Chapter 77: Functions of Natural Killer Cells

DEFINITION

Natural killer (NK) cells were identified in the blood and lymphoid organs of humans and experimental animals as cells capable of killing tumors, virus-infected cells, and, in some instances, normal cells, in the absence of previous deliberate or known sensitization.^1,2 NK cells are now considered to belong to the cellular subgroup that is characterized by the production of interferon (IFN)-γ within the family of innate lymphoid cells (ILCs), a family of developmentally related cells involved in innate immunity and tissue development.³ NK cells are defined as cytotoxic cells with the predominant morphology of large granular lymphocytes (LGLs) that: (1) neither productively rearrange any of the genes encoding the T-cell receptor (TCR) chains nor express on their surface the CD3-TCR complex; (2) express the CD56 (N-CAM), CD335 (NKp46), and CD16 (FcγRIIIA), antigens in humans, the NK1.1 (NKR-P1C), NKp46, and DX5 (VLA-2/CD49d) antigens in mice, and the NKR-P1 antigen in rats; and (3) can kill cells not expressing major histocompatibility complex (MHC) class I or class II antigens. Thus, target-cell recognition by NK cells is distinct from cytotoxic T lymphocytes (CTLs), which recognize specific antigenic peptides bound to MHC class I molecules. Nonetheless, the presence of MHC class I on target cells affects NK cell recognition, in some cases inhibiting a NK cell response.

Certain T lymphocytes that express either αβ or a γδ TCR may exhibit, particularly upon activation, TCR-independent cytolytic activity that resembles that of NK cells and often express many of the same surface receptors as NK cells. Among the T lymphocytes in humans and mice that coexpress many of the NK cell antigens, invariant natural killer T (iNKT) cells express an invariant TCR that recognizes glycolipids presented by CD1d, a nonclassical MHC molecule, and upon stimulation rapidly produce large amounts of IFN-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and IL-13.⁴

MORPHOLOGY

Human LGLs are medium- to large-size lymphocytes with round or indented nuclei, condensed chromatin, and usually prominent nucleoli. The cytoplasm is abundant and contains a variety of organelles and granules (primary lysosomes) that, in addition to lysosomal enzymes, contain proteins important for cytotoxic function, such as serine esterases (granzymes) and pore-forming proteins (perforin).⁵ Although many NK cells have the morphology typical of LGL, a significant proportion of NK cells are agranular and indistinguishable from other lymphocytes.⁶

ORIGIN AND TISSUE DISTRIBUTION

NK cells originate in the marrow from the common lymphoid progenitor cell.⁷ Most have a life span ranging from a few days to a few weeks,⁸ but some may persist for months after exposure to viral challenge.⁹ In mice, the cytokine IL-15 plays a particularly important role in the differentiation and expansion of NK cells.¹⁰ NK cell differentiation does not require the thymus, although NK cell progenitors can differentiate in the thymus from precursors expressing IL-7Rα CD127.¹¹ Secondary lymphoid tissues may also be a site of NK cell development in humans.¹² The increased number of NK cells and altered anatomical distribution in response to infection or other stimuli are the result of increased NK cell production in the marrow and proliferation of peripheral NK cells.

NK cells represent approximately 5 to 20 percent of blood lymphocytes.¹³ Immature NK cells express high amounts of CD56, lack CD16, and have low cytolytic capacity, whereas mature NK cells in blood express low amounts of CD56, high levels of CD16, and mediate potent lytic activity.¹⁴ NK cells are present in the red pulp of the spleen and are found at a low frequency in other lymphoid organs and marrow.² NK cells with a phenotype resembling the CD56^high peripheral subset have been detected in lymph nodes.¹⁵ Small numbers of NK cells can be identified in the liver (pit cells), lung, and intestinal mucosa.^16,17 In response to type I IFN or viral or bacterial infections, NK cells accumulate in organs in which they normally are rare, particularly the liver, marrow, and lymph nodes where they may produce large amounts of cytokines.¹⁸ CD56^bright CD16^– NK cells are the predominant cell type present in the human early pregnancy decidua.¹⁹ Decidual NK cells produce cytokines that have tissue remodeling capacity, facilitate embryonic implantation, monitor mucosal integrity throughout the menstrual cycle, control trophoblast invasion during pregnancy, and modulate the maternal immune response against embryo antigens.¹⁹

CELL-MEDIATED CYTOTOXICITY

Cytotoxicity mediated by NK cells depends on binding to the target cells, followed by activation of the lytic mechanism, which usually involves release of perforin and granzymes from the granules.⁵ Cytotoxicity can also be mediated through the interaction of surface molecules, for example, the interaction of Fas ligand, membrane tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL) on NK cells with their death-inducing receptors on target cells. Lysis of the target cells results from alteration of membrane permeability and induction of apoptosis.⁵

Several surface molecules on NK cells have been identified that activate the cytotoxic mechanism and induce cytokine secretion (Fig. 77–1).²⁰ One of these molecules is the low-affinity receptor for the Fc fragment of immunoglobulin (Ig) G (FcγRIIIA or CD16), which is expressed on most human circulating NK cells in association with the signal-transducing CD3ξ or FcεRIγ chains. When CD16 is crosslinked by IgG antibodies bound to a target cell surface, it triggers antibody-dependent cell-mediated cytotoxicity (ADCC). Natural killing can also be activated by several other receptors that recognize relevant ligands on the potential target cell. NKG2D, a receptor expressed on all NK cells, has been implicated in NK cell recognition of transformed and virus-infected cells.²¹ This receptor recognizes a family of MHC class I-related glycoproteins (including MICA, MICB, ULBP1–ULBP6), which are absent or expressed at only low levels on healthy cells but are induced or upregulated upon cell transformation or viral infection.²¹ Viruses, such as cytomegalovirus, have devised strategies to prevent the expression of NKG2D ligands in the infected cells,²² presumably to escape NK cell-mediated immunity. NK cells express many other activating receptors that have been implicated in their recognition of tumors, including DNAM-1 (CD226) and the “natural cytotoxicity” receptors NKp30, NKp44, and NKp46.²⁰

NK cells preferentially kill certain tumor cells lacking expression of MHC class I molecules.²³ NK cells are regulated by positive signals initiated by activating receptors and negative signals transmitted by interactions between inhibitory receptors for MHC class I on the NK cells and autologous MHC class I molecules on potential target cells. NK cells may mediate immune surveillance against cells that lose expression of MHC class I. Numerous viruses inhibit the synthesis or transport of MHC class I proteins, presumably to avoid detection by CTL.²⁴ In addition, frequent loss of MHC class I expression on tumor cells has been documented.²⁵ However, NK cells are capable of killing cells expressing MHC class I if they receive sufficiently strong activation signals.

Two families of NK cell receptors for MHC class I have been identified in humans. The Killer cell Ig-like receptors (KIRs) are encoded by approximately 15 genes present on human chromosome 19q13.4.²⁶ KIR genes are highly polymorphic and evolve rapidly, diversifying by gene duplication and conversion events. Certain KIRs bind human leukocyte antigen (HLA)-C ligands, whereas other KIRs recognize certain alleles of HLA-B or HLA-A. Another class of NK cell receptors for MHC class I are heterodimeric glycoproteins composed of a CD94 subunit that is disulfide bonded to an NKG2A molecule.²⁰ The genes encoding CD94 (KLRD1) and NKG2A (KLRC1) are on human chromosome 12p12-p13 and are members of the C-type lectin superfamily. The CD94-NKG2A receptors bind to HLA-E, a unique MHC class I protein that displays peptides derived from leader segments from HLA-A, HLA-B, HLA-C, or HLA-G proteins.²⁷ When synthesis of HLA-A, HLA-B, HLA-C, or HLA-G is disrupted, possibly by viral infection or transformation of the host cell, HLA-E cannot be transported to the cell surface for presentation to the CD94-NKG2A receptor. The various KIR and CD94-NKG2A receptors are expressed on overlapping subsets within the NK cell population and on certain memory T cells, usually CD8⁺ T cells, although a minor subset of CD4⁺ T cells also express KIR. The inhibitory KIR and CD94-NKG2A receptors have an immunoreceptor tyrosine-based inhibitory motif (ITIM) sequence in their cytoplasmic domains, which binds to the cytoplasmic tyrosine phosphatase SHP-1, resulting in suppression of cytotoxicity and cytokine secretion.²⁰ Therefore, the functional behavior of NK and T cells expressing KIR or CD94-NKG2A is regulated by the balance of positive signals transmitted by a variety of activating receptors and negative signals provided by the inhibitory MHC class I receptors. Although the expression of NK cell inhibitory receptors is variegated and polymorphic, most NK cells express at least one inhibitory receptor recognizing self MHC and thus are not self-reactive. This is accomplished at least in part by the requirement of interaction of an inhibitory receptor with its ligands during NK cell development for full functional maturation and possibly expansion (NK cell “licensing”).²⁸

Certain receptors of the KIR and CD94-NKG2 families do not possess ITIM sequences and activate, rather than suppress, NK- and T-cell responses.²⁰ These receptors noncovalently associate with the homodimeric adapter protein DAP12.²⁹ Like the CD3ξ and the FcεRIγ subunits, DAP12 contains an immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain. Upon receptor ligation, DAP12 becomes tyrosine phosphorylated, recruits the ZAP70 and Syk cytoplasmic tyrosine kinases, and induces cellular activation.²⁹ The physiologic role of activating NK cell receptors for MHC class I has not been determined, but these receptors may have consequences in allogeneic marrow transplantation. In mice, an activating receptor in the Ly49 family (the functional counterpart of KIRs in humans) has been shown to recognize a viral glycoprotein encoded by cytomegalovirus and protects the mice from this pathogen.^30,31 This finding suggests that certain activating KIRs in humans may also recognize pathogens.

Although resting blood NK cells are cytotoxic, their activity can be greatly enhanced both in vivo and in vitro by exposure to cytokines such as IFN-α/β, IL-2, IL-12, IL-15, and IL-18.^32,33,34 Resting NK cells constitutively express intermediate-affinity IL-2 receptors, and IL-2 induces the progression of most NK cells into the cell cycle.³⁵

PRODUCTION OF CYTOKINES

Many of the physiologic functions of NK cells are mediated at least partly by their ability to secrete cytokines. NK cells are powerful producers of IFN-γ and GM-CSF, and other cytokines and chemokines. Stimulation by cytokines, such as IL-2, IL-12, IL-18, TNF-α, and IL-1,^2,33,36,37 and triggering by activating receptors, such as CD16 interacting with immune complexes, are among the stimuli that, acting individually or often in synergistic combination, induce NK cells to produce cytokines.^2,38,39

INNATE RESISTANCE

Together with myeloid cells, NK cells are effectors of the innate or natural resistance, which represents the first line of defense against infection (Fig. 77–2). The ability of NK cells to participate in the resistance against infection by certain viruses is well documented in experimental animals and is strongly suggested by the recurrent viral infections in the rare patients with a selective deficiency of NK cells.⁴⁰ NK cells selectively kill virus-infected cells by a mechanism that is at least partly dependent on the production of IFN-α, a potent stimulator of NK cell activity.^41,42 In vivo viral infection and type I IFN production usually are accompanied by rapid activation of, and increase in the number of, NK cells.¹⁸ The NK cell response to virus infection is followed by an antigen-specific T helper and CTL response, which peaks 7 to 9 days after infection.¹⁸ The early NK cell response induces a significant reduction in the titer of certain viruses, including mouse cytomegalovirus (MCMV).⁴³ NK cell activation induced by viral infection may have beneficial or pathogenic effects.⁴⁴ After expansion in response to MCMV infection, NK cells expressing the activating Ly49H receptor persist for several months and respond rapidly to rechallenge with MCMV, thus possessing immunologic memory.⁹ Similarly, a subset of liver-resident NK cells can mediate antigen-specific contact hypersensitivity responses and immunologic memory.^45,46

NK cells enhance the response of phagocytic cells to microorganisms, especially intracellular bacteria and parasites, by producing high levels of the phagocyte-activating cytokines IFN-γ and GM-CSF in response to the microorganisms themselves or to factors, such as IL-12 and TNF-α, produced by infected phagocytic cells.^47,48

REGULATION OF ADAPTIVE IMMUNITY

NK cells, by interacting with infectious agents and antigens early during the immune response, have either stimulatory or inhibitory effects on the function of B and T cells and antigen-presenting cells.² Evidence for an enhancing effect of NK cells on B-cell responses has been shown both in vitro and in vivo by studies demonstrating that NK cells in the absence of T cells support antigen-specific B-cell responses, partly by producing IFN-γ.^49,50 In certain infections, NK cells may be necessary for optimal induction of both a CD4⁺ and CD8⁺ T-cell response.^51,52 NK cells stimulated by microorganisms or by cytokines, such as IL-12 and IL-18, produce large amounts of IFN-γ and other cytokines that facilitate T-helper cell type 1 development.^53,54 The reciprocal activating interaction between NK cells and the antigen-presenting dendritic cells is important for the regulation of both innate resistance and the downstream adaptive response to pathogens.^55,56

MODULATION OF HEMATOPOIESIS

Experimental and clinical studies have demonstrated that NK cells are involved in the regulation of hematopoiesis.⁵⁷ The effect of NK cells is mostly mediated by secretion of soluble factors. NK cells, constitutively or upon activation, produce several lymphokines, some with mostly inhibitory effects on hematopoiesis, such as TNF and IFN-γ, and some with mostly stimulatory effects, such as GM-CSF.^36,58 The effector role of NK cells in rejection of parental marrow graft in irradiated F1 mice⁵⁹ and in suppressing erythropoiesis and phagocytopoiesis in mice infected with lymphocytic choriomeningitis virus (LCMV)⁶⁰ demonstrate that in vivo activated NK cells can affect both allogeneic and syngeneic hematopoietic progenitor cells. Because of the ability of NK cells to kill transformed hematopoietic cells, NK cells have been postulated to play an important role in the graft-versus-leukemia reaction in allogeneic marrow transplantation but only a modest, if any, role in graft-versus-host disease.⁶¹ In haploidentical or mismatched hematopoietic transplantation, the presence on donor NK cells of the KIR not recognizing inhibiting ligands on host hematopoietic and malignant cells results in protection from leukemia relapse.⁶² A reduced incidence of graft-versus-host disease has also been observed and thought to result from the elimination of recipient antigen-presenting cells by donor NK cells.⁶²

NK cell function and NK cell numbers are often decreased in pathologic conditions, including cancer and AIDS.^63,64 The reduced activity or number of NK cells may contribute to disease pathology by decreasing the innate resistance against tumor growth and metastasis in cancer patients or against opportunistic infections in AIDS patients. NK cell (and cytotoxic T-cell) hyporesponsiveness is observed in patients with Chédiak-Higashi syndrome,⁶⁵ a rare autosomal recessive disease associated with cellular dysfunction, including fusion of cytoplasmic granules and defective degranulation of neutrophil lysosomes. NK cell numbers are normal in these patients, but the NK cells present a single, large granule in the cytoplasm and have a severely reduced ability to mediate cytotoxicity.⁶⁵

Malignant acute expansion of NK cells is rare, more frequent in Asians than in whites, and often associated with Epstein-Barr virus infection.⁶⁶ Extranodal NK cell lymphomas occur in both the nasopharyngeal region and in nonnasal areas as an NK cell (CD2⁺, CD3^–, CD56⁺, CD16^–, CD57^–) leukemia or lymphoma that mostly affects extranodal tissues.⁶⁷ It affects predominantly men in their fifth decade and usually has an extremely aggressive clinical course. Aggressive NK cell leukemia is a catastrophic disease that affects young adults and is characterized by the systemic presence of neoplastic NK cells in blood and marrow.⁶⁷ An entity previously known as “blastic NK cell lymphoma,” characterized by CD4⁺, CD56⁺ cells with dermal tropism, is now recognized to represent an expansion of plasmacytoid dendritic cells rather than NK cells.⁶⁷ A chronic monoclonal proliferative disorder of LGL with a clinical course that is often relatively indolent is more commonly observed.⁶⁸ Most patients have lymphocytic infiltration of the marrow. Severe neutropenia and anemia often are observed. Associated diseases, most commonly rheumatoid arthritis, hepatitis, or cancer, are present in up to half of patients.⁶⁸ Although cells from all these patients are characterized by a LGL morphology, in approximately two-thirds of the cases they represent a monoclonal expansion of CD8⁺ T cells, and in only less than one-third of cases they have the typical phenotype and genotype of CD3^–, CD56⁺, CD57⁺, and, in some patients, CD16⁺ NK cells.⁶⁸

TARGETING NK CELL RECEPTORS

Data showing recipients of KIR-incompatible donors have reduced relapse of acute myelogenous leukemia (AML) following mismatched allogeneic stem cell transplantation remain the most compelling evidence to support the antitumor activity of NK cells.⁶¹ Further, studies in mice showing Ly49-incompatible NK cells mediate graft-versus-tumor effects have led to clinical trials exploring adoptively infused allogeneic NK cells in patients with cancer. In humans, pilot studies have shown that adoptive transfer of IL-2-activated MHC-mismatched allogeneic NK cells can proliferate in vivo and can induce tumor regression in patients with AML and solid tumors.⁶⁹

A strategy to disrupt inhibitory KIRs to enhance NK tumor killing involves blocking KIRs with monoclonal antibodies or genetically silencing their expression. IPH2101 is a fully human IgG₄ antibody that binds to KIR2D, thus blocking its ability to suppress NK cell function. Studies in a mouse model showing that IPH2101 enhances NK-cell killing of KIR ligand-matched tumor cells has led to phase I studies evaluating the efficacy of IPH2101-mediated KIR2D blockade in patients with AML and other hematologic disorders.⁷⁰ Genetic disruption of the inhibitory receptor NKG2A has been shown to enhance NK cell killing of HLA-E–expressing tumors ex vivo and in vivo following their infusion into tumor-bearing mice.⁷¹

ADOPTIVE NATURAL KILLER CELL TRANSFER

Recently, methods have been developed to expand large numbers of human NK cells ex vivo, which provides the opportunity to study the efficacy of adoptive NK-cell immunotherapy in patients with cancer.⁷² Most expansion cultures utilize irradiated feeder cells such as Epstein-Barr virus–transformed lymphoblastoid cell lines or the human erythromyeloblastoid leukemia cell line K562 cells genetically modified to express membrane bound 4–1BB ligand, IL-15, or IL-21. Expanded NK cells have increased surface expression of NK activating receptors and cytotoxicity effector molecules. Compared to resting NK cells, expanded NK cells express higher amounts of IFN-γ, Fas ligand, and TRAIL, and have markedly enhanced cytotoxicity against K562 and other tumor cells compared to resting or short-term IL-2–activated NK cells. Phase I trials of infusing large numbers of these ex vivo expanded autologous NK cells are now ongoing in patients with a variety of different cancers.⁷³

Studies suggest that lenalidomide, an immunomodulatory drug derived from thalidomide, and several monoclonal antibodies blocking immune checkpoints, such as anti–PD-1, anti–PD-L1, and anti–CTLA-4, augment NK-cell–mediated ADCC.^74,75 The advances in our ability to expand NK cells ex vivo now makes it feasible to combine these and other monoclonal antibodies with adoptive NK cell transfer to potentially augment their antitumor effects.

OVERCOMING DEFICIENT NATURAL KILLER CELL HOMING

Expanded NK cells are inefficient at homing to the marrow and lymph nodes where hematologic malignancies reside. A variety of techniques to improve NK cell homing to these target organs have been described.⁷² NK cell trogocytosis (the transfer of plasma membrane fragments from the presenting cell to the lymphocyte) of the membrane-bound chemokine receptor CCR7 expressed on K562 cells can be used to increase NK cell CCR7 surface expression, improving their homing into the lymph nodes of athymic mice.⁷⁶ Nicotinamide, a specific inhibitor of nicotinamide adenine dinucleotide–dependent enzymes, substantially increases surface expression of CD62L on NK cells when added to cell cultures, improving their homing into the spleens and marrow of immunodeficient mice.

Recruitment of leukocytes to marrow is largely dependent on E-selectin binding.⁷⁷ Ex vivo expanded NK cells primarily express nonglycosylated ligands for E-selectin, potentially limiting their homing ability to the marrow. Ex vivo forced fucosylation of NK cells with fucosyltransferase VI enhances their E-selectin binding capacity in vitro and in mice improves their homing to the marrow.⁷² Forced fucosylation of NK cells is currently being studied as a novel approach to improve NK homing to the marrow to enhance NK cell killing of hematologic malignancies.

AUGMENTING NATURAL KILLER CELL TUMOR KILLING

Exposing tumors to drugs that enhance caspase activity or upregulate death receptors for the apoptosis-inducing ligands TRAIL or Fas-L is an alternative strategy to bolster the antitumor effects of NK cells.⁷⁸ The proteasome inhibitor bortezomib upregulates surface expression of the TRAIL receptor DR5, enhancing tumor susceptibility to NK-cell TRAIL-mediated apoptosis in vitro and in vivo.⁷⁹ In mice, eradicating regulatory T cells prior to adoptive NK cell infusions further potentiates this antitumor effect. Clinical trials evaluating the antitumor activity of ex vivo expanded adoptively infused NK cells following bortezomib treatment are ongoing.

Methods to transfect chimeric antigen receptors (CARs) into NK cells have been optimized to induce tumor-specific NK cell killing in clinical applications. CARs specific for antigens expressed on tumors such as CD19 on B-cell malignancies, HER2/ErbB2 on breast carcinomas, and GD2 on neuroblastoma tumors when transduced into NK cells enhance tumor killing by autologous NK cells.^72,80 These findings, as well as studies showing the efficacy of T-cell based CAR therapy targeting CD19 in B-cell malignancies, suggest CAR-modified NK cells are worthy of exploration in the clinic (Chap. 26).