NK cells and apoptosis

Introduction

Natural killer (NK) cells are a sophisticated cell of the innateimmune system with the capacity to kill cells and producecytokines through the interaction of a variety of cell surfacereceptors with their cognate ligands. Natural killer cells arisefrom a bi-potential T/NK progenitor cell (review1), lack CD3and do not rearrange or express any of the TCR genes. Tra-ditionally NK cells are described as large granular lympho-cytes that are central in the innate immune response againsttumours, parasites and cells infected with viruses and intra-cellular bacteria.2–4 They also are of importance in immuneregulation. The ability of NK cells to produce IFN-γ rapidlyafter infection influences the subsequent clonal expansion ofantigen-specific T cells.5 Natural killer cell cytotoxicity isconsidered to be important in tumour surveillance, bonemarrow rejection and antiviral activity. The major mechanismof target cell apoptosis involves exocytosis of cytoplasmicgranules from the NK cell towards the target cells, but alsocan involve engagement of Fas ligand (FasL) or TNF on theNK cell with Fas (CD95) or TNFR on the target cell. Naturalkiller cells also can undergo apoptosis following target celland NK cell receptor engagement, providing a mechanism fordown-regulating the NK cell response. The present reviewfocuses on the major families of NK cell receptors and theeffector mechanisms used by NK cells in immune responses.

NK cell surface receptors regulating effector function

Natural killer cells express a number of receptors that activateor inhibit effector function. These receptors belong to eitherthe Ig-superfamily of type I membrane proteins (Table 1) orthe C-type lectin superfamily of type II membrane proteins(Table 2).

Activation receptors

The first activation antigen described on NK cells was CD16(FcγRIIIA), the low affinity receptor for IgG.2 CD16 is a70-kDa glycoprotein of the Ig superfamily and is expressedas a transmembrane protein on the majority of human periph-eral blood NK cells, on mouse NK cells and also on mono-cytes and subsets of T cells. Natural killer cells in the liverand decidua lack CD16.6,7 Ligation of CD16 with mAb orimmune complexes stimulates NK cell cytotoxicity andcytokine secretion,2 a process involving phosphorylation ofimmunoreceptor tyrosine-based activation motifs (ITAM) onthe covalently associated signal transducing ζ (CD3 ζ) and γ (FcεRIγ) chains.8

CD2 is a 50-kDa receptor of the Ig-superfamily expressedon a subset of NK cells and all T cells. On NK cells, CD2 isinvolved in activating NK cell cytotoxicity and cytokinesecretion. CD2 mAb stimulates cytotoxicity of FcRII+ NK-resistant target cells,9 and NK-resistant P815 cells transfectedwith the CD2 ligand CD58 become susceptible to lysis byhuman NK cells.10 CD2 is involved in spontaneous cyto-toxicity against the prototypic target cell K562 by interactingwith a novel carbohydrate ligand CD2L.11

NKR-P1 (CD161) are a family of C-type lectin type IImembrane receptors encoded by genes in the NK genecomplex (NKC) on rat chromosome 4,12 murine chromosome6,13 and human chromosome 12.14,15 NKRP-1 is a disulphide-linked homodimer expressed by NK cells and a subset of T cells in rat,16 mouse17 and human.14 Murine T cells thatexpress NKR-P1, termed NK1.1+ T cells, are characterizedby a unique TCR composed of the invariant Vα14-Jα281associated with polyclonal Vβ8, Vβ7 and Vβ2 that recognizethe monomorphic class Ib molecule, CD1 (review18). Inrodents NKR-P1 is an activation molecule that triggerscytolytic activity and cytokine secretion.19,20 However, onhuman NK cells, NKR-P1 does not trigger cytolytic activitybut mAb blocks spontaneous killing of sensitive target cells14

and inhibits CD16-activated killing of IL-12 cultured NKcells.21 IL-12 up-regulates NKR-P1 expression on human NK cells, suggesting a role for NKR-P1 on NK cells at sitesof inflammation.21

Immunology and Cell Biology (1999) 77, 64–75

Special Feature

NK cells and apoptosis

HILARY S WARREN1 and MARK J SMYTH2

1Cancer Research Unit, Canberra Hospital, Australian Capital Territory and 2Cellular Cytotoxicity Laboratory,Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia

Summary Natural killer (NK) cells are a cell of the innate immune system that play an important role in the earlyresponse to viral infections and tumours. Natural killer cells are cytolytic, and secrete cytokines that influence thedeveloping antigen-specific immune response. In the present article the NK cell surface molecules regulating effec-tor function, the NK cell effector mechanisms involved in apoptosis, and the role of NK cell effector mechanismsin immune responses are reviewed.

Key words: apoptosis, Fas, granzymes, killer Ig-like receptors, NK cells, perforin, TNF.

Correspondence: Dr M Smyth, Cellular Cytotoxicity Laboratory,Austin Research Institute, Austin Hospital, Studley Road, Heidel-berg, Vic. 3084, Australia. Email: <[email protected]>

Received 9 October 1998; accepted 9 October 1998.

Class I MHC receptors

NK cells and subsets of T cells express clonally distributedreceptors that recognize class I MHC. Individual NK cellsexpress more than one receptor, and receptors are expressedfor both self and non-self class I MHC. There are two cate-gories of receptors. The Ig superfamily killer Ig-like recep-tors (KIR) in humans are encoded by genes on chromosome19, and C-type lectin family receptors are encoded by genesin the NKC (the CD94/NKG2 family in humans and rodentsand the Ly-49 receptor family in mice).

Killer Ig-like receptors were originally described asinhibitory receptors that recognized particular groups ofHLA-C or -B alleles, resulting in inhibition of NK cell cyto-toxicity and cytokine secretion (review22). Molecular charac-terization of KIR revealed heterogeneity in both theextracellular and intracellular regions.23,24 Killer Ig-like recep-tors contain either two or three Ig-like domains in the extra-cellular region, and either a long or short cytoplasmic tail.Killer Ig-like receptors with a long cytoplasmic tail containan immunoreceptor tyrosine-based inhibitory motif (ITIM)that confers inhibitory properties of this receptor. The ITIM islacking in the short form of the receptor, termed the non-inhibitory KIR. The non-inhibitory KIR contain a chargedresidue (K) in the transmembrane region, allowing covalentassociation with a signal tranduction molecule called DAP-12that contains an ITAM.25–27 Ligation of the non-inhibitoryKIR on NK cells and T cells stimulates NK cell cytotoxicityand cytokine secretion.28,29 Serological identification of KIRhas proven unsatisfactory in defining the complexity of these

receptors. Killer Ig-like receptors are best defined by analysisof genomic DNA and mRNA transcripts,30 and can bedescribed by the number of Ig-like domains (KIR2D orKIR3D) and by the presence of a long (L) or short (S) cyto-plasmic tail. In contrast to the relatively conserved phenotypeof the inhibitory KIR, there is substantial variation in thenumber and type of non-inhibitory KIR in the population.30

The inhibitory KIR are functionally dominant over the non-inhibitory KIR,28 and over other receptors activating NK cellcytotoxicity.31

The CD94 and NKG2 receptors are C-type lectinsencoded by genes in the NKC.32–34 CD94 and the NKG2family were initially described in human NK cells and T cellsubsets where their association as disulphide-bonded hetero-dimeric receptors was first appreciated.35–38 These receptorshave recently been described in mouse and rat.39–41 TheCD94/NKG2 receptors recognize HLA-E that becomessurface expressed following binding of signal peptides ofparticular class I alleles,42 explaining the apparent broad classI allele specificity of these receptors. CD94 is an invariantchain with virtually no cytoplasmic domain,33 with functionof the heterodimeric receptor conferred by the NKG2family.36,38 These receptors exist as inhibitory (CD94/NKG2A or B) and non-inhibitory (CD94/NKG2C, E) forms,explaining the original description of CD94 as a receptor thatinhibits or activates NK cell cytotoxicity and cytokine secre-tion.43,44 For the inhibitory receptors, inhibition is conferredthrough ITIM in NKG2A or B.36 For non-inhibitory recep-tors, activation is conferred by association of NKG2C withDAP-12 containing an ITAM.45

Table 1 Ig-superfamily receptors

ITAM, immunoreceptor tyrosine-based activating motif; ITIM, immunoreceptor tyrosine-based inhibitory motif.

NK cells and apoptosis 65

Receptor type Activating Activating Activating Inhibitory

Nomenclature CD16 CD2 KIR2DS1-S5 KIR2DL1-L4KIR3DS1 KIR3DL1-L2

Species Human and rodent Human and rodent Human Human

Gene localization Chromosome 1q23 Chromosome 1p13.1 Chromosome 19q13.4 Chromosome 19q13.4

Structure Two Ig-like domains, Two Ig-like domains, Two Ig-like domains (2D), Two Ig-like domains (2D),∼ 70 kDa ∼ 50 kDa ∼ 50 kDa ∼ 58 kDa

Three Ig-like domains Three Ig-like domains (3D),(3D), ∼ 70 kDa ∼ 70-kDa monomer

(3DL1) or 140-kDa dimer(3DL2)

Structural requirements Covalent association of Association with ζ Covalent association of Cytoplasmic domainfor signal transduction cytoplasmic domain cytoplasmic domain contains two ITIM per

with ζ:ζ or ζ:γ dimers with DAP-12 containing monomercontaining ITAM an ITAM

Ligands Fc of Ab/Ag complexes CD58 (human) HLA class I alleles HLA class I allelesCD48 (mouse) 2DS1: Cw-2,-4,-5,-6,-15 2DL1: Cw-2,-4,-5,-6,-15CD59 (human) 2DS2: Cw-1,-3,-7,-8 2DL2: Cw-1,-3,-7,-8CD2L (human) 2DS3: unknown 2DL3: unknown

2DS4: unknown 2DL4: unknown2DS5: unknown3DS1: unknown 3DL1: Bw4

3DL2: A(?)

Ly-49 is a highly polymorphic family of C-type lectinreceptors that is identified on mouse NK cells and subsets ofT cells and encoded by genes in the NKC that comprise Ly-49-A through Ly-49-I.46 The Ly-49 gene family hasrecently been identified in the rat.12 In the mouse, functionalstudies describe the inhibitory receptors Ly-49-A and -C, andmore recently the activating receptors Ly-49-D and -H. Ly-49-A interacts with the polymorphic α1/α2 domains ofMHC class I,47 and it appears that Ly-49 receptors interactwith a peptide-induced conformational determinant of classI MHC,48 and that binding is strengthened by carbohydraterecognition.49,50 Interaction of Ly-49-A or Ly-49-G receptorswith class I MHC inhibits NK cell cytotoxicity,51 T cellproliferation51 and cytokine secretion.52 Granule release andcytokine secretion stimulated by immobilized mAb to NKR-P1 is prevented by co-immobilizing Ly-49-A mAb,53 demon-strating that the Ly-49 inhibitory receptors are dominant over activation receptors as shown for human KIR. Ly-49-Dwas the first member of the Ly-49 gene family reported as anactivating receptor. Addition of mAb augments lysis of FcγR+

target cells by Ly-49-D+ NK cells.54 Activation is effectedthrough covalent association with a signal transducing mole-cule termed pp1655 or DAP-1256 that contains an ITAMmotif. A second Ly-49 activating receptor is Ly-49-H, whichalso associates with DAP-12.56 There is some evidence thatrejection of allogeneic bone marrow grafts involves recogni-tion of class I MHC through activating receptors,57 includingLy-49-D.58

Other NK cell receptors

Novel NK cell receptors are continuing to be identified,including the heavily glycosylated 110-kDa disulphide-linked homodimeric molecule described in the rat,59 a mole-cule in the rat called NKR-P2 that may form a distinct lectinfamily that includes human NKG2D,60 and a molecule in thehuman termed ‘activation-induced C-type lectin’ (AICL),61

all encoded in the NKC. No specific receptors for spon-taneous cytotoxicity have been identified, but this processmay involve a cohort of adhesion receptors,10 as well as otherreceptors whose ligands are not defined, such as a p38molecule,62 and the NK cell-specific p4663 and NKp44 mole-cules.64 A number of novel inhibitory receptors that are notNK cell-specific and which do not recognize class I MHCinclude LAIR-1, a 32-kDa molecule structurally similar toKIR with a single Ig-like domain and a cytoplasmic tail con-taining two ITIM,65 and a p40 molecule.66,67

NK cell effector molecules

For a long time it has been clear that several mechanisms ofcytolysis may be used by NK cells. These mechanisms canbe dissected in vitro on the basis of target cell sensitivity, thepresence or absence of extracellular Ca2+, the need foreffector cell de novo protein synthesis, or requisite killer cell granule exocytosis. The features of the granule exocy-tosis and the Fas ligand (FasL)/TNF and Fas/TNFR pathwaysof apoptosis are summarized in Table 3, and illustrated inFig. 1.

HS Warren and MJ Smyth66

Tab

le 2

C-t

ype

lect

in s

uper

fam

ily

NK

C, n

atur

al k

ille

r ce

ll g

ene

com

plex

; IT

AM

, im

mun

orec

epto

r ty

rosi

ne-b

ased

act

ivat

ing

mot

if;

ITM

, im

mun

orec

epto

r ty

rosi

ne-b

ased

inh

ibit

ory

mot

if.

Rec

epto

r ty

peA

ctiv

atin

g or

inh

ibit

ing

Act

ivat

ing

Inhi

bito

ryU

nkno

wn

Act

ivat

ing

Inhi

bito

ry

Nom

encl

atur

eN

KR

-P1A

, -B

, -C

(ro

dent

s)C

D94

/NK

G2C

,EC

D94

/NK

G2A

/BC

D94

/NK

G2D

Ly-

49D

; L

y-49

HL

y-49

A, B

, C, E

, F, G

2, I

NK

R-P

1A (

hum

an)

Spe

cies

Hum

an a

nd r

oden

tH

uman

Hum

anR

oden

tM

ouse

Mou

se

Gen

e lo

cali

zati

onC

hrom

osom

e 12

p12-

p13

Chr

omos

ome

Chr

omos

ome

Chr

omos

ome

4 (r

at)

Chr

omos

ome

6 (m

ouse

)C

hrom

osom

e 6

(mou

se)

(hum

an);

chr

omos

ome

412

p12.

3-p1

3.1

12p1

2.3-

p13.

1C

hrom

osom

e 6

(in

the

NK

C)

(in

the

NK

C)

(rat

); c

hrom

osom

e 6

(in

the

NK

C)

(in

the

NK

C)

(mou

se)

(in

the

NK

C)

(mou

se)

(in

the

NK

C)

Str

uctu

reD

isul

phid

e-bo

nded

dim

ers

CD

94 (

∼30

kDa)

CD

94 (

∼30

kDa)

Dim

er o

f 44

-kD

a su

buni

tsD

imer

of

44-k

Da

subu

nits

of ∼

40kD

aN

KG

2C,E

(∼

39kD

a)N

KG

2A (

∼43

kDa)

Str

uctu

ral

requ

irem

ents

Cyt

opla

smic

dom

ain

of r

oden

tN

KG

2 as

soci

ates

wit

hN

KG

2 co

ntai

ns I

TIM

Cyt

opla

smic

dom

ain

Cyt

opla

smic

dom

ain

for

sign

alN

KR

-P1

asso

ciat

es w

ith

p56lc

k ;D

AP

-12

cont

ains

ITA

Mas

soci

ates

wit

h D

AP

-12

cont

ains

an

ITIM

tran

sduc

tion

cyto

plas

mic

dom

ain

of r

oden

tco

ntai

ning

an

ITA

MN

KR

-P1B

con

tain

s an

IT

IM

Lig

ands

Unk

now

nH

LA

-EH

LA

-EU

nkno

wn

Unk

now

nL

y-49

A:

H-2

Dd , H

-2D

k

(sur

face

exp

ress

ed a

fter

(sur

face

exp

ress

ed a

fter

Ly-4

9C:

broa

d H

-2 s

peci

fici

tybi

ndin

g si

gnal

pep

tide

sbi

ndin

g si

gnal

pep

tide

sof

a r

ange

of

HL

Aof

a r

ange

of

HL

Acl

ass

I al

lele

s)cl

ass

I al

lele

s)

Granule-mediated cell death

The basic mechanism of granule exocytosis and the evi-dence supporting this process has been reviewed else-where.68 Natural killer cell cytotoxic granules are vectoriallysecreted into the intercellular space formed during con-jugation of the NK cell and target cell, and lysis is oftenassociated with the formation of membrane lesions on thetarget cell.69 The granules of NK cells contain a number ofproteins, including a pore-forming protein termed per-forin,70 and a family of serine proteases called granzymes.71

Perforin causes osmotic damage due to its binding of phos-phorylcholine headgroups, polymerization and subsequentpore formation in the lipid bilayer of the target cell.70,72,73

Perforin is found essentially in CTL (including γδ T cells)and NK cell granules.74,75 NK cell-mediated target cell deathgenerally involves changes such as chromatin condensation,extensive membrane blebbing and ultimately nuclear DNAfragmentation (apoptosis).76,77 These events clearly occursometime before appreciable perforin-mediated cell lysis,and purified perforin alone is incapable of causing DNAfragmentation. Indeed, a supplementary role is played bygranzymes in target cell killing as supported by much invitro and in vivo experimental evidence.78–80 Granzymes arethe major protein components of CTL and NK cell granulesand they synergize with perforin to trigger an ‘internal dis-integration’ pathway in the target cell.78,81 Most notably, NKcell granzyme B shares Asp-ase specificity with an increas-ingly large family of cell death cysteine proteases (termedcaspases). Granzyme B appears to trigger an endogenouscell death cascade by activating key target cell caspases.82–85

Collectively, these data indicate that granzyme B can greatlyamplify the activation of key signalling components sharedby other cell death stimuli (including the Fas/TNFR path-ways), and directly contribute to apoptotic nuclear mor-phology. Importantly however, granzyme-mediated celldeath is not strictly caspase-dependent, and other keypathways remain to be elucidated.86 Intracellular for mostgranzyme-family members are yet to be defined, and somedata support a role for granzyme A and another granuletryptase serine protease in NK cell-mediated cytotoxic-ity.78,79

TNF superfamily-mediated cell death

Previous observations of target cell death in the absence ofCa2+, granule exocytosis or perforin suggested the existenceof alternative pathways of NK cell-mediated cytotoxicity. TheTNF superfamily of molecules includes several members thatcan deliver an apoptotic signal to target cells expressing acorresponding TNF receptor superfamily molecule. Thesedeath receptors have been recently identified as a subgroupwith a predominant function in the induction of apoptosis.The receptors are characterized by an intracellular region,called the death domain, which is required for the transmis-sion of the cytotoxic signal.87 Currently, five different suchdeath receptors are known including TNF receptor-1, CD95(Fas/APO-1), TNF-receptor-related apoptosis-mediatedprotein (TRAMP) and TNF-related apoptosis-inducing ligand(TRAIL) receptor-1 and -2. Signalling via these receptorsleads to an apoptotic cell death, with characteristic cytoplas-mic and nuclear condensation and DNA fragmentation.87–89

This death process is rapid (within several h), occurs in theabsence of extracellular Ca2+, RNA or protein synthesis, andalso can be triggered in target cells by monoclonal anti-bodies (mAbs).89 Triggering of these pathways requirescross-linking of the receptor, and the ligand (e.g. FasL orTNF) expressed by the NK cell has a trimeric structure insolution. Ligand binding induces receptor oligomerization,followed by the recruitment of an adaptor protein to the deathdomain through homophilic interaction. Caspases, includingthose with adaptor function (i.e. can associate with the recep-tor (e.g. Fas) via receptor-associated death domains (e.g. Fas-associated death domain, FADD) and Asp-ase activity (e.g.FADD-like interleukin-1-β converting enzyme (FLICE)proteases) are involved in receptor-mediated death pathways,and many of the signalling molecules in these apoptotic path-ways have been characterized.90–92 In summary, the signallingpathways by which these receptors induce apoptosis arerather similar. In addition, further pathways have been linkedto death receptor-mediated apoptosis, such as sphingomyeli-nases, Jun kinase pathways, and oxidative stress. Fas/FasL(primarily CD4+ T) and TNFR/TNF (primarily CD8+ T) inter-actions are involved in the clonal deletion of autoreactive T cells in peripheral lymphoid organs.93–95 However, FasL


Table 3 Dual mechanisms of NK cell-mediated apoptosis

Feature Granule exocytosis FasL/TNF–Fas/TNFR

Nature Exocytosis of perforin/granzymes Signalling via death receptorFunction Clearance of Unknown in NK cells

some virus-infected cellsintracellular bacteria infected cellsmalignant/transformed cellstransplanted cells

Ca2+ requirement Yes NoKinetics Rapid (< 4 h) Rapid (FasL< 4 h, TNF> 4 h)Morphology Nuclear DNA fragmentation Nuclear DNA fragmentation

Chromatin condensation Chromatin condensationExtensive membrane blebbing Extensive membrane blebbingVacuolation of cytoplasm Vacuolation of cytoplasm

Involvement of caspases caspase 3,7 caspases 8, effector caspases (e.g. 3,7)Protein synthesis requirement No No

expression appears constitutive in NK cells, and NK cells candisplay FasL- or TNF-mediated cytotoxicity.96 We now appre-ciate that in mice the lpr mutation is a loss-of-function muta-tion of the Fas gene and that gld represents a point mutationin the FasL gene, abolishing the ability of FasL to bind Fas.97

A careful analysis of NK cell development in either gld or lprmice is yet to be reported. These mice, combined with othergene knockout mice, such as those for TNF,98 perforin99 and

granzymes,80,100 provide useful animals in which to study NKcell function and the relative importance of their variouseffector functions in immune responses.

Role of NK cell effector mechanisms in immuneresponses

NK cells and virus infection

The essential components of immune responses to virusrange from direct cytotoxicity to the secretion of solublefactors and antibodies, depending on the type and life cycleof the virus. Natural killer cells are involved in limiting viralreplication during the initial stage of an infection, and theimportance of NK cells in viral infection has been mostdefinitively shown following murine cytomegalovirus(MCMV) infection.101 Natural killer cells can control CMVinfections via two different mechanisms: by the secretion ofantiviral cytokines (e.g. IFN-γ)102 or via the direct lysis ofvirus-infected cells by using perforin.103,104 Spleen NK cellsexert their effects in a perforin-dependent manner,104 whereasin the liver, NK cell IFN-γ regulates MCMV synthesis, andthus distinct organ-dependent mechanisms are used by NKcells to control virus infection.101 Tumour necrosis factor-αalso can control viral infections, although the role of NK cell-secreted TNF in vivo is less clear.105 Natural killer cells wereshown to be responsible for focal inflammation, and to beinduced to migrate at high levels, in MCMV-infectedlivers.106 The NK cell gene complex (NKC) contains genesthat encode molecules that confer resistance or sensitivity toseveral viruses.107 An MCMV class I homologue, m144,appears to be important in protection against NK cell-medi-ated immune responses,108 but at this stage, the ligand form144 and what effector functions it regulates remainunknown. In infections with certain non-cytopathic viruses,such as lymphocytic choriomeningitis virus (LCMV), the NKcells’ primary role is probably via IFN-γ secretion, and theirdirect cytotoxicity is less relevant than that mediated by per-forin-dependent CTL that clear the virus.99,109–112 Theiler’svirus, a murine picornavirus, infects the central nervoussystems (CNS) of mice and is cleared by a process whichrequires NK cells and CD8+ CTL. Clearance of Theiler’svirus from the CNS in mice is perforin-dependent.113

Protection against pox viruses such as vaccinia virus arenot affected by the lack of either perforin-or FasL-dependentcytotoxicity.110 The effect of IFN-γ and TNF-α on vacciniavirus replication also was independent of NK cell cytotox-icity mediated by perforin.99,111 Using mice deficient forgranzyme A, it has been shown that granzyme A plays acrucial role in recovery from the mouse pox virus,ectromelia, by mechanism(s) other than cytolytic activity.114

Granzyme A-deficient mice have an increased virus titre andincreased mortality and morbidity. The mechanism(s)remains undefined, but highlights the possibility that in viralinfection, NK cell granzymes may act somewhat indepen-dently of perforin. The control of hepatitis B virus (HBV)infection is thought to be mediated by class I-restricted CTLand less is known concerning the role of NK cells. To limitkiller cell-mediated destruction of infected hepatocytes,CTL-secreted IFN-γ and TNF-α evidently play an importantrole in suppressing HBV gene expression.115 These antiviral

Figure 1 Natural killer cells may utilize two basic mechanismsof apoptosis. (1) The first involves exocytosis of cytotoxic gran-ules containing perforin and granzymes (including granzyme B,GrB) from the NK cell. Once exocytosed, perforin homopoly-merises in the membrane in a calcium-dependent manner andenables the release of GrB into the target cell cytosol. GranzymeB first activates caspase-3 (C-3) which in turn cleaves the N-peptide of procaspase-7 (C-7), making it accessible to matura-tion by the GrB. The executioner C-7 then causes proteolysis ofkey cytosolic and nuclear substrates. The compartmentalizationof some C-3 to mitochondria suggests that the C-3 there may beactivated via caspase-9 (C-9). Notably, GrB also may act inde-pendently of the caspase cascade. (2) The second pathwayinvolves members of the TNF superfamily (FasL/TNF) and theircorresponding ligands (Fas/TNFR). Ligand binding inducesreceptor oligomerization, followed by the recruitment of anadaptor protein (e.g. Fas-associated death domain (FADD)) to thedeath domain through homophilic interaction. Apical caspases,such as caspase-8 (C-8), with adaptor function and caspaseactivity are involved in receptor-mediated death pathways. C-8activation following Fas/TNFR ligation does not process othercaspases in the absence of C-3. A similar cascade to that in (1) istriggered and cellular proteolysis and nuclear degradation ensue.


cytokines may limit virus replication and viral antigen pre-sentation in hepatocytes without killing them, thereby min-imizing potential killer cell damage by direct lysis. Recentexperiments with perforin-deficient mice have shown thatcytotoxicity is not crucial for the resolution of infection withseveral cytopathic viruses including vesicular stomatitisvirus, Semliki forest virus, and influenza virus.110,116 Thesefindings may reflect the general pattern that infections withcytopathic viruses are mainly controlled by soluble mediatorssuch as antibodies and interferons released by NK cells.117

NK cells and antibacterial function

The most likely way that NK cells control bacterial infectionsin vivo is by producing cytokines that activate macrophagesto degrade the bacteria.118,119 Little information exists con-cerning the importance of NK cell cytotoxicity with respectto bacterial infection. Significantly, IFN-γ production by NKcells is critical in the prevention of overwhelming infectionby obligate intracellular microbial pathogens in severalexperimental animal models, and monocyte-derived IL-15 iscritical for optimal NK cell production of IFN-γ.120 In murineCMV models of retinitis, NK cells have been shown toprevent infection,121 but it remains to be determined by whicheffector mechanisms NK cells control infection. Wherebacteria-infected cells are lysed directly by NK cells it islikely that perforin is relevant. Nevertheless, LPS and bacte-rial carbohydrates induce bone marrow NK cell cytotoxicityby increasing FasL expression on NK cells,122 and thereforesome bacterial infections may be controlled by Fas-mediatedcytotoxicity.

NK cells and antiparasite function

NK cells are now recognized as major effectors of innateresistance to Toxoplasma gondii, Leishmania major andSchistosoma mansoni. The principal mechanism by whichNK cells control the growth of these pathogens is indirect,involving cytokine production (IFN-γ) rather than cytolyticactivity.101,123 Cytokine production limits parasite replicationand promotes the development of adaptive cell-mediatedimmunity. In the case of Eimeria papillata, resistance to rein-fection does not require IFN-γ and appears to be mediated atleast in part by a perforin-dependent mechanism.124 Othermodels of parasite infection in vivo remain to be evaluated inperforin-deficient or FasL mutant mice. The recent observa-tion that NK cells promote growth of the human filarialparasite Brugia malayi in mice suggests that the interactionof the host immune system with the parasite can be doubleedged.125

NK cells and tumour surveillance

Experiments in perforin gene knockout mice indicate thatperforin is critical for the cytotoxicity of lymphokine acti-vated killer and NK cells.99,111 Both NK1.1+ T cells and CD3–

NK1.1+ NK cells can provide protection from tumour, theformer in an IL-12-dependent manner.126 Natural killer cellsrecognize target cells lacking MHC class I, and syngeneiclymphoid tumours that are MHC class I– are controlled byNK cells in a perforin-dependent manner.127–129 Tumours have

been induced in perforin-deficient, FasL mutant gld, andwild-type mice by (i) injection of different syngeneic tumourcell lines of different tissue origin in naive mice; (ii) admin-istration of the chemical carcinogens (e.g. methylcholan-threne); or (iii) by injection of acutely oncogenic Moloneysarcoma virus. In all models, tumours were eliminated orcontrolled 10–100-fold better by wild-type mice in anunprimed situation by NK1.1+ cells. A role for NK cell FasLin tumour control in vivo has yet to be demonstrated, andindeed seems unlikely based on experiments with Fas-sensitive tumours and FasL mutant mice.129 Thus, perforin-dependent cytotoxicity is the only crucial mechanism of NKcell-dependent resistance to tumour described thus far invivo. Future experiments using targets that have various classI and Fas levels will resolve this issue further.

NK cells and graft rejection

Natural killer cells can mediate acute rejection of bonemarrow cell (BMC) grafts.130 Natural killer cells are able toefficiently reject BMC grafts that lack expression of MHCantigens.131 It is well established that some hybrid F1 micehave the ability to reject parental grafts and that rejection ismediated by radioresistant host NK cells (hybrid resis-tance).132 The discovery of NK cell subsets with non-overlapping inhibitory receptors for parental class I mole-cules has provided an explanation for hybrid resistance.133

Natural killer cells also reject allogeneic and semi-allogeneicBMC grafts. In both cases a role for NK cells and the Ly-49Dactivating receptor is implicated.58 However, as yet the effec-tor function of NK cells responsible for this phenomenon hasnot been described.

Natural killer cells may use both cytokine, (e.g. IFN-γ,TNF-α and IL-12) and cytotoxic (e.g. perforin and Fas-FasL), pathways to reject incompatible BMC grafts,134 sinceall mice bearing deletional mutations of IFN-γ, TNF-RI/II orperforin, or mice treated with mAb to IL-12, or IFN-γrejected BMC, whereas those treated with anti-NK1.1 mAbdid not. Interestingly, perforin-deficient mice maintained in aconventional breeding facility failed to reject class I-deficientBMC,134 thus the breeding colony environment can compro-mise mechanisms that normally compensate for perforin-deficient NK cells. In models of allogeneic BMT, Tcell-depleted allogeneic BM transplanted into perforin-defi-cient, gld, and normal mice revealed that strong allogeneicresistance remains largely intact in perforin-deficient andFas-ligand-defective recipient mice.135 Thus, perforin- andFas-mediated cytotoxic pathways are not required for resis-tance to BM allografts in mice. Thus alternative pathways ofcytotoxicity and/or soluble factors can mediate NK cell resis-tance to allogeneic BM. In situations where NK cell-medi-ated allo-and xeno-lysis have been evaluated, killing appearsto be strictly via perforin and granule exocytosis.136

Other NK cell effector functions in immune responses

Natural killer cells are present in the uterine mucosa ofrodents137,138 and in the human decidua.139 Human decidualNK cells express activation antigens (CD69, class II MHCand CD45R0),7,140 probably responding to cytokinesproduced by neighbouring monocytes. Decidual NK cells


express perforin, granzyme A and TIA-1,141 implying anactive lytic function, consistent with a role for these NK cellsin limiting trophoblast invasion.142 Decidual NK cells expressKIR and CD94/NKG2 receptors143–146 that recognize HLA-Gon trophoblasts,147 suggesting an important role for thesereceptors in regulating NK cell function at the maternal–foetal interface.

It is likely that NK cells contribute to inflammatoryresponses as a consequence of antigen recognition by T cells.Recent studies show that NK cells secrete IL-5, an IL-2-dependent process,148 and that IL-5 production by NK cellscontributes to the allergic response in the lung.149

Amplifying and limiting NK cell responses

Natural killer cell responses can be amplified by NK cell pro-liferation and down-regulated through receptor-mediatedapoptosis. Natural killer cell proliferation for some NKsubsets is effected solely by T cell-derived IL-2 or monocyte-derived IL-15. In most cases NK cell proliferation requirescostimulation through membrane contact with activated T cells, B cells or certain stromal cell lines (review150),implying that interaction with other cells in the micro-environment influences the effectiveness of NK cellresponses. That combinations of monocyte-derived cytokines(IL-15, IL-12 and IL-10) or T cell-derived IL-2 can effect NKcell proliferation suggests that proliferation is an early mono-cyte-dependent response to infectious agents, as well as alater response following generation of antigen-specific T cells. Natural killer cell proliferation is promoted throughCD16,151 and the CD94152 and p50.3153 activating receptors inthe human and through NKR-P1 in rodents,154 and there isevidence that KIR can inhibit proliferation stimulatedthrough the p50.3 receptor.153 Natural killer cell proliferationnot only increases the available number of NK cells but, asshown with human NK cells, also results in the differentia-tion of their effector function, in particular qualitative andquantitative changes in cytokine production.150 Thus prolif-erating NK cells produce up to 50-fold higher levels of IFN-γ, TNF-α and GM-CSF, and acquire the ability toproduce IL-5.148 The particular combination of cytokines thatcostimulate NK cell proliferation dramatically influenceswhich cytokines the proliferating NK cells produce,155 withIL-12 up-regulating IFN-γ secretion, and IL-4 up-regulatingIL-5 secretion.148 Thus there is probably considerable inter-action between NK cells and monocytes, and between NKcells and T cells, that influence the type of immune responseultimately generated against infectious agents.

Natural killer cell interaction with susceptible targetcells,156 activating cytokines157 and the activating receptorsCD16,158 CD94159 and CD2160 also leads to rapid NK celldeath by apoptosis. The mechanism of receptor-stimulatedcell death is controversial and may157,161 or may not159,160,162

involve endogenously produced TNF-α and Fas/FasL.163 Inrodents, activation of NK cells through NKR-P1164 and theLy-49D-activating receptor54 stimulates NK cell apoptosis.

The question of what regulates the balance between NKcell proliferation and NK cell apoptosis stimulated throughthe same activation receptors, is intriguing. It is likely that thesignal requirements for CD16-stimulated NK cell prolifera-tion are more demanding than for apoptosis, because

although cell division is stimulated by CD16 ligation, NKcell growth is not sustained.165 The affinity of NK cell acti-vation receptors for their ligands and the effect of cellularcostimulation is likely to determine the balance betweenapoptosis and proliferation. Of interest are recent studies onthe TCR166 and the mast cell and basophil FcεRI167 showingquantitative and qualitative differences in intracellular acti-vation depending on the affinity of ligands for these recep-tors.

Conclusions

While the basic mechanisms that NK cells use to killinfected, malignant or foreign target cells have become wellcharacterized, some key experiments concerning the impor-tance of each particular mechanism in immune responses invivo remain to be performed. Thus far it would appear thatNK cells strictly use perforin and granule exocytosis in lyticresponses to tumour and virus-infected cells. This distin-guishes their in vivo lytic activity from CTL that more obvi-ously also use TNF superfamily molecules to control virusinfection and tumour growth. However, the relative role ofNK cell cytotoxicity and cytokine secretion in effectorresponses to these and other immune responses still needs tobe clearly delineated. We also know that NK cells are veryheterogeneous and that different subsets may be stimulatedby different innate cytokine networks. A good example is thepotentially different roles of NK cells and NKT cells ininnate antitumour immunity, particularly with respect to thecytokine networks that drive the proliferation, migration andeffector function of these cells. Natural killer cells in differ-ent tissues will encounter and interact with different APC,and to better understand the eventual immune effectorresponse we must first seek to understand the relationshipbetween the stimulus, NK cells and other innate cells (e.g.tissue dendritic cells). Similarly, our current knowledge of thedevelopment, proliferation, maturation and eventual apopto-sis of NK cells is sparse. The derivation of many gene knock-out and transgenic mice has helped us understand the role ofapoptosis in the ontogeny and immunoregulation of T cellsand B cells. Some of these mice will be useful tools toaddress the importance of apoptosis in the shaping of an NKcell repertoire. In addition, now that we have a better grasp ofthe key activation and inhibitory molecules that regulate NK cell effector function, we are poised to use these markersto dissect the signals that drive NK cells to differentiate,proliferate or die.

Acknowledgements

HSW is supported by a National Health and Medical ResearchCouncil of Australia (NH & MRC) Senior Research Fellow-ship, and MJS by a Wellcome Trust Senior Research Fellowship in Medical Science.

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NK cells and apoptosis