Moraes, M.D. et al. (1995) MHC class I-selected

CD4CD8TCR- T cells are a potential source of IL-4 during

primary immune response.J. Immunol. 155, 4544455061 Yoshimoto, T. and Paul, W.E. (1994) CD4, NK1.1 T cells promptly

produce interleukin 4 in response to in vivo challenge with anti-CD3.

J. Exp. Med. 179, 12851295

62 Arase, H. et al. (1996) Interferon production by natural killer (NK) cells

and NK1.1 T cells upon NKR-P1 cross-linking.J. Exp. Med. 183, 23912396

63 Carlyle, J.R. et al. (1999) Mouse NKR-P1B, a novel NK1.1 antigen with

inhibitory function.J. Immunol. 162, 59175923

64 Exley, M. et al. (1998) CD161 (NKR-P1a) costimulation of CD1d-

dependent activation of human T cells expressing invariant V--24J-

-Q TCR- chains.J. Exp. Med. 188, 867876

65 Leite de Moraes, M.C. et al. (1998) IL-4-producing NK T cells are biased

towards IFN- production by IL-12. Influence of the

microenvironment on the functional capacities of NK T cells. Eur. J.

Immunol. 28, 1507151566 Kawamura, T. et al. (1998) Critical role of NK1 T cells in IL-12-

induced immune responses in vivo.J. Immunol. 160, 1619

67 Arase, H. et al. (1994) Cytotoxicity of fresh NK1.1 T cell receptor

thymocytes against a CD4CD8 thymocyte population associated

with intact fas antigen expression.J. Exp. Med. 180, 423

68 Smyth, M.J. et al. (2000) Differential tumor surveillance by natural

killer (NK) and NKT cells.J. Exp. Med. 191, 661668

69 Burdin, N. et al. (1999) Immunization with -galactosylceramide

polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis.

Eur. J. Immunol. 29, 20142025

70 Kitamura, H. et al. (2000) -galactosylceramide induces early B-cell

activation through IL-4 production by NKT cells. Cell. Immunol. 199, 3742

71 Singh, N. et al. (1999) Cutting edge: Activation of NK T cells by CD1d

and -galactosylceramide directs conventional T cells to the

acquisition of a Th2 phenotype.J. Immunol. 163, 23732377

72 Cui, J.Q. et al. (1999) Inhibition of T helper cell type 2 cell

differentiation and immunoglobulin E response by ligand-activated

V 14 natural killer T cells.J. Exp. Med. 190, 783792

73 Tomura, M. et al. (1999) A novel function of V 14 CD4 NKT cells:

Stimulation of IL-12 production by antigen-presenting cells in the

innate immune system.J. Immunol. 163, 93101

74 Nishimura, T. et al. (2000) The interface between innate and acquired

immunity: glycolipid antigen presentation by CD1d-expressing

dendritic cells to NKT cells induces the differentiation of antigen-

specific cytotoxic T lymphocytes.Int. Immunol. 12, 987994

75 Kitamura, H. et al. (1999) The natural killer T (NKT) cell ligand -

galactosylceramide demonstrates its immunopotentiating effect by

inducing interleukin (IL)-12 production by dendritic cells and IL-12

receptor expression on NKT cells.J. Exp. Med. 189, 11211127

76 Carnaud, C. et al. (1999) Cutting edge: cross-talk between cells of the

innate immune system: NKT cells rapidly activate NK cells.

J. Immunol. 163, 46474650


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11/11

77 Eberl, G. and MacDonald, H.R. (2000) Selective induction of NK cell

proliferation and cytotoxicity by activated NKT cells. Eur. J. Immunol.

30, 985992

78 Nakagawa, R. et al. (1998) Treatment of hepatic metastasis of thecolon26 adenocarcinoma with an -galactosylceramide, KRN7000.

Cancer Res. 58, 12021207

79 Kawano, T. et al. (1998) Natural killer-like nonspecific tumor cell lysis

mediated by specific ligand-activated V 14 NKT cells. Proc. Natl.

Acad. Sci. U. S. A. 95, 56905693

80 Gonzalez-Aseguinolaza, G. et al. (2000) -galactosylceramide-activated

V14 natural killer T cells mediate protection against murine malaria.

Proc. Natl. Acad. Sci. U. S. A. 97, 84618466

81 Osman, Y. et al. (2000) Activation of hepatic NKT cells and subsequent

liver injury following administration of -galactosylceramide.Eur. J.

Immunol. 30, 19191928

82 Bendelac, A. et al. (1996) Increased interleukin 4 and immunoglobulin

E production in transgenic mice overexpressing NK1 T cells.J. Exp.

Med. 184, 1285129383 Hammond, K.J.L. et al. (1998) -T cell receptor (TCR) CD4CD8

(NKT) thymocytes prevent insulin-dependent diabetes mellitus in

nonobese diabetic (NOD/Lt) mice by the influence of interleukin

(IL)-4 and/or IL-10.J. Exp. Med. 187, 10471056

84 Sonoda, K.H. et al. (1999) CD1-reactive natural killer T cells are

required for development of systemic tolerance through an immune-

privileged site.J. Exp. Med. 190, 12151225

85 Wang, Y.F. et al. (1997) Direct thymic involvement in anterior chamber-

associated immune deviation evidence for a nondeletional

mechanism of centrally induced tolerance to extrathymic antigens in

adult mice.J. Immunol. 158, 21502155

86 Zeng, D.F. et al. (1999) Bone marrow NK1.1 and NK1.1 T cells

reciprocally regulate acute graft versus host disease.J. Exp. Med. 189,

1073108187 Sumida, T. et al. (1995) Selective reduction of T cells bearing invariant

V--24J--Q antigen receptor in patients with systemic sclerosis.

J. Exp. Med. 182, 11631168

88 Shivakumar, S. et al. (1989) T cell receptor expressing double-

negative (CD4/CD8) and CD4 T helper cells in humans augment

the production of pathogenic anti-DNA autoantibodies associated

with lupus nephritis.J. Immunol. 143, 103112

89 Funauchi, M. et al. (1999) Increased interleukin 4 production by NK T

cells in systemic lupus erythematosus. Clin. Immunol. 92, 197202

90 Chen, Y.C. et al. (1997) Establishment and characterization of cloned

CD4 CD8 -T cell receptor (TCR)-bearing autoreactive T cells

from autoimmune NZB NZW F1 mice. Clin. Exp. Immunol. 108, 5257

91 Zeng, D. et al. (2000) Cutting edge: a role for CD1 in the pathogenesis

of lupus in NZB/NZW mice.J. Immunol. 164, 50005004

92 Mieza, M.A. et al. (1996) Selective reduction of V--14 NKT cells

associated with disease development in autoimmune-prone mice.

J. Immunol. 156, 40354040

93 Takeda, K. and Dennert, G. (1993) The development of autoimmunity

in C57BL/6 lpr mice correlates with the disappearance of natural

killer type-1 positive cells: evidence for their suppressive action on

bone marrow stem cell proliferation, B cell immunoglobulin

secretion, and autoimmune symptoms.J. Exp. Med. 177, 155164

94 Gombert, J.M. et al. (1996) Early quantitative and functional deficiency of

NK1-like thymocytes in the NOD mouse. Eur. J. Immunol. 26, 29892998

95 Godfrey, D.I. et al. (1997) Flow cytometric study of T cell development

in NOD mice reveals a deficiency in TCRCD4CD8 thymocytes.

J. Autoimmunity 10, 279285

96 Lehuen, A. et al. (1998) Overexpression of natural killer T cells protects

V--14-J--281 transgenic nonobese diabetic mice against diabetes.

J. Exp. Med. 188, 18311839

97 Iwakoshi, N.N. et al. (1999) Diabetes prone BB rats are severely

deficient in natural killer T cells.Autoimmunity 31, 114

98 Wilson, S.B. et al. (1998) Extreme TH1 bias of invariant V--24J--Q T

cells in type 1 diabetes. Nature 391, 177181

99 Grant, E.P. et al. (1999) Molecular recognition of lipid antigens by T

cell receptors.J. Exp. Med. 189, 195205100 Behar, S.M. et al. (1999) Susceptibility of mice deficient in CD1D or TAP1

to infection withMycobacterium tuberculosis .J. Exp. Med. 189, 19731980

101 Emoto, M. et al. (1999) Induction of IFN--producing CD4 natural

killer T cells byMycobacterium bovis bacillus CalmetteGuerin. Eur.

J. Immunol. 29, 650659

102 Pied, S. et al. (2000) Liver CD4CD8 NK1.1 TCR intermediate

cells increase during experimental malaria infection and are able to

exhibit inhibitory activity against the parasite liver stage in vitro.

J. Immunol. 164, 14631469

103 Matsuzaki, G. et al. (1995) Early appearance of T cell receptor

CD4 CD8 T cells with a skewed variable region repertoire after

infection with Listeria monocytogenes. Eur. J. Immunol. 25, 19851991

104 Kadena, T. et al. (1997) TCR CD4 CD8 T cells differentiate

extrathymically in an lck-independent manner and participate inearly response against Listeria monocytogenes infection through

interferon- production.Immunology 91, 511519

105 Emoto, M. et al. (1997) Bacille CalmetteGuerin and interleukin-12

downmodulate interleukin-4-producing CD4 NK1 T lymphocytes.

Eur. J. Immunol. 27, 183188

106 Flesch, L.E.A. et al. (1997) IL-4 secretion by CD4 NK1 T cells induces

monocyte chemoattractant protein-1 in early listeriosis.J. Immunol. 159, 710

107 Hashimoto, W. et al. (1995) Cytotoxic NK1.1 T cells with intermediate

TCR induced in the liver of mice by IL-12.J. Immunol. 154, 43334340

108 Takahashi, M. et al. (1996) LPS induces NK1.1 T cells with potent

cytotoxicity in the liver of mice via production of IL-12 from kupffer

cells.J. Immunol. 156, 24362442

109 Cui, J.Q. et al. (1997) Requirement for V14 NKT cells in IL-12-

mediated rejection of tumors. Science 278, 16231626110 Takeda, K. et al. (2000) Relative contribution of NK and NKT cells to

the anti-metastatic activities of IL-12.Int. Immunol. 12, 909914

111 Smyth, M.J. et al. (2000) The anti-tumor activity of IL-12: mechanisms

of innate immunity that are model and dose dependent.J. Immunol.

165, 26652670

112 Nakamura, E. et al. (1997) Involvement of NK1CD4CD8 T cells

and endogenous IL-4 in non-MHC-restricted rejection of embryonal

carcinoma in genetically resistant mice.J. Immunol. 158, 53385348

113 Prussin, C. and Foster, B. (1997) TCR V--24 and V--11 coexpression

defines a human NK1 T cell analog containing a unique Th0

subpopulation.J. Immunol. 159, 58625870

114 Illes, Z. et al. (2000) Differential expression of NK T cell V 24J Q

invariant TCR chain in the lesions of multiple sclerosis and chronic

inflammatory demyelinating polyneuropathy.J. Immunol. 164, 43754381

115 Emoto, M. et al. (1995) Interleukin-4-producing CD4 NK1.1 TCR-

(intermediate) liver lymphocytes are downregulated by Listeria

monocytogenes. Eur. J. Immunol. 25, 33213325

116 Denkers, E.Y. et al. (1996) A role for CD4NK1.1 T lymphocytes as

major histocompatibility complex class II independent helper cells in

the generation of CD8 effector function against intracellular

infection.J. Exp. Med. 184, 131139

117 Enomoto, A. et al. (1997) Predominant appearance of NK1.1 T cells

producing IL-4 might be involved in the increased susceptibility of

mice with the beige mutation during Salmonella infection.J. Immunol.

158, 22682277

118 Naiki, Y. et al. (1999) Regulatory role of peritoneal NK1.1 T cells in

IL-12 production during Salmonella infection.J. Immunol. 163, 20572063

119 Takeda, K. et al. (1996) Liver NK1.1 CD4 T cells activated by

IL-12 as a major effector in inhibition of experimental tumor

metastasis.J. Immunol. 156, 33663373

120 Matsuda, J.L. et al. (2000) Tracking the response of natural killer T cells

to a glycolipid antigen using Cd1D tetramers.J. Exp. Med. 192, 741754


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