Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E.

The lytic function of human natural killer (NK) cells is markedly influenced by recognition of class I major histocompatibility complex (MHC) molecules, a process mediated by several types of activating and inhibitory receptors expressed on the NK cell. One of the most important of these mechanisms of regulation is the recognition of the non-classical class I MHC molecule HLA-E, in complex with nonamer peptides derived from the signal sequences of certain class I MHC molecules, by heterodimers of the C-type lectin-like proteins CD94 and NKG2. Using soluble, recombinant HLA-E molecules assembled with peptides derived from different leader sequences and soluble CD94/NKG2-A and CD94/NKG2-C proteins, the binding of these receptor-ligand pairs has been analysed. We show first that these interactions have very fast association and dissociation rate constants, secondly, that the inhibitory CD94/NKG2-A receptor has a higher binding affinity for HLA-E than the activating CD94/NKG2-C receptor and, finally, that recognition of HLA-E by both CD94/NKG2-A and CD94/NKG2-C is peptide dependent. There appears to be a strong, direct correlation between the binding affinity of the peptide-HLA-E complexes for the CD94/NKG2 receptors and the triggering of a response by the NK cell. These data may help to understand the balance of signals that control cytotoxicity by NK cells.     

Role that each NKG2A immunoreceptor tyrosine-based inhibitory motif plays in mediating the human CD94/NKG2A inhibitory signal

The human NKG2A chain of the CD94/NKG2A receptor contains two immunoreceptor Tyr-based inhibitory motifs (ITIMs) in its cytoplasmic tail. To determine the relative importance of membrane-distal (residues 6-11) and membrane-proximal (residues 38-43) ITIMs in mediating the inhibitory signal, we made site-directed mutants of NKG2A at the Y (Y8F, Y40F, Y8F/Y40F) and the residues two positions N-terminal (Y-2) of Y (V6A, I38A, V6A/I38A) in each motif. Wild-type (wt) and mutated NKG2A were then cotransfected with CD94 into rat basophilic leukemia 2H3 cells. Immunochemical analyses after pervanadate treatment showed that each of the mutant molecules could be phosphorylated to expected levels relative to wt NKG2A and that all the mutations significantly reduced the avidity of SH2 domain-bearing tyrosine phosphatase-1 for NKG2A. Confocal microscopy was used to determine whether SH2 domain-bearing tyrosine phosphatase-1 and CD94/NKG2A colocalized intracellularly after receptor ligation. Only the Y8F/Y40F and Y8F mutant NKG2A molecules failed to show a dramatic colocalization. In agreement with this result, the Y8F/Y40F mutant was unable to inhibit FcepsilonRI-mediated serotonin release and the Y8F mutant was relatively ineffective compared with wt NKG2A. In contrast, the Y40F mutant was 70% as effective as wt in mediating inhibition, and the Y-2 mutations did not remarkably affect inhibitory function. These results show that, like KIR, both NKG2A ITIMs are required for mediating the maximal inhibitory signal, but opposite to KIR, the membrane-distal ITIM is of primary importance rather than the membrane-proximal ITIM. This probably reflects the opposite orientation of the ITIMs in type II vs type I proteins.     

Chicken CD69 and CD94/NKG2-like genes in a chromosomal region syntenic to mammalian natural killer gene complex

In mammals, natural killer (NK) cell C-type lectin receptors were encoded in a gene cluster called natural killer gene complex (NKC). The NKC is not reported in chicken yet. Instead, NK receptor genes were found in the major histocompatibility complex. In this study, two novel chicken C-type lectin-like receptor genes were identified in a region on chromosome 1 that is syntenic to mammalian NKC region. The chromosomal locations were validated with fluorescent in situ hybridization. Based on 3D structure modeling, sequence homology, chromosomal location, and phlylogenetic analysis, one receptor is the orthologue of mammalian cluster of differentiation 69 (CD69), and the other is highly homologous to CD94 and NKG2. Like CD94/NKG2 gene found in teleostean fishes, chicken CD94/NKG2 has the features of both human CD94 and NKG2A. Unlike mammalian NKC, these two chicken C-type lectin receptors are not closely linked but separated by 42 million base pairs according to the chicken draft genome sequence. The arrangement of several other genes that are located outside the mammalian NKC is conserved among chicken, human, and mouse. The chicken NK C-type lectin-like receptors in the NKC syntenic region indicate that this chromosomal region existed before the divergence between mammals and aves. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequences have been submitted to the GenBank nucleotide sequence database under the accession number chicken CD69 (DQ156495), CD94/NKG2 (DQ156496), and CD94/NKG2 variant (DQ241793).     

HLA-E is the ligand for the natural killer cell CD94/NKG2 receptors

CD94/NKG2 is a recently described receptor present on natural killer (NK) cells and certain T cells that is composed of the CD94 chain covalently associated with a member of the NKG2 family of molecules. Both chains are glycosylated members of the C-type lectin superfamily. The CD94/NKG2 receptors are functionally heterogenous depending on which NKG2 family member is associated with CD94. Initially, it was thought that CD94/NKG2 receptors recognized a broad array of HLA-A, -B and -C (classical), as well as the nonclassical HLA-G, MHC class I molecules. Instead, recent data have suggested that this receptor is specific for HLA-E complexed with a peptide derived from the signal sequence (residues 3–11) of certain classical MHC class I molecules. Position 2 (residue 4) in the signal sequence derived peptides appears pivotal in determining whether the HLA-E/peptide complex confers resistance to NK-mediated lysis. The potential roles that the CD94/NKG2-HLA-E receptor ligand interaction might play in infection and tumor development are discussed.     

CD94/NKG2-A inhibitory complex blocks CD16-triggered Syk and extracellular regulated kinase activation, leading to cytotoxic function of human NK cells.

The CD94/NKG2-A complex is the inhibitory receptor for the nonclassical MHC class I molecule HLA-E on human NK cells. Here we studied the molecular mechanisms underlying the inhibitory activity of CD94/NKG2-A on NK cell functions by analyzing its interference on CD16-initiated signaling pathways involved in the control of cytolytic activity. Both tyrosine phosphorylation and activation of Syk kinase together with tyrosine phosphorylation of CD16 receptor zeta subunit are markedly inhibited by the coengagement of CD94/NKG2-A complex. As a downstream consequence, CD94/NKG2-A cross-linking impairs the CD16-induced activation of extracellular regulated kinases (ERKs), a pathway involved in NK cytotoxic function. The block of ERK activation is exerted at an early, PTK-dependent stage in the events leading to p21ras activation, as the CD16-induced tyrosine phosphorylation of Shc adaptor protein and the formation of Shc/Grb-2 complex are abrogated by CD94/NKG2-A simultaneous engagement. Our observations indicate that CD94/NKG2-A inhibits the CD16-triggered activation of two signaling pathways involved in the cytotoxic activity of NK cells. They thus provide molecular evidence to explain the inhibitory function of CD94/NKG2-A receptor on NK effector functions.     

HLA-E and HLA-G expression on porcine endothelial cells inhibit xenoreactive human NK cells through CD94/NKG2-dependent and -independent pathways

Human NK cells contribute a significant role to host defense as well as xenogeneic cytotoxicity. Previous studies using human 721.221 cell line have shown that peptides derived from the leader sequence of the HLA-G binds and up-regulates the surface expression of HLA-E molecules, which was considered to consequently provide negative signals to human NK cells. However, the direct role of HLA-G in inhibiting human NK cells remains controversial. In this study, we showed that the expression of HLA-G or HLA-E in porcine endothelial cells directly protected sensitive porcine cells from human NK cell-mediated xenogeneic cytotoxicity. Ab blocking assays using F(ab')2 of the HLA class I-specific mAb PA2.6 indicated that the protection was directly mediated by the expression of HLA-G and HLA-E on the porcine cells. The HLA-E-mediated protection was blocked by anti-human CD94 Ab. In addition, the engagement of HLA-E lead to the phosphorylation of the CD94/NKG2 complex and the recruitment of SH2 domain-containing protein phosphatase 1 (SHP-1) to the complex. Therefore, HLA-E protected porcine cells from xenoreactive human NK cells through a CD94/NKG2-dependent pathway. In contrast, HLA-G inhibited human NK cells in the absence of CD94/NKG2 phosphorylation or SHP-1 recruitment, and the inhibition was not blocked by anti-CD94 Ab. Therefore, HLA-G protected porcine cells from human NK cells through a CD94/NKG2-independent pathway. These results demonstrated that both HLA-E and HLA-G could directly inhibit human NK cells in the absence of other endogenous HLA class I molecules. These results also have practical implications in preventing xenograft rejection mediated by human NK cells.     

Molecular determinants regulating the pairing of NKG2 molecules with CD94 for cell surface heterodimer expression

The lytic capacity of a NK cell is regulated, in part, by the balance in cell surface expression between inhibitory CD94/NKG2A and activating CD94/NKG2C heterodimers. We demonstrate that, in the absence of DAP12, rhesus monkey NKG2A is preferentially expressed at the cell surface with CD94 due to a single amino acid difference in the transmembrane of NKG2A and NKG2C. Furthermore, in the context of an NKG2A transmembrane, the stalk domain of NKG2C was found to enhance heterodimer formation with CD94 compared with the stalk domain of NKG2A. In the presence of DAP12, the ability of NKG2C to compete for cell surface CD94 heterodimerization is enhanced and approaches that of NKG2A. Finally, allelic differences that affect the ability of rhesus NKG2A to reach the cell surface with CD94 could also be mapped to the transmembrane. These differences in the ability of inhibitory and activating NKG2 molecules to reach the cell surface provide a mechanism for the regulation of NK cell activity.     

Expression of CD94/NKG2-A on human T lymphocytes is induced by IL-12: implications for adoptive immunotherapy

NK cell receptors (NKRs) are expressed on a subset of human T cells, predominantly CD8(+), within which they can modulate TCR-mediated functions. In an attempt to identify the mechanisms leading to NKR expression, we analyzed the capacity of IL-12 to modulate the expression by T cells of the components of the CD94/NKG2-A inhibitory receptor, a member of the C-type lectin-like family of NKR. We show that IL-12 induces the expression of NKG2-A and/or CD94 by CD8(+) T cells in culture, and that this induction was mediated neither by IFN-gamma nor by IL-15. We also show, using the redirected killing assay, that IL-12-induced expression of both CD94 and NKG2-A led to the acquisition by T cells of a functional inhibitory receptor. Expression of the CD94/NKG2-A inhibitory receptor was also induced by IL-12 during T cell Ag stimulation so that in the presence of this cytokine a high proportion of melanoma-reactive CTL induced from PBL by melanoma peptide stimulation expressed this receptor. This study emphasizes the implication of IL-12 in the modulation of immune responses through NKR induction.     

Cytotoxicity of CD56(bright) NK cells towards autologous activated CD4+ T cells is mediated through NKG2D, LFA-1 and TRAIL and dampened via CD94/NKG2A

In mouse models of chronic inflammatory diseases, Natural Killer (NK) cells can play an immunoregulatory role by eliminating chronically activated leukocytes. Indirect evidence suggests that NK cells may also be immunoregulatory in humans. Two subsets of human NK cells can be phenotypically distinguished as CD16(+)CD56(dim) and CD16(dim/-)CD56(bright). An expansion in the CD56(bright) NK cell subset has been associated with clinical responses to therapy in various autoimmune diseases, suggesting an immunoregulatory role for this subset in vivo. Here we compared the regulation of activated human CD4(+) T cells by CD56(dim) and CD56(bright) autologous NK cells in vitro. Both subsets efficiently killed activated, but not resting, CD4(+) T cells. The activating receptor NKG2D, as well as the integrin LFA-1 and the TRAIL pathway, played important roles in this process. Degranulation by NK cells towards activated CD4(+) T cells was enhanced by IL-2, IL-15, IL-12+IL-18 and IFN-α. Interestingly, IL-7 and IL-21 stimulated degranulation by CD56(bright) NK cells but not by CD56(dim) NK cells. NK cell killing of activated CD4(+) T cells was suppressed by HLA-E on CD4(+) T cells, as blocking the interaction between HLA-E and the inhibitory CD94/NKG2A NK cell receptor enhanced NK cell degranulation. This study provides new insight into CD56(dim) and CD56(bright) NK cell-mediated elimination of activated autologous CD4(+) T cells, which potentially may provide an opportunity for therapeutic treatment of chronic inflammation.     

Analysis of HLA-E peptide-binding specificity and contact residues in bound peptide required for recognition by CD94/NKG2

The MHC class Ib molecule HLA-E is the primary ligand for CD94/NKG2A-inhibitory receptors expressed on NK cells, and there is also evidence for TCR-mediated recognition of this molecule. HLA-E preferentially assembles with a homologous set of peptides derived from the leader sequence of class Ia molecules, but its capacity to bind and present other peptides remains to be fully explored. The peptide-binding motif of HLA-E was investigated by folding HLA-E in vitro in the presence of peptide libraries derived from a nonameric leader peptide sequence randomized at individual anchor positions. A high degree of selectivity was observed at four of five total anchor positions, with preference for amino acids present in HLA-E-binding peptides from class Ia leader sequences. Selectivity was also observed at the nonanchor P5 position, with preference for positively charged amino acids, suggesting that electrostatic interactions involving the P5 side chain may facilitate assembly of HLA-E peptide complexes. The observed HLA-E peptide-binding motif was strikingly similar to that previously identified for the murine class Ib molecule, Qa-1. Experiments with HLA-E tetramers bearing peptides substituted at nonanchor positions demonstrated that P5 and P8 are primary contact residues for interaction with CD94/NKG2 receptors. A conservative replacement of Arg for Lys at P5 completely abrogated binding to CD94/NKG2. Despite conservation of peptide-binding specificity in HLA-E and Qa-1, cross-species tetramer-staining experiments demonstrated that the interaction surfaces on CD94/NKG2 and the class Ib ligands have diverged between primates and rodents.     

NK cell CD94/NKG2A inhibitory receptors are internalized and recycle independently of inhibitory signaling processes

Human CD94/NKG2A is an inhibitory receptor that recognizes HLA-E and is expressed by NK cells and a subset of T cells. We have analyzed the cellular trafficking of the CD94/NKG2A receptor using the NKL cell line and peripheral blood NK cells. Flow cytometric, confocal microscopic, and biochemical analyses show that CD94/NKG2A continuously recycles in an active process that requires the cytoskeleton between the cell surface and intracellular compartments that are distinguishable from recycling compartments used by well-characterized receptors, such as transferrin receptor (CD71). CD94/NKG2A, an inhibitory receptor, traffics differently from the closely related CD94/NKG2C molecule, an activating receptor. Using transfection/expression analyses of wild-type and mutant CD94/NKG2A molecules in the HLA-E negative rat basophilic cell line RBL-2H3, we demonstrate that CD94/NKG2A internalization is independent of ligand cross-linking or the presence of functional immunoreceptor tyrosine-based inhibition motifs. Thus, the mechanisms that control cell surface homeostasis of CD94/NKG2A are independent of functional signaling.     

Expression of p58.2 or CD94/NKG2A inhibitory receptors in an NK-like cell line,YTINDY, leads to HLA Class I-mediated inhibition of cytotoxicity in the p58.2- but not the CD94/NKG2A-expressing transfectant

Natural killer cytotoxicity is down-regulated by HLA Class I-specific inhibitory receptors classified as killer inhibitory receptors (KIRs) or C-type lectins. The regulation of their inhibitory signaling pathways is not completely understood. The YTINDY NK-like cell line was transfected to express p58.2 KIR (YT/C143 transfectant) or CD94/NKG2A C-type lectin (YT/CD94 transfectant); and YT/C143, but not YT/CD94, cytotoxicity was down-regulated by Class I. YT/C143 and YT/CD94 expressed equally low p56(lck) levels, suggesting that p56(lck) is not absolutely required for p58.2 signaling but may be required for CD94/NKG2A signaling. Lower SHP-1 levels and activity were observed in YT/CD94 compared to YT/C143. However, increasing SHP-1 to equivalent levels in YT/C143 did not restore inhibition in YT/CD94. Our results suggest that the combination of low p56(lck) and SHP-1 levels may be responsible for the absent inhibitory signal in YT/CD94. In addition, the possible expression of CD94/NKG2C activating receptor may override inhibitory signals transduced through CD94/NKG2A.     

CD94/NKG2A expression is associated with proliferative potential of CD8 T cells during persistent polyoma virus infection

Memory CD8 T cells comprise a critical component of durable immunity because of their capacity to rapidly proliferate and exert effector activity upon Ag rechallenge. During persistent viral infection, memory CD8 T cells repetitively encounter viral Ag and must maintain a delicate balance between limiting viral replication and minimizing immunopathology. In mice infected by polyoma virus, a natural mouse pathogen that establishes long-term persistent infection, the majority of persistence-phase antiviral CD8 T cells express the inhibitory NK cell receptor CD94/NKG2A. In this study, we asked whether CD94/NKG2A expression is associated with Ag-specific recall of polyoma virus-specific CD8 T cells. During the persistent phase of infection, polyoma virus-specific CD8 T cells that express CD94/NKG2A were found to preferentially proliferate; this proliferation was dependent on cognate Ag both in vitro and in vivo. In addition, CD94/NKG2A(+) polyoma-specific CD8 T cells have a markedly enhanced capacity to produce IL-2 upon ex vivo Ag stimulation compared with CD94/NKG2A(-) polyoma-specific CD8 T cells. Importantly, CD94/NKG2A(+) anti-polyoma virus CD8 T cells appear to be essential for Ag-specific recall responses in mice persistently infected by polyoma virus. Because of its higher proliferative potential and capacity to produce IL-2, we propose that the CD94/NKG2A(+) subpopulation represents a less differentiated state than the CD94/NKG2A(-) subpopulation. Identification of proliferation-competent subpopulations of memory CD8 T cells should prove valuable in designing therapeutic vaccination strategies for persistent viral infections.