Natural killer (NK) cells are key components of the innate immune response and can mediate cellular cytotoxicity without previous antigen exposure. The mechanisms underlying NK cell activation have been uncovered in a stepwise fashion from the early 1980s (for a detailed review of the field and its development see (Lanier 2008)). The role of the CD16 FcγRIII cell surface receptor in antibody-dependent NK cell activation was described in 1983. In 1986, Karre and colleagues proposed the “missing self-hypothesis” hypothesis and demonstrated the presence of inhibitory NK cell surface receptors responsive to the self-MHC (major histocompatibility complex) molecules on normal host cells. The search for antibody-independent NK cell activating receptors led to the identification of the NKG2 family of NK cell receptors in 1991. Among these was NKG2D, a molecule that shared limited homology with other NKG2 family members and was expressed in both NK and T cells as a predicted type 2 transmembrane C-type lectin receptor. In 1994, Bahram et al. identified two polymorphic genes encoding molecules related to classical MHC class I molecules which they named major histocompatibility complex I chain-related genes A and B (MICA, MICB) (Bahram et al. 1994). In 1999, NKG2D was identified as the activating receptor for MICA and MICB (Bauer et al. 1999).
NKG2D is recognized as a dominant activating NK cell receptor, capable of activating NK cells (Bauer et al. 1999), and influencing T cell activation. It is expressed constitutively in human NK cells, γδ T cells, and CD8+ T cells and may also be found in some invariant natural killer T (iNKT) cell and CD4+ T cell populations. In mice, NKG2D is constitutively expressed in NK cells, but is only expressed in CD8+ T cells when they have been activated. NKG2D has been identified in a wide range of mammals, including nonhuman primates, pigs, cattle, and rats.
The cytoplasmic portion of human NKG2D does not possess intrinsic signaling capability. Instead, a charged amino acid (arginine) in the transmembrane portion of NKG2D interacts with an aspartate residue in the adaptor molecule DAP10 (DNAX-activation protein 10). Each NKG2D molecule binds to a DAP10 dimer in this fashion to form a hexameric structure capable of signal transduction. The YINM motif in the cytoplasmic region of DAP10 recruits downstream signaling molecules. DAP10 lysine 84 is also necessary for ubiquitinylation and intracellular signaling (Quatrini et al. 2015). Murine NKG2D interacts with both DAP10 and DAP12, an ITAM (immunoreceptor tyrosine-based activation motifs)-containing adaptor molecule, through similar transmembrane region interactions. Both long and short NKG2D isoforms are capable of interacting with and signaling through either DAP10 or DAP12 in vitro, but in vivo the long form of NKG2D only interacts with DAP10 (O’Callaghan 2009). Human NKG2D does not interact with DAP12.
Signal transduction from NKG2D is complex (Fig. 1). An important early event in the NKG2D-DAP10 signal cascade is tyrosine phosphorylation of the YINM motif in DAP10 by the src family tyrosine kinase; Lck and src kinase inhibitors abrogate downstream signal transduction. The phosphorylated YINM motif recruits further intermediates: the p85 subunit of PI3K (phosphatidylinositide-3 kinase) and the adaptor protein Grb-2 (growth factor receptor-bound protein 2). Both binding events are essential for signal transmission. The GNEF (guanine nucleotide exchange factor) Vav1, which plays an important role in actin reorganization, is recruited to Grb-2, where it is phosphorylated and activated. From here, signal transmission diverges, but it is known that Vav1 acts in part by binding the γ2 isoform of phospholipase C (PLCγ2), which among other downstream targets activates MAPK8 (mitogen-activated protein kinase 8, JNK1). In contrast, MAPK1 (ERK2) appears to be an important downstream target of PI3K. Ubiquitinylation of the NKG2D-DAP10 complex is also essential for internalization of the complex, degradation, and signal transduction via ERK1/2 (Quatrini et al. 2015). These signaling pathways are reviewed by (Lanier 2008).
While human NKG2D does not interact with DAP12, NKG2D-DAP12 signaling remains an important consideration in murine NKG2D models. Unlike DAP10, NKG2D-DAP12 signaling is dependent on the ITAM domain of the cytoplasmic portion of DAP12. Engagement of ligand by NKG2D leads to the phosphorylation of two tyrosine residues in the DAP12 ITAM by src family tyrosine kinases. This enables recruitment of the syk tyrosine kinase, which is necessary for NKG2D-DAP12 function. It is likely that the downstream components of this pathway are similar to those in other ITAM-associated receptors, which have been studied in greater detail in different contexts.
A striking characteristic of NKG2D is the wide range of distinct ligands with which it can productively interact. Such ligand diversity indicates that NKG2D has evolved as an important receptor receiving input from the various stimuli which act through the upregulation of the different ligands. For further detailed review, see Mistry and O’Callaghan (2007) and Lanier (2015).
Humans express eight distinct functional NKG2D ligands: MICA and MICB (major histocompatibility complex I chain-related genes A and B) and the UL16 binding proteins (ULBP) 1–6. MICA and MICB are distant homologues of classical MHC class I molecules. Both are highly polymorphic, a feature of much interest, with 105 and 42 known alleles, respectively, identified to date (http://hla.alleles.org/alleles/classo.html). MICA and MICB are transmembrane molecules with MHC class I-like α1, α2, and α3 domains. However, in contrast to classical MHC class I molecules, they do not associate with β2-microglobulin, and the cleft separating the α2 and α3 domains is closed and does not bind peptides.
In contrast, the ULBPs have limited polymorphism and lack an α3 domain. ULBP 1–3 were originally identified as proteins that were bound by the cytomegalovirus protein UL16 and that interacted with NKG2D to induce NK cell cytokine secretion and cytotoxicity. Seven related gene sequences were subsequently described in the same region of chromosome 6q. Three of these encode functional molecules, ULBP 4 (RAET1E, LETAL), ULBP 5 (RAET1G), and ULBP 6 (RAET1L). ULBPs 1–3 and ULBP 6 are GPI-linked cell surface proteins, while ULBP 4 and 5 exist principally in transmembrane forms. A GPI-linked splice variant of ULBP 5 has been described, while ULBP 4 has three functional splice variants, each encoding transmembrane receptors.
Mice do not have MICA or MICB genes. There are three families of murine NKG2D ligands: the retinoic acid early (RAE) 1 family including α, β, γ, δ, and ε; H60 a, b, and c; and murine ULBP-like transcript 1 (Mult1). RAE1 α, β, and γ were described in 1996 as glycophosphatidyl-inositol (GPI)-linked cell surface molecules of unknown function, isolated from retinoic acid-treated mouse embryonal carcinoma cells. The murine NKG2D ligands show limited homology with murine classical MHC class I molecules and have an α1, α2 domain structure which does not present peptide and does not associate with β2 microglobulin. H60a, H60b, and Mult1 are transmembrane proteins, while H60c is a GPI-linked cell surface protein. The binding reactions between murine NKG2D and its ligands have been studied in detail, and binding affinities vary distinctly between NKG2D and its various ligands ((O’Callaghan et al. 2001); (Mistry and O’Callaghan 2007)).
The ligands for NKG2D are expressed on some normal cells, including gastrointestinal epithelial cells, and activated T cells, but are primarily displayed in pathological settings, notably in response to viral infection, some intracellular bacterial infections, and in many cancers. While several stimuli, broadly described as physiological stressors, are known to increase cell surface expression of NKG2D ligands on target cells, the mechanisms underlying regulation of NKG2D ligand expression remains poorly understood (Mistry and O’Callaghan 2007).
NKG2D may play significant roles in viral immunity, tumor immunity, autoimmune diseases, and transplant rejection (Lanier 2015). These roles are mediated through specific cellular processes, especially the induction of natural killer cell cytotoxicity and cytokine production. An understanding of the role of NKG2D in T cell costimulation is evolving.
Natural cytotoxicity. Natural cytotoxicity involves the coordination of several complex cellular processes including cytoskeletal reorganization, immune synapse formation, and perforin translocation. Freshly isolated human NK cells are capable of mounting a cytotoxic response to a target expressing NKG2D ligands without further experimental manipulation. Signaling through NKG2D can override inhibitory signaling through the interaction of HLA-E with CD94/NKG2A (Bauer et al. 1999) although in vivo NK cell activation is likely to reflect a broad balance between activating and inhibitory signaling. NKG2D may mediate TCR-independent cytotoxicity in IL-2-treated CD8+ T cells, γδ-TCR T cells, and some CD4+ T cell populations, but the in vivo significance of this is less certain and remains to be firmly established.
Cytokine and chemokine production. Early studies suggested that isolated ligation of NKG2D on NK cells and γδ-TCR T cells is sufficient to induce secretion of cytokines, primarily IFNγ, TNFα, and to a lesser extent, GMCSF and TNFβ (Fauriat et al. 2010). In one study, isolated human NKG2D ligation induced little cytokine secretion, but IFNγ and TNFα, prototypical NK cell cytokines, in addition to MIP-1α, MIP-1β, and RANTES, were produced when NKG2D was triggered together with CD16 (FCγRIII), natural cytotoxicity receptors, 2B4, or LFA-1(Fauriat et al. 2010) .
Costimulation. CD28 is the prototypical TCR costimulatory molecule, signaling through the p85 subunit of PI3K, which is recruited to a YMNM motif in the cytoplasmic domain of CD28. Given this shared signaling pathway with NKG2D, it was hypothesized that NKG2D might costimulate TCR-dependent T cell activation. While some early studies using cytokine-supported freshly isolated naïve (CD62L+, CD45RO−) human αβ TCR CD8+ T cells provided evidence suggestive of NKG2D-mediated co-stimulation (Maasho et al. 2005), a direct comparison of the co-stimulating capacities of NKG2D and CD28 found that NKG2D ligation was insufficient to mediate costimulation in contrast to CD28 ligation (Ehrlich et al. 2005). Further studies suggest that NKG2D activation can costimulate effector CD8+ αβ T cells, while CD28 signaling is necessary for naïve T cell costimulation (Rajasekaran et al. 2010). This functional divergence is also suggested by differential intracellular signaling pathway activity following CD28 or NKG2D activation (McQueen et al. 2016).
Murine knockouts. NKG2D knockout mice have provided further insights into NKG2D function (Guerra et al. 2008; Zafirova et al. 2009). Crossbreeding of NKG2D deficient mice with mice which develop prostate cancer or lymphoma demonstrates earlier and more rapid progression of these cancers in NKG2D-deficient mice (Guerra et al. 2008). In this lymphoma model, NKG2D signaling is necessary for NK cell mediated lymphoma cell death (Belting et al. 2015). Furthermore, NK cells from NKG2D-deficient mice have a diminished ability to kill NKG2D ligand expressing target tumor cells (Zafirova et al. 2009). NK cell development also appears to be affected, with more rapid NK cell division, and enhanced cytokine responses to NK cell stimulation (Zafirova et al. 2009).
Several factors modify the cell surface expression of NKG2D (Mistry and O’Callaghan 2007; O’Callaghan 2009). Cytokines appear to be the principle regulators of NKG2D cell surface concentration on both NK cells and T cells. Altering NKG2D cell surface density may alter the influence of NKG2D on cellular activation. TGFβ decreases cell surface NKG2D expression in NK cells, while IL-21 decreases NKG2D cell surface expression in both NK cells and CD8+ T cells. IL-15 increases NKG2D expression on CD8+ T cells, CD4+ T cells, and NK cells, while IL-2, IL-12, and IFNα have been shown to increase NKG2D expression on NK cells.
Excessive exposure to NKG2D ligands is reported to lower NKG2D cell surface concentrations in both physiological and pathological settings. In pregnancy, syncytiotrophoblast cells of the placenta produce exosomes containing soluble NKG2D ligands, which decrease NK cell NKG2D cell surface expression and reduce NK cell cytotoxicity (Hedlund et al. 2009).
At the transcriptional level, binding of the STAT3 transcription factor to a consensus site upstream of the NKG2D receptor gene is important for NKG2D receptor expression in both mouse and human cells (Zhu et al. 2014).
NKG2D is a homodimeric C-type lectin-like molecule which acts as an activating receptor in NK cells and can play an activating and potentially costimulating role in γδ T cells and CD8+ T cells, and some CD4+ T cell and iNKT cell populations. It is widely conserved in mammals, suggesting an important immune role. It is a promiscuous receptor with multiple diverse ligands, which are now well characterized in humans and mice. Future work is needed to further elucidate NKG2D signaling mechanisms, define functional interactions between NKG2D and other immune signaling molecules, and identify regulatory mechanisms that govern NKG2D ligand expression in target cells.
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