Natural killer (NK) cells selectively lyse cells without requiring prior activation. NK cells belong to a family of innate lymphoid cells (ILCs), which are critical for immune surveillance and subsequent host defense against virus-infected and cancerous cells as part of the innate immune system. In humans, NK cells represent 8–20% of circulating lymphocytes; in inbred laboratory mice, NK cells represent 2–5% of lymphocytes found in the spleen and bone marrow [1,2]. Unlike other lymphocytes—including B cells, T cells, and natural killer T (NKT) cells—NK cells do not express an antigen-specific receptor such as the clonotypic B cell receptor or T cell receptor/CD3ε complex. Instead, NK cells function in antigen-independent manner that generally does not give rise to immunological memory or long-term protective immunity. Recent studies have implicated NK cells as potential therapeutic targets in cancer treatment .
Human NK cells develop and mature in bone marrow (BM) and secondary lymphoid tissues (SLTs), including tonsils, spleen, and lymph nodes . Development takes place in six distinct stages (Table 1). Stage 1 occurs exclusively in the BM, whereas stages 2 through 6 can occur either in BM or SLTs. Hematopoietic stem cells develop into multipotent progenitor (MPP) cells with either a lymphoid or a myeloid bias. From MPP cells, a common lymphoid progenitor emerges that develops into a precursor NK cell, then to an immature NK cell, and finally to a mature NK cell. Precursor NK cells (NKPs) can only differentiate into mature NK cells and not into T, B, myeloid, or erythroid cells . The stages of human NK development are based primarily on the expression levels of CD34, CD117, CD56, and CD94. An increase in CD56 expression is critical for NK maturation and is closely followed by expression of CD94/NKG2A. The interleukin 2/15 receptor beta (CD122) is also an important marker for the later stages of NK cell development.
|Stage 1||Stage 2||Stage 3||Stage 4||Stage 5||Stage 6||Stage 7||Stage 8|
|Abbreviations: CD, cluster of differentiation; ILR, IL-1 receptor; NKG, natural killer group. Hi: high expression levels, Low: low expression levels.|
Mouse NK cells resemble human NK cell in function, but they express different developmental markers and mainly develop in specialized bone marrow niches . NK cell development begins with CD122 expression on precursor NK cells (NKPs) and transitions through six specific stages (Table 2). The sequential acquisition of the NK cell receptors NKG2D/A/C, CD62L, Ly49, and CD117 (c-Kit) leads to the maturation of mouse NK cells. Expression of CD51 and CD49b defines the initial stage of mouse NK cell maturation [5,6], whereas expression of CD43 (leukosialin) and CD11b (Mac1) and acquisition of Ly49 receptors define the terminal stages. Expression of KLRG1 induces a subset of mouse NK cells to migrate into SLTs. Additional functional classifications of mouse NK cells are made using CD27 and CD11b [5,6].
|NKP||Stage A||Stage B||Stage C||Stage D||Stage E||Stage F|
|Abbreviations: CD, cluster of differentiation; KLRG, killer cell immunoglobulin–like receptor; NCR, natural cytotoxicity triggering receptor; NKP, NK cell progenitors; NKG, natural killer group.|
Most normal healthy cells express major histocompatibility complex (MHC) class I molecules, which mark these cells as “self”. When the NK cell inhibitory receptors recognize the cognate MHC class I molecules, the NK cell’s cytotoxicity function is switched off, preventing it from killing these cells. To acquire the capacity to recognize “nonself” target cells with low MHC I expression, NK cells must be educated to detect host MHC class I molecules using their cognate inhibitory receptors, a process also referred to as tuning or licensing.
NK cells also adapt to their environment through a process of priming by cytokines, such as IL-2, IL-15 (trans-presented by dendritic cells), IL-18, IL-12, and IFNα (Figure 1) [7,8]. The integration of various pathways (inhibitory and activating) following interaction with neighboring cells governs the dynamic equilibrium that regulates NK cell activation and determines whether or not NK cells are activated to kill target cells and produce cytokines. The innate immune system is generally thought to lack the capacity for immunological memory; however, recent findings show that some NK cells can be long lived and mount a robust recall response to aberrant cells or viruses [5,6,7].
Figure 1. NK cell education. NK cells interact with potential target cells to educate what is “self” through the interaction of MHC class I molecules expressed on normal healthy cells. NK cells are also primed through the interaction with cytokines such IL-12, IL-18, IL-21, and IFN alpha. Abbreviations: MHC, major histocompatibility complex, IL, interleukin; IFN, interferon.
As NK cells are capable of immune surveillance and host defense without the need for prior activation, the expression of cell surface receptors is critical. NK cells express both activating and inhibitory receptors, as well as adhesion receptors. These NK cell receptors function as a sensory system, and the balance of the engagement and subsequent downstream signaling from these receptors determines the cellular response.
Mature, circulating NK cells (human: CD56+, mouse: CD49b+) constitutively express multiple activating receptors (Table 3). NK cells are unique among immune cells in that all mature circulating NK cells constitutively express FCRγ, CD3ζ, and DAP12 type I transmembrane-anchored proteins that exist as either disulfide-bonded homodimers or, in the case of FCRγ and CD3ζ, as disulfide-bonded heterodimers. Most importantly, these proteins contain immunoreceptor tyrosine-based activation motifs (ITAMs)—defined by the sequence (D/E)XXYXX(L/I)X6–8YXX(L/I), where X represents any amino acid, slashes separate alternative amino acids that may occupy a given position, and X6–8 denotes any 6 to 8 amino acids between the two YXX(L/I) elements—in their cytoplasmic domains. DAP12 and FCRγ have a single ITAM, and CD3ζ has three ITAMs per chain. There are no other known signaling motifs in the rather short cytoplasmic domains of these proteins, and mutation of the ITAM tyrosine residues abolishes their signaling function. Ligand engagement of activating receptors promotes downstream signaling through these ITAM-containing adapter molecules. ITAMs are phosphorylated by Src kinases upon engagement with an NK activating receptor, allowing the binding and activation of the tyrosine kinases Syk and Zap70 [9,10,11,12].
One important family of activating NK receptors are the natural cytotoxicity receptors (NCRs), which include NKp30, NKp44, and NKp46. Upon stimulation, these receptors deliver potent signals to NK cells in order to lyse harmful cells and produce inflammatory cytokines such as IFNγ. The NCRs are expressed on mature circulating NK cells and play a critical role in the control of various viral infections and cancer.
A second important activating receptor, NKG2D, is unique in that, when expressed on human NK cells, it is associated with the cytoplasmic adaptor protein DAP10 to initiate distinct signaling cascades. DAP10 possesses a YINM motif, which allows for PI3K binding and activation. In addition, DAP10 binds Grb2, which associates with Vav1. NKG2D is expressed from the earliest NK cell precursor stages onward and, in addition to a role in cytolytic activity, NKG2D engagement induces the upregulation of other activating NK receptors .
|KIR2DS4||Viral Helicases, HLA-Cw4||DAP12||H|
|NKG2D||MICA, MICB, ULBP1||DAP10||H, M|
|CD122||IL-2, IL-15||SHC1||H, M|
|Abbreviations: DAP12, DNAX activating protein of 12 kD; FCRγ, Fc fragment of IgE receptor Ig; HA, hemagglutinin; H, human; HLA, human leukocyte antigen; IL, interleukin; KLRG, killer cell immunoglobulin-like receptor; M, mouse; MICA, major histocompatibility complex (MHC) class I chain-related protein A; MICB, major histocompatibility complex (MHC) class I chain-related protein B; NKP, NK cell progenitors; NKG, natural killer group; SHC1, SHC Adaptor Protein 1; ULBP1; UL16 binding protein 1.|
NK cell inhibitory receptors (Table 4) maintain NK cells in an inactive state. Some inhibitory NK receptors are specific for MHC class I molecules, whereas others bind non-MHC ligands. Some of these inhibitory NK receptors, such as killer cell immunoglobulin-like receptors (KIRs) and leukocyte immunoglobulin-like receptors (LILRs), are monomeric type I glycoproteins of the immunoglobulin superfamily, whereas others, such as the Ly49 and CD94-NKG2A receptors, are type II glycoproteins with a C-type lectin–like scaffold. All inhibitory receptors share a common immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic regions [10,11,12].
|Abbreviations: H; human, HLA; human leukocyte antigen, IL; interleukin, KLRG; killer cell immunoglobulin like receptor, LIR; leukocyte Ig-like receptor, M; mouse, NKG; natural killer group, SIGLEC; Sialic acid-binding immunoglobulin-type lectins|
As most NK cells reside in the vasculature, they must undergo extravasation and migrate to the site of inflammation, infection, or tumorigenesis. Adhesion receptors (Table 5) are critical for this process and, upon activation of the NK cell, increase the expression of these receptors [9,10,11].
During immune surveillance, NK cells constantly contact other cells. As NK cells can induce cytolytic activity on virally infected cells and tumor cells without prior sensitization or activation, the decision to kill or not depends on a balance of signals from activating receptors and inhibitory receptors on the NK cell surface. As discussed above in NK Cell Education, NK cells interact with “self” cells to learn how to recognize these cells, and this interaction is mediated by inhibitory signaling by NK receptors that specifically bind to MHC (HLA) class I molecules on healthy cells (Figure 2). When MHC class I molecules are present on potential target cells in the absence of any activation signal, the NK cells recognize the cell as “immunological self”, and cytolytic activity is attenuated through ITIM-mediated signaling. When MHC class I molecules are absent or down regulated on tumor or virally infected cells (“missing self”), inhibitory receptor signaling is lost, resulting in NK cell activation and subsequent cytolytic activity. The killing process of “induced self” occurs when NK cell activating receptors receive activation signaling from ligands expressed on target cells in response to viral infection or tumorigenesis. This activation signaling overrides the inhibitory signaling, resulting in NK cell activation and subsequent cytolytic activity [9,10,11,12].
Figure 2. NK cell activation. The integration of various pathways (inhibitory and activating) following interaction with neighboring cells governs the dynamic equilibrium that regulates NK cell activation and determines whether NK cells are activated to kill target cells and produce cytokines. “Immunological self” occurs when NK cells interact with normal healthy cells that express MHC class I molecules. “Missing self” occurs when NK cells interact with cells that do not express MHC class I molecules due to viral infection or tumorigenesis. This lack of “self” inhibitory signaling promotes NK-mediate cytolytic activity. “Induced self” occurs when cells express ligands, such as viral antigens, that interact with activating receptors. This activating signal overrides any inhibitory signaling and promotes NK-mediate cytolytic activity. Abbreviations: MHC, major histocompatibility complex; MICA/B, major histocompatibility complex (MHC) class I chain-related protein A/B.
As NK cells reside predominantly in the peripheral circulation, they must exit the vasculature to encounter potential target cells. Chemoattractants released by virally infected cells or tumor cells promote inflammation of vascular endothelium, and detection of the altered endothelium promotes NK extravasation and migration to the site of chemoattractant release. NK cell arrest from circulation, transendothelial migration (TEM), chemotaxis, and haptotaxis to the inflammatory locus, and adhesion at the inflammatory site are mediated by the β1 and β3 integrins. At the loci of inflammation or tumorigenesis, the NK cell interacts with possible target cells through interaction of integrins, in particular, LFA-1.
During interaction with a potential target cell, the NK cell receptor repertoire determines the outcome of the interaction through the balance of inhibitory and activation signals. If the NK cell signaling balance favors activation, the NK cell proceeds with the signaling and cell architectural changes necessary for NK cell mediated killing. This signaling decision occurs when the NK cell is in loose contact with the potential target cell. When the decision to kill is made, the NK cell firmly attaches to the target cell. NK activation receptor–mediated signaling promotes inside-out signaling to integrin LFA-1, which promotes integrin clustering and signaling. LFA-1-mediated attachment induces actin cytoskeletal reorganization to create tight lytic synapse between the NK and target cells (Figure 3). Subsequent MTOC polarization towards the lytic synapse and tubulin reorganization and assembly occurs that allows lytic granules to be secreted into the lytic synapse (Figure 4). Lytic granule convergence and polarization represents the final step at which NK cells are susceptible to inhibitory signaling before a commitment to cytotoxicity. After polarizing towards the target cell and before degranulation, perforin and granzyme B containing lytic granules dock at the lytic synapse and fuse with the plasma membrane. Perforin is released and induces membrane flipping and rapid apoptosis of the target cell (Video 1) [13,14,15].
Granzymes (granule-secreted enzymes) are a family of serine proteases stored within lysosomal-granules of natural killer (NK) cells and cytotoxic lymphocytes (CTLs). Critical for the induction of target cell apoptosis, granzymes cleave intracellular substrates, triggering many apoptotic pathways to ensure target cells die. Granzymes also play a significant role in the immune defense against viruses, tumors, and intracellular bacteria. More recent research demonstrated that granzymes have an extracellular role resulting in the destruction of tissues and vascular integrity that implicates granzymes in a number of inflammatory and age-related diseases. This family is emerging as an important group of proteins involved in immune function and surveillance.
Granzyme A is the most abundant serine protease in NK-cytotoxic and CD8+ cytotoxic T cells granules. It not only activates a novel programmed cell death pathway that begins in the mitochondrion and generates reactive oxygen species (ROS) to ultimately activate single-stranded DNA damage, but also targets other important nuclear proteins for degradation, including histones, lamins and several key DNA damage repair proteins such as Ku70 and PARP-1. Granzyme A also displays proinflammatory activity; it activates IL-1s, TNF- α, and IL-6 and may have other effects not yet understood.
Granzyme B (GrB) is found in the granules of both NK cells and cytotoxic T cells. Granzyme B has also been described as CGL1 (cathepsin G-like-1) and CTLA-1 (cytotoxic T lymphocyte-associated serine esterase 1) based on identification of mRNA in various cytotoxic T cells, but not observed in non-cytotoxic lymphoid cells. Granzyme B is crucial for the rapid induction of target cell death by apoptosis, induced by interaction with cytotoxic T cells. Granzyme B activates the intracellular cascade of caspases finally resulting in the killing of the target cells.
Like Granzyme A, Granzyme K is a tryptase serine protease with overlapping, but not identical, substrate specificity. Although Granzyme K induces ROS generation, it also induces caspase-independent cell death through Bid-dependent mitochondrial damage. Data have also shown that Granzyme-K binds and cleaves p53. This cleavage is reported to incite a mediated-killing of tumor cells. It appears that by activating multiple cell death pathways, killer cells provide better protection against a variety of intracellular pathogens and tumors. Granzyme K too displays pro-inflammatory potential as it has been shown that an extracellular form is capable of inducing cytokine production from human lung fibroblasts through the activation of PAR-1.
Figure 3. LFA-1 and actin reorganization in NK cells promotes a tight lytic synapse. Primary NK cells were adhered to ICAM-1 and formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5–10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with LFA-1 antibody in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4°C in a humidified chamber. Cells were washed with PBST and incubated with Invitrogen goat anti–mouse IgG secondary antibody, DyLight 488 conjugate in PBS at room temperature. Cells were then stained with Invitrogen Alexa Fluor 568 Phalloidin. This fluorescence image shows a dense peripheral actin ring colocalized with a ring of LFA-1 contacts, forming a tight lytic synapse.
Primary human NK cells can be isolated from peripheral blood mononuclear cells (PBMCs). PBMCs can be isolated from human blood or peripheral leukocytes. NK cells can be isolated from PBMCs using immunomagnetic beads such as the Invitrogen Dynabeads Untouched Human NK Cells Kit. Cell sorting for NK cells is an alternative to immunomagnetic beads. CD56 and CD16 are specific human NK cell markers and allows for the identification and isolation of NK cells without contamination from other lymphocytes (Figure 5).
CD56 status on NK cells confers phenotypic properties . CD56Low CD16Hi NK cells constitute most circulating NK cells; CD56HiCD16−/Low NK cells constitute a smaller portion. CD56Hi NK cells proliferate and produce cytokines in response to stimulation with IL-12. CD56Low NK cells are more cytolytic and produce significant amounts of cytokines when their activating receptors are engaged. The CD56Low NK cells differentiate from the CD56Hi NK cells. NK cells are a heterogeneous population with respect to the expression of activating and inhibitory receptor.
NK cells in culture behave differently and express different markers from cells isolated in vivo. NK cell cultured with IL-2 will induce CD57 expression on CD57−CD56Low NK cell subsets. CD57+ NK cells are highly mature and may demonstrate multiple properties including, cytotoxic capacity, sensitivity to stimulation via CD16, a decreased responsiveness to cytokines and capacity to proliferate.
Mouse NK cells can be isolated from mouse spleens either by immunomagnetic beads or cell sorting . Single cell suspension can be sorted for CD49b+, NK1.1+, and CD3-. All mouse NK cells should be maintained in media supplemented with IL-2.
Primary human NK cells can be cultured for up to 8 weeks. NK cells can be maintained in Gibco Advanced RPMI 1640 Medium supplemented with 10% human AB+ serum, 2 mM Gibco CTS GlutaMAX-I, 1 mM sodium pyruvate and 100 U/mL rIL-2 and 25U/ml rIL-15. Expansion of NK cells can be accomplished by co-culture with irradiated K562 cells in the presence of phytohaemagglutinin (100 ng/mL) for 6–9 days. NK cells should be resorted for CD56+/ NKG2D+HI and cultured with irradiated K562 in complete RPMI with 100 ng/mL phytohemaglutinin. K562 cells should be irradiated (6000 rad) to prevent proliferation of target cell during primary NK cell proliferation.
Quantitative determination of the cytotoxic activity of human NK cells can be determined using target cells- K562 (Figure 6). Target cells and NK cells should be co-plated in cell culture treated plates, such as Thermo Scientific Nunc MicroWell 96-Well, Cell Culture-Treated, Flat-Bottom, Optical Polymer Base Microplates, coated with 5 µg/mL fibronectin overnight in Gibco Hanks Buffered Saline Solution (HBSS) at 4˚C. Target cells at a density of 1 x 105 (K562) cells are then added to each well and allowed to adhere for 1 hour at 37˚C in the presence of 10 ng/mL SDF-1α. Wells are then washed with HBSS. NK cells at a density of 5 x 105 in HBSS are added to each well, in triplicate, and incubated at 37˚C. Cytotoxicity can detected with Invitrogen LIVE/DEAD Cell-Mediated Cytotoxicity Kit by fluorescence imaging. For imaging, cells can be imaged using the Invitrogen EVOS M7000 Imaging system with EVOS Onstage Incubator, and data analyzed using Invitrogen Celleste Image Analysis Software. Alternatively, cells can be imaged and analyzed using high-content analysis platforms.
Alternatively, for flow cytometry analysis, target cells can be stained with Invitrogen CellTrace dyes and then incubated with effector NK cells in the presence of Invitrogen SYTOX dyes. As NK cells attach and kill target cells, the cell membrane becomes permeant and the SYTOX dye enters, binds to nucleic acids, and fluoresces, which can be quantified. For flow cytometry researchers can use the Invitrogen Attune NxT Flow Cytometer.
In addition, the following flow cytometry protocols for evaluating NK cells can be used: OMIP-029: Human NK-Cell Phenotypization and OMIP‐027: Functional Analysis of Human Natural Killer Cells.
Figure 6. Quantitative determination of the cytotoxic activity of human NK cells. Target K562 cells are preincubated with the green-fluorescent membrane stain DiOC18 and then mixed with effector cells in the presence of the red-fluorescent, membrane-impermeant dye propidium iodide. Live and dead target cells retain the green-fluorescent membrane stain; target and effector cells with compromised membranes exhibit red-fluorescent nucleic acid staining; live effector cells are nonfluorescent. Cells were imaged on an Invitrogen EVOS M7000 Imaging System with EVOS Onstage Incubator.
While individual cytokines exert some effects, a combination of different cytokines is usually required for full activation, proliferation, survival, and effector functions of NK cells. Positive cytokine regulators of NK cell function include IL-2, IL-15, IL-12, IL-18, IL-21, and IFG-g that are produced mainly by Th1 cells, macrophages, and dendritic cells. In contrast, the cytokines IL-4, IL-5, and IL-10 secreted by Th2 cells or IL-1β, IL-6, IL-10, IL-23, IL-35, and TGFβ secreted by tumor cells can suppress NK cell function.
|Cytokines and chemokines||IL-2, IL-15, IL-12, IL-18, and IL-21 IFG-g||IL-4, IL-5, IL-1β, IL-6, IL-10, IL-23, IL-35, TGF-β||IFN-γ, TNF-α, GM-CSF, IL-10, IL-5, and IL-13, MIP-1α, MIP-1β, IL-8, RANTES|
|Other factors||Granzyme-A, -B, & -M, Perforin|
There are at least ﬁve distinct stages in NK cell development, and the surface expression of various cytokine receptors during these stages of NK cell development suggests that diﬀerent cytokines are relevant for transition from one development stage to the next. In addition, as new opportunities to use cancer immunotherapy based on natural killer cells develop, understanding the role of cytokines and immune checkpoint inhibitors in the tumor microenvironment and their effect on NK cells will become critical. Activated NK cells secrete a wide variety of cytokines and chemokines, including GM-CSF, IL-10, IL-5, IL-13, IFN-γ, RANTES, TNF-α, MIP-1α, MIP-1β, and IL-8 which can be measured using immunoassays. In addition, immune checkpoint markers expressed on NK cell such as LAG-3, CD96, TIM-3, TGIT, and PD-1 all have soluble forms that can be measured in serum or plasma.
|Human||Cytokine/Chemokine/Growth Factor Convenience 45-Plex Human Panel 1||GM-CSF, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A (CTLA-8), IL-18, IL-21, IL-22, IL-23, IL-27, IL-31, LIF, SCF, TNF alpha, TNF beta, Eotaxin (CCL11), GRO alpha (CXCL1), IP-10 (CXCL10), MCP-1 (CCL2), MIP-1 alpha (CCL3), MIP-1 beta (CCL4), RANTES (CCL5), SDF-1 alpha, BDNF, EGF, FGF-2, HGF, NGF beta, PDGF-BB, PlGF-1, SCF, VEGF-A, VEGF-D||EPXR450-12171-901|
|Immuno-Oncology Checkpoint 14-Plex Human ProcartaPlex Panel 1||D27, CD28, CD137 (4-1BB), GITR, HVEM, BTLA, CD80, CD152 (CTLA4), IDO, LAG-3, PD-1, PD-L1, PD-L2, TIM-3||EPX14A-15803-901|
|Immuno-Oncology Checkpoint 14-Plex Human ProcartaPlex Panel 2||MICA, MICB, Perforin, ULBP-1, ULBP-3, ULBP-4, Arginase-1, CD73 (NT5E), CD96 (Tactile), E-Cadherin, Nectin-2, PVR, Siglec-7, Siglec-9||EPX140-15815-901|
|Mouse||Immune Monitoring 48-Plex Mouse ProcartaPlex Panel||BAFF, G-CSF (CSF-3), GM-CSF, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-15/IL-15R, IL-17A (CTLA-8), IL-18, IL-19, IL-22, IL-23, IL-25 (IL-17E), IL-27, IL-28, IL-31, IL-33, LIF, M-CSF, RANKL, TNF alpha, ENA-78 (CXCL5), Eotaxin (CCL11), GRO alpha (CXCL1), IP-10 (CXCL10), MCP-1 (CCL2), MCP-3 (CCL7), MIP-1 alpha (CCL3), MIP-1 beta (CCL4), MIP-2, RANTES (CCL5), Betacellulin (BTC), Leptin, VEGF-A, IL-2R, IL-7R alpha, IL-33R (ST2)||EPX480-20834-901|
|Immuno-Oncology Checkpoint 7-Plex Mouse ProcartaPlex Panel 2||CD137L (4-1BBL), CD152 (CTLA4), CD276 (B7-H3), CD80, PD-1, PD-L1, PD-L2||EPX070-20835-901|
|Immuno-Oncology Checkpoint 4-Plex Mouse ProcartaPlex Panel 1||BTLA, CD27, LAG-3, TIM-3||EPX040-20830-901|
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