NK lymphocytes recognize abnormal or aberrant cells through multiple receptors that detect normal host molecules, as well as stress-induced or pathogen-expressed motifs (Lanier 2005; Long et al. 2013). Individual NK cells express both activating and inhibitory receptors, which together drive the specificity towards target cells.
The NK cell inhibitory receptors have been classified into three groups, namely, the heterodimeric CD94/NKG2A, the Ig-like transcript (ILT) receptors, and the members of the killer cell Ig-like receptors (KIRs). All of them bind to classical or nonclassical MHC-class I molecules. A common characteristic of the inhibitory receptors is the presence of immunoreceptor tyrosine-based inhibition motif(s) (ITIM) within their intracellular tail. Following engagement by their ligands, the inhibitory receptors become phosphorylated on the tyrosine residue(s) present in the ITIM(s), creating docking sites for the SH2-domains of the cytoplasmic protein tyrosine phosphatases SHP1 and SHP2. Their recruitment further results in the downregulation of the intracellular activation cascade. In contrast, activating receptors recognize a large variety of ligands, mostly distinct from MHC-class I molecules, and exhibit more complex but well-characterized signaling pathways. Natural cytotoxicity receptors (NCRs) and NKG2D are the major receptors involved in NK cytotoxicity. The NCRs (namely NKp46, NKp44 and NKp30) belong to the Ig-superfamily and represent non-MHC-class I-specific receptors whose cellular ligands still have to be confirmed (Arnon et al. 2006). In contrast to NKp46 and NKp30, constitutively expressed on circulating NK lymphocytes, NKp44 expression is activation-dependent (Vitale et al. 1998). The NCRs transduce signals through their association with ITAM-containing molecules such as CD3ζ, FcεRIγ, and DAP12 (Moretta et al. 2000; Vivier et al. 2004). Besides the NCRs, NKG2D is a C-type lectin-like receptor shown to recognize the MHC-class I homologues MICA and MICB and the family of UL16-binding proteins (ULBPs) (Gonzalez et al. 2006). NKG2D uses the transmembrane polypeptide DAP10 for signaling, which interacts with the PI3-kinase once phosphorylated. Interestingly NKp80, an additional C-type lectin-like activating receptor exclusively expressed by human NK cells has been identified. A search for NKp80 ligands led to the identification of activation-induced C-type lectin (AICL) (Welte et al. 2006). However, NKp80 signaling pathway remains enigmatic as this receptor does not contain a transmembrane charged residue (a feature allowing association with ITAM-containing adaptor proteins) or any intracellular consensus activation motifs. Finally, beside these MHC-class Ia/Ib molecule-independent activating receptors, it is important to mention the well-characterized DAP12-associated CD94/NKG2C and KIRs activating isoforms (Olcese et al. 1997; Lanier et al. 1998), although the precise events leading to their specific recruitment still have to be better defined. Here we review our knowledge on a unique NK cell receptor, named CD160, which delineates the CD56dimCD16brightCD3− subset within peripheral blood NK lymphocytes.
Structure, Specificity, and Function of CD160
CD160 receptor was initially identified by the monoclonal antibody (mAb) BY55 generated following mice immunization with YT2C2, a human leukemia cell line with NK activity. BY55 mAb was shown to immunoprecipitate an 80-kDa cell surface structure expressed mainly on a subset of circulating NK and T lymphocytes and on all intestinal intraepithelial T lymphocytes (Maiza et al. 1993; Anumanthan et al. 1998). Subsequently, CD160 expression was detected on mast cells (Ortonne et al. 2011), activated endothelial cells (Fons et al. 2006), a subset of skin CD4+ T lymphocytes (Sako et al. 2014), and finally found increased in activated T lymphocytes (Abecassis et al. 2007; Nikolova et al. 2009). The increased expression of CD160 during T cell activation was found inhibited in CD4+CD25brightFox P3+ T lymphocytes (A.B.., personal communication).
The molecular cloning of CD160 molecule recognized by BY55 mAb revealed that it is a glycosylphosphatidylinositol (GPI)-anchored receptor, unique in its nucleotide and aminoacid sequences, encompassing a single Ig-like extracellular domain (Anumanthan et al. 1998). The CD160 gene was found to be located on human chromosome 1q21.1 (in the thrombocytopenia absent radius (TAR) syndrome area). The mature molecule corresponds to a cysteine-rich 134-aminoacids polypeptide, with six cysteines and two potential sites for N-linked glycosylation. The size predicted for the mature N-glycosylated CD160 polypeptide would be approximately 25-kDa, indicating that the 80-kDa cell surface structure immunoprecipitated by BY55 mAb corresponds to multimers (most likely trimers).
In NK lymphocytes or mast cells, the membrane expression of the GPI-anchored CD160 is downmodulated upon activation with cytokines. Indeed, IL-15 treatment converts the membrane-bound receptor to a soluble form through a proteolytic cleavage involving a metalloprotease (Giustiniani et al. 2007). The CD160 transcript remains highly synthesized during the process of protein shedding, its synthesis being even induced in CD56bright NK cells upon interleukin treatment. The soluble CD160 that is released by NK lymphocytes or mast cells impairs the MHC class I-specific cytotoxic CD8+ T lymphocytes and NK cells function. This observation is most likely related to the ability of soluble CD160 to interact with MHC-class I molecule and to eventually compete with activating NK receptors for ligand binding (Giustiniani et al. 2007), resulting in the downmodulation of NK cell cytotoxic activity (Fig. 2).
CD160 Transmembrane Isoform (CD160-TM): Structure and Function
Three additional RNA encoding putative CD160 isoforms were identified in NK cells. These transcripts originate from the alternative splicing of the CD160 gene and their translation would lead to the synthesis of proteins sharing high level of homology with CD160 (Fig. 1) (Giustiniani et al. 2009). The first one corresponds to a CD160 mRNA that lacks the coding region for the Ig domain; this deletion would therefore result in a GPI-anchored molecule devoid of extracellular Ig domain (CD160ΔIg-GPI). The second transcript presents a high level of identity with the CD160ΔIg-GPI coding sequence and would lead to a CD160 molecule with no extracellular Ig domain but possessing a transmembrane and an intracellular domain (CD160ΔIg-TM). The third mRNA is the larger one and codes for a putative receptor having the complete extracellular moiety of CD160, as well as a transmembrane and an intracellular part, and would thus correspond to a transmembrane isoform of the original GPI-anchored molecule (CD160-TM). CD160-TM encompasses a transmembrane charged aminoacid, a feature usually found in activating KIRs and allowing their interaction with DAP10 or DAP12, as well as two potential tyrosine phosphorylation sites within its intracellular domain. The two transcripts coding for the GPI-linked molecules are detected in resting NK cells, while the synthesis of the transmembrane receptors mRNA is only induced upon NK cell activation (Giustiniani et al. 2009). While the proper expression of the two isoforms lacking an Ig domain cannot be assessed because of lack of the appropriate antibodies, the translation of the CD160-TM isoform is specifically detected in activated NK cells but not in activated mast cells or endothelial cells. In fact, the appearance of CD160-TM at the plasma membrane parallels the partial disappearance of the GPI-bound isoform that occurs through a phospholipase-dependent proteolysis (Giustiniani et al. 2007). Interestingly, in paroxysmal nocturnal hemoglobinuria (PNH), a disease characterized by the lack of GPI-linked receptors expression, circulating NK lymphocytes express CD160-TM instead of GPI-anchored CD160(Giustiniani et al. 2012).
Biochemical analyses further reveal that CD160-TM is expressed as a multimeric molecule with an apparent molecular weight of 100 kDa. Unlike CD160-GPI, it seems to interact with MHC-class I molecules, but not with HVEM (Giustiniani et al. 2012). This suggests that conformational changes may be induced by the presence of a transmembrane and an intracellular domain, driving different ligand specificity for CD160-TM. This possibility is sustained by the observation that CD160-TM isoform is not recognized by the anti-CD160 mAb BY55 (Giustiniani et al. 2009).
Functionally, CD160-TM behaves as an activating receptor on NK cells (Fig. 2). Indeed, its triggering leads to the generation of cytotoxic activity towards target cells and enhances NK cells degranulation. This activating function is under the control of an intracellular tyrosine residue located within the intracellular domain of CD160-TM and requires the expression of the Src-family kinase p56lck, as assessed by the loss of function upon tyrosine mutation or kinase depletion (Giustiniani et al. 2007). However, the protein intermediates involved in the downstream signaling pathways remain to be identified.
Among the NK receptors, CD160 and its NK cell lineage highly restricted isoform CD160-TM present unique structural and functional characteristics. Work performed so far suggests that the tightly regulated expression of CD160 isoforms might be an important step in the cascade of events leading to a specific and efficient NK cell response. The existence of at least a GPI-linked and a transmembrane CD160 receptor, each expressed at different stage of NK cell activation and signaling through distinct intracellular pathways, together with the generation of a soluble form of CD160 that mediates immuno-regulatory functions, opens new perspectives regarding the cellular events involved in the regulation of NK cell functions. Finally, in mice, CD160-TM receptor does not exist, while the GPI-anchored CD160 is expressed but only as a dimeric molecule (Maeda et al. 2005). Therefore, the functional specificity of CD160 has to be considered according to its various structural features and its degree of multimerization.
CD160 is a multimeric Ig-like 134-aminoacid glycoprotein expressed as a GPI-anchored receptor on CD56dimCD16+ circulating NK lymphocytes, resting and activated T lymphocytes subsets, activated endothelial cells, and mast cells. In NK lymphocytes only, an additional transmembrane CD160 isoform, with two putative tyrosine phosphorylation sites within its cytoplasmic tail, is expressed upon activation. Both isoforms share identical extracellular region with an Ig-like V-domain, that is weakly homologous to the one of KIR2DL4/CD158d, and behave as activating NK receptors upon engagement. MHC class I molecules have been shown to bind with low affinity to CD160 in both mice and human. The two receptors are described as participating to the generation of an appropriate NK cell effector response through a tightly regulated expression process. Thus, while the transmembrane CD160 isoform is exclusively expressed on NK cells upon activation, the GPI-anchored receptor is concomitantly cleaved, leading to the release of a soluble CD160 molecule that exhibits immunoregulatory function.
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