Natural killer (NK) cells' activating and inhibiting receptors KIR, Ly-49 and NCR families

Natural kller (NK) cells, unlike natural killer T (NKT) cells, do not express an antigen-specific receptor. Regulation of the cytotoxic activity of NK cells is mediated by several activating and inhibiting receptors expressed on the cell surface including natural cytotoxicity receptors (NCR), lectin-like receptors and CD158 family of killer immunoglobulin-like receptors (KIR) that bind to specific components present on the surface of bacteria, virally-infected cells, stressed cells, or cancer cells. Rodents lack KIR and instead express functionally equivalent lectin-like Ly-49 receptors. These receptors can be activating or inhibiting depending upon semi-conserved motifs (ITAM and ITIM) found in the receptor’s intracellular domain allowing a unique and controlled response by the NK cell.

Human receptors Mouse receptors
CD94 (NKG2) CD94 (NKG2)
CD158a (KIR2DL1/S1) CD314 (NKG2D)
CD158f (KRI2DL5A) CD335 (NKp46)
CD314 (NKG2D) Ly-49A
CD335 (NKp46) Ly-49A/D
CD336 (NKp44) Ly-49D
CD337 (NKp30) Ly-49C/I/F/H
  Ly-49E/F
  Ly-49G
  Ly-49G2
  Ly-49H
  Ly-49I
  Ly-108
  NKG2A/C/E
  NKG2AB6

 

Activating natural cytotoxicity receptors (NCR)

Natural killer cells (NK) are central players in the vertebrate immune system. These cells rapidly eliminate tumor or virally-infected cells through recognition of different cellular and pathogen- associated ligands, with no requirement for prior antigen recognition. Regulation of the NK killing process is mediated by several activating and inhibiting receptors on the NK cell surface (see table above). Natural killer cells are now recognized as a subset of cytotoxic innate lymphoid cells (ILC) that express the transcription factor E4BP4.

One important family of engaged NK mediators is the activating natural cytotoxicity receptors (NCR), which include NKp30, NKp44 and NKp46. Upon stimulation, they deliver potent signals to NKs in order to lyse harmful cells and produce inflammatory cytokines such as IFNγ. With the exception of NKp46, mice lack a homolog of NKp44 and contain only a pseudogene of human NKp30.

NKp46 (NCR1/ CD335)

  • Highly conserved NK marker in mammals, particularly in blood and bone marrow but also expressed lymphoid tissue inducer cells; associates with CD3zeta and the gamma-chain of FceRI
  • Implicated in the control of various viral infections, cancer and diabetes and the absence of NKp46 results in an impaired eradication of certain tumors, such as lymphoma and melanoma

NKp44 (NCR2/ CD336)

  • Expressed only on activated circulating NK cells, stimulation with cytokines such as IL-2 or IL-15 increases it’s constitutive expression and cytolytic potential
  • Distant member of the family of triggering receptors expressed on myeloid cells (TREM), which are activating receptors involved in the innate inflammatory response and in sepsis

NKp30 (NCR3/ CD337)

  • Recognizes the ligand B7-H6, expressed on tumor cells but so far not on healthy cells, leading to NK killing of tumor cells during immunosurveillance activities
  • Binds BAG6, present on the membrane of immature dendritic cells (iDCs), triggering NK cell immunoediting of iDCs while preserving the more immunogenic mature DCs during a protective immune response

 

Natural killer cell activation

Natural Killer (NK) cells are activated in response to IL-2, IL-15, IL-15/IL-15RA complex, IL-18, and IL-12, and produce and secrete a variety of cytokines, chemokines (including IFN-gamma, TNF-alpha, IL-17A, and IL-22) and death-eliciting proteins (perforin and granzymes). Similar to cytotoxic CD8+ T cells, activated NK cells contain cytoplasmic granules that contain proteins such as perforin and granzymes that create pores in the cell membrane and initiate apoptosis via a caspase cascade in target cells, respectively. Of the Granzyme family, granzyme B is the best-characterized, but granzymes A through M are also involved and have been shown to have unique ligand specificity.

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Cytokine Role

IL-2

Augments NK cell activity and boosts its cytolytic activity by activating various kinase pathways

IL-12 p70

Induces activation, stimulates cytotoxicity and production of IFN-gamma and TNF

IL-15/IL-15R

Supports proliferation, accumulation and survival

IL-15

BALB/c splenocytes were stimulated for 4 days with Mouse IL-2 Recombinant Protein. Cells were harvested and stained with Anti-Mouse CD8a eFluor™ 450 followed by fixation and permeabilization using the Intracellular Fixation & Permeabilization Buffer Set and protocol. Cells were subsequently stained intracellularly with Rat IgG2a K Isotype Control APC (left) or Anti-Mouse Perforin APC (right). Cells in the lymphocyte gate were analyzed.

 

Role of granzymes

Granzymes (granule-secreted enzymes) are a family of serine proteases stored within lysosomal-granules of natural killer (NK) cells and cytotoxic lymphocytes (CTLs). Critical for the induction of target cell apoptosis, granzymes cleave intracellular substrates, triggering many apoptotic pathways to ensure target cells die. Granzymes also play a significant role in the immune defense against viruses, tumors and intracellular bacteria. More recent research demonstrated that granzymes have an extracellular role resulting in the destruction of tissues and vascular integrity that implicates granzymes in a number of inflammatory and age-related diseases. This family is emerging as an important group of proteins involved in immune function and surveillance.

Granzyme A

Granzyme A is the most abundant serine protease in CD8+ CTL- and NK-cytotoxic granules. It not only activates a novel programmed cell death pathway that begins in the mitochondrion and generates reactive oxygen species (ROS) to ultimately activate single-stranded DNA damage, but also targets other important nuclear proteins for degradation, including histones, lamins and several key DNA damage repair proteins such as Ku70 and PARP-1. Granzyme A also displays proinflammatory activity; it activates IL-1s, TNF- α, and IL-6 and may have other effects not yet understood.

Granzyme B

Granzyme B (GrB) is found in the granules of cytotoxic T cells and NK cells. Granzyme B has also been described as CGL1 (cathepsin G-like-1), a serine protease expressed only in cytotoxic T-lymphocytes after cell activation. Granzyme B has been called CTLA-1 (cytotoxic T lymphocyte-associated serine esterase 1) based on identification of mRNA in various cytotoxic T cells, but not observed in non-cytotoxic lymphoid cells. Granzyme B is crucial for the rapid induction of target cell death by apoptosis, induced by interaction with cytotoxic T cells. Granzyme B activates the intracellular cascade of caspases finally resulting in the killing of the target cells.

Granzyme K

Like Granzyme A, Granzyme K is a tryptase serine protease with overlapping, but not identical, substrate specificity. Although Granzyme K induces ROS generation, it also induces caspase-independent cell death through Bid-dependent mitochondrial damage. Data have also shown that Granzyme-K binds and cleaves p53. This cleavage is reported to incite a mediated-killing of tumor cells. It appears that by activating multiple cell death pathways, killer cells provide better protection against a variety of intracellular pathogens and tumors. Granzyme K too displays pro-inflammatory potential as it has been shown that an extracellular form is capable of inducing cytokine production from human lung fibroblasts through the activation of PAR-1.

 

NKT cells comprise a distinct T cell lineage

Natural Killer T (NKT) cells represent a distinct lineage of T cells that express an invariant T cell receptor (TCR) and share a number of cell surface markers in common with NK cells. NKT cells are non-polymorphic CD1d molecule restricted T cells and are activated by glycolipid antigens presented by CD1d. Expression of CD160 and Vα24Jα18 TCR can be used for the identification of mouse and human NKT cells, respectively.

Expression of transcriptional repressor, Promyelocytic Leukemia Zinc Finger (PLZF), in immune cells differs between mice and humans. In mice, PLZF is highly expressed in immature CD1d-resricted invariant NKT (iNKT) cells and a subset of gamma delta (Vg1.1+Vd6.3+) T cells. In humans, PLZF is expressed in NK cells, gamma delta T cells, as well as CD4 and CD8+ T cells. PLZF exists as a homodimer or in complex with PLZP, has been shown to be involved in the development of NKT cells, NK cell function, cellular quiescence, and growth suppression and has been shown to inhibit gene expression induced by retinoic acid receptor.

 

Staining of normal human peripheral blood cells with Anti-Human CD3 eFluor™ 450 and Mouse IgG1 K Isotype Control PE-Cyanine7 (left) or Anti-Human V alpha 24 J alpha 18 TCR PE-Cyanine7 (right). Cells in the lymphocyte gate were used for analysis.