Biologia Molecular e Imunologia 03

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Basic Science for Clinicians

Molecular Biology and Immunologyfor Clinicians 22Natural Killer Cell Receptors and ActivationMechanismsLeonard H. Sigal

Natural killer (NK) cells (called “third population cells” many years ago because they did not bear surface markers of the first two defined popu-lations, B cells and T cells) are now known to occupy a pivotal position inthe immune system, straddling the “divide” between the innate and adap-tive responses. Natural killer cells are capable of production of manycytokines, both pro- and anti-inflammatory, and induction of target celldeath by lysis and/or programmed cell death (apoptosis). Some of thesecytokines are pivotal in the autoimmune and antipathogenic immuneresponses, implicating NK cells in the pathogenesis of many human dis-eases. Multiple detection systems allow tight control of the potent effectorsystems that mediate NK cells’ effects. Recent studies have shown that NKcell function is under tight control, with complex inhibitory and activating

signaling assuring that these cells can accurately detect intracellular infec-tion and malignant degeneration without damaging healthy cells. Al-though NK cell receptors do not have antigenic specificity, they do detectcertain patterns on the surface of target cells. Their ability to make manycytokines that alter antigen-specific immune responses mediated by othercells puts NK cells in a unique position to influence both innate andadaptive responses. (J Clin Rheumatol 2003;9:55–59)

Key words: Natural killer cells, Target cells, Immune system

Natural killer (NK) cells arehighly proficient cytolyticcells and prodigious

sources of many cytokines. Naturalkiller cells are a crucial part of theinnate immune system, with onefoot crossing the threshold into theadaptive immune response. Theyare bone marrow derived and ap-pear early in the developing hu-man immune system. They firstpresent by 6 to 8 weeks of gesta-

tion, appearing before organizationof the thymus is accomplished.Natural killer cells constitute 5% to20% of all blood lymphocytes andabout 5% of splenic lymphocytes

but are uncommon in uninfectedlymphoid tissues. Their killing ac-tivity can be increased 100- to 1000-fold by cytokines like interferon al-pha and beta and interleukin (IL)-12; these and other cytokines areproduced early in the innate re-

sponse to infection, assuring en-hanced NK-cell activity within 1 to2 days of exposure, long before anantigen-specific immune responsecan be generated to a pathogen.Natural killer cells are crucial indefense against many intracellularpathogens (e.g., herpes viruses, Lis-teria monocytogenes, Leishmania). Inaddition to killing tumor cells andcells containing intracellular patho-gens, there is now reason to believe

NK cells may be able to influenceautoimmune disease. By the natureof cytokines produced, NK cellstake part in the regulation of T-cell– dependent antibody produc-tion in both health and autoim-mune disease. Thus, decreases inNK-cell activity seen in lupus,Crohn’s disease, and rheumatoidarthritis may mediate not only theautoimmune disease but also theincreased risk these patients haveof microbial infection. Thus, ma-nipulation of NK-cell activity may

Division of Rheumatology and Connective TissueResearch, Department of Medicine, Department ofPediatrics, and Department of Molecular Genetics& Microbiology, University of Medicine and Den-tistry of New Jersey-Robert Wood Johnson Medi-cal School, New Brunswick, New Jersey.Address correspondence to: Leonard H. Sigal,MD, 1 Robert Wood Johnson Place—MEB 484,New Brunswick, NJ 08903-0019 E-mail: [email protected] © 2003 by Lippincott Williams &Wilkins, Inc.

Sigal • Natural Killer Cell Receptors 55



be important in the pathogenesisand control of autoimmune, infec-tious, and neoplastic disease.

Natural killer cells do not bearthe surface markers of T cells (likeCD3, CD4, or CD8 or the T-cell an-tigen receptor) or B cells (mem-

brane-bound immunoglobulin). In-

stead, NK cells usually bear CD56(an isoform of human neural celladhesion molecule, whose functionin NK cells is unclear) and CD16(the low-affinity Fc gamma-III re-ceptor, which best binds IgG1 andIgG3) (Table 1). Natural killer cellshave at least four different familiesof NK-cell receptors, although noNK-cell receptor has the heteroge-neity of the B- or T-cell surface an-tigen receptors we have explored

in earlier articles (Table 2).The ligands for some of thesereceptors have been identified. Killercell immunoglobulin-like receptors(KIRs) recognize major histocompat-ibility complex (MHC) class I humanleukocyte antigen (HLA)-A, -B,and -C markers (see Table 2). TheCD94/NKG2C disulfide-linked het-erodimer binds to the nonclassicMHC class I molecule HLA-E, whichreaches the cell surface by hitching a

ride with classic class I molecules.Somatic cells infected with certainviruses or after malignant degenera-

tion have decreased expression ofHLA-A, -B, and -C class I molecules(this may be a strategy to decreasethe antigen-presenting activity of theinfected cell; were infected cells toexpress normal amounts of class I,they could present pathogen/can-cer-specific antigens and elicit a

CD8 T-cell response that woulddestroy the infected cell). Naturalkiller cells use HLA-E as a surrogatemarker for class I expression andthereby monitor the internal statusof their potential target. Once as-sured that the cell is fine, the NK cellcurbs its murderous potential andgoes on about its patrolling.

NKG2D is another C-type lec-tin (also expressed on gamma/delta T cells and some CD8 al-

pha/beta T cells) that binds to cellsurface MHC class I chain–relatedA (MICA) and B (MICB) molecules.MICA and MICB are distantly re-lated to MHC class I alpha chains(MICA and MICB consist of twoIgSF-related domains but do notassociate with beta2 microglobu-lin). MICA and MICB expression isminimal on normal cells but in-creases with cellular stress or ma-lignant degeneration. This is prob-

ably because the promoter thatdrives the MICA and MICB genesis similar to the promoter of the

heat shock protein 70 gene so thatintracellular stresses drive bothpromoters. Mice do not expressmolecules similar to MICA andMICB, but murine NK2GD binds toa series of proteins induced by reti-noic acid, known as retinoic acidearly proteins (Raes) and to minor

histocompatibility antigen (H60).Both Raes and H60 are upregulatedin tumors; expression of these mol-ecules makes tumors more vulner-able to NK-cell–mediated cytolysisand to rechallenge with tumorcells, where killing is mediated byCD8 alpha/beta T cells, on whichNKG2D acts as a coreceptor, pro-viding a costimulatory signal thatenhances T-cell efficacy. This issimilar to the costimulatory signal

CD28 delivers by interacting withCD80 and CD86 in T-cell activa-tion. On activation of T cells, anintracellular area of CD28 binds tothe p85 subunit of phosphatidyl-inositol 3 kinase (PI3K), starting acascade of intracellular effects.Continuing the parallelism, an in-tracellular segment of NKG2D alsoengages and activates PI3K.

NKG2D is also expressed byintraepithelial lymphocytes in the

skin and gut, most of which aregamma/delta T cells and some ofwhich are CD8 alpha/beta cells.

TABLE 1. Types of natural killer cells

CD56dim and CD16bright CD56bright and CD16dim

90% of all NK cells* 10% of all NK cells*

Low cytokine production† High cytokine production†

High natural cytotoxicity/ADCC Low natural cytotoxicity/ADCC

High ADCC Low ADCCLymphocyte activated killer (LAK) cell Lymphocyte activated killer (LAK) cell

High KIR and ILT expression Low KIR and ILT expression

Low IL-1R and IL-18R expression High IL-1R and IL-18R expression

Low CD94/NKG2A High CD94/NKG2A

CXCR1, CX3CR1 CCR7, CXCR3 and I-TAC

Low affinity IL-2R (heterodimer) (beta/gamma) High affinity IL-2R (hetero-trimer) (alpha/beta/gamma)

*50 –70% of these cells may actually have non-detectable levels of CD16.†Cytokines include interferongamma, TNFalpha, lymphotoxin (TNF beta), GM-CSF, IL-3, 5, 8, 10, and 13 and thechemokines MIP 1alpha, MIP 1beta and lymphotactin.

56 Journal of Clinical Rheumatology • Volume 9, Number 1 • February 2003



NKG2D may be able to detect pre-cancerous epithelial changes in the

gut and skin, although the sensingcells are T cells rather than NKcells. In human beings, MICA andMICB are expressed almost solelyon proliferating cells, not solely inthe gut but also in endothelial cellsand on fibroblasts as well as intransformed cells. Another set ofligands for NKG2D is represented

by the UL16 binding proteins dis-tantly related to class I MHC mol-ecules. UL16 is a protein expressed

by human cytomegalovirus; theclinical relevance of this is not yetclear.

As noted, NK cells, especiallythe CD56 bright subset, are potentproducers of certain cytokines (Ta-

ble 1). Natural killer cells stimu-lated by the combination of IL-12and tumor necrosis factor-alphaproduce large amounts of interfer-on-gamma early in the immune re-sponse; this cytokine may be piv-otal in controlling certain infections

before the elaboration of antigen-specific T cells. Natural killer cellsconstitutively express the interme-diate affinity receptor for IL-2 (aheterodimer consisting of the betaand gamma chains); CD56 bright

cells are probably the only lympho-cyte population also to express thehigh-affinity heterotrimer (a com-plex of the alpha, beta, and gammachains) constitutively. This is prob-ably why NK cells proliferate so

readily even to low levels of IL-2.Natural killer cells also bear other

surface receptors, including IL-1,IL-10, IL-12, IL-15, and IL-18; theCD56 bright population expressesmore IL-1R and IL-18R. There isdifferential expression of chemo-kine receptors; CD56 bright cells ex-press high levels of CCR7 (a T-cellhoming receptor) and CXCR3 (re-ceptor for the interferon-inducibleproteins such as interferon-gammainducible protein-10 and interfer-on-inducible T-cell alpha chemoat-

tractant), whereas CD56

dim

cellslack CCR7 but have high levelsof CXCR1 (ligand is IL-8) andCX3CR1 (bound by fractalkine).

As their name implies, NKcells are potent killers of infectedand malignant cells. Once their re-ceptors bind to activating ligands,NK cells activate their killing ca-pacity by release of preformedpackaged molecules known as per-forins and granzymes found in cy-toplasmic granules. The former po-lymerize to form a pore in thetarget cell membrane, leading tocell lysis. Does this strategy (i.e.,the complement “membrane attackcomplex”) sound familiar? Humanand murine perforin are 30% ho-mologous with complement com-ponent C9. Perforin is also part ofthe armamentarium of cytolyticCD8 T cells.

There are at least three gran-zymes, which are serine proteases

related to the digestive enzymestrypsin and chymotrypsin—yet an-

other example of how disparate ef-fector functions use molecules di-verging from a common sourceobscured by the mists of evolution.Granzymes have been implicatedin inducing apoptosis in the targetcell. Granzyme B cleaves an en-zyme known as cysteine proteaseprotein (CPP-32) or caspase 3,which is an early caspase in theapoptotic cascade. ActivatedCPP-32 in turn activates a nuclease

caspase-activated deoxyribonucle-ase (CAD) by the degradation ofthe inhibitory protein referred to asthe inhibitor of CAD. As a bonus,some of the nucleases involved inthe destruction of the mammaliantarget cell also degrade viral nu-cleic acids. Once apoptosis occurs,the dead cell is recognized byphagocytic cells, probably becauseof the expression by the dying cellof phosphatidylserine on its sur-face, which is normally found onlyon the inner aspect of the cell’slipid bilayer membrane. The ad-vantage of apoptosis with subse-quent phagocytosis is that withoutnecrosis, there is no liberation oftoxic chemicals, cytokines, and che-motactic factors and no influx ofinflammatory cells and hyperemia(i.e., no inflammation to disrupt lo-cal tissue).

Balancing forces, yin and yang,are a repeated motif in biology in

TABLE 2. Families of natural killer cells

Name Acronym/example Found on Designation Ligand

KIR killer cell immunoglobulin-like receptors

NK-cells, some T-cells CD158a-k & CD158z Class I-AB & C

NCR natural cytotoxicity receptors

ILT (LIR) immunoglobulin-liketranscripts (leukocyteimmunoglobulin-likereceptor)

Monocytes, MacrophagesDendritic cells, B-cells, NK-cells

CD85a-m

C-type lectin CD94/NKG2A heterodimerNKG2D

Gamma/delta T-cells, alpha/beta T-cells, intra-epitheliallymphocytes

HLA-E




general and in immunology specif-ically. Natural killer cell cytolyticactivity must be tightly controlled.There are NK-cell surface receptorsthat result in inhibition of cytolyticactivity and receptors that activatethe cell; the former usually have a

higher affinity for their ligand thando the latter, assuring a net inhibi-tory “tone.”

Intracellular control systemsare found in receptor molecules orassociated “adaptor” molecules.The inhibitory receptors have animmunoreceptor tyrosine-based in-hibitory motif (ITIM) or a basicamino acid residue (lysine or argi-nine) within their transmembraneregion that allows the receptor to

associate with an adaptor (contain-ing a positively charged aminoacid in its transmembrane region)that delivers the suppressive sig-nal. On activation of the receptor,the ITIM region recruits a tyrosinephosphatase, either SH1 or SH2domain– bearing tyrosine phospha-tase, which in turn inhibits the NKcell. For inhibitory receptors thatdo not contain an intracellularITIM, an adaptor protein that doescontain an ITIM associates with thereceptor molecule and the adaptormolecule. ITIMs are also found inB-cell and T-cell surface molecules(CD22 and Fc-gamma R IIB-1 in Bcells and CTLA-4 in T cells).

On the other side of the equa-tion is activation, mediated by animmunoreceptor tyrosine-basedactivation motif (ITAM). These arethe receptors that prompt cytolyticactivity and the release of cyto-kines. Both B- and T-cell antigen

receptors contain multiple subunitswith the receptor and effector typ-ically on separate molecules: the B-cell antigen receptor is linked toIg-alpha and beta (CD79a and b,respectively), and the T-cell anti-gen receptor is linked to alpha,

beta, gamma, delta, and epsilon as

well as zeta chains of its CD3 com-plex, all adaptor molecules. Natu-ral killer cells contain the same zetachain, the gamma chain of the Fc-epsilon I receptor, and DAP12.These adaptors all share the basicscheme: a small extracellular seg-ment, an aspartic acid residue inthe transmembrane segment, andan intracytoplasmic ITAM. Whenmultiple ligated receptors cluster,there is phosphorylation of ty-

rosines within the ITAM that pro-vides a docking and activation sitefor cytoplasmic kinases (Zap-70and Syk). The CD16 molecule asso-ciates with homo- or heterodimersof zeta or Fc-epsilon I receptor-gamma chains. On ligation of the

CD16, the p56lck tyrosine kinase isactivated, which in turn activatesSyk or Zap-70; the ultimate effect isactivation of PI3K, p21ras, nuclearfactor of activated T cells, phospho-lipase Cgamma1, and mitogen-acti-vated protein kinase with modifi-cation of the NK cell’s function.

Recent studies have identifiedother activating receptors. NKp46,NKp30, and NKp44 are membersof the IgSF, the first with two ex-tracellular domains and the latterwith one. Zeta and Fc-epsilon I re-ceptor-gamma chains associatewith the NKp46 receptor; NKp30associates with zeta homodimersand NKp44 associates with DAP12homodimers. NKp80 is a C-typelectin without intracellular ITAMor a polar amino acid residue in its

transmembrane region; NKp80may be a costimulatory moleculeinvolved in cytolysis.

The 2B4 (CD244) receptor (an-other member of the IgSF) foundon all NK cells activates cytokine

production and cytotoxicity. It binds the adaptor protein calledthe SLAM-associated protein (SAP)

(the signaling lymphocyte activa-tion molecule [SLAM (CD150)] ispresent on all activated T cells andnow known to be a receptor for themeasles virus). A defect in SAP isthe cause of X-linked lymphopro-liferative syndrome (Duncan’s syn-drome), whose sufferers die ofEpstein-Barr virus (EBV)–relatedmalignancies. The precise linkage

between SAP dysfunction, uncon-trolled EBV growth, and malignant

degeneration is not yet clear.Epstein Barr virus has acquiredan ITAM sequence in its LMP2Agene that probably mediates EBVpolyclonal B-cell activation. Otherviruses (e.g., human herpes virus 8[Kaposi sarcoma virus]) also con-tain ITAM sequences that may beinvolved in virus-induced transfor-mation of the targeted cell.

Receptor molecules that do notcontain an ITAM have a basic aminoacid residue (usually arginine or ly-sine for those of you keeping score)in their transmembrane region thatassociates with a positive residue inan adaptor molecule called DAP12 (Icontacted the scientist who namedDAP; originally, the D was to signifythe company [DNAX] where thework was done, but the name wasultimately just a convenient mono-syllable for a 12-kDa protein).DAP12 (also called killer activity re-ceptor–associated protein) is a ho-

. . .highly proficient

cytolytic cells and

prodigious sources of

many cytokines.

. . .a crucial part of the

innate immune system. . .

. . .crucial in defense

against many

intracellular pathogens. . .

58 Journal of Clinical Rheumatology • Volume 9, Number 1 • February 2003



modimer and contains an ITAM.DAP12 is found on many myeloidcells elsewhere in the body. There isa human disease caused by a muta-tion of DAP12. Nass-Hakola diseaseconsists of lipomembranous os-teodysplasia with bony cysts, a pro-gressive presenile frontal lobe

dementia with sclerosing leukoen-cephalitis, and death by the age of 40to 50 years. Both microglia and oste-oclasts are myeloid derived, so localdysfunction of these cells may becausative.

The NKG2D receptor (recog-nizing MICA and MICB) containsno ITAM itself but associates withDAP10, which engages PI3K bymeans of its interaction with thep85 PI3K subunit.

Some KIRs contain one or moreITIMs; the rest associate withDAP12. There are at least 12 KIRs,named by size and the number ofITIMs they contain. There are a se-ries of killer cell immunoglobulin-like receptors (KIR2DL and KIR3DL)immunoglobulin-like domains andlong cytoplasmic domain receptorsas well as killer cell immunoglobu-lin-like receptors (KIR2DS andKIR3DS) with two and three immu-

noglobulin-like domains and shortcytoplasmic domain receptors.

Some of the 13 immunoglob-ulin-like transcripts (ILT)/leuko-cyte immunoglobulin-like recep-tors (LIR) have one or moreITAMs, whereas the others asso-ciate with Fc-epsilon I receptor-gamma chains. ILT6 (LIR4) has notransmembrane or cytoplasmicregion and may be a soluble pro-tein.

Natural killer cells have beenidentified as a major component ofthe “large granular lymphocytes”seen in the blood of some patientswith Felty’s syndrome; these cells

were identified by their lack ofCD3, CD4, or CD8 surface markers

but their expression of CD16 andCD56. Their role in suppression of

bone marrow function and otherfeatures of Felty’s syndrome is stillunclear, but they are on the scene;

based on the contents of this article,

one could come up with potential

mechanisms mediated by secretedfactors and/or cell surface recep-tors. Although I cannot find refer-ence to a direct contribution of NKcells to the pathogenesis of lupusor rheumatoid arthritis, once yougive free rein to your imagination(as I suggested in the previous sen-tence), it is interesting to ponderhow NK cells, with their incrediblearmamentarium, might contributeto autoimmunity and how possiblechanges in ITIM-bearing receptorfunction might unleash the killerinside. Stay tuned—there may bemore on this in the literatureshortly.

CONCLUSIONThis will be among the longest

of my contributions to this seriesand little wonder. When I recall

what I learned about NK cells inmedical school (next to nothing)and what we included in my text-

book of immunology publishedonly 8 years ago (only a few pages),it is staggering how much has beenlearned about these cells and theirdelicately balanced controls ( yin

and yang) and how pivotal NKcells are in the early immune re-sponse, especially to intracellularorganisms, and in the response totumors (and perhaps in autoimmu-nity?). They truly do stand as a

bridge between innate and adap-tive immune responses. Study ofNK cells may lead to therapeuticinterventions valuable in infec-tious, inflammatory, and malig-nant diseases. The length of this

article reminds me of the old say-ing: “Absence of proof is not proofof absence”; just because we didnot know much about NK cells didnot mean they were not important.

SUGGESTED READINGCooper MA, Fehniger TA, Caligiuri MA. The bi-ology of human natural killer-cell subsets. TrendsImmunol 2001;22:633– 40.Horowitz DA, Dixon Gary J, Ohtsuka K. Role ofNK cells and TGF beta in the regulation of T-cell-dependent antibody production in health and au-toimmune disease. Microbe Infect 1999;1:1305–11.Kogure T, Niizawa A, Hai LX, Fujinaga H, Shi-

mada Y, Ochiai H, et al. Effect of interleukin 2 onkiller cell inhibitory receptors in patients withrheumatoid arthritis. Ann Rheum Dis2001;60:166–9.Lanier LL. NK cell receptors. Annu Rev Immunol1998;16:359–93.Lanier LL. On guard-activating NK cell receptors.Nat Immunol 2001;1:23–7.Lanier LL, Bakker ABH. The ITAM-bearing trans-membrane adaptor DAP12 in lymphoid and my-eloid cell function. Immunol Today 2000;21:611– 4.Lopez-Boter M, Bellon T, Llano M, Navarro F,Garcia P, deMiguel M. Paired inhibitory and trig-gering NK cell receptors for HLA class I mole-cules. Hum Immunol 2000;61:7–17.Moretta A, Bottino C, Vitale M, Pende D, CantoniC, Mingari MC, et al. Activating receptors andcoreceptors involved in human natural killer cell-

mediated cytolysis. Annu Rev Immunol 2001;19:197–224.Raulet DH, Vance RE, McMahon CW. Regulationof the natural killer cell receptor repertoire. AnnuRev Immunol 2001;19:291–330.Young NT, Uhrberg M. KIR expression shapescytotoxic repertoires: a developmental program ofsurvival. Trends Immunol 2002;23:71– 4.

Natural killer cells have

been identified as a major

component of the “large

granular lymphocytes”

seen in the blood of some

patients with Felty’s

syndrome. . .


Biologia Molecular e Imunologia 03

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