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Natural killer cells (or NK cells) are a type of cytotoxic lymphocyte critical to the innate immune system. The role NK cells play is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virally infected cells and respond to tumor formation, acting at around 3 days after infection. Typically immune cells detect MHC presented on infected cell surfaces, triggering cytokine release causing lysis or apoptosis. NK cells are unique, however, as they have the ability to recognise stressed cells in the absence of antibodies and MHC, allowing for a much faster immune reaction. They were named “natural killers” because of the initial notion that they do not require activation in order to kill cells that are missing “self” markers of major histocompatibility complex (MHC) class 1 .

NK cells are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation. . NK cells differ from Natural Killer T cells (NKT) phenotypically, by origin and by respective effector functions; often NKT cell activity promotes NK cell activity by secreting IFN&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 CD56 in humans, NK1.1 or NK1.2 in C57BL/6 mice. Up to 80% of human NK cells also express CD8.

In addition to the knowledge that natural killer cells are effectors of innate immunity, recent research has uncovered information on both activating and inhibitory NK cell receptors which play important function roles including self tolerance and sustaining NK cell activity. NK cell also play a role in adaptive immune response , numerous experiments have worked to demonstrate 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. The ability for NK cells to act in both the innate and adaptive immune response is becoming increasingly important in research utilizing NK cell activity and potential cancer therapies.

NK cell receptors


NK cell receptors can also be differentiated based on function. Natural cytotoxicity receptors directly induce apoptosis after binding to ligands that directly indicate infection of a cell. The MHC dependent 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 i.e. if the inhibitory receptor signaling is more prominent then NK cell activity will be inhibited, similarly if the activating signal if dominant then NK cell activation will result .

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

Inhibitory Receptors

 * KIR (Killer-cell immunoglobulin-like receptors) — belong to a multigene family of more recently-evolved Ig-like extracellular domain receptors; are present in non-human primates; and are the main receptors for both classical MHC I (HLA-A, HLA-B, HLA-C) and also non-classical HLA-G in primates. Some KIRs are specific for certain HLA subtypes. Most KIR are inhibitory and dominant.  Regular cells express MHC class 1 and therefore are recognised by KIR receptors and NK cell killing is inhibited.
 * ILT or LIR (leukocyte inhibitory receptors) — are recently-discovered members of the Ig receptor family.

Activating Receptors

 * Ly49 (homodimers) — a relatively ancient, C-type lectin family receptor; 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), upon stimulation, mediate NK killing and release of IFNϒ.
 * CD94 : NKG2 (heterodimers) — a C-type lectin family receptor, conserved in both rodents and primates and identifies non-classical (also non-polymorphic) MHC I molecules like 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.
 * CD16 (Fc&gamma;IIIA) play a role in antibody-dependent cell-mediated cytotoxicity (ADCC), in particular they bind IgG.

NK cell receptors can also be differentiated based on function. Natural cytotoxicity receptors directly induce apoptosis after binding to ligands that directly indicate infection of a cell. The MHC dependent receptors (described above) use an alternate pathway to induce apoptosis in infected cells.

Cytolytic Granule Mediated Cell Apoptosis
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 would only release the virions, whereas apoptosis leads to destruction of the virus inside. &alpha;defensins, an antimicrobial is also secreted by NK cells, it directly kills bacteria by disrupting their cell walls analogous to neutrophils.

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
Infected cells are routinely opsonised with antibodies for detection by immune cells. Antibodies that bind to antigens can be recognised by FcϒRIII (CD16) receptors expressed on NK cells resulting in NK activation, release of cytolytic granules and consequent cell apoptosis .

Cytokine induced NK and CTL activation
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. 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 is generating antigen-specific cytotoxic T cells that can clear the infection. NK cells work to control viral infections by secreting IFN&gamma; and TNF&alpha;, IFN&gamma; activates macrophages for phagocytosis and lysis and TNF&alpha; acts promote direct NK tumor cells killing. Patients deficient in NK cells prove to be highly susceptible to early phases of herpes virus infection.

Missing 'self' hypothesis
In order 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. Most of these receptors are not unique to NK cells and can be present in some T cell subsets as well.

These inhibitory receptors recognize MHC class I alleles, which could explain why NK cells kill cells possessing low levels of MHC class I molecules. This inhibition is crucial to the role played by NK cells. MHC class I molecules consist of the main mechanism by which cells display viral or tumor antigens to cytotoxic T-cells. A common evolutionary adaptation to this seen in both intracellular microbes and tumours is a chronic down-regulation of these MHC I molecules, rendering the cell impervious to T-cell mediated immunity. It is believed that NK cells, in turn, evolved as an evolutionary response to this adaptation, as the loss of the MHC would deprive these cells of the inhibitory effect of MHC and render these cells vulnerable to NK cell mediated apoptosis .

Tumor cell surveillance
Natural Killer Cells (NK) often lack antigen specific cell surface receptors and therefore 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 immuno-surveillance by directly inducing the death of tumor cells (NKs act as cytolytic effector lymphocytes) even with the absence of surface adhesion molecules and antigenic peptides, this role of NK cells is critical for immune success particularly because T cells are unable to recognize pathogens in the absence of surface antigens. Tumor cell detection results in activation of NK cells and consequent cytokine production and release.

If the tumor cells do not cause inflammation they will also be regarded as self and therefore will not induce a T cell response. A number of cytokines are produced by NKs including Tumor Necrosis Factor &alpha; (TNF&alpha;), IFN&gamma; and Interleukin (IL-10); TNF&alpha; and IL-10 act as pro-inflammatory and inmmuo-supressors respectively. The activation of NK cells and subsequent production of cytolytic effector cells impacts macrophages, dendritic cells and neutrophils which subsequently affects 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. NKG2D is a disulphide linked homodimer which recognizes a number of ligands such as DAP-10, MICA which are typically expressed on tumor cells.

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 antibodies. This allows NK cells to target cells against which a humoral response has been mobilized and to lyse cells through Antibody-dependent cellular cytotoxicity (ADCC).NK cells promote the expression of FAS on cancer cells, FAS is not normally expressed on tumor cells, and it therefore aids FAS-dependent apoptosis upon binding with FASL expressing NK cells.

NK cell function in adaptive response
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 T and B cells play in the adaptive immune response. Despite prior belief that NK cells play no role in the adaptive immune responses, they have since been found to undergo expansion, contraction, memory maintenance and recall .

NK cell function in pregnancy
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 proccess. 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. These uNK cells are the most abundant leukocytes present in the uterus in early pregnancy, representing approximately 70% of leukocytes here, however where they originate from remains controversial.

These NK cells have been shown to have the ability to elicit cell cytotoxicity in vitro, however at a lower level than peripheral NK cells, despite containing perforin. 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 in order to defend against cytotoxic T cell-mediated death. This would normally trigger NK cells by missing self recognition, however these cells survive. It is thought that 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 defends it against NK cell-mediated death.

NK cells secrete a high level of cytokines which help mediate their function. Some important cytokines they secrete include TNF-α, IL-10, IFN-γ and TGF-β, among others.

NK cell evasion by tumor cells
By shedding decoy NKG2D soluble ligands tumor cells have evolved a process by which they are able to 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. This method of evasion is existent in prostate cancer. In addition, prostate cancer tumors can evade CD8 cell recognition due to the ability to lose expression of MHC class 1 molecules, this example of immune evasion actually highlights NK cell 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) .

History
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 lyse tumor cells without having been previously sensitized to them. As these discoveries were incompatible with the established model at the time, many initially considered that these observations were artifacts. However, 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 post-doctoral fellow Hugh Pross, in the mouse, and by Hugh Pross and doctoral student Mikael Jondal in the human. 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 lyse tumor cells against which they had been previously immunized. 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. The human data were confirmed, for the most part, by West et al. 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. 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, was the first time that NK cells had been visualized microscopically and was a major breakthrough in the field.

Anti-cancer therapy
Tumor specific antibodies are being used to target and destroy tumors with specific antigens. These antibodies employ effector cells such as NK cells activating them via their FC regions to target and lyse the specific pathogen. This has proven successful in treatment against breast cancer. Also trialed is combining monoclonal antibodies with KIR on NK cells in human cancer patients, the success of this has not yet been 100% confirmed but studies are aimed in myeloid leukemia and multiple myeloma. Understanding the importance of NK cells in tumor-immuno-surveillance is key to finding new and advancing existing cancer therapy.

NK cells in a study in Children's Hospital Boston in coordination with Dana-Farber Cancer Institute, whereby 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 quite capable of reducing tumor growth and prolonging survival of the recipients.

Innate Resistance to HIV?
Recent research suggests that specific KIR-MHC class 1 gene interactions could control innate genetic resistance to certain viral infections including HIV and its consequent development of AIDS. 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 defer progression of HIV to AIDS, this is evident due to 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 decrease HIV and AIDS susceptibility. Future research would aim to pinpoint relevant KIR/HLA interactions with aim to produce a vaccine against HIV/AIDS. NK cells can impose immune pressure on HIV, something that had previously been described only for T cells and antibodies and that HIV mutates to avoid NK cell activity.

Literature

 * Cellular and Molecular Immunology by Abbul K. Abbas & Andrew Lichtman Saunders Copyright 2003
 * How the Immune System Works, 2nd edition, by Lauren Sompayrac, PhD Blackwell Publishing 2003
 * Immunobiology: The Immune System In Health And Disease by Janeway, Travers, Walport & Shlomchik Churchchill Livingstone Copyright 2005
 * Kuby Immunology, 6th edition, by Thomas J. Kindt, Richard A. Goldsby,and Barbara A.Kuby W.H. Freeman and Company,New York
 * Regulation of interferon-gamma during innate and adaptive immune response, by Schoenborn J.R., and Wilson C.B. Advances in Immunology, 96:41-101, 2007.