Biologia Molecular E Imunologia 03

Basic Science for Clinicians

Molecular Biology and Immunology for Clinicians 22 Natural Killer Cell Receptors and Activation Mechanisms Leonard 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 populations, B cells and T cells) are now known to occupy a pivotal position in the immune system, straddling the “divide” between the innate and adaptive responses. Natural killer cells are capable of production of many cytokines, both pro- and anti-inflammatory, and induction of target cell death by lysis and/or programmed cell death (apoptosis). Some of these cytokines are pivotal in the autoimmune and antipathogenic immune responses, implicating NK cells in the pathogenesis of many human diseases. Multiple detection systems allow tight control of the potent effector systems that mediate NK cells’ effects. Recent studies have shown that NK cell function is under tight control, with complex inhibitory and activating signaling assuring that these cells can accurately detect intracellular infection and malignant degeneration without damaging healthy cells. Although NK cell receptors do not have antigenic specificity, they do detect certain patterns on the surface of target cells. Their ability to make many cytokines that alter antigen-specific immune responses mediated by other cells puts NK cells in a unique position to influence both innate and adaptive responses. (J Clin Rheumatol 2003;9:55–59) Key words: Natural killer cells, Target cells, Immune system

atural killer (NK) cells are highly proficient cytolytic cells and prodigious sources of many cytokines. Natural killer cells are a crucial part of the innate immune system, with one foot crossing the threshold into the adaptive immune response. They are bone marrow derived and appear early in the developing human immune system. They first present by 6 to 8 weeks of gesta-

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tion, appearing before organization of the thymus is accomplished. Natural killer cells constitute 5% to 20% of all blood lymphocytes and about 5% of splenic lymphocytes but are uncommon in uninfected lymphoid tissues. Their killing activity can be increased 100- to 1000fold by cytokines like interferon alpha and beta and interleukin (IL)12; these and other cytokines are produced early in the innate re-

sponse to infection, assuring enhanced NK-cell activity within 1 to 2 days of exposure, long before an antigen-specific immune response can be generated to a pathogen. Natural killer cells are crucial in defense against many intracellular pathogens (e.g., herpes viruses, Listeria monocytogenes, Leishmania). In addition to killing tumor cells and cells containing intracellular pathogens, there is now reason to believe NK cells may be able to influence autoimmune disease. By the nature of cytokines produced, NK cells take part in the regulation of Tcell– dependent antibody production in both health and autoimmune disease. Thus, decreases in NK-cell activity seen in lupus, Crohn’s disease, and rheumatoid arthritis may mediate not only the autoimmune disease but also the increased risk these patients have of microbial infection. Thus, manipulation of NK-cell activity may

Division of Rheumatology and Connective Tissue Research, Department of Medicine, Department of Pediatrics, and Department of Molecular Genetics & Microbiology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical 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: sigallh@ umdnj.edu Copyright © 2003 by Lippincott Williams & Wilkins, Inc.

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be important in the pathogenesis and control of autoimmune, infectious, and neoplastic disease. Natural killer cells do not bear the surface markers of T cells (like CD3, CD4, or CD8 or the T-cell antigen receptor) or B cells (membrane-bound immunoglobulin). Instead, NK cells usually bear CD56 (an isoform of human neural cell adhesion molecule, whose function in NK cells is unclear) and CD16 (the low-affinity Fc gamma-III receptor, which best binds IgG1 and IgG3) (Table 1). Natural killer cells have at least four different families of NK-cell receptors, although no NK-cell receptor has the heterogeneity of the B- or T-cell surface antigen receptors we have explored in earlier articles (Table 2). The ligands for some of these receptors have been identified. Killer cell immunoglobulin-like receptors (KIRs) recognize major histocompatibility complex (MHC) class I human leukocyte antigen (HLA)-A, -B, and -C markers (see Table 2). The CD94/NKG2C disulfide-linked heterodimer binds to the nonclassic MHC class I molecule HLA-E, which reaches the cell surface by hitching a ride with classic class I molecules. Somatic cells infected with certain viruses or after malignant degenera-

tion have decreased expression of HLA-A, -B, and -C class I molecules (this may be a strategy to decrease the antigen-presenting activity of the infected cell; were infected cells to express normal amounts of class I, they could present pathogen/cancer-specific antigens and elicit a CD8⫹ T-cell response that would destroy the infected cell). Natural killer cells use HLA-E as a surrogate marker for class I expression and thereby monitor the internal status of their potential target. Once assured that the cell is fine, the NK cell curbs its murderous potential and goes on about its patrolling. NKG2D is another C-type lectin (also expressed on gamma/ delta T cells and some CD8⫹ alpha/beta T cells) that binds to cell surface MHC class I chain–related A (MICA) and B (MICB) molecules. MICA and MICB are distantly related to MHC class I alpha chains (MICA and MICB consist of two IgSF-related domains but do not associate with beta2 microglobulin). MICA and MICB expression is minimal on normal cells but increases with cellular stress or malignant degeneration. This is probably because the promoter that drives the MICA and MICB genes is similar to the promoter of the

heat shock protein 70 gene so that intracellular stresses drive both promoters. Mice do not express molecules similar to MICA and MICB, but murine NK2GD binds to a series of proteins induced by retinoic acid, known as retinoic acid early proteins (Raes) and to minor histocompatibility antigen (H60). Both Raes and H60 are upregulated in tumors; expression of these molecules makes tumors more vulnerable to NK-cell–mediated cytolysis and to rechallenge with tumor cells, where killing is mediated by CD8⫹ alpha/beta T cells, on which NKG2D acts as a coreceptor, providing a costimulatory signal that enhances T-cell efficacy. This is similar to the costimulatory signal CD28 delivers by interacting with CD80 and CD86 in T-cell activation. On activation of T cells, an intracellular area of CD28 binds to the p85 subunit of phosphatidylinositol 3 kinase (PI3K), starting a cascade of intracellular effects. Continuing the parallelism, an intracellular segment of NKG2D also engages and activates PI3K. NKG2D is also expressed by intraepithelial lymphocytes in the skin and gut, most of which are gamma/delta T cells and some of which are CD8⫹ alpha/beta cells.

TABLE 1. Types of natural killer cells CD56dim and CD16bright 90% of all NK cells* Low cytokine production† High natural cytotoxicity/ADCC High ADCC Lymphocyte activated killer (LAK) cell High KIR and ILT expression Low IL-1R and IL-18R expression Low CD94/NKG2A CXCR1, CX3CR1 Low affinity IL-2R (heterodimer) (beta/gamma)

CD56bright and CD16dim 10% of all NK cells* High cytokine production† Low natural cytotoxicity/ADCC Low ADCC Lymphocyte activated killer (LAK) cell Low KIR and ILT expression High IL-1R and IL-18R expression High CD94/NKG2A CCR7, CXCR3 and I-TAC 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 (TNFbeta), GM-CSF, IL-3, 5, 8, 10, and 13 and the chemokines MIP 1alpha, MIP 1beta and lymphotactin.

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TABLE 2. Families of natural killer cells Name KIR NCR ILT (LIR)

C-type lectin

Acronym/example killer cell immunoglobulinlike receptors natural cytotoxicity receptors immunoglobulin-like transcripts (leukocyte immunoglobulin-like receptor) CD94/NKG2A heterodimer NKG2D

NKG2D may be able to detect precancerous epithelial changes in the gut and skin, although the sensing cells are T cells rather than NK cells. In human beings, MICA and MICB are expressed almost solely on proliferating cells, not solely in the gut but also in endothelial cells and on fibroblasts as well as in transformed cells. Another set of ligands for NKG2D is represented by the UL16 binding proteins distantly related to class I MHC molecules. UL16 is a protein expressed by human cytomegalovirus; the clinical relevance of this is not yet clear. As noted, NK cells, especially the CD56bright subset, are potent producers of certain cytokines (Table 1). Natural killer cells stimulated by the combination of IL-12 and tumor necrosis factor-alpha produce large amounts of interferon-gamma early in the immune response; this cytokine may be pivotal in controlling certain infections before the elaboration of antigenspecific T cells. Natural killer cells constitutively express the intermediate affinity receptor for IL-2 (a heterodimer consisting of the beta and gamma chains); CD56bright cells are probably the only lymphocyte population also to express the high-affinity heterotrimer (a complex of the alpha, beta, and gamma chains) constitutively. This is probably why NK cells proliferate so Sigal • Natural Killer Cell Receptors

Found on

Designation

NK-cells, some T-cells

CD158a-k & CD158z

Monocytes, Macrophages Dendritic cells, B-cells, NKcells

CD85a-m

Gamma/delta T-cells, alpha/ beta T-cells, intra-epithelial lymphocytes

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; the CD56bright population expresses more IL-1R and IL-18R. There is differential expression of chemokine receptors; CD56bright cells express high levels of CCR7 (a T-cell homing receptor) and CXCR3 (receptor for the interferon-inducible proteins such as interferon-gamma inducible protein-10 and interferon-inducible T-cell alpha chemoattractant), whereas CD56dim cells lack CCR7 but have high levels of CXCR1 (ligand is IL-8) and CX3CR1 (bound by fractalkine). As their name implies, NK cells are potent killers of infected and malignant cells. Once their receptors bind to activating ligands, NK cells activate their killing capacity by release of preformed packaged molecules known as perforins and granzymes found in cytoplasmic granules. The former polymerize to form a pore in the target cell membrane, leading to cell lysis. Does this strategy (i.e., the complement “membrane attack complex”) sound familiar? Human and murine perforin are 30% homologous with complement component C9. Perforin is also part of the armamentarium of cytolytic CD8⫹ T cells. There are at least three granzymes, which are serine proteases

Ligand Class I-A B&C

HLA-E

related to the digestive enzymes trypsin and chymotrypsin—yet another example of how disparate effector functions use molecules diverging from a common source obscured by the mists of evolution. Granzymes have been implicated in inducing apoptosis in the target cell. Granzyme B cleaves an enzyme known as cysteine protease protein (CPP-32) or caspase 3, which is an early caspase in the apoptotic cascade. Activated CPP-32 in turn activates a nuclease caspase-activated deoxyribonuclease (CAD) by the degradation of the inhibitory protein referred to as the inhibitor of CAD. As a bonus, some of the nucleases involved in the destruction of the mammalian target cell also degrade viral nucleic acids. Once apoptosis occurs, the dead cell is recognized by phagocytic cells, probably because of the expression by the dying cell of phosphatidylserine on its surface, which is normally found only on the inner aspect of the cell’s lipid bilayer membrane. The advantage of apoptosis with subsequent phagocytosis is that without necrosis, there is no liberation of toxic chemicals, cytokines, and chemotactic factors and no influx of inflammatory cells and hyperemia (i.e., no inflammation to disrupt local tissue). Balancing forces, yin and yang, are a repeated motif in biology in 57

general and in immunology specifically. Natural killer cell cytolytic activity must be tightly controlled. There are NK-cell surface receptors that result in inhibition of cytolytic activity and receptors that activate the cell; the former usually have a

. . .highly proficient cytolytic cells and prodigious sources of many cytokines.

higher affinity for their ligand than do the latter, assuring a net inhibitory “tone.” Intracellular control systems are found in receptor molecules or associated “adaptor” molecules. The inhibitory receptors have an immunoreceptor tyrosine-based inhibitory motif (ITIM) or a basic amino acid residue (lysine or arginine) within their transmembrane region that allows the receptor to associate with an adaptor (containing a positively charged amino acid in its transmembrane region) that delivers the suppressive signal. On activation of the receptor, the ITIM region recruits a tyrosine phosphatase, either SH1 or SH2 domain– bearing tyrosine phosphatase, which in turn inhibits the NK cell. For inhibitory receptors that do not contain an intracellular ITIM, an adaptor protein that does contain an ITIM associates with the receptor molecule and the adaptor molecule. ITIMs are also found in B-cell and T-cell surface molecules (CD22 and Fc-gamma R IIB-1 in B cells and CTLA-4 in T cells). On the other side of the equation is activation, mediated by an immunoreceptor tyrosine-based activation motif (ITAM). These are the receptors that prompt cytolytic activity and the release of cytokines. Both B- and T-cell antigen 58

receptors contain multiple subunits with the receptor and effector typically on separate molecules: the Bcell antigen receptor is linked to Ig-alpha and beta (CD79a and b, respectively), and the T-cell antigen receptor is linked to alpha, beta, gamma, delta, and epsilon as well as zeta chains of its CD3 complex, all adaptor molecules. Natural killer cells contain the same zeta chain, the gamma chain of the Fcepsilon I receptor, and DAP12. These adaptors all share the basic scheme: a small extracellular segment, an aspartic acid residue in the transmembrane segment, and an intracytoplasmic ITAM. When multiple ligated receptors cluster, there is phosphorylation of tyrosines within the ITAM that provides a docking and activation site for cytoplasmic kinases (Zap-70 and Syk). The CD16 molecule associates with homo- or heterodimers of zeta or Fc-epsilon I receptorgamma chains. On ligation of the

. . .a crucial part of the innate immune system. . .

CD16, the p56lck tyrosine kinase is activated, which in turn activates Syk or Zap-70; the ultimate effect is activation of PI3K, p21ras, nuclear factor of activated T cells, phospholipase Cgamma1, and mitogen-activated protein kinase with modification of the NK cell’s function. Recent studies have identified other activating receptors. NKp46, NKp30, and NKp44 are members of the IgSF, the first with two extracellular domains and the latter with one. Zeta and Fc-epsilon I receptor-gamma chains associate with the NKp46 receptor; NKp30 associates with zeta homodimers and NKp44 associates with DAP12 homodimers. NKp80 is a C-type lectin without intracellular ITAM or a polar amino acid residue in its

transmembrane region; NKp80 may be a costimulatory molecule involved in cytolysis. The 2B4 (CD244) receptor (another member of the IgSF) found on all NK cells activates cytokine

. . .crucial in defense against many intracellular pathogens. . .

production and cytotoxicity. It binds the adaptor protein called the SLAM-associated protein (SAP) (the signaling lymphocyte activation molecule [SLAM (CD150)] is present on all activated T cells and now known to be a receptor for the measles virus). A defect in SAP is the cause of X-linked lymphoproliferative syndrome (Duncan’s syndrome), whose sufferers die of Epstein-Barr virus (EBV)–related malignancies. The precise linkage between SAP dysfunction, uncontrolled EBV growth, and malignant degeneration is not yet clear. Epstein Barr virus has acquired an ITAM sequence in its LMP2A gene that probably mediates EBV polyclonal B-cell activation. Other viruses (e.g., human herpes virus 8 [Kaposi sarcoma virus]) also contain ITAM sequences that may be involved in virus-induced transformation of the targeted cell. Receptor molecules that do not contain an ITAM have a basic amino acid residue (usually arginine or lysine for those of you keeping score) in their transmembrane region that associates with a positive residue in an adaptor molecule called DAP12 (I contacted the scientist who named DAP; originally, the D was to signify the company [DNAX] where the work was done, but the name was ultimately just a convenient monosyllable for a 12-kDa protein). DAP12 (also called killer activity receptor–associated protein) is a ho-

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modimer and contains an ITAM. DAP12 is found on many myeloid cells elsewhere in the body. There is a human disease caused by a mutation of DAP12. Nass-Hakola disease consists of lipomembranous osteodysplasia with bony cysts, a progressive presenile frontal lobe dementia with sclerosing leukoencephalitis, and death by the age of 40 to 50 years. Both microglia and osteoclasts are myeloid derived, so local dysfunction of these cells may be causative. The NKG2D receptor (recognizing MICA and MICB) contains no ITAM itself but associates with DAP10, which engages PI3K by means of its interaction with the p85 PI3K subunit. Some KIRs contain one or more ITIMs; the rest associate with DAP12. There are at least 12 KIRs, named by size and the number of ITIMs they contain. There are a series of killer cell immunoglobulinlike receptors (KIR2DL and KIR3DL) immunoglobulin-like domains and long cytoplasmic domain receptors as well as killer cell immunoglobulin-like receptors (KIR2DS and KIR3DS) with two and three immunoglobulin-like domains and short cytoplasmic domain receptors. Some of the 13 immunoglobulin-like transcripts (ILT)/leukocyte immunoglobulin-like receptors (LIR) have one or more ITAMs, whereas the others associate with Fc-epsilon I receptorgamma chains. ILT6 (LIR4) has no transmembrane or cytoplasmic region and may be a soluble protein. 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; these cells

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were identified by their lack of CD3, CD4, or CD8 surface markers but their expression of CD16 and CD56. Their role in suppression of bone marrow function and other features of Felty’s syndrome is still unclear, but they are on the scene; based on the contents of this article, one could come up with potential

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. . .

mechanisms mediated by secreted factors and/or cell surface receptors. Although I cannot find reference to a direct contribution of NK cells to the pathogenesis of lupus or rheumatoid arthritis, once you give free rein to your imagination (as I suggested in the previous sentence), it is interesting to ponder how NK cells, with their incredible armamentarium, might contribute to autoimmunity and how possible changes in ITIM-bearing receptor function might unleash the killer inside. Stay tuned—there may be more on this in the literature shortly. CONCLUSION This will be among the longest of my contributions to this series and little wonder. When I recall

what I learned about NK cells in medical school (next to nothing) and what we included in my textbook of immunology published only 8 years ago (only a few pages), it is staggering how much has been learned about these cells and their delicately balanced controls (yin and yang) and how pivotal NK cells are in the early immune response, especially to intracellular organisms, and in the response to tumors (and perhaps in autoimmunity?). They truly do stand as a bridge between innate and adaptive immune responses. Study of NK cells may lead to therapeutic interventions valuable in infectious, inflammatory, and malignant diseases. The length of this article reminds me of the old saying: “Absence of proof is not proof of absence”; just because we did not know much about NK cells did not mean they were not important. SUGGESTED READING Cooper MA, Fehniger TA, Caligiuri MA. The biology of human natural killer-cell subsets. Trends Immunol 2001;22:633– 40. Horowitz DA, Dixon Gary J, Ohtsuka K. Role of NK cells and TGF beta in the regulation of T-celldependent antibody production in health and autoimmune disease. Microbe Infect 1999;1:1305–11. Kogure T, Niizawa A, Hai LX, Fujinaga H, Shimada Y, Ochiai H, et al. Effect of interleukin 2 on killer cell inhibitory receptors in patients with rheumatoid arthritis. Ann Rheum Dis 2001;60:166 –9. Lanier LL. NK cell receptors. Annu Rev Immunol 1998;16:359 –93. Lanier LL. On guard-activating NK cell receptors. Nat Immunol 2001;1:23–7. Lanier LL, Bakker ABH. The ITAM-bearing transmembrane adaptor DAP12 in lymphoid and myeloid cell function. Immunol Today 2000;21:611– 4. Lopez-Boter M, Bellon T, Llano M, Navarro F, Garcia P, deMiguel M. Paired inhibitory and triggering NK cell receptors for HLA class I molecules. Hum Immunol 2000;61:7–17. Moretta A, Bottino C, Vitale M, Pende D, Cantoni C, Mingari MC, et al. Activating receptors and coreceptors involved in human natural killer cellmediated cytolysis. Annu Rev Immunol 2001;19: 197–224. Raulet DH, Vance RE, McMahon CW. Regulation of the natural killer cell receptor repertoire. Annu Rev Immunol 2001;19:291–330. Young NT, Uhrberg M. KIR expression shapes cytotoxic repertoires: a developmental program of survival. Trends Immunol 2002;23:71– 4.

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