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Natural killer cell
Natural killer cell
Natural killer cell
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Natural killer cell
Human natural killer cell, colorized scanning electron micrograph
Details
SystemImmune system
FunctionCytotoxic lymphocyte
Identifiers
MeSHD007694
FMA63147
Anatomical terms of microanatomy

Natural killer cells, also known as NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system. They are a kind of large granular lymphocyte[1][2] (LGL), belong to the rapidly expanding family of known innate lymphoid cells (ILC), and represent 5–20% of all circulating lymphocytes in humans.[3] The role of NK cells is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected cells, stressed cells, tumor cells, and other intracellular pathogens based on signals from several activating and inhibitory receptors. Most immune cells detect the antigen presented on major histocompatibility complex I (MHC-I) on infected cell surfaces, but NK cells can recognize and kill stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named "natural killers" because of the notion that they do not require activation to kill cells that are missing "self" markers of MHC class I.[4] This role is especially important because harmful cells that are missing MHC I markers cannot be detected and destroyed by other immune cells, such as T lymphocyte cells.

NK cells can be identified by the presence of CD56 and the absence of CD3 (CD56+, CD3−).[5] NK cells differentiate from CD127+ common innate lymphoid progenitor,[6] which is downstream of the common lymphoid progenitor from which B and T lymphocytes are also derived.[6][7] NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter into the circulation.[8] NK cells differ from natural killer T cells (NKTs) phenotypically, by origin and by respective effector functions; often, NKT cell activity promotes NK cell activity by secreting interferon gamma. In contrast to NKT cells, NK cells do not express T-cell antigen receptors (TCR) or pan T marker CD3 or surface immunoglobulins (Ig) B cell receptors, but they usually express the surface markers CD16 (FcγRIII) and CD57 in humans, NK1.1 or NK1.2 in C57BL/6 mice. The NKp46 cell surface marker constitutes, at the moment, another NK cell marker of preference being expressed in both humans, several strains of mice (including BALB/c mice) and in three common monkey species.[9][10]

Outside of innate immunity, both activating and inhibitory NK cell receptors play important functional roles in self tolerance and the sustaining of NK cell activity. NK cells also play a role in the adaptive immune response:[11] numerous experiments have demonstrated their ability to readily adjust to the immediate environment and formulate antigen-specific immunological memory, fundamental for responding to secondary infections with the same antigen.[12] The role of NK cells in both the innate and adaptive immune responses is becoming increasingly important in research using NK cell activity as a potential cancer therapy and HIV therapy.[13][14]

Early history

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In early experiments on cell-mediated cytotoxicity against tumor target cells, both in cancer patients and animal models, investigators consistently observed what was termed a "natural" reactivity; that is, a certain population of cells seemed to be able to destroy tumor cells without having been previously sensitized to them. The first published study to assert that untreated lymphoid cells were able to confer a natural immunity to tumors was performed by Dr. Henry Smith at the University of Leeds School of Medicine in 1966,[15] leading to the conclusion that the "phenomenon appear[ed] to be an expression of defense mechanisms to tumor growth present in normal mice." Other researchers had also made similar observations, but as these discoveries were inconsistent with the established model at the time, many initially considered these observations to be artifacts.[16]

By 1973, 'natural killing' activity was established across a wide variety of species, and the existence of a separate lineage of cells possessing this ability was postulated. The discovery that a unique type of lymphocyte was responsible for "natural" or spontaneous cytotoxicity was made in the early 1970s by doctoral student Rolf Kiessling and postdoctoral fellow Hugh Pross, in the mouse,[17] and by Hugh Pross and doctoral student Mikael Jondal in the human.[18][19] The mouse and human work was carried out under the supervision of professors Eva Klein and Hans Wigzell, respectively, of the Karolinska Institute, Stockholm. Kiessling's research involved the well-characterized ability of T lymphocytes to attack tumor cells which they had been previously immunized against. Pross and Jondal were studying cell-mediated cytotoxicity in normal human blood and the effect of the removal of various receptor-bearing cells on this cytotoxicity. Later that same year, Ronald Herberman published similar data with respect to the unique nature of the mouse effector cell.[20] The human data were confirmed, for the most part, by West et al.[21] using similar techniques and the same erythroleukemic target cell line, K562. K562 is highly sensitive to lysis by human NK cells and, over the decades, the K562 51chromium-release assay has become the most commonly used assay to detect human NK functional activity.[22] Its almost universal use has meant that experimental data can be compared easily by different laboratories around the world.

Using discontinuous density centrifugation, and later monoclonal antibodies, natural killing ability was mapped to the subset of large, granular lymphocytes known today as NK cells. The demonstration that density gradient-isolated large granular lymphocytes were responsible for human NK activity, made by Timonen and Saksela in 1980,[23] was the first time that NK cells had been visualized microscopically, and was a major breakthrough in the field.

Types

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NK cells can be classified as CD56bright or CD56dim.[24][25][5] CD56bright NK cells are similar to T helper cells in exerting their influence by releasing cytokines.[25] CD56bright NK cells constitute the majority of NK cells, being found in bone marrow, secondary lymphoid tissue, liver, and skin.[5] CD56bright NK cells are characterized by their preferential killing of highly proliferative cells,[26] and thus might have an immunoregulatory role. CD56dim NK cells are primarily found in the peripheral blood,[5] and are characterized by their cell killing ability.[25] CD56dim NK cells are always CD16 positive (CD16 is the key mediator of antibody-dependent cellular cytotoxicity, or ADCC).[25] CD56bright can transition into CD56dim by acquiring CD16.[5]

NK cells can eliminate virus-infected cells via CD16-mediated ADCC.[27]

Receptors

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The HLA ligand for KIR

NK cell receptors can also be differentiated based on function. Natural cytotoxicity receptors directly induce apoptosis (cell death) after binding to Fas ligand that directly indicate infection of a cell. The MHC-independent receptors (described above) use an alternate pathway to induce apoptosis in infected cells. Natural killer cell activation is determined by the balance of inhibitory and activating receptor stimulation. For example, if the inhibitory receptor signaling is more prominent, then NK cell activity will be inhibited; similarly, if the activating signal is dominant, then NK cell activation will result.[28]

Protein structure of NKG2D

NK cell receptor types (with inhibitory, as well as some activating members) are differentiated by structure, with a few examples to follow:

Protein structure of NKp44

Activating receptors

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  • Ly49 (homodimers), relatively ancient, C-type lectin family receptors, are of multigenic presence in mice, while humans have only one pseudogenic Ly49, the receptor for classical (polymorphic) MHC I molecules.
  • NCR (natural cytotoxicity receptors), type 1 transmembrane proteins of the immunoglobulin superfamily, upon stimulation mediate NK killing and release of IFNγ. They bind viral ligands such as hemagglutinins and hemagglutinin neuraminidases, some bacterial ligands and cellular ligands related to tumour growth such as PCNA.
  • CD16 (FcγIIIA) plays a role in antibody-dependent cell-mediated cytotoxicity; in particular, they bind immunoglobulin G.
  • TLR – Toll-like receptors are receptors that belong in the group of pattern recognition receptors (PRR) which are typical for the cells of innate immunity but are expressed also on NK cells. They recognize PAMPs (pathogen-associated molecular patterns) and DAMPs (damage-associated molecular patterns) as their ligands. These receptors are crucial for the induction of the immune response. TLR induction amplifies the immune response by promoting the production of inflammatory cytokines and chemokines and ultimately leads to the activation of NK cell effector functions.[29] So NK cells directly react to the presence of pathogens in their surroundings. Apart from TLR-10, NK cells express all of the human TLR although in various levels. NK cells express high levels of TLR-1, moderate levels of TLR-2, TLR-3, TLR-5 and TLR-6, low levels of TLR-4, TLR-8 and TLR-9 and very low levels of TLR-7.[30] TLR receptors are constitutionally expressed independently of their state of activation and they cooperate with cytokines and chemokines on the activation of the natural killer cells.[31] These receptors are expressed extracellularly on the cell surface or endosomally inside the endosomes. Apart from TLR-3 and TLR-4, all TLR signal through adaptor protein MyD88 which ultimately leads mainly to the activation of NF-κB. TLR-3 signals through the adaptor protein TRIF and TLR-4 can switch between signaling through MyD88 and TRIF respectively. Induction of different TLR leads to distinct activation of NK cell functions.[32]

Inhibitory receptors

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  • Killer-cell immunoglobulin-like receptors (KIRs) belong to a multigene family of more recently evolved Ig-like extracellular domain receptors; they are present in nonhuman primates, and are the main receptors for both classical MHC I (HLA-A, HLA-B, HLA-C) and nonclassical Mamu-G (HLA-G) in primates. Some KIRs are specific for certain HLA subtypes. Most KIRs are inhibitory and dominant. Regular cells express MHC class 1, so are recognised by KIR receptors and NK cell killing is inhibited.[8]
  • CD94/NKG2 (heterodimers), a C-type lectin family receptor, is conserved in both rodents and primates and identifies nonclassical (also nonpolymorphic) MHC I molecules such as HLA-E. Expression of HLA-E at the cell surface is dependent on the presence of nonamer peptide epitope derived from the signal sequence of classical MHC class I molecules, which is generated by the sequential action of signal peptide peptidase and the proteasome. Though indirect, this is a way to survey the levels of classical (polymorphic) HLA molecules.
  • ILT or LIR (immunoglobulin-like receptor) – are recently discovered members of the Ig receptor family.
  • Ly49 (homodimers) have both activating and inhibitory isoforms. They are highly polymorphic on the population level; though they are structurally unrelated to KIRs, they are the functional homologues of KIRs in mice, including the expression pattern. Ly49s are receptor for classical (polymorphic) MHC I molecules.

Function

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Cytolytic granule mediated cell apoptosis

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NK cells are cytotoxic; small granules in their cytoplasm contain proteins such as perforin and proteases known as granzymes. Upon release in close proximity to a cell slated for killing, perforin forms pores in the cell membrane of the target cell, creating an aqueous channel through which the granzymes and associated molecules can enter, inducing either apoptosis or osmotic cell lysis. The distinction between apoptosis and cell lysis is important in immunology: lysing a virus-infected cell could potentially release the virions, whereas apoptosis leads to destruction of the virus inside. α-defensins, antimicrobial molecules, are also secreted by NK cells, and directly kill bacteria by disrupting their cell walls in a manner analogous to that of neutrophils.[8]

Antibody-dependent cell-mediated cytotoxicity (ADCC)

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Infected cells are routinely opsonized with antibodies for detection by immune cells. Antibodies that bind to antigens can be recognised by FcγRIII (CD16) receptors expressed on NK cells, resulting in NK activation, release of cytolytic granules and consequent cell apoptosis. This is a major killing mechanism of some monoclonal antibodies like rituximab (Rituxan), ofatumumab (Azzera), and others. The contribution of antibody-dependent cell-mediated cytotoxicity to tumor cell killing can be measured with a specific test that uses NK-92, an immortal line of NK-like cells licensed to NantKwest, Inc.: the response of NK-92 cells that have been transfected with a high-affinity Fc receptor are compared to that of the "wild type" NK-92 which does not express the Fc receptor.[33]

Cytokine-induced NK and Cytotoxic T lymphocyte (CTL) activation

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Cytokines play a crucial role in NK cell activation. As these are stress molecules released by cells upon viral infection, they serve to signal to the NK cell the presence of viral pathogens in the affected area. Cytokines involved in NK activation include IL-12, IL-15, IL-18, IL-2, and CCL5. NK cells are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response generates antigen-specific cytotoxic T cells that can clear the infection. NK cells work to control viral infections by secreting IFNγ and TNFα. IFNγ activates macrophages for phagocytosis and lysis, and TNFα acts to promote direct NK tumor cell killing. Patients deficient in NK cells prove to be highly susceptible to early phases of herpes virus infection. [Citation needed]

Missing 'self' hypothesis

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Schematic diagram indicating the complementary activities of cytotoxic T cells and NK cells

For NK cells to defend the body against viruses and other pathogens, they require mechanisms that enable the determination of whether a cell is infected or not. The exact mechanisms remain the subject of current investigation, but recognition of an "altered self" state is thought to be involved. To control their cytotoxic activity, NK cells possess two types of surface receptors: activating receptors and inhibitory receptors, including killer-cell immunoglobulin-like receptors. Most of these receptors are not unique to NK cells and can be present in some T cell subsets, as well.

The inhibitory receptors recognize MHC class I alleles, which could explain why NK cells preferentially kill cells that possess low levels of MHC class I molecules. This mode of NK cell target interaction is known as "missing-self recognition", a term coined by Klas Kärre and co-workers in the late 90s. MHC class I molecules are the main mechanism by which cells display viral or tumor antigens to cytotoxic T cells. A common evolutionary adaptation to this is seen in both intracellular microbes and tumors: the chronic down-regulation of MHC I molecules, which makes affected cells invisible to T cells, allowing them to evade T cell-mediated immunity. NK cells apparently evolved as an evolutionary response to this adaptation (the loss of the MHC eliminates CD4/CD8 action, so another immune cell evolved to fulfill the function).[34]

Tumor cell surveillance

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Natural killer cells often lack antigen-specific cell surface receptors, so are part of innate immunity, i.e. able to react immediately with no prior exposure to the pathogen. In both mice and humans, NKs can be seen to play a role in tumor immunosurveillance by directly inducing the death of tumor cells (NKs act as cytolytic effector lymphocytes), even in the absence of surface adhesion molecules and antigenic peptides. This role of NK cells is critical to immune success particularly because T cells are unable to recognize pathogens in the absence of surface antigens.[4] Tumor cell detection results in activation of NK cells and consequent cytokine production and release.

If tumor cells do not cause inflammation, they will also be regarded as self and will not induce a T cell response. A number of cytokines are produced by NKs, including tumor necrosis factor α (TNFα), IFNγ, and interleukin (IL-10). TNFα and IL-10 act as proinflammatory and immunosuppressors, respectively. The activation of NK cells and subsequent production of cytolytic effector cells impacts macrophages, dendritic cells, and neutrophils, which subsequently enables antigen-specific T and B cell responses. Instead of acting via antigen-specific receptors, lysis of tumor cells by NK cells is mediated by alternative receptors, including NKG2D, NKp44, NKp46, NKp30, and DNAM.[28] NKG2D is a disulfide-linked homodimer which recognizes a number of ligands, including ULBP and MICA, which are typically expressed on tumor cells. The role of dendritic cell—NK cell interface in immunobiology have been studied and defined as critical for the comprehension of the complex immune system.[citation needed]

NK cells, along with macrophages and several other cell types, express the Fc receptor (FcR) molecule (FC-gamma-RIII = CD16), an activating biochemical receptor that binds the Fc portion of IgG class antibodies. This allows NK cells to target cells against which there has been a humoral response and to lyse cells through antibody-dependant cytotoxicity (ADCC). This response depends on the affinity of the Fc receptor expressed on NK cells, which can have high, intermediate, and low affinity for the Fc portion of the antibody. This affinity is determined by the amino acid in position 158 of the protein, which can be phenylalanine (F allele) or valine (V allele). Individuals with high-affinity FcRgammRIII (158 V/V allele) respond better to antibody therapy. This has been shown for lymphoma patients who received the antibody Rituxan. Patients who express the 158 V/V allele had a better antitumor response. Only 15–25% of the population expresses the 158 V/V allele. To determine the ADCC contribution of monoclonal antibodies, NK-92 cells (a "pure" NK cell line) has been transfected with the gene for the high-affinity FcR.

Clearance of senescent cells

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Natural killer cells (NK cells) and macrophages play a major role in clearance of senescent cells.[35] Natural killer cells directly kill senescent cells, and produce cytokines which activate macrophages which remove senescent cells.[35]

Natural killer cells can use NKG2D receptors to detect senescent cells, and kill those cells using perforin pore-forming cytolytic protein.[36] CD8+ cytotoxic T-lymphocytes also use NKG2D receptors to detect senescent cells, and promote killing similar to NK cells.[36] For example, in patients with Parkinson's disease, levels of Natural killer cells are elevated as they degrade alpha-synuclein aggregates, destroy senescent neurons, and attenuate the neuroinflammation by leukocytes in the central nervous system.[37]

Adaptive features of NK cells—"memory-like", "adaptive" and memory NK cells

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Main article: Adaptive NK cells

The ability to generate memory cells following a primary infection and the consequent rapid immune activation and response to succeeding infections by the same antigen is fundamental to the role that T and B cells play in the adaptive immune response. For many years, NK cells have been considered to be a part of the innate immune system. However, recently increasing evidence suggests that NK cells can display several features that are usually attributed to adaptive immune cells (e.g. T cell responses) such as dynamic expansion and contraction of subsets, increased longevity and a form of immunological memory, characterized by a more potent response upon secondary challenge with the same antigen.[38][39] In mice, the majority of research was carried out with murine cytomegalovirus (MCMV) and in models of hapten-hypersensitivity reactions. Especially, in the MCMV model, protective memory functions of MCMV-induced NK cells were discovered[40] and direct recognition of the MCMV-ligand m157 by the receptor Ly49 was demonstrated to be crucial for the generation of adaptive NK cell responses.[40] In humans, most studies have focused on the expansion of an NK cell subset carrying the activating receptor NKG2C (KLRC2). Such expansions were observed primarily in response to human cytomegalovirus (HCMV),[41] but also in other infections including Hantavirus, Chikungunya virus, HIV, or viral hepatitis. However, whether these virus infections trigger the expansion of adaptive NKG2C+ NK cells or whether other infections result in re-activation of latent HCMV (as suggested for hepatitis [42]), remains a field of study. Notably, recent research suggests that adaptive NK cells can use the activating receptor NKG2C (KLRC2) to directly bind to human cytomegalovirus-derived peptide antigens and respond to peptide recognition with activation, expansion, and differentiation,[43] a mechanism of responding to virus infections that was previously only known for T cells of the adaptive immune system.

NK cell function in pregnancy

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As the majority of pregnancies involve two parents who are not tissue-matched, successful pregnancy requires the mother's immune system to be suppressed. NK cells are thought to be an important cell type in this process.[44] These cells are known as "uterine NK cells" (uNK cells) and they differ from peripheral NK cells. They are in the CD56bright NK cell subset, potent at cytokine secretion, but with low cytotoxic ability and relatively similar to peripheral CD56bright NK cells, with a slightly different receptor profile.[44] These uNK cells are the most abundant leukocytes present in utero in early pregnancy, representing about 70% of leukocytes here, but from where they originate remains controversial.[45]

These NK cells have the ability to elicit cell cytotoxicity in vitro, but at a lower level than peripheral NK cells, despite containing perforin.[46] Lack of cytotoxicity in vivo may be due to the presence of ligands for their inhibitory receptors. Trophoblast cells downregulate HLA-A and HLA-B to defend against cytotoxic T cell-mediated death. This would normally trigger NK cells by missing self recognition; however, these cells survive. The selective retention of HLA-E (which is a ligand for NK cell inhibitory receptor NKG2A) and HLA-G (which is a ligand for NK cell inhibitory receptor KIR2DL4) by the trophoblast is thought to defend it against NK cell-mediated death.[44]

Uterine NK cells have shown no significant difference in women with recurrent miscarriage compared with controls. However, higher peripheral NK cell percentages occur in women with recurrent miscarriages than in control groups.[47]

NK cells secrete a high level of cytokines which help mediate their function. NK cells interact with HLA-C to produce cytokines necessary for trophoblastic proliferation. Some important cytokines they secrete include TNF-α, IL-10, IFN-γ, GM-CSF and TGF-β, among others.[44] For example, IFN-γ dilates and thins the walls of maternal spiral arteries to enhance blood flow to the implantation site.[48]

NK cell evasion by tumor cells

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By shedding decoy NKG2D soluble ligands, tumor cells may avoid immune responses. These soluble NKG2D ligands bind to NK cell NKG2D receptors, activating a false NK response and consequently creating competition for the receptor site.[4] This method of evasion occurs in prostate cancer. In addition, prostate cancer tumors can evade CD8 cell recognition due to their ability to downregulate expression of MHC class 1 molecules. This example of immune evasion actually highlights NK cells' importance in tumor surveillance and response, as CD8 cells can consequently only act on tumor cells in response to NK-initiated cytokine production (adaptive immune response).[49]

Excessive NK cells

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Experimental treatments with NK cells have resulted in excessive cytokine production, and even septic shock. Depletion of the inflammatory cytokine interferon gamma reversed the effect.[citation needed]

Applications

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Anticancer therapy

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Tumor-infiltrating NK cells have been reported to play a critical role in promoting drug-induced cell death in human triple-negative breast cancer.[50] Since NK cells recognize target cells when they express nonself HLA antigens (but not self), autologous (patients' own) NK cell infusions have not shown any antitumor effects. Instead, investigators are working on using allogeneic cells from peripheral blood, which requires that all T cells be removed before infusion into the patients to remove the risk of graft versus host disease, which can be fatal. This can be achieved using an immunomagnetic column (CliniMACS). In addition, because of the limited number of NK cells in blood (only 10% of lymphocytes are NK cells), their number needs to be expanded in culture. This can take a few weeks and the yield is donor-dependent.

CAR-NK cells

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Chimeric antigen receptors (CARs) are genetically modified receptors targeting cell surface antigens that provide a valuable approach to enhance effector cell efficacy. CARs induce high-affinity binding of effector cells carrying these receptors to cells expressing the target antigen, thereby lowering the threshold for cellular activation and inducing effector functions.[51]

CAR T cells are now a fairly well-known cell therapy. However, wider use is limited by several fundamental problems: The high cost of CAR T cell therapy, which is due to the need to generate specific CAR T cells for each patient; the necessity to use only autologous T cells, due to the high risk of GvHD if allogeneic T cells are used; the inability to reinfuse CAR T cells if the patient relapses or low CAR T cell survival is observed; CAR T therapy also has a high toxicity, mainly due to IFN-γ production and subsequent induction of CRS (cytokine release syndrome) and/or neurotoxicity.[52]

The use of CAR NK cells is not limited by the need to generate patient-specific cells, and at the same time, GvHD is not caused by NK cells, thus obviating the need for autologous cells.[53] Toxic effects of CAR T therapy, such as CSR, have not been observed with the use of CAR NK cells. Thus, NK cells are considered an interesting "off-the-shelf" product option. Compared to CAR T cells, CAR NK cells retain unchanged expression of NK cell activating receptors. Thus, NK cells recognize and kill tumor cells even if, due to a tumor-escape strategy on tumor cells, ligand expression for the CAR receptor is downregulated.[52]

NK cells derived from umbilical cord blood have been used to generate CAR.CD19 NK cells. These cells are capable of self-producing the cytokine IL-15, thereby enhancing autocrine/paracrine expression and persistence in vivo. Administration of these modified NK cells is not associated with the development of CSR, neurotoxicity, or GvHD.[51]

The FT596 product is the first "Off-the-Shelf", universal, and allogenic CAR NK cellular product derived from iPSCs to be authorized for use in clinical studies in the USA.[54] It consists of an anti-CD19 CAR optimized for NK cells with a transmembrane domain for the NKG2D activation receptor, a 2B4 costimulatory domain and a CD3ζ signaling domain. Two additional key components were added: 1) a high-affinity, non-cleavable Fc receptor CD16 (hnCD16) that enables tumor targeting and enhanced antibody-dependent cell cytotoxicity without negative regulation, combined with 2) a therapeutic monoclonal antibody targeting tumor cells and an IL-15/IL-15 receptor fusion protein (IL-15RF) promoting cytokine-independent persistence.[55]

NK-92 cells

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A more efficient way to obtain high numbers of NK cells is to expand NK-92 cells, an NK cell line with all the characteristics of highly active blood Natural Killer (NK) cells but with much broader and higher cytotoxicity. NK-92 cells grow continuously in culture and can be expanded to clinical-grade numbers in bags or bioreactors.[56] Clinical studies have shown NK-92 cells to be safe and to exhibit anti-tumor activity in patients with lung or pancreatic cancer, melanoma, and lymphoma.[57][58] When NK-92 cells originate from a patient with lymphoma, they must be irradiated prior to infusion.[59][60] Efforts, however, are being made to engineer the cells to eliminate the need for irradiation. The irradiated cells maintain full cytotoxicity. NK-92 are allogeneic (from a donor different from the recipient), but in clinical studies have not been shown to elicit significant host reaction.[61][62]

Unmodified NK-92 cells lack CD-16, making them unable to perform antibody-dependent cellular cytotoxicity (ADCC); however, the cells have been engineered to express a high affinity Fc-receptor (CD16A, 158V) genetically linked to IL-2 that is bound to the endoplasmic reticulum (ER).[63][64] These high affinity NK-92 cells can perform ADCC and have greatly expanded therapeutic utility.[65][66][67][68]

NK-92 cells have also been engineered to expressed chimeric antigen receptors (CARs), in an approach similar to that used for T cells. An example of this is an NK-92 derived cell engineered with both a CD16 and an anti-PD-L1 CAR; currently in clinical development for oncology indications.[69][70][71] A clinical grade NK-92 variant that expresses a CAR for HER2 (ErbB2) has been generated[72] and is in a clinical study in patients with HER2 positive glioblastoma.[73] Several other clinical grade clones have been generated expressing the CARs for PD-L1, CD19, HER-2, and EGFR.[74][66] PD-L1 targeted high affinity NK cells have been given to a number of patients with solid tumors in a phase I/II study, which is underway.[75]

NKG2D-Fc fusion protein

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In a study at Boston Children's Hospital, in coordination with Dana–Farber Cancer Institute, in which immunocompromised mice had contracted lymphomas from EBV infection, an NK-activating receptor called NKG2D was fused with a stimulatory Fc portion of the EBV antibody. The NKG2D-Fc fusion proved capable of reducing tumor growth and prolonging survival of the recipients. In a transplantation model of LMP1-fueled lymphomas, the NKG2D-Fc fusion proved capable of reducing tumor growth and prolonging survival of the recipients.

In Hodgkin lymphoma, in which the malignant Hodgkin Reed-Sternberg cells are typically HLA class I deficient, immune evasion is in part mediated by skewing towards an exhausted PD-1hi NK cell phenotype, and re-activation of these NK cells appears to be one mechanism of action induced by checkpoint-blockade.[76]

TLR ligands

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Signaling through TLR can effectively activate NK cell effector functions in vitro and in vivo. TLR ligands are then potentially able to enhance NK cell effector functions during NK cell anti-tumor immunotherapy.[30]

Trastuzumab is a monoclonal anti-HER2 antibody that is used as a treatment of the HER2+ breast cancer.[77] NK cells are an important part of the therapeutical effect of trastuzumab as NK cells recognize the antibody coated cancer cells which induces ADCC (antibody-dependent cellular cytotoxicity) reaction. TLR ligand is used in addition to trastuzumab as a means to enhance its effect. The polysaccharide krestin, which is extracted from Trametes versicolor, is a potent ligand of TLR-2 and so activates NK cells, induces the production of IFNg and enhances the ADCC caused by recognition of trastuzumab-coated cells.[78]

Stimulation of TLR-7 induces the expression of IFN type I and other pro-inflammatory cytokines like IL-1b, IL-6 and IL-12. Mice suffering with NK cell-sensitive lymphoma RMA-S were treated with SC1 molecule. SC1 is novel small-molecule TLR-7 agonist and its repeated administration reportedly activated NK cells in TLR-7- and IFN type I- dependent manner thus reversing the NK cell anergy which ultimately lead to lysis of the tumor.[79]

VTX-2337 is a selective TLR-8 agonist and together with monoclonal antibody cetuximab it was used as a potential therapy for the treatment of recurrent or metastatic SCCHN. Results have shown that the NK cells had become more reactive to the treatment with cetuximab antibody upon pretreatment with VTX-2337. This indicates that the stimulation of TLR-8 and subsequent activation of inflammasome enhances the CD-16 mediated ADCC reaction in patients treated with cetuximab antibody.[80]

NK cells play a role in controlling HIV-1 infection. TLR are potent enhancers of innate antiviral immunity and potentially can reverse HIV-1 latency. Incubation of peripheral blood mononuclear cells with novel potent TLR-9 ligand MGN1703 have resulted in enhancement of NK cell effector functions, thus significantly inhibiting the spread of HIV-1 in culture of autologous CD4+ T-cells. The stimulation of TLR-9 in NK cells induced a strong antiviral innate immune response, an increase in HIV-1 transcription (indicating the reverse in latency of the virus) and it also boosted the NK cell-mediated suppression of HIV-1 infections in autologous CD4+ T cells.[81]

New findings

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Innate resistance to HIV

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Recent research suggests specific KIR-MHC class I gene interactions might control innate genetic resistance to certain viral infections, including HIV and its consequent development of AIDS.[8] Certain HLA allotypes have been found to determine the progression of HIV to AIDS; an example is the HLA-B57 and HLA-B27 alleles, which have been found to delay progression from HIV to AIDS. This is evident because patients expressing these HLA alleles are observed to have lower viral loads and a more gradual decline in CD4+ T cells numbers. Despite considerable research and data collected measuring the genetic correlation of HLA alleles and KIR allotypes, a firm conclusion has not yet been drawn as to what combination provides decreased HIV and AIDS susceptibility.

NK cells can impose immune pressure on HIV, which had previously been described only for T cells and antibodies.[82] HIV mutates to avoid NK cell detection.[82]

Tissue-resident NK cells

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Most of our current knowledge is derived from investigations of mouse splenic and human peripheral blood NK cells. However, in recent years tissue-resident NK cell populations have been described.[83][84] These tissue-resident NK cells share transcriptional similarity to tissue-resident memory T cells described previously. However, tissue-resident NK cells are not necessarily of the memory phenotype, and in fact, the majority of the tissue-resident NK cells are functionally immature.[85] These specialized NK-cell subsets can play a role in organ homeostasis. For example, NK cells are enriched in the human liver with a specific phenotype and take part in the control of liver fibrosis.[86][87] Tissue-resident NK cells have also been identified in sites like bone marrow, spleen and more recently, in lung, intestines and lymph nodes. In these sites, tissue-resident NK cells may act as reservoir for maintaining immature NK cells in humans throughout life.[85]

Adaptive NK cells against leukemia targets

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Natural killer cells are being investigated as an emerging treatment for patients with acute myeloid leukemia (AML), and cytokine-induced memory-like NK cells have shown promise with their enhanced antileukemia functionality.[88] It has been shown that this kind of NK cell has enhanced interferon-γ production and cytotoxicity against leukemia cell lines and primary AML blasts in patients.[88] During a phase 1 clinical trial, five out of nine patients exhibited clinical responses to the treatment, and four patients experienced a complete remission, which suggests that these NK cells have major potential as a successful translational immunotherapy approach for patients with AML in the future.[88]

See also

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References

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Further reading

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Natural killer (NK) cells are large granular lymphocytes that form a critical component of the , specializing in the rapid recognition and destruction of virus-infected cells, tumor cells, and other abnormal targets without requiring prior antigen-specific sensitization or (MHC) restriction. Originating from hematopoietic stem cells in the , NK cells undergo multilineage differentiation through a series of developmental stages, including common lymphoid progenitors and NK cell precursors, maturing primarily in the and secondary lymphoid tissues before entering circulation. Their effector functions are governed by a dynamic balance of activating and inhibitory receptors on the cell surface, such as natural cytotoxicity receptors (e.g., NKp46, NKp30) for and killer cell immunoglobulin-like receptors (KIRs) for inhibition, allowing NK cells to distinguish healthy "self" cells from stressed or altered targets via the "missing-" . In humans, NK cells are phenotypically divided into two main subsets: the cytokine-producing CD56bright population, which predominates in lymph nodes and supports adaptive immunity through interferon-gamma (IFN-γ) secretion, and the highly cytotoxic CD56dimCD16+ subset, which circulates in blood and mediates (ADCC). Beyond direct killing via perforin and granzymes, NK cells bridge innate and adaptive responses by interacting with dendritic cells, T cells, and macrophages, while dysregulation of NK cell activity is implicated in viral infections, autoimmune disorders, and cancer progression.

Discovery and history

Early observations

In the , pioneering experiments in transplantation using irradiated mice established the feasibility of rescuing lethally irradiated animals through bone marrow infusion, laying the groundwork for understanding hematopoietic reconstitution. However, subsequent studies revealed complexities, including unexpected resistance to allograft engraftment in certain settings, highlighting an innate mechanism of rejection independent of adaptive immunity. These findings distinguished the effectors from macrophages, which primarily phagocytose rather than lyse target cells, and from T cells, which require prior . The phenomenon of hybrid resistance was first systematically documented in the early 1960s by Georges Cudkowicz, who reported that semi-allogeneic mice rejected parental grafts despite lacking classical (MHC) barriers that typically permit graft acceptance. Later collaborations with Milton Bennett further explored this. In these studies, irradiated s resisted transplantation of as few as 10^4 parental marrow cells, a resistance that developed rapidly without prior immunization and was radioresistant, further differentiating it from T cell-mediated immunity. This H-2-linked but non-classical rejection mechanism provided early evidence of specialized innate effectors capable of surveilling hematopoietic cells. Concurrent 1960s experiments in mice uncovered spontaneous, non-antibody-dependent against tumor cells, particularly lymphomas, in unprimed and peripheral blood populations. Eva Klein and colleagues observed that normal mouse lysed certain syngeneic and allogeneic tumor targets without complement or , a activity resistant to treatments that depleted T cells or macrophages, such as anti-theta serum or silica particles. These observations underscored an innate population distinct from adaptive effectors, active against transformed cells lacking normal regulatory signals. In humans, similar non-antibody-dependent killing was noted in the late through assays showing peripheral blood lymphocytes spontaneously lysing cultured tumor cell lines, such as those derived from Burkitt's lymphoma, without prior exposure or humoral factors. These human studies paralleled murine findings and suggested a conserved innate cytotoxic pathway, later recognized as precursors to formal NK cell identification.

Identification and nomenclature

Natural killer (NK) cells were first identified in 1975 through independent studies in mice and s, marking a pivotal moment in understanding innate immune cytotoxicity. In mice, Rolf Kiessling, Eva Klein, and Hans Wigzell at the described a population of bone marrow-derived lymphoid cells capable of spontaneously lysing Moloney virus-induced tumor cells without prior immunization or antigen-specific priming. Concurrently, Ronald B. Herberman and colleagues at the reported similar spontaneous cytotoxic activity in human peripheral blood lymphocytes against a human cell line, distinguishing these effectors from conventional T and B lymphocytes. These early discoveries characterized the cells as "null lymphocytes" or "null cells," a term reflecting their lack of established surface markers for T cells (such as theta antigen in mice or E-rosette formation in humans) or B cells (such as surface immunoglobulin). The null cell designation arose from functional assays showing enrichment of in lymphocyte fractions depleted of T and B cells via rosetting techniques or antibody panning, confirming their distinct identity within the lymphoid lineage. The nomenclature "natural killer" was coined in these 1975 studies to emphasize the cells' innate, non-adaptive killing mechanism, which occurred spontaneously against tumor targets without the need for or deliberate . This term gained widespread acceptance by the late , as evidenced in key publications reviewing the phenomenon, and it highlighted the cells' role in immediate host defense. During the 1980s, phenotypic identification advanced with the recognition of specific surface markers: (FcγRIII, the low-affinity IgG ) was established as a defining feature of NK cells in 1983, enabling flow cytometric isolation of cytotoxic effectors. Subsequently, CD56 () was identified in 1986 as another key marker, allowing delineation of NK cell subsets such as CD16+ CD56dim and CD56bright populations based on expression levels. These markers, validated through studies, solidified the formal identification of NK cells distinct from other lymphocytes.

Origin and development

Hematopoietic lineage

Natural killer (NK) cells originate from hematopoietic stem cells (HSCs) within the bone marrow, where they derive specifically from common lymphoid progenitors (CLPs). These CLPs represent a committed stage in lymphoid differentiation, giving rise to all lymphoid lineages, including NK cells, but NK cell precursors diverge early by committing to the NK lineage without the need for antigen receptor gene rearrangements characteristic of T and B cell development. This distinction ensures that NK cells remain part of the innate immune system, bypassing the adaptive processes of V(D)J recombination required for T cell receptors and B cell immunoglobulins. Commitment to the NK cell lineage is tightly regulated by key transcription factors, including E4BP4 (also known as NFIL3), which is essential for initiating NK progenitor specification from CLPs by promoting the expression of downstream genes critical for development. TOX, a member of the high-mobility group box family, further supports this commitment by enabling the survival and differentiation of early NK progenitors, independent of pathways leading to T or B cells. Additionally, Id2, a helix-loop-helix inhibitor of basic helix-loop-helix transcription factors, is indispensable for maintaining NK cell fate by suppressing alternative differentiation programs, such as those toward B cells.00533-6) During embryonic development, NK cell origins differ from those in adults, with the fetal liver serving as the primary site of hematopoiesis and NK progenitor emergence as early as gestational week 9 in humans. Fetal liver-derived progenitors contribute significantly to the initial pool of circulating NK cells, transitioning to dominance postnatally as the primary hematopoietic niche for sustained NK cell production in adults. This shift reflects the sequential colonization of hematopoietic sites during , ensuring robust innate immunity from fetal stages onward. NK cells develop through thymus-independent pathways, primarily within the microenvironment.

Maturation and education

Natural killer (NK) cells arise from common lymphoid progenitors derived from hematopoietic stem cells and undergo maturation primarily within specialized niches to acquire functional competence. In humans, NK cell maturation follows a sequential progression from an immature stage characterized by high expression of CD56 (CD56bright NK cells) to a mature stage with low CD56 expression (CD56dim NK cells). CD56bright NK cells represent an early developmental phase, featuring high levels of CD94/NKG2 receptors and potent production capacity but limited cytotoxic potential. As maturation advances, CD56dim NK cells emerge, marked by acquisition of (FcγRIII) for enhanced and increased expression of perforin and granzymes for direct killing. Mature CD56dim CD16+ NK cells express medium to high levels of CD11b, an integrin associated with adhesion and cytotoxic activity, while CD56bright subsets express low or none; subsets can be further distinguished by CD27 expression, with NK cells comprising 10-15% of lymphocytes and most being CD11b+. This transition also involves downregulation of CD117 (c-Kit) and upregulation of maturation markers like CD57. In mice, NK cell maturation parallels the human process but is delineated by CD27 and CD11b expression levels, with immature stages identified as CD27high CD11blow and mature stages as CD27low CD11bhigh, the latter expressing the NK1.1 marker in relevant strains. Immature murine NK cells exhibit interferon-γ production similar to CD56bright cells, while mature ones gain robust akin to CD56dim counterparts. Maturation occurs predominantly in the bone marrow, where NK cell precursors interact with stromal cells and cytokines like IL-15 to support differentiation, though secondary lymphoid tissues such as lymph nodes, , and tonsils also serve as critical niches for further development and terminal maturation in both humans and mice. These peripheral sites provide additional microenvironmental cues, including IL-15 trans-presentation, to refine NK cell subsets. A pivotal aspect of NK cell maturation is the education or licensing , which ensures self-tolerance by calibrating responsiveness through interactions between germline-encoded inhibitory receptors and self-major histocompatibility complex (MHC) class I molecules. In humans, killer-cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A recognize specific HLA class I alleles, while in mice, Ly49 receptors interact with H-2 MHC class I; this engagement during development "licenses" NK cells to respond effectively to target cells lacking self-MHC (missing-self recognition). Unlicensed NK cells, which fail to engage self-MHC due to mismatched inhibitory receptors, remain hyporesponsive or anergic, thereby preventing but limiting their effector functions. This rheostat-like calibration tunes NK cell potency proportionally to the strength of self-MHC interactions.

Characteristics and subtypes

Morphological features

Natural killer (NK) cells are a subset of large granular lymphocytes (LGLs), distinguished by their medium-to-large size and prominent cytoplasmic inclusions. In humans and mice, these cells typically range from 12 to 15 μm in diameter, larger than typical small lymphocytes (7–10 μm), with a round to reniform nucleus occupying about half the cell volume and eccentric placement. The abundant pale blue , visible under light microscopy, contains 5–20 azurophilic granules that stain positively with Wright-Giemsa, reflecting their lysosomal origin. These granules primarily store perforin, which forms pores in target cell membranes, and granzymes, serine proteases that induce . NK cells are phenotypically defined by the absence of T cell receptor (TCR) and B cell receptor (BCR) complexes, lacking CD3 expression that is characteristic of T lymphocytes. In humans, mature NK cells express CD56 (neural cell adhesion molecule) as a pan-marker, with subsets further delineated by CD16 (FcγRIII) expression: CD56bright CD16− cells predominate in lymphoid tissues and emphasize cytokine production, while CD56dim CD16+ cells are more abundant in blood and exhibit enhanced cytotoxicity. In mice, NK cells are identified by CD3− expression combined with NK1.1 (in certain strains like C57BL/6) or DX5 (CD49b, an integrin α2 subunit), markers that highlight their innate lymphoid identity without antigen-specific receptors.01326-4/fulltext) Ultrastructural analysis via electron microscopy reveals distinctive cytoplasmic features that underpin NK cell function. The is rich in free ribosomes and polyribosomes, facilitating rapid protein synthesis, alongside a well-developed Golgi apparatus and rough involved in the and trafficking of cytokines and lytic granule components. Mitochondria are sparse but functional, supporting energy demands during activation. These organelles collectively enable NK cells to form and release cytotoxic granules efficiently, contributing to their role in innate immune surveillance.

Tissue-resident populations

Tissue-resident natural killer (NK) cells represent specialized subsets that persist in non-lymphoid organs, distinct from circulating NK cells, and exhibit adaptations to local microenvironments for sustained immune surveillance. These populations maintain tissue-specific phenotypes and functions, often self-renewing locally without reliance on continuous input. In the liver, tissue-resident NK cells (LrNK) are notable for their expression of tumor necrosis factor-related apoptosis-inducing ligand (), which enables them to target and eliminate activated hepatic stellate cells, thereby regulating liver progression. This TRAIL-mediated is NKG2D-dependent and plays a protective role against fibrotic diseases. LrNK cells constitute a significant proportion of hepatic NK cells, displaying enhanced IFN-γ production and longevity compared to circulating counterparts. Salivary gland-resident NK cells form a unique subset characterized by hyporesponsiveness to viral infections, with distinct surface markers like low and high expression, allowing them to prioritize tolerance in this mucosal site while retaining cytotoxic potential. In the gut, intraepithelial NK cell variants reside within the epithelial layer, exhibiting heightened cytolytic activity in early life and contributing to barrier immunity; these cells often overlap with ILC1-like profiles but maintain classical NK features such as perforin and granzyme expression.02231-6/fulltext) Transcriptionally, tissue-resident NK cells differ from circulating NK cells, often showing lower Eomesodermin (Eomes) expression, which correlates with immature yet tissue-adapted states, particularly in murine models where liver trNK cells exhibit Eomes^low T-bet^high profiles. This transcriptional divergence supports their residency and specialized effector functions, such as localized over systemic responses. Epigenetically, these cells display distinct patterns and accessibility at loci regulating tissue-specific genes, including those for molecules like CD49a, ensuring stable residency and functional plasticity in diverse organs. Developmentally, many tissue-resident NK populations originate during fetal stages from progenitors in the or fetal liver, seeding organs early in . For instance, liver-resident NK cells emerge from fetal liver hematopoietic waves independent of adult contributions, enabling lifelong self-maintenance through local proliferation. While conventional NK cells mature primarily in the , tissue-resident subsets like those in the liver and gut can develop via hematopoietic stem cell-independent pathways in the , highlighting their ontogenic divergence.00388-5)

Receptors and recognition

Activating receptors

Natural killer (NK) cells express a variety of activating receptors that recognize stress-induced or altered self-ligands on target cells, such as virally infected or transformed cells, thereby triggering cytotoxic responses and production. These receptors initiate signaling cascades that promote NK cell activation, including and target cell lysis. Key families include the natural cytotoxicity receptors (NCRs) and other germline-encoded receptors like , DNAM-1, and CD16. The NCRs, comprising NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1), are immunoglobulin-like transmembrane glycoproteins primarily expressed on NK cells and are critical for natural cytotoxicity against tumor and virus-infected cells. NKp46, the first identified NCR, binds to ligands such as viral hemagglutinins on influenza-infected cells and proteoglycans on tumor cells, facilitating direct recognition and lysis. NKp30 engages tumor-associated ligands like B7-H6, a member of the B7 family expressed on various malignancies, and certain viral proteins, contributing to antitumor and antiviral responses. NKp44, predominantly found on activated or decidual NK cells, interacts with (PCNA) on tumor cells, which inhibits NK cell-mediated killing, and hemagglutinins from , which enhances NK cell-mediated killing. Additional activating receptors include , a C-type lectin-like receptor that pairs with the DAP10 in humans, binding to class I-related molecules such as , MICB, and the UL16-binding proteins (ULBPs), which are upregulated on stressed or malignant cells. DNAM-1 (CD226), an member, recognizes nectin-like molecules including CD112 (nectin-2) and ( receptor), which are overexpressed on tumor cells, thereby promoting NK cell adhesion and cytotoxicity. (), a low-affinity , mediates (ADCC) by binding the Fc portion of IgG antibodies coating target cells, enabling NK cells to lyse opsonized pathogens or tumors. Upon ligand engagement, these receptors transduce signals through immunoreceptor tyrosine-based activation motifs (ITAMs) or other motifs that recruit proteins. For instance, NCRs and certain others associate with DAP12, which contains ITAMs that, upon , recruit Syk family kinases, leading to activation of Cγ and downstream pathways. NKG2D and DNAM-1 primarily signal via DAP10, which lacks ITAMs but engages (PI3K), resulting in AKT activation and cytoskeletal reorganization for . Collectively, these pathways culminate in calcium mobilization, granule , and perforin/granzyme release, enabling target cell elimination.

Inhibitory receptors

Inhibitory receptors on natural killer (NK) cells deliver negative signals upon engagement with self-major histocompatibility complex ( molecules, thereby suppressing NK cell activation to preserve immune and self-tolerance. These receptors dominate NK cell regulation in both humans and mice, with their ligation overriding activating signals to prevent . In humans, killer-cell immunoglobulin-like receptors (KIRs) constitute the primary family of MHC-specific inhibitory receptors expressed on NK cells. Inhibitory KIRs such as KIR2DL1, KIR2DL2, and KIR2DL3 possess three immunoglobulin-like domains and bind to distinct epitopes on allotypes: KIR2DL1 recognizes HLA-C alleles with a at position 80 (C2 group), while KIR2DL2 and KIR2DL3 bind HLA-C alleles with at position 80 (C1 group). Additionally, leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), also known as CD85j or ILT-2, functions as an inhibitory receptor on a subset of NK cells by binding , a nonclassical HLA class I molecule expressed at immune-privileged sites like the . In mice, the orthologous inhibitory receptors include members of the Ly49 family, which are C-type lectin-like proteins encoded in the natural killer complex on chromosome 6. Inhibitory Ly49 receptors, such as Ly49A, Ly49C/I, and Ly49G2, specifically recognize alleles of classical molecules (H-2D, H-2K, or H-2L), with each subtype exhibiting distinct specificities that calibrate NK cell responses during development. The NKG2A/CD94 heterodimer, a conserved inhibitory receptor across , binds to the nonclassical MHC molecule in humans (Qa-1^b in mice), which displays peptides derived from the leader sequences of other HLA class I proteins. The cytoplasmic tails of these inhibitory receptors contain one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Ligand binding induces ITIM tyrosine phosphorylation by Src family kinases, enabling recruitment of Src homology 2 (SH2) domain-containing protein tyrosine phosphatases SHP-1 (PTPN6) and SHP-2 (PTPN11). SHP-1 predominantly dephosphorylates key activatory signaling intermediates, such as Vav1 and phospholipase Cγ, while SHP-2 modulates ERK and PI3K pathways; together, they counteract proximal signals from activating receptors, inhibiting NK cell cytotoxicity and cytokine release. This inhibitory signaling underpins missing-self recognition, whereby NK cells are licensed to target MHC-deficient cells while sparing healthy self-expressing ones.

Mechanisms of action

Direct cytotoxicity

Natural killer (NK) cells mediate direct cytotoxicity against virus-infected and transformed cells primarily through two antibody-independent pathways: the release of cytotoxic granules containing perforin and granzymes, and the expression of death receptor ligands such as Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL). These mechanisms enable rapid elimination of aberrant cells without prior sensitization, distinguishing NK cells from adaptive cytotoxic lymphocytes. The granule-mediated pathway predominates in mature NK cells, accounting for the majority of lysis in many experimental models, while death ligand engagement provides an alternative or complementary route, particularly against certain resistant targets. In the perforin/granzyme pathway, upon recognition of a target, NK cells polarize their lytic granules toward the immunological synapse and release their contents via exocytosis. Perforin, a pore-forming protein discovered in the early 1980s, oligomerizes in the target cell membrane to create transmembrane pores approximately 10-20 nm in diameter, allowing entry of serine proteases like granzyme B. Once inside, granzyme B cleaves and activates Bid, leading to mitochondrial outer membrane permeabilization, and directly processes caspases such as caspase-3 and -7 to induce apoptosis. This process is calcium-dependent and highly efficient, with perforin-deficient NK cells exhibiting severely impaired cytotoxicity in vivo. Studies in perforin-knockout mice have demonstrated that this pathway is essential for NK cell control of certain viral infections and tumors, highlighting its non-redundant role in innate immunity. The death ligand pathway involves surface expression or soluble release of FasL and TRAIL by activated NK cells, which engage corresponding receptors on target cells to trigger extrinsic apoptosis. FasL binds Fas (CD95) to recruit the death-inducing signaling complex (DISC), activating caspase-8 and downstream effector caspases, while TRAIL interacts with death receptors DR4 and DR5 to similarly initiate caspase cascades. Seminal work identified functional FasL expression on freshly isolated human NK cells, enabling lysis of Fas-sensitive targets independently of granules. Likewise, NK cells were established as major producers of TRAIL, with its expression upregulated upon activation and contributing to cytotoxicity against TRAIL-sensitive cells like certain tumor lines. This pathway is particularly relevant for targets lacking susceptibility to granzymes, such as some immature dendritic cells or virally infected cells expressing death receptors. Target selection for direct cytotoxicity relies on the integration of activating and inhibitory signals, where stressed cells upregulate ligands for NK activating receptors while downregulating to evade inhibition. For instance, , a key activating receptor, binds stress-inducible ligands such as and MICB, which are expressed on infected or transformed cells due to DNA damage or oncogenic stress. This recognition, first demonstrated in 1999, triggers NK cell activation and or death ligand upregulation, ensuring selective killing of unhealthy cells. Cytokines like IL-15 can briefly prime resting NK cells, enhancing receptor expression and granule content for more potent responses.

Antibody-dependent cytotoxicity

Antibody-dependent cytotoxicity (ADCC) is a key mechanism by which natural killer (NK) cells eliminate antibody-opsonized target cells, such as virus-infected or tumor cells coated with (IgG). This process is primarily mediated by the low-affinity Fcγ receptor IIIa (FcγRIIIa, also known as ), which is expressed on the surface of most human NK cells and binds to the Fc domain of IgG antibodies bound to target cells. Upon ligation, CD16 clusters and initiates signaling cascades that activate NK cell . The signaling pathway downstream of CD16 involves immunoreceptor tyrosine-based activation motifs (ITAMs) present in associated adaptor proteins, specifically the CD3ζ chain and the FcεRIγ chain. These adaptors form homo- or heterodimers with , and of their ITAMs by Src family kinases recruits and activates spleen tyrosine kinase (Syk) and ζ-chain-associated protein kinase 70 (ZAP-70), leading to downstream events such as calcium mobilization, cytoskeletal reorganization, and . This targeted activation bridges adaptive and innate immunity, enhancing the efficiency of antibody-mediated responses against pathogens and malignancies. A functional polymorphism in the FCGR3A encoding influences ADCC potency; the V158 variant exhibits higher affinity for the IgG Fc domain compared to the F158 variant, resulting in more robust NK cell activation and greater therapeutic efficacy in antibody-based treatments like rituximab for . Individuals homozygous for V158 (V/V) show enhanced ADCC responses, which has implications for personalized strategies. Engineered NK cell lines expressing the high-affinity V158 , such as haNK cells, demonstrate superior cytotoxicity against antibody-coated targets in preclinical models. In addition to standalone ADCC, CD16 engagement synergizes with NK cell direct cytotoxicity mechanisms on antibody-opsonized targets, amplifying overall killing efficiency against virus-infected or tumor cells through enhanced granule exocytosis containing perforin and granzymes. This cooperative action underscores ADCC's role in bolstering antitumor and antiviral defenses.

Cytokine secretion

Natural killer (NK) cells play a key role in innate immunity through the secretion of immunomodulatory cytokines, which help shape the early immune response. The primary cytokine produced by NK cells is interferon-gamma (IFN-γ), which enhances macrophage activation by upregulating their antimicrobial activity and promotes Th1-biased responses by favoring the differentiation of T helper 1 cells. This IFN-γ production is potently induced by synergistic stimulation with interleukin-12 (IL-12) and interleukin-18 (IL-18), cytokines often released by antigen-presenting cells during infection. In addition to IFN-γ, activated NK cells secrete tumor necrosis factor-alpha (TNF-α) and (GM-CSF), both of which contribute to the initiation and amplification of early by recruiting and activating other immune cells. TNF-α promotes pro-inflammatory signaling, while GM-CSF supports the differentiation and of myeloid cells involved in the inflammatory milieu. Cytokine secretion is particularly pronounced in the CD56bright subset of human NK cells, which prioritizes immunomodulatory functions over direct , leading to higher output of , , and GM-CSF compared to the CD56dim subset. This subset bias enables CD56bright NK cells to rapidly modulate the immune environment upon activation.

Physiological roles

Antiviral defense

Natural killer (NK) cells serve as a critical component of the innate immune system's early response to viral infections, rapidly eliminating infected cells and limiting viral dissemination through direct cytotoxicity and cytokine production. Many viruses, such as herpesviruses, downregulate class I (MHC-I) molecules on infected cells to evade recognition by cytotoxic T cells; however, this "missing-self" phenotype renders the cells susceptible to NK cell-mediated killing via activating receptors like and DNAM-1, which detect stress-induced ligands upregulated on infected surfaces. Additionally, NK cells produce interferon-gamma (IFN-γ) and other cytokines that induce an antiviral state in neighboring cells, inhibit , and promote the and of adaptive immune components. This dual mechanism enables NK cells to bridge innate and adaptive immunity during the initial 24–48 hours post-infection, before antigen-specific responses mature. A prominent example of NK cell specificity in antiviral defense is observed in mice infected with murine cytomegalovirus (MCMV), where genetic resistance in strains like is conferred by the activating receptor Ly49H on NK cells, which binds the viral MHC-I homolog m157 expressed on infected cells. This recognition triggers selective proliferation and IFN-γ production by Ly49H-positive NK cells, leading to efficient control of viral replication in the and liver during acute infection; mice lacking Ly49H exhibit markedly increased viral titers and mortality. In humans, analogous interactions occur with human (HCMV), where killer immunoglobulin-like receptors (KIRs) on NK cells interact with HLA class I alleles to enhance antiviral activity; for instance, activating KIR2DS2 in combination with HLA-C1 ligands is associated with better control of HCMV replication in transplant recipients, reducing through enhanced NK cell and secretion. NK cell licensing, or education, further refines their antiviral efficacy by calibrating responsiveness based on self-MHC-I recognition during development. Licensed NK cells, which express inhibitory receptors (e.g., Ly49 or KIR) that bind self-MHC-I, exhibit heightened functionality against MHC-I-low targets like virus-infected cells, displaying superior and IFN-γ production compared to unlicensed counterparts.00289-4) In the context of viral infections, this education ensures that licensed NK subsets preferentially target infected cells with downregulated MHC-I while sparing healthy tissues; studies in MCMV-infected mice demonstrate that unlicensed NK cells drive more effective viral clearance than licensed Ly49+ ones, particularly when MHC-I modulation is prominent. This process underscores the adaptive-like tuning of NK cells for precise antiviral responses.

Tumor surveillance

Natural killer (NK) cells play a critical role in tumor surveillance by recognizing and eliminating malignant cells through a balance of activating and inhibitory signals. This process allows NK cells to detect early signs of cellular transformation, such as oncogenic stress, before tumors establish themselves. Unlike adaptive immune cells, NK cells can act rapidly without prior , providing an innate barrier against cancer and progression. A key mechanism in NK-mediated tumor surveillance involves the upregulation of stress-induced ligands on transformed cells, which engage activating receptors on NK cells. Specifically, major histocompatibility complex class I-related chain A (MICA) and unique long 16-binding proteins (ULBPs) are frequently overexpressed on the surface of tumor cells due to cellular stress from DNA damage or oncogenic signaling. These ligands bind to the NKG2D receptor on NK cells, triggering degranulation and cytokine release to induce target cell apoptosis. This NKG2D-MICA/ULBP axis is particularly effective against a broad range of solid and hematological malignancies, where ligand expression correlates with enhanced NK cytotoxicity. Another pivotal aspect of NK tumor surveillance is the "missing-self" recognition, where NK cells target cells that have downregulated class I (MHC-I) molecules. Many tumors reduce MHC-I expression to evade cytotoxic T lymphocytes, but this loss removes inhibitory signals from NK cell receptors like KIRs and NKG2A, thereby licensing NK activation and attack. This mechanism ensures that MHC-I-deficient variants, common in cancers such as and colorectal , are selectively eliminated by NK cells, preventing immune escape. Seminal studies in MHC-deficient mouse models confirmed that NK cells reject such tumor cells , underscoring the hypothesis's relevance to oncogenesis. In controlling , liver-resident NK cells are instrumental in intercepting circulating tumor cells (CTCs) that disseminate from primary tumors. These NK cells patrol the hepatic sinusoids and rapidly lyse CTCs expressing stress ligands or lacking MHC-I, thereby reducing the seeding of distant . Experimental models demonstrate that depletion of liver NK cells significantly increases and liver in mice challenged with or colon cells, highlighting their frontline role in limiting systemic tumor spread. This surveillance is enhanced by the unique microenvironment of the liver, where NK cells constitute up to 15% of lymphocytes and exhibit heightened cytotoxic potential against blood-borne malignancies.

Reproductive immunology

Uterine natural killer (uNK) cells represent a specialized subset of NK cells that predominate in the during early , comprising 50-90% of decidual lymphocytes. These cells are primarily of the CD56bright , which distinguishes them from peripheral blood CD56dim NK cells by their high expression of CD56 and low cytotoxic potential, enabling supportive roles in reproductive processes. Abundant in the decidua basalis, uNK cells accumulate during the implantation window and peak in number during the first trimester, facilitating key events in placental formation. uNK cells contribute to embryo implantation and placental development by promoting and vascular remodeling at the maternal-fetal interface. They secrete angiogenic factors such as (VEGF) and (PlGF), which stimulate endothelial and spiral artery modification to ensure adequate nutrient and oxygen supply to the developing . These cytokines enhance decidual vascularization, supporting trophoblast invasion and preventing shallow . To maintain maternal-fetal tolerance, cells interact with non-classical HLA class I molecules, particularly expressed on extravillous s, through inhibitory receptors like LILRB1. This engagement suppresses uNK , preventing attack on fetal cells while allowing controlled migration. The LILRB1-HLA-G axis promotes an immunosuppressive environment, balancing immune surveillance with protection of the semi-allogeneic . Dysfunctions in cells are associated with reproductive disorders, including and . In , reduced numbers of decidual NK cells correlate with impaired spiral artery remodeling and placental ischemia. Similarly, alterations in uNK populations, such as decreased levels of specific subsets like uNK1, have been observed in , contributing to implantation failure and loss.

Pathological involvement

Immune deficiencies and disorders

Natural killer (NK) cell deficiencies represent a group of rare primary immunodeficiencies characterized by impaired NK cell numbers, maturation, or function, leading to increased susceptibility to viral infections and malignancies. Classical NK cell deficiency, also known as NK cell deficiency type 1, is defined by the selective absence or severe reduction of circulating NK cells, often resulting in recurrent or severe herpesviral infections such as or , as well as higher rates of papillomavirus-associated and certain cancers like . A key genetic cause is heterozygous germline mutations in the GATA2 gene, which encodes a essential for hematopoietic development; these mutations lead to a profound loss of the CD56bright NK cell subset while sparing the CD56dim subset to varying degrees, disrupting NK cell maturation and homeostasis. Patients with GATA2 mutations often present with a broader MonoMAC syndrome, including monocytopenia, B-cell deficiency, and myelodysplasia, but the NK cell defect contributes specifically to the infectious vulnerability. Classical NK cell deficiency type 2 (CNKD2) is caused by biallelic mutations in the MCM4 gene, which encodes a DNA replication component; this leads to partial depletion particularly of the CD56dim NK cell subset, severe functional impairment, and associated features such as growth retardation and , predisposing to life-threatening viral infections like disseminated varicella or severe EBV disease. Beyond numerical deficiencies, functional NK cell defects occur in conditions like type 1 (XLP1), a severe triggered primarily by Epstein-Barr virus infection. XLP1 is caused by mutations in the SH2D1A gene, which encodes the adaptor protein (signaling lymphocytic activation molecule-associated protein), essential for signaling through SLAM family receptors on NK cells. In affected individuals, NK cells exhibit impaired against EBV-infected B cells due to defective 2B4 receptor signaling, as SAP fails to associate properly with this activating receptor, leading to , , or dysgammaglobulinemia upon viral challenge. NK cell numbers may be normal, but their functional impairment underscores the role of SAP in NK-mediated immune surveillance. NK cell dysfunction also manifests in autoimmune disorders such as systemic lupus erythematosus (SLE), where reduced NK cell numbers and activity contribute to immune dysregulation. In SLE patients, peripheral blood NK cell counts are significantly decreased compared to healthy controls, accompanied by diminished cytotoxic function and impaired responsiveness, which may exacerbate production and tissue damage. This low NK cell activity correlates with disease activity, potentially due to increased or sequestration in inflamed tissues, thereby hindering the clearance of apoptotic cells and apoptotic debris that drive . In contrast to hyperactivation states, these deficiencies highlight NK cells' regulatory role in preventing excessive immune responses.

Hyperactivation and autoimmunity

Hyperactivation of natural killer (NK) cells can contribute to severe pathological conditions, including life-threatening hyperinflammatory syndromes and disorders, where uncontrolled and production lead to tissue damage and immune dysregulation. In (HLH), a rare but fatal syndrome, NK cells exhibit excessive activation alongside T cells and macrophages, driving a characterized by elevated levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). This hypercytokinemia is often fueled by dysregulated signaling through IL-2 and IL-15, that potently stimulate NK cell , survival, and effector functions, resulting in uncontrolled immune responses that overwhelm regulatory mechanisms and cause multiorgan failure. In adult-onset HLH, NK cells display an activated phenotype with preserved cytotoxic capacity, contrasting with the NK deficiencies typical in familial forms, and contribute to the syndrome's hallmark hemophagocytosis and hyperinflammation. In autoimmune diseases such as (RA), NK cell overactivation can promote pathology by targeting self-tissues, particularly in the synovium where inflammatory subsets accumulate. Activated NK cells in RA joints produce pro-inflammatory mediators like (GM-CSF), (M-CSF), and (RANKL), which exacerbate synovial inflammation, activation, and joint destruction. This overactivation arises from an imbalance in NK cell receptor signaling, where diminished inhibitory receptor function (e.g., via killer-cell immunoglobulin-like receptors) fails to restrain responses to self-antigens, leading to aberrant against autologous cells and amplification of adaptive autoimmune responses through IFN-γ secretion. Although peripheral NK cell numbers may be reduced in RA, the tissue-resident activated NK populations drive chronic , highlighting their dual role in both protection and pathogenesis. Rare clonal expansions of NK cells manifest as NK-type large granular lymphocyte (LGL) leukemia, a chronic lymphoproliferative disorder characterized by persistent proliferation of mature NK cells, often exceeding 2 × 10^9/L in peripheral blood. This clonal overgrowth leads to hyperactivation-like symptoms, including cytopenias, recurrent infections, and associated (e.g., or pure red cell aplasia), driven by somatic mutations in genes like that enhance NK survival and responsiveness. In NK-LGL leukemia, the expanded cells exhibit a mature immunophenotype (CD3^− ^+ CD56^+) with constitutive activation markers, contributing to immune dysregulation through excessive IFN-γ production and impaired immune surveillance. This condition underscores how dysregulated NK proliferation can mimic hyperactivation, bridging lymphoproliferation and autoimmune features.

Evasion strategies by targets

Pathogens and tumor cells employ various mechanisms to evade detection and elimination by natural killer (NK) cells, which rely on the balance between activating and inhibitory signals for target recognition. One prominent strategy used by viruses involves mimicry, where human (HCMV) encodes the glycoprotein UL18, a structural homolog of molecules that binds to inhibitory receptors such as leukocyte immunoglobulin-like receptor 1 (LILRB1) on NK cells. This interaction delivers inhibitory signals that suppress NK cell activation and cytotoxicity against HCMV-infected cells, thereby promoting viral persistence. UL18's high-affinity binding to LILRB1 mimics the engagement of self-, exploiting the NK cell's "missing-self" recognition paradigm to avoid . Tumor cells counteract NK-mediated surveillance through the proteolytic shedding of activating ligands, particularly releasing soluble forms of chain-related protein A (s) from the cell surface. This soluble binds to the receptor on NK cells, leading to its internalization and downregulation, which impairs NK cell recognition and cytotoxic responses against the tumor. Elevated levels of sMICA in the serum of cancer patients correlate with reduced expression and diminished antitumor immunity, facilitating tumor escape. In the tumor microenvironment, immunosuppressive factors like transforming growth factor-β (TGF-β) further hinder NK cell function by inhibiting their maturation and effector capabilities. TGF-β signaling suppresses the development of mature NK cells, maintaining them in an immature state with reduced and production, a process exacerbated by high TGF-β levels in solid tumors. This also directly impairs perforin and granzyme expression in mature NK cells, contributing to an overall immunosuppressive milieu that shields tumors from NK attack.

Therapeutic applications

Adoptive NK cell therapies

Adoptive natural killer (NK) cell therapies involve the expansion and infusion of NK cells to harness their innate cytotoxic potential against malignancies, particularly in hematologic cancers like (AML). These therapies typically utilize unmodified NK cells derived from the patient (autologous) or donors (allogeneic), with allogeneic approaches preferred due to enhanced alloreactivity against tumors lacking inhibitory ligands. In autologous infusions, NK cells are isolated from the patient's peripheral , activated and expanded using cytokines such as interleukin-2 (IL-2), and reinfused following lymphodepleting to reduce endogenous immune suppression and improve engraftment. However, autologous NK therapies have shown limited antitumor efficacy in clinical settings, primarily due to the patient's immunosuppressive , with phase I trials in solid tumors reporting safety but modest response rates below 20%. Allogeneic adoptive NK cell therapy, particularly from haploidentical donors, has emerged as a more promising strategy for AML, leveraging mismatched killer immunoglobulin-like receptor (KIR) interactions to promote NK cell licensing and tumor targeting. Seminal work demonstrated that IL-2-activated haploidentical NK cells, infused after lymphodepleting regimens like and , could expand for up to 12 days and mediate complete remissions in 5 of 19 poor-prognosis AML patients (26%), with no observed. Subsequent trials refined this approach by incorporating recombinant human IL-15 (rhIL-15) to support NK cell survival without the toxicity of high-dose IL-2, achieving remissions in 35% of AML patients. Haploidentical donors are selected for KIR incompatibility to maximize alloreactive potential, and cells are typically expanded overnight or for short periods to preserve functionality. Clinical trials have highlighted the utility of adoptive NK cell therapy in the post-hematopoietic transplant (HSCT) setting for relapse prevention or treatment. In a phase II trial, haploidentical NK cells infused after haploidentical HSCT in pediatric AML patients resulted in event-free survival improvements, with response rates around 50% in intermediate-risk cases. Similarly, donor-derived NK cells administered post-HSCT in AML achieved complete remission in approximately 57% of patients at one month, underscoring their role in bridging to long-term remission. Overall, across multiple trials in AML, response rates range from 20% to 50%, with higher efficacy in settings, though durable responses often require subsequent allogeneic HSCT. These outcomes establish adoptive NK as a safe bridge , with infusion-related toxicities minimal and primarily limited to transient release. Despite these advances, challenges persist in adoptive NK cell therapies, notably the short in vivo persistence of infused cells, typically lasting only 1-3 weeks without sustained support, which limits long-term tumor control and contributes to rates exceeding 50% in cases. Lymphodepleting is essential to enhance NK cell homing to and lymphoid tissues by depleting regulatory T cells and competing lymphocytes, thereby creating an immunological niche; without it, engraftment is negligible. Heterogeneity in donor NK cell quality and patient responses further complicates outcomes, prompting ongoing efforts to optimize expansion protocols and briefly explore engineered enhancements for improved . Future refinements aim to address these hurdles through better supportive care to extend NK cell functionality.

Engineered NK cells

Engineered natural killer (NK) cells involve genetic modifications to enhance their specificity, persistence, and antitumor activity, primarily through the introduction of chimeric antigen receptors (), use of immortalized cell lines, or bispecific antibody constructs that redirect NK cell killing. These approaches aim to overcome limitations of primary NK cells, such as short lifespan and variable activation, while maintaining their inherent safety profile, including reduced risk of compared to T cell therapies. Early clinical data indicate that engineered NK cells can achieve complete remissions in cancers without severe adverse events. CAR-NK cells express synthetic receptors that recognize tumor-associated antigens, enabling targeted cytotoxicity independent of restriction. A prominent example targets , a marker on B-cell malignancies like and , where CAR-NK therapy has shown objective response rates of up to 73% in phase I/II trials involving 11 patients with relapsed or refractory disease, with no instances of GVHD or observed. This GVHD resistance stems from the alloreactive nature of NK cells and lack of T cell contamination in NK preparations, contrasting with CAR-T cells that often require lymphodepleting . Preclinical studies further demonstrate that CD19-CAR-NK cells, often derived from or induced pluripotent stem cells, exhibit potent of CD19-positive tumor cells while sparing healthy tissues. The NK-92 cell line, derived from a with , serves as a renewable source for engineered NK therapies due to its perpetual proliferation and high baseline . To mitigate risks of uncontrolled growth, NK-92 cells are irradiated before , rendering them replication-incompetent while preserving effector functions for off-the-shelf administration without patient-specific . In clinical settings, irradiated NK-92 cells expressing against targets like HER2 or have been tested in solid tumors, including and , demonstrating safety with no significant side effects and partial responses in some patients across multiple phase I trials. For instance, NK-92-CD33-CAR therapy in achieved stable disease in 6 out of 9 (67%) treated individuals. Bispecific engagers enhance NK cell targeting by simultaneously binding activating receptors on NK cells and tumor antigens, forming an that triggers and release. Bispecific killer engagers (BiKEs), such as those linking (the FcγRIII receptor) to tumor markers like or HER2, have shown preclinical efficacy in redirecting NK cells against and cells, with enhanced compared to monoclonal antibodies alone. -Fc fusion proteins, which dimerize NKG2D ligands overexpressed on stressed tumor cells to activate NKG2D receptors, promote NK-mediated tumor clearance and depletion of immunosuppressive cells like myeloid-derived suppressor cells in the . These constructs, often Fc-optimized for prolonged half-life, have demonstrated antitumor activity in models without off-target effects on healthy cells. Ongoing trials explore BiKEs and TriKEs (trispecific variants incorporating IL-15 for NK expansion) in hematologic and solid malignancies, highlighting their potential for rapid deployment. These engineered strategies are increasingly combined with checkpoint inhibitors or cytokines to further boost efficacy, though standalone applications remain the focus of current advancements. Recent 2025 data from early-phase trials, such as the off-the-shelf CAR-NK SENTI-202, have shown complete remissions in relapsed or cancers, including AML, in a subset of patients (e.g., 4 out of 7), underscoring ongoing progress as of April 2025.

Combination with immunomodulators

Natural killer (NK) cells can be enhanced through combination therapies involving immunomodulators such as cytokines, checkpoint inhibitors, and Toll-like receptor (TLR) agonists, which amplify their cytotoxic activity and persistence in clinical settings. Cytokine preconditioning with interleukin-15 (IL-15) and interleukin-21 (IL-21) promotes NK cell expansion and activation by stimulating proliferation and enhancing effector functions like cytotoxicity and cytokine production. IL-15 supports NK cell survival and memory-like differentiation, while IL-21 synergistically boosts expansion yields up to eightfold when combined with IL-15, leading to improved antitumor responses. In clinical protocols for neuroblastoma, ex vivo preconditioning of NK cells with IL-15 and IL-21 has been employed to generate highly cytotoxic populations for adoptive transfer, demonstrating safety and preliminary efficacy in relapsed/refractory patients. Checkpoint inhibitors targeting inhibitory receptors on NK cells, such as killer-cell immunoglobulin-like receptors (KIRs), relieve suppression and enhance NK-mediated killing. The anti-KIR monoclonal antibody lirilumab blocks multiple KIRs (e.g., KIR2DL1, KIR2DL2/3, KIR3DL2), promoting NK cell activation against HLA-expressing targets without causing significant toxicity. When combined with PD-1 inhibitors like nivolumab, lirilumab augments NK cell function in solid tumors, such as non-small cell lung cancer, by concurrently disrupting PD-1/PD-L1 and KIR/HLA inhibitory axes, resulting in improved tumor control in preclinical models and early-phase trials. TLR agonists like CpG oligodeoxynucleotides (CpG-ODN) and polyinosinic:polycytidylic acid (poly I:C) indirectly activate NK cells by inducing cytokine storms, including type I s and IL-12, which drive NK maturation and . CpG, a TLR9 agonist, triggers NK cell and IFN-γ production via plasmacytoid dendritic cells, enhancing antitumor immunity in models of infection and cancer. Poly I:C, a TLR3 agonist, similarly promotes NK cell activation through signaling, with combined CpG/poly I:C formulations showing synergistic effects on NK against tumors and virus-infected cells, though careful dosing is required to mitigate excessive release.

Emerging research

Adaptive and memory-like properties

Natural killer (NK) cells exhibit memory-like properties characterized by epigenetic modifications following human cytomegalovirus (HCMV) infection, enabling enhanced functional responses upon re-encounter with the . These changes involve and modifications that silence genes encoding signaling adapters such as FcRγ, SYK, and EAT-2, leading to a distinct adaptive NK cell subset with altered signaling pathways and improved (ADCC). In HCMV-seropositive individuals, this epigenetic reprogramming results in the selective expansion of NK cells with reduced expression of these adapters, promoting a memory-like state that persists long-term. Memory-like NK cells demonstrate enhanced responses, producing higher levels of interferon-γ (IFN-γ) and other cytokines upon secondary stimulation compared to naive NK cells. This capacity is particularly evident in HCMV-specific contexts, where pre-exposed NK cells exhibit amplified effector functions, contributing to improved control of viral reinfection. Epigenetic inheritance of these modifications ensures clonal expansion and maintenance of the , as shown in mouse models of infection where encounter drives pronounced remodeling. Adaptive NK cells, a specialized subset, are defined by the absence of FcRγ expression and are predominantly found in HCMV-seropositive individuals. These FcRγ-negative NK cells display expanded populations expressing multiple killer-cell immunoglobulin-like receptors (KIRs), particularly those with inhibitory specificities for self-HLA class I alleles, which correlate with heightened ADCC against HCMV-infected targets. The expansion of KIR+ FcRγ-negative NK cells is driven by HCMV infection and results in a reprogrammed signaling axis that favors antibody-mediated responses over natural . NK cell licensing, or education through self-MHC class I interactions, plays a critical role in enabling memory formation, with pre-educated (licensed) NK cells showing antigen-specific boosting upon exposure. Licensed NK cells, which have received inhibitory signals via KIR or NKG2A during development, exhibit enhanced responsiveness to cytokines like IL-12, IL-15, and IL-18, leading to amplified IFN-γ production in recall scenarios. This pre-education ensures that only functional NK cells contribute to memory-like responses, linking classical licensing to adaptive features without altering core inhibitory receptor expression.

Tissue-specific functions

Natural killer (NK) cells exhibit specialized functions tailored to the unique immunological demands of different tissues, reflecting adaptations in their activation, production, and interactions with local cell types. In the liver, gut, and , distinct NK cell subsets contribute to , defense, and tissue repair, often through organ-specific mechanisms that modulate and . In the liver, NK cells play a critical anti-fibrotic role by targeting activated hepatic stellate cells (HSCs), the primary producers of during , through direct and secretion of interferon-gamma (IFN-γ). This IFN-γ production inhibits HSC activation and proliferation while promoting their , thereby limiting fibrotic progression in models of chronic liver injury induced by toxins or . Liver-resident NK cells, which constitute a significant proportion of hepatic lymphocytes, further enhance this function by expressing high levels of activating receptors like , enabling rapid responses to fibrogenic stimuli. Additionally, these cells provide immune surveillance against hepatic tumors, such as , by recognizing stress-induced ligands on malignant hepatocytes and eliminating them via perforin- and granzyme-mediated , which helps control early tumor dissemination in the hepatic microenvironment. In the gut, ILC1-like NK cells, characterized by their expression of T-bet and production of IFN-γ, contribute to regulation by influencing epithelial barrier integrity and modulating bacterial translocation. These cells respond to microbial signals at the mucosal interface, promoting the production of and cytokines that maintain symbiotic balance while preventing dysbiosis-associated . At the intestinal mucosa, NK cells exert potent antiviral effects by recognizing infected epithelial cells through natural cytotoxicity receptors and (ADCC), leading to the clearance of pathogens like noroviruses and , which is particularly vital in the where viral entry occurs frequently. In the brain, NK cells interact closely with microglia during neuroinflammation, where they infiltrate the central nervous system (CNS) and modulate microglial activation via IFN-γ secretion, which can amplify pro-inflammatory responses in conditions like multiple sclerosis or infection but also facilitate debris clearance. These interactions occur primarily in the perivascular space and parenchyma, with NK cells upregulating to home to inflamed sites and influencing microglial polarization toward an M1-like state that enhances control. Post-stroke, NK cells contribute to repair processes by promoting and in the peri-infarct region; brain endothelial cells secrete to recruit protective NK subsets that release growth factors and limit excessive infiltration, thereby supporting vascular remodeling and neuronal recovery in ischemic lesions. Liver, gut, and brain NK cells largely originate from precursors that seed tissues during development, with local proliferation sustaining resident pools.

Clinical trial advancements

Recent advancements in chimeric antigen receptor (CAR)-NK cell therapies have progressed to phase II trials targeting lymphomas since 2023, demonstrating improved safety profiles and response rates compared to earlier phases. For instance, off-the-shelf CAR-NK therapies incorporating logic-gated mechanisms have elicited complete remissions in patients with relapsed or refractory blood cancers, including lymphomas, as reported at the 2025 AACR meeting. To address antigen escape and relapse, multi-antigen targeting strategies in CAR-NK designs, such as dual CD19/CD20 approaches, have shown enhanced antitumor activity and persistence in preclinical and early clinical models for B-cell lymphomas. Allogeneic NK cell therapies derived from have expanded into trials for tumors, with phase I studies in 2023-2024 reporting favorable safety and preliminary efficacy in advanced pediatric tumors when expanded . These off-the-shelf products avoid HLA matching requirements, enabling broader application, and ongoing trials as of 2024 include combinations with checkpoint inhibitors for and other malignancies. In 2024-2025, the FDA granted fast-track designations to several allogeneic NK combinations, Biomarkers like KIR-HLA mismatch have emerged as predictors of response in NK cell therapies, with mismatched donor-recipient pairs correlating to heightened antitumor activity in recent analyses of adoptive transfer trials. Specifically, inhibitory KIR ligand mismatches have been linked to reduced relapse risk post-therapy in 2024 retrospective studies. Complementing this, AI-driven modeling for patient selection, including machine learning-derived NK cell signatures, has improved prognostic accuracy in trial enrollment for NK therapies from 2023 onward, enabling personalized matching based on features.

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