CHAPTER 9

Immunological Sculpting:Natural Killer-CellReceptors and Ligands

Sarah S. Donatelli, Julie Y. DjeuH. Lee Moffitt Cancer Center, Tampa, FL USA

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I. INTRODUCTIONThe innate immune system is a crucial component of host defense against both foreignpathogenic invaders and aberrant self cells, such as tissues that have undergone malignant

transformation. Natural Killer (NK) cells are integral members of the innate arm of immunity,

and cancer patients who are deficient in NK cell number or function exhibit a weakenedantitumoral response and ultimately poor prognosis. NK cells are among the first to recognize

and kill virally or bacterially infected and cancerous, or “stressed,” cells.

NK cells differentiate healthy from stressed cells through a vast array of germline-encoded

receptors that confer either activating or inhibitory signals. NK-cell receptors differ from T-cell

receptors in that they are recombinase activating gene (RAG) independent; that is, they do notsomatically rearrange variable diversity or joining (VDJ) gene segments to form endless

combinations of antigen-specific receptors. Rather, NK cells express genes for a finite number

of receptors that recognize self antigens, and these ligands provide the basis of the NK cell’sreaction to the target cell. Distinct NK clones that exhibit varied combinations of both

inhibitory and activating receptors result from the stochastic, or probabilistically determined,

expression of the receptors.

The expressed receptors potentiate either stimulatory “kill” or inhibitory “no kill” signals upon

contact with ligands provided by the target cell. Major histocompatibility class I (MHC I)molecules constitutively expressed by healthy tissues provide ligands for receptors that transmit

inhibitory “no kill” signals to the NK cell. Consequently, a lack of MHC I fails to provide the NK

cell with inhibitory signals and thus indirectly potentiates an activation signal. On the otherhand, stress-induced proteins normally absent on healthy cells emerge as ligands for receptors

that provide the NK cell with an activating “kill” signal. During a given target interaction, the

NK-cell’s receptors will associate with a combination of MHC I molecules and stress-inducedligands. The balance of signals that emanates from these interactions dictates the NK-cell’s

response to the stressed cell. This chapter introduces the concepts of NK-cell receptor acquisition

and signaling, and reviews current knowledge of individual receptors and their ligands.

II. NK EDUCATION, LICENSING, AND PRIMINGNK cells acquire the capacity to recognize and kill stressed cells through distinct receptor-

ligand interactions deemed education and licensing [1]. Before NK cells develop into potent

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effectors that kill damaged cells, they must first become tolerant to healthy tissues through

interaction with and recognition of self MHC I molecules to prevent the occurrence ofautoimmunity [2]. In mice, this education occurs through cis interactions; that is, through the

association of an MHC I molecule with an MHC I-specific receptor expressed on the surface of

the NK cell itself [3]. This interaction requires phosphorylation of the tyrosine in theimmunotyrosine-based inhibitory motif (ITIM) located within the cytoplasmic tail of the

MHC I-binding receptor [4]. NK cells that do not express at least one MHC I-specific receptor

or NK cells that cannot engage at least one MHC I molecule in such cis interactions do notacquire the ability to kill stressed cells and thus remain “unlicensed” [3,4]. Therefore, it is only

after the NK cell is tolerant to self MHC that it can be licensed to kill.

The mechanism by which NK cells are licensed remains incompletely described; however, the

prevailing theory, or the “arming” of NK cells, has been experimentally supported. The basis

for the arming theory is that a positive signal is conferred through the inhibitory ITIM, asablation of the ITIM abrogates NK functionality [1,4]. This postulation seemed counterintu-

itive until recent experiments in mice revealed MHC I-specific receptor variants that exclusively

bind MHC I in cis (on the surface of the NK cell itself) are utilized for licensing. Association ofthese variants with cis MHC I results in sequestration of the inhibitory receptors, allowing for

a physical separation between the activating and inhibitory receptors. The activating receptors

then have the freedom to contact their ligands and transmit the resulting positive signalsrequired for the mobilization of lytic granules. Therefore, such spatial segregation positively

regulates NK-cell cytolysis, suggesting that physical distance from inhibitory molecules may be

required for effective function of activating receptors [3]. This requirement may apply to bothMHC I-specific and non-MHC I-specific activating receptors, as their signaling pathways are

shared and conserved, with the tyrosine phosphatases recruited by ITIM signaling

dephosphorylating kinases crucial early in the lytic cascade. These experiments resolved theseemingly counterintuitive fact that positive regulation can ultimately be conferred by

inhibitory ITIM signaling.

NK-cell licensing can occur in both the bone marrow during development, and in peripheral,

mature NK cells. Importantly, the capacity to kill can be revoked in the absence of MHC I,

suggesting that NK development is a constant and malleable process [5]. The licensing signalvaries with respect to the diversity of the host MHC haplotype, the number of MHC I specific

receptors per NK cell interacting with host MHC, and the affinity of said receptors for their

cognate MHC molecule [1]. Importantly, licensing is a steady-state process involved in NKcell maintenance, and under inflammatory conditions, unlicensed NK cells can kill stressed

cells, as cytokine stimulation overrides MHC I-dependent licensing in both mice [1] and

humans [6].

During steady-state maintenance as well as inflammatory siege, the killing capacity of NK

cells is enhanced by cytokines in the immune milieu. Cytokines particularly important to

NK-cell development and function are interleukin (IL) IL-2, IL-15, IL-12, IL-18, IL-21, andtype I interferons (IFN). Most of these cytokines are produced by other members of the

immune system, such as T cells, activated macrophages, and dendritic cells. IL-2 and IL-15,

produced by T cells and dendritic cells respectively, promote NK survival, proliferation,cytokine production, and cytotoxicity [7e12], likely by increase of activating surface killing

receptors [13]. Similarly, IL-12 and IL-21, sourced from activated macrophages and T cells

respectively, promote proliferation [14,15], cytokine production [16], and cytotoxicity[17,18], while macrophage and dendritic cell-derived IL-18 promotes survival [19]. IL-6

produced by T cells and macrophages increases IFNg and IL-17 production and cytotoxicity[20,21]. Virally induced type I interferons a/b also induce potent cytokine production and

cytotoxicity [22,23]. Therefore, interplay of NK cells with other members of the immune

system, as well as with their putative targets, is essential for a healthy NK-mediated hostdefense.

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III. HOW RECEPTOR: LIGAND INTERACTIONS TRIGGER CELL LYSISA. Cell-to-cell interactions

NK-cell receptors interact with a combination of stimulating and repressive ligands when they

investigate unfamiliar cells, and the balance of signals resulting from these interactions

determines the fate of the target. If the activation signals overcome the inhibitory signals, theNK cell will lyse the target cell. Inhibitory signals emanate primarily from MHC I ligands,

although there are also non-MHC-related inhibitory ligands [24]. All healthy cells (with the

exception of erythrocytes and platelets) express MHC I and thus, will only engage the MHCI-specific inhibitory receptor present on all functionally competent NK cells, signaling the NK

cell to spare the cell. Stressed cells, on the other hand, often downregulate MHC I, thus

preventing transduction of the inhibitory signal, allowing for triggering of NK-cell activation.The process of NK-mediated rejection of MHC I-deficient targets is known as “missing self.” In

addition to MHC I dysregulation, stressed cells can also display non-MHC I-related activatingligands, such as stress proteins induced by viral infections or malignant transformation, that

will trigger NK-cell-mediated cytolysis through their activating receptors. The specific receptors

and their ligands are discussed in detail in the following sections. There is evidence thata stronger signal (determined by either the strength or quantity of the receptor-ligand

interactions) is required for cytotoxicity of the target, whereas a weaker signal may only elicit

inflammatory cytokines such as IFNg that will recruit members of the adaptive immuneresponse for a closer investigation of the putative invader. As mentioned earlier, in mice, MHC

I-specific receptors can associate with MHC I molecules in cis, that is, MHC I molecules

expressed on the NK cell’s own plasma membrane. These cis interactions enhanceNK-mediated cytolytic activity of target cells by reducing the necessity for trans binding, or

NK-expressed MHC I-specific inhibitory receptor binding to target cell MHC I, and lowering

the threshold for activation through triggering receptors [25,26]. This caveat suggests that theNK-cell-intrinsic MHC repertoire can contribute to determination of the signal strength

required to kill the target cell, and NK cells diverse in MHC alleles may be more innately

“active” than NK cells that do not exhibit MHC diversity.

B. Signaling

When an NK cell encounters a potential target, intracellular signals reverberate via the surface

receptors. The major stimulatory or inhibitory signaling pathways are conserved among

respective receptors (Figure 9.1). Activating receptors have short cytoplasmic tails that lack anycapacity for signaling. Therefore, activating receptors generally associate with

immunotyrosine-based activation motif (ITAM)-containing adaptor molecules that can be

phosphorylated to activate downstream signals that end in lytic granule and/or cytokinesecretion. ITAM-containing adaptor proteins associate with the short cytoplasmic tails by

binding to charged transmembrane amino acids.

The most frequently used adaptor molecules are dynax activating protein 12kD (DAP12),

CD3z, FcRg, and DAP10. DAP12 is associated with NK activating receptors through charged

residues in the transmembrane region of the receptor’s cytoplasmic tail, and upon receptor-ligand interaction, Src family kinases such as lymphocyte-specific protein tyrosine kinase (Lck)

and Fyn phosphorylate the tyrosine in the adaptor’s ITAM. Spleen tyrosine kinase (Syk) [27] or

zeta-chain-associated protein kinase 70 (Zap70)[28] is recruited by binding to thephosphotyrosine in the ITAM and activating phosphoinositide-3 kinase (PI3K) that leads to

sequential Rac1, PAK1, and MAPK/ERK activation to mobilize lytic granules that kill the target

cell [29,30]. Recently, protein kinase C-Ө (PKC-Ө) has emerged as a requirement for IFNg

release, but not cytolysis, during sustained ITAM signaling [31]. Other adaptors, such as CD3z

and FcRg, function in the same manner as DAP12, as they contain the same ITAM (Figure 9.1).

DAP10, on the other hand, does not express an ITAM but contains a PI3K-binding domain.The Fyn/Lck phosphorylation of this tyrosine-containing domain recruits PI3K which results

FIGURE 9.1Conserved signaling pathways of NK receptors.Activating receptor ligation leads to association of

ITAM-containing adaptor proteins and subsequent entry

into the PI3K signaling pathway, ultimately leading to IFNgand lytic granule secretion. Inhibitory receptor ligation

leads to activating/inhibitory receptor colocalization, ITIM

phosphorylation and subsequent recruitment of SHP-1 and

SHP-2 to the SH2 domain, which dephosphorylates critical

signaling proteins at the activating receptor/adaptor

protein complex. Activating receptors are depicted in

green, while inhibitory receptors are depicted as red.

Signaling proteins are blue. Green arrows depict

sequential activation by phosphorylation of the previous

signaling protein.

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in mobilization of granules via the same pathway as DAP12 (Figure 9.1)[32]. In addition,

antibody-dependent cell cytotoxicity by NK cells can occur via this pathway through FcgRIII(CD16), which couples to CD3z, or FcεRg. Overall, a multitude of ligands can trigger NK cells

and the initial signaling molecules activated may vary depending on the receptor, but they

merge downstream into a conserved common cascade ending in ERK activation andsubsequent lytic granule mobilization.

In contrast, inhibitory receptors have long cytoplasmic tails that contain one or moreimmunotyrosine inhibitory motifs (ITIM). Upon receptor-ligand interaction, these ITIMs are

phosphorylated by Lck and Fyn (Figure 9.1). Src homology-2 domain containing phosphatase

1 and 2 (SHP-1 and SHP-2) then bind to the phosphotyrosine in the ITIM through their SH2domains [33,34]. SHP-1 and SHP-2 then function to disrupt the lytic cascade by specific

dephosphorylation of Syk and Zap70, crucial early signaling proteins necessary for NK

activation tyrosines [35].

IV. RECEPTORS FOR MHC I AND MHC I-RELATED MOLECULESA. Killer immunoglobulin-like receptors (KIR)

MHC Imolecules provide the most robust and abundant signals to NK cells. It is throughMHC

molecules that NK cells can sample the health of the environment. These interactions are

crucial to NK function, providing both inhibitory and activating signals. The largest family ofmolecules expressed in human NK cells that recognize MHC I on target cells are the killer

immunoglobulin-like receptors (KIR) and can be inherited as two haplotypes, A and B

(Figure 9.2). All KIR contain either two (KIR2D) or three (KIR3D) extracellular immuno-globulin (Ig)-like domains and may exhibit either short (KIR2/3DS) or ITIM-containing long

(KIR2/3DL) cytoplasmic tails that play activating or inhibitory roles in NK cell target signaling,

respectively (Figure 9.3). For simplicity, in this review, all stimulatory KIR gene products arereferred to as “aKIR,” and all inhibitory KIR gene products are referred to as “iKIR.”

As stated earlier, aKIR provide stimulatory signals to NK cells when they contact their targetligands, causing lytic granule mobilization that induces target cell lysis or cytokine secretion to

FIGURE 9.2KIR haplotypes. Schematic depiction of the KIR genotypesof haplotypes A and B. The A haplotype is fixed in gene

content, while the B haplotype varies extensively in gene

content. The boxes on and below the line are KIRs all

variably expressed in a given B haplotype. Framework

genes present on all haplotypes are outlined in bold black

line, activating KIR are depicted in green and inhibitory KIR

in red. The numbers below the series of boxes in haploype

B represent the various KIR combinations that can be

expressed in a given B haplotype.

FIGURE 9.3NK-cell surface receptors. Receptors contributingto NK activation are green and grouped with a solid

green line, while receptors contributing to NK

inhibition are red and grouped with a solid red line.

Activating co-receptors are grouped with a dashed,

color-coded line. ITIMs are depicted as yellow

slashes in the red cytoplasmic tails. Abbreviated

receptor names are displayed proximal to the

receptor depiction.

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amplify the immune response. They contain no intrinsic signaling capacity in their shortcytoplasmic tails, but they do contain a charged lysine or arginine that facilitates association

with DAP12. Six aKIR that contain two extracellular domains exhibiting different MHC I allelic

preferences have been identified. Like their homologous inhibitory relatives, KIR2DS1,KIR2DS2, and KIR2DS3 bind human leukocyte antigen (HLA)-C, MHC I, alleles. This caveat

implies that when an NK cell sees a target expressing HLA-C, it can receive both inhibitory and

activating signals from the same ligand and adds to the complexity of the ensuing reaction.Furthermore, it suggests that the surface ratio of activating-to-inhibitory KIR may be a deter-

mining factor in the fate of the target cell. KIR2DS4 is exceptional in that it is the only KIR

expressed in Haplotype A individuals. Furthermore, proteins other than MHC molecules havebeen identified as ligands. In addition to HLA-C1, -C2, and -A11 [36], KIR2DS4 is triggered

by unidentified melanoma proteins [37], and unknown proteins expressed by chronic

lymphocytic leukemia (CLL) cells [38], suggesting that this aKIR may play a distinct role inantitumoral defense. Ligands for KIR2DS5 and KIR2DS3 remain unidentified.

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In contrast to aKIR, iKIR provide negative intracellular signals by recruiting SHP-1 and SHP-2to the ITIMs in their long cytoplasmic tails upon ligand binding. Eight iKIR that exhibit

different MHC allelic preferences have been identified. KIR2DL1, KIR2DL2, and KIR2DL3

bind HLA-C alleles [39], KIR3DL1 binds HLA-B alleles [40,41], and KIR3DL2 binds HLA-A3and A11 [42]. The ligand for KIR2DL5 remains unidentified. KIR2DL4 is distinct in that it is

expressed in all humans, and it has been experimentally shown to exhibit both activating andinhibitory signals. Furthermore, KIR2DL4 recognizes soluble HLA-G, which is expressed in the

placenta, where classical HLAmolecules are absent, and functions during pregnancy to prevent

spontaneous abortion of the fetus.

It has been documented that KIR expression is controlled epigenetically by DNA methylation

of cytosines in CpG dinucleotides rather than by genetic sequence differences in promoters

[43]. These DNA methylation patterns are maintained over numerous cell divisions, andcontribute to the diversity of gene expression in individual NK clones and their progeny [44].

KIRs are expressed stochastically leading to a repertoire of NK clones that express different

combinations of activating and inhibitory KIRs. Genotyping and epidemiological studies haveyielded two predominant patterns of KIR expression (Figure 9.2). Haplotype A individuals

exhibit a relatively fixed gene content consisting of five iKIRs along with one aKIR, KIR2DS4,

and KIR2DL4, which can exhibit both inhibitory and activating functions. Conversely,Haplotype B individuals express variable gene content consisting of multiple aKIR and iKIR

combinations [45]. The frequencies of these haplotypes vary significantly with ethnicity;

furthermore, correlations between KIR haplotypes and susceptibility to certain cancers andinfections are beginning to emerge.

B. Natural cytotoxicity receptors

Apart from MHC-dependent interactions, NK cells rely on interactions with non-MHC-related

proteins to distinguish stressed from healthy cells. The presence of non-MHC-related ligandswas first noted when NK cells readily lysedMHC I-deficient tumor cells. We now know that this

feature is a function due both to a lack of MHC I-mediated inhibition (“missing self”) as well

as engagement of activating receptors that recognize non-MHC I molecules. The receptorsdiscovered to play a large role in this MHC-independent target lysis were termed the natural

cytotoxicity receptors (NCR) (Figure 9.3). Deletion of even one NCR reduces the ability of NK

cells to kill tumor targets in vivo [46]; this weakness is important because tumor-producedtransforming growth factor b-1 (TGFb) downregulates NCR family members to evade NK

cytolysis [47]. Additionally, NCRs can potentiate interactions with antigen-presenting cells,

such as dendritic cells, that can aid in cellular maturation for recruitment of the adaptive armof immunity [48].

The NCR consist of NKp30, NKp44, NKp46, and NKp80 [49e51] and are named for their

respective molecular weights. NKp30, NKp46, and Nkp80 are expressed on activated andresting NK cells while NKp44 is upregulated by IL-2. NKp46 ligands include hemagglutinin

and hemagglutinin-neuraminidase of influenza and parainfluenza [52], as well as heparin

sulfate proteoglycans, which also bind NKp30 [53]. Other ligands for NKp30 are nuclear factorHLA-B-associated transcript 3, a factor released from tumor cells [54], B7-H6 [55], CMV pp65,

and BAT3 [56].

Of the NCRs, only NKp44 associates with and signals through DAP12 (Figure 9.1)[49]. NKp30

and NK46 couple to CD3z and FcεRg adaptor molecules, which form heterotrimeric adaptor

complexes [50,51]. NKp80 is an activating receptor that binds to the genetically linkedactivation-induced C-type lectin (AICL) and stimulates NK cytotoxicity against malignant

myeloid cells [57]. NKp80 does not require an adaptor protein; rather, it signals through

a hemi-ITAM in its cytoplasmic tail through recruitment of Syk [58] and likely progressionthrough the PI3K activation pathway.

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C. CD94/NKG2 and NKG2D

The NKG2 family is similar to KIR in that it contains both inhibitory and activating receptors;

however, either MHC I or non-MHC I molecules can function as ligands. Furthermore, somefamily members, NKG2A, NKG2C, and NKG2E, heterodimerize with CD94, while NKG2D

associates with itself to form a homodimer (Figure 9.3).

NKG2D is distantly related to the other NKG2 family members and does not dimerize withCD94, but rather forms homodimers that associate with DAP10 to yield an activation signal. In

humans, NKG2D complexes bind stress-inducible MHC class I chain-related gene A and B

(MICA/ MICB), whose steady-state distribution is restricted to intestinal epithelia, but isupregulated in many epithelial tumors [59,60]. Additional NKG2D ligands are UL16-binding

proteins (ULBP) 1/2/3/4 which are induced by cytomegalovirus infection [61e63] and in

mice, NKG2D recognizes the retinoic acid-inducible early gene-1 (Rae-1)-like proteins [62]and H-60, a minor MHC derived antigen. NKG2D mediates direct tumor target killing as well

as induction of antitumoral T-cell responses [64], and is therefore a crucial component of the

NK defense. However, tumors have demonstrated evasion of NKG2D-mediated killing bysecretion of TGFb, which downregulates NKG2D on NK cells [65], and NKG2D ligands on

tumor cells [66]. Additionally, tumors shed soluble MICA/B, which functions as a decoy

receptor [67].

The other three NKG2 family members, NKG2A, NKG2C, and NKG2E, form heterodimeric

complexes with CD94, another lectin-like protein. These complexes recognize the nonclassicalMHCmolecule, HLA-E. HLA-E is deemed nonclassical in part because it presents only peptides

derived from classical MHC I molecules. This feature allows NK cells to indirectly explore the

MHC I environment of the putative target [68]. NKG2A contains an ITIM; thus upon bindingto HLA-E it elicits an inhibitory signal. Interestingly, NKG2C/E-CD94 also binds HLA-E, yet it

does not contain an ITIM. Rather, it associates with DAP12, transmitting an activation signal.

D. CD160 (BY55)

CD160 is an activating receptor expressed in the CD56dimCD16þ cytotoxic NK subset that

constitutes the major NK fraction in circulating human NK cells (Figure 9.3)[69,70].Engagement of CD160 recruits PI3K to activate the lytic cascade [71]. Both

glycosylphosphatidylinositol (GPI)-anchored and transmembrane isoforms are found on NK

cells. The major GPI-anchored form exhibits homology to KIR2DL4; however, its signalingconstituents have not yet been elucidated. On the other hand, the transmembrane isoform,

which is transiently expressed on cytokine-activated NK cells, contains a transmembrane

region that activates Erk1/2 [72]. CD160 is a promiscuous receptor that binds a variety ofligands including MHC class 1a and 1b molecules HLA-C [69], soluble HLA-G [73], HLA-A2,

HLA-B7, and HLA-E [69], as well as the herpesvirus entry mediator protein [74].

E. SLAM family receptors

In vivo, NK cells not only contact tissue cells, they also contact other NK cells. To prevent

unmitigated self killing, NK express signaling lymphocyte activation molecule (SLAM)-relatedreceptors. SLAM-family receptors expressed on human NK cells are NTB-A and CRACC, as well

as 2B4 (CD244) (Figure 9.3). NTB-A and CRACC serve as self-ligands during homotypic

interactions between NK cells, while the 2B4 ligand is CD48, a glycophosphatidylinositol(GPI)-linked member of the CD2 family of receptors expressed on hematopoietic cells [75].

Engagement of 2B4, NTB-A and CRACC onMHC I-negative targets (i.e., stressed cells) activates

NK-cell cytotoxicity, while 2B4, NTB-A, or CRACC-expressing, MHC I-positive cells(i.e., neighboring NK cells) are spared [76].

Signaling of SLAM-family members is distinct from that of other NK-cell receptors as they donot recruit traditional ITAM-containing adaptor proteins such as DAP10 or DAP12. Rather, the

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cytoplasmic domains contain tyrosine-based motifs that undergo phosphorylation to recruitSH2-domain containing SLAM-associated protein (SAP) or Ewing’s sarcoma-activated

transcripts-2 (EAT-2). In mice, the SLAM-family receptors can exhibit either activating or

inhibitory functions, depending on the adaptor that they recruit; however, in humans,SLAM-family receptors have only been shown to be stimulatory [77].

F. Additional NK receptors

In addition to directly lysing target cells, NK also work in concert with B lymphocytes to

mediate antibody-dependent cellular cytotoxicity (ADCC) [78]. NK express CD16 which binds

IgG-opsonized cells (Figure 9.3). The activating CD16 then associates with ITAM-containingCD3z and FCRg to bind Syk and proceed through the NK activation pathway to ultimately lyse

the target cell [79].

NK cells also express an array of receptors that promote the formation of NK-celletarget cellconjugates and therefore act as coreceptors for the lytic granule inducing receptors. Some of

these receptors have signaling capacity that promotes activation and cytotoxicity in addition to

enhancing adhesion. Others primarily function to form andmaintain the contacts between theNK cell and the target cell. Typically, these receptors recognize adhesion molecules present on

the target cells. The primary adhesion receptor that is required for stable conjugate formation is

leukocyte function associated antigen 1 (LFA-1) (Figure 9.3)[80], an integrin that binds tointercellular adhesion molecule 1 (ICAM-1). The binding of LFA-1 to ICAM-1 results in actin

polymerization for enhanced adhesion to the target cell [81]. LFA-1 has been demonstrated as

a co-activator during NCR-mediated stromal cell killing and NKG2D-mediated killing ofdendritic cells [82]. Another important activating receptor is DNAX accessory molecule-1

(DNAM-1), which also potentiates adhesion and cytotoxicity (Figure 9.3) [31,83] and mayhave a role in NK-cell migration [84]. The ligands for DNAM-1 in humans are CD155 (polio

virus receptor (PVR) or Nectin-5), and CD112 (Nectin-2), both of which are upregulated on

tumor cells [85], suggesting a role for the receptor in tumor cell lysis [86]. DNAM-1 signaling isdependent upon Fyn [83], and downstream signaling is dependent upon association with

LFA-1 [87]. Two additional receptors that recognize Nectins are class I-restricted T-cell-

associated molecule (CRTAM) which binds Necl-2 and enhances NK activation, and CD96(Tactile), which recognizes Necl-5 and primarily promotes adhesion, but not activation

(Figure 9.3). Another molecule that promotes NK-celletarget cell interactions is CD100 (or

Semaphorin 4D (SEMA4D)) (Figure 9.3), which potentiates enhanced cytotoxicity and cyto-kine production upon CD72 ligation by enhancing the adhesion between NK cells and their

targets [88]. CD72 is primarily expressed on B cells [89], implicating this interaction in

NK-mediated killing of stressed B cells.

In addition to receptors that bind adhesion molecules on target cells and thus promote

NK-target cell cytotoxicity, NK cells also exhibit receptors that recognize adhesion molecules,

yet are inhibitory. KLRG1 is an ITIM-containing inhibitory receptor that recognizes the clas-sical cadherins, E-, N-, and R-cadherin (Figure 9.3)[90]. Reduced levels of E-cadherin in

malignant epithelial tumors correlate with tumor metastasis, suggesting a potential role for NK

detection of malignant epithelia [91]. LAIR-1 recognizes a common collagen motif andcontains a single extracellular Ig-like domain and two cytoplasmic tyrosine-based inhibitory

motifs (ITIMs) (Figure 9.3)[92]. Cross-linking of LAIR-1 on NK cells delivers a potent inhib-

itory signal that is capable of inhibiting target cell lysis mediated by resting and activated NKcells [92,93] and B cells [94].

Other receptors can help to differentiate subsets of NK cells. While some have knownsignaling functions, others have only been identified as developmental markers. A major

marker for NK subsets is CD56 (Figure 9.3); CD56lo cells constitute the majority of circu-

lating NK cells and exhibit high cytotoxicity capacity to target cells. Conversely, CD56hi cellsconstitute approximately 10% of circulating NK cells and produce IFNg but have low

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cytolytic capacity. In addition, CD27 is expressed on the CD56hi subset of NK and bindsCD70 (Figure 9.3)[95], and this interaction promotes NK-mediated cytotoxicity and primes

CD8þ T-cell responses [96]. Moreover, NK-cell differentiation is accompanied by expression

of a post-translation modification of P-selectin glycoprotein ligand-1 (PSGL-1), deemed thePEN5 epitope, that allows for binding to the integrin L-selectin, suggesting that PEN5/PSGL-1

potentially serves as a NK-homing/trafficking receptor [97]. Of note, NK cells also expressnon-KIR MHC I-specific inhibitory receptors. For example, Lilr1b (ILT2/CD85j /LIR-1),

which is a potent MHC I-binding inhibitory receptor, possesses four ITIMs in its cytoplasmic

tail (Figure 9.3)[98].

Aberrantly expressed glycoproteins and glycolipids have recently been characterized as tumor

markers and can be engaged by sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs)

which are broadly expressed on hematopoietic cells. Of the receptors, Siglec 3 (CD33) isexpressed on activated NK cells and contains two ITIMs that negatively regulate NK-cell

activation (Figure 9.3)[99,100]. Siglec-7 (p70/AIRM) and Siglec-9 are CD33-related Siglecs

that are also expressed on NK cells (Figure 9.3) [101] and have been implicated in clinicalpathogenesis. Decreased Siglec-7 expression is an early marker of NK dysfunction and high

viral load in HIV infections [102]. Furthermore, Siglec-7 expressed on NK preferentially binds

internally branched a2,6-linked disialic gangliosides such as disialosyl globopentaosylcer-amide (DSGb5), a ganglioside upregulated on renal cell carcinoma that contributes to

metastases [103]. Siglec-9’s ligand is MUC-16 [104], which is overexpressed in cancer [105] and

functions to inhibit NK-cell-mediated antitumor responses [106] by acting as an anti-adhesivemolecule and preventing the formation of the immunological synapse between target cells

and NK cells [107].

V. CONCLUSIONNK cells are a crucial part of the first line immune response, particularly in the context of

cancer. In contrast to T cells, which are negatively selected and clonally deleted in the thymusif they exhibit self-reactivity, NK cells are genetically primed to tolerate self MHC and yet

recognize and kill malignant tissue through detection of aberrantly expressed self-proteins.Through an array of receptors, NK cells can differentiate stressed cancerous cells from healthy

tissue, and the balance of inhibitory and activating signals that result from the interactions

determines the fate of the putative malignancy (Figure 9.3). Unfortunately, tumors can evadeNK-cell-mediated destruction by secretion of immunosuppressive molecules and/or shedding

soluble variants of activating receptor ligands. Therefore, in the tumor microenvironment,

NK-cell function is compromised. Identifying mechanisms that maintain the expression ofstimulatory receptors may provide therapeutic benefit by shifting the intratumoral NK cell

towards an aggressive and active phenotype that is ready to detect and destroy invading

malignancies.

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