The CD94/NKG2 C-type lectin receptor complex

A multigene family of human Ig-SF receptors and members of the murine Ly49 C-type lectin family are involved in natural killer (NK) cell-mediated recognition of MHC class I molecules. The human CD94 glycoprotein covalently assembles with different C-type lectins of the NKG2 family. By functional criteria, the CD94/ NKG2-A (kp43) receptor complex appears also involved in NK cell-mediated recognition of different HLA class I allotypes. Similarly to the other NK inhibitory receptors, NKG2-A contains cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). By contrast, NK clones bearing a different receptor complex (CD94/ p39) are triggered upon ligation by CD94-specific monoclonal antibodies (MAbs); the p39 subunit is likely encoded by other member(s) of the NKG2 family. Expression of the different CD94/ NKG2 complexes is warranted to precisely assess their specific interaction with HLA class I molecules, and the molecular basis for their divergent functional properties.     

HLA-E-bound peptides influence recognition by inhibitory and triggering CD94/NKG2 receptors: preferential response to an HLA-G-derived nonamer

The HLA-E class Ib molecule constitutes a major ligand for the lectin-like CD94/NKG2 natural killer (NK) cell receptors. Specific HLA class I leader sequence-derived nonapeptides bind to endogenous HLA-E molecules in the HLA-defective cell line 721.221, inducing HLA-E surface expression, and promote CD94/NKG2A-mediated recognition. We compared the ability of NK clones which expressed either inhibitory or activating CD94/NKG2 receptors to recognize HLA-E molecules on the surface of 721.221 cells loaded with a panel of synthetic nonamers derived from the leader sequences of most HLA class I molecules. Our results support the notion that the primary structure of the HLA-E-bound peptides influences CD94/NKG2-mediated recognition, beyond their ability to stabilize surface HLA-E. Further, CD94/NKG2A⁺ NK clones appeared more sensitive to the interaction with most HLA-E-peptide complexes than did effector cells expressing the activating CD94/NKG2C receptor. However, a significant exception to this pattern was HLA-E loaded with the HLA-G-derived nonamer. This complex triggered cytotoxicity very efficiently over a wide range of peptide concentrations, suggesting that the HLA-E/G-nonamer complex interacts with the CD94/NKG2 triggering receptor with a significantly higher affinity. These results raise the possibility that CD94/NKG2-mediated recognition of HLA-E expressed on extravillous cytotrophoblasts plays an important role in maternal-fetal cellular interactions.     

Regulation of FasL expression in natural killer cells

Fas ligand (FasL)-mediated cytotoxicity is initiated in natural killer (NK) cells through ligation of their activating receptors. The CD16 receptor has been shown to induce FasL expression and cytotoxicity in NK cells. In this study, we made the novel observation that FasL expression was upregulated in NKL cells stimulated through 2B4 and LFA-1 activating receptors, implying a role for FasL-mediated cytotoxicity early in the immune response. Coligation with CD94/NKG2A human leukocyte antigen (HLA) class I inhibitory receptor did not block the induced FasL expression; therefore, these opposing pathways appear to function independently. We also showed, however, that FasL-mediated cytotoxicity was downregulated in CD94/NKG2A-expressing LAK cells in response to the HLA-E ligand, suggesting a mechanism by which aberrant cells expressing class I may evade FasL-mediated cytotoxicity. Thus we show for the first time that 2B4, LFA-1, and CD94/NKG2A receptors are involved in modulating FasL expression and, therefore, cytotoxicity mediated by NK cells.     

Molecular characterization of human CD94: A type II membrane glycoprotein related to the C-type lectin superfamily

Natural killer (NK) cells preferentially express several genes of the C-type lectin superfamily which have been implicated in the regulation of NK cell function. We demonstrate that CD94 is a type II membrane protein encoded by a unique gene of the C-type lectin superfamily. While homology of CD94 with the NK cell-associated NKR-P1 and NKG2 C-type lectin genes is limited to the structural motifs conserved in the carbohydrate recognition domain, all of these genes are on human chromosome 12, the syntenic of mouse chromosome 6, where genes of the NK complex (NKR-P1 and Ly-49) are located. An unexpected feature of CD94 is the essential absence of a cytoplasmic domain, implying that association with other receptors may be necessary for the function of this molecule.     

Soluble HLA-A,-B,-C and -G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8 ligation

There is convincing evidence that soluble HLA-A,-B,-C (sHLA-A,-B,-C) and soluble HLA-G (sHLA-G) antigens can induce apoptosis in CD8(+) activated T cells although there is scanty and conflicting information about the mechanism(s) by which sHLA-A,-B,-C antigens and sHLA-G antigens induce apoptosis. In this study we have compared the apoptosis-inducing ability of sHLA-A,-B,-C antigens with that of sHLA-G1 antigens in CD8(+) T lymphocytes and CD8(+) NK cells. Furthermore we have compared the inhibitory effect of sHLA-A,-B,-C antigens and of sHLA-G1 antigens on the activity of EBV-specific CD8(+) cytotoxic T lymphocytes (CTL). sHLA molecules were purified from serum and from the supernatant of HLA class I-negative cells transfected with one gene encoding either classical or non-classical HLA class I antigens. Both classical and non-classical sHLA class I molecules trigger apoptosis in CD8(+) T lymphocytes and in CD8(+) NK cells, which lack the T cell receptor, and their apoptotic potency is comparable. The binding of sHLA-A,-B,-C and sHLA-G1 molecules to CD8 leads to Fas ligand (FasL) up-regulation, soluble FasL (sFasL) secretion and CD8(+) cell apoptosis by Fas/sFasL interaction. Moreover, classical and non-classical sHLA class I molecules inhibit the cytotoxic activity of EBV-specific CD8(+) CTL. As the amount ofsHLA-G molecules detectable in normal serum is significantly lower than that of sHLA-A,-B,-C molecules, the immunomodulatory effects of sHLA class I molecules purified from serum are likely to be mainly attributable to classical HLA class I antigens. As far as the potential in vivo relevance of these findings is concerned, we suggest that classical sHLA class I molecules may play a major immunoregulatory role in clinical situations characterized by activation of the immune system and elevated sHLA-A,-B,-C serum levels. In contrast, non-classical HLA class I molecules may exert immunomodulatory effects in particular conditions characterized by elevated sHLA-G levels such as pregnancy and some neoplastic diseases.     

The CD94 and NKG2-A C-type lectins covalently assemble to form a natural killer cell inhibitory receptor for HLA class I molecules

CD94, a type II membrane protein containing a C-type lectin domain, has been shown to be involved in natural killer (NK) cell-mediated recognition of different HLA allotypes. The inhibitory form of the CD94 receptor has recently been identified by the specific monoclonal antibody (mAb) Z199. Herein, we demonstrate that the inhibitory receptor is in fact a complex formed by the covalent association of CD94 with the NKG2-A molecule (Mr ～ 43 kDa), another member of the C-type lectin superfamily, and that Z199 mAb specifically recognize NKG2-A molecules. Although the NKG2-A-encoding cDNA has been known for several years, the corresponding protein and its possible function remained undefined. Moreover, we show that the NKG2-B protein, an alternatively spliced product of the NKG2-A gene, can also assemble with CD94. Remarkably, both NKG2-A and NKG2-B proteins contain cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIM). This may provide the molecular basis of the inhibitory function mediated by the CD94/NKG2-A receptor complexes.     

Inhibition of antigen-induced T cell response and antibody-induced NK cell cytotoxicity by NKG2A: association of NKG2A with SHP-1 and SHP-2 protein-tyrosine phosphatases

Subsets of T and natural killer (NK) lymphocytes express the CD94-NKG2A heterodimer, a receptor for major histocompatibility complex class I molecules. We show here that engagement of the CD94-NKG2A heterodimer inhibits both antigen-driven tumor necrosis factor (TNF) release and cytotoxicity on melanoma-specific human T cell clones. Similarly, CD16-mediated NK cell cytotoxicity is extinguished by cross-linking of the CD94-NKG2A heterodimer. Combining in vivo and in vitro analysis, we report that both I/VxYxxL immunoreceptor tyrosine-based inhibition motifs (ITIM) present in the NKG2A intracytoplasmic domain associate upon tyrosine phosphorylation with the protein tyrosine phosphatases SHP-1 and SHP-2, but not with the polyinositol phosphatase SHIP. Determination of the dissociation constant, using surface plasmon resonance analysis, indicates that NKG2A phospho-ITIM interact directly with the SH2 domains of SHP-1 and SHP-2 with a high affinity. Engagement of the CD94-NKG2A heterodimer therefore appears as a protein-tyrosine phosphatase-based strategy that negatively regulates both antigen-induced T cell response and antibody-induced NK cell cytotoxicity. Our results suggest that this inhibitory pathway sets the threshold of T and NK cell activation.     

The role of CD94/NKG2 in innate and adaptive immunity

CD94/NKG2 is a heterodimer expressed on natural killer (NK) and a small subset of T cells. This receptor varies in function as an inhibitor or activator depending on which isoform of NKG2 is expressed. The ligand for CD94/NKG2 is HLA-E in human and its homolog, Qa1 in mouse, which are both nonclassical class I molecules that bind leader peptides from other class I molecules. Although <5% of CD8 T cells express the receptor in a naïve mouse, its expression is upregulated upon specific recognition of antigen. Similar to NK cells, most CD8 T cells that express high levels of CD94 co-express NKG2A, the inhibitory isoform. The engagement of this receptor can lead to a blocking of cytotoxicity. However, these receptors have also been implicated in the cell survival of both NK and CD8T cells. The level of CD94 expression is inversely correlated with the level of apoptosis in culture. Thus, CD94/NKG2 receptors may regulate effector functions and cell survival of NK cells and CD8 T cells, thereby playing a crucial role in the innate and adaptive immune response to a pathogen.     

The leukocyte Ig-like receptor (LIR)-1 for the cytomegalovirus UL18 protein displays a broad specificity for different HLA class I alleles: analysis of LIR-1 + NK cell clones

Leukocyte Ig-like receptor (LIR)-1 is a member of the Ig superfamily which has been shown to bind the human cytomegalovirus MHC class I homologue UL-18 protein. In this study, we have analyzed the expression and function of LIR-1 in human NK cells. We show that LIR-1 is expressed by a subset of NK cells variable in size among different donors. When compared to the known HLA class I-specific NK receptors, the expression of LIR-1 was found to be partially overlapped with that of CD94-NKG2A or with that of killer inhibitory receptors (KIR) belonging to the Ig superfamily. The use of the soluble form of UL-18 molecule revealed, in double fluorescence analysis, a selective binding to LIR-1 + cells while no correlation was observed between expression of either KIR or CD94-NKG2A molecules and ability to bind UL18. We further determined whether LIR-1 could also function as receptor for HLA class I molecules. To this end, we assessed the capability of LIR-1 + NK cell clones of lysing HLA class I- target cells transfected with different class I alleles, including HLA-A, -B, -C and -G alleles. Data revealed that LIR-1 functions as a broad HLA class I-specific inhibitory receptor recognizing different alleles coded for by different HLA loci.     

Human T cell receptor-mediated recognition of HLA-E

The HLA-E class Ib molecule presents hydrophobic peptides derived from the leader sequences of other class I molecules, constituting the ligands for CD94/NKG2 lectin-like receptors. Along the course of our studies on human CD94+ T cells, we characterized an alpha beta CD8+CD94/NKG2C+ CTL clone (K14). In cytolytic assays against the murine TAP-deficient RMA-S cells transfected with human beta2 microglobulin and HLA-E (RMA-S/HLA-E), loaded with different synthetic peptides, K14 displayed a pattern of specific recognition distinct to that observed in CD94/NKG2C+ NK clones tested in parallel. RMA-S/HLA-E cells loaded with some but not all HLA class I leader sequence peptides were efficiently recognized by K14 but not by CD94/NKG2C clones, andvice versa. Remarkably, K14 also reacted with HLA-E loaded with a peptide derived from the BZLF-1 Epstein-Barr virus protein. Anti-CD94 mAb did not prevent K14 cytotoxicity against RMA-S/HLA-E cells, whereas incubation with anti-clonotypic mAb specific for the K14 TCR markedly inhibited lysis. Soluble HLA-E tetramers refolded with different peptides (i.e. VMAPRTVLL, VMAPRTLIL, VMAPRTLFL) specifically stained K14 cells. HLA-E tetramer binding was minimally reduced by pretreatment with anti-CD94 mAb alone, but was completely prevented in combination with anti-clonotypic mAb. Altogether, the data unequivocally imply the generation of human T cells potentially recognizing through the alpha beta TCR HLA-E molecules that bind to class I- and virus-derived peptides.     

HLA-E is expressed on trophoblast and interacts with CD94/NKG2 receptors on decidual NK cells

Non-classical MHC class I molecule HLA-E is the ligand for CD94/NKG2 NK cell receptors. Surface expression of HLA-E requires binding of specific HLA class I leader sequences. The uterine mucosa in early pregnancy (decidua) is infiltrated by large numbers of NK cells, which are closely associated with placental trophoblast cells. In this study we demonstrate that trophoblast cells express HLA-E on their cell surface in addition to the previously reported expression of HLA-G and HLA-C. Furthermore, we show that the vast majority of decidual NK cells bind to HLA-E tetrameric complexes and this binding is inhibited by mAb to CD94. Thus, recognition of fetal HLA-E by decidual NK cells may play a key role in regulation of placentation. The functional consequences of decidual NK cell interaction were investigated in cytotoxicity assays using polyclonal decidual NK cells. The overall effect of CD94/NKG2 interaction with HLA-E is inhibition of cytotoxicity by decidual NK cells. However, since decidual NK cells are unable to kill trophoblast even in the presence of mAb to MHC class I molecules and NK cell receptors, HLA-E interaction with CD94/NKG2 receptors may regulate other functions besides cytolysis during implantation.     

Two genes in the rat homologous to human NKG2

Two different lectin-like receptors for MHC class I molecules have so far been identified on natural killer (NK) cells, the Ly-49 homodimeric receptors in mice and the NKG2/CD94 heterodimeric receptors in humans. The recent identification of a rat CD94 orthologue implied that NK cell receptors equivalent to NKG2/CD94 also exist in rodents. Here we describe the cDNA cloning of two rat genes homologous to members of the human NKG2 multigene family. The deduced rat NKG2A protein contains a cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM), whereas the cytoplasmic tail of rat NKG2C lacks ITIM. The genes map to the rat NK gene complex and are selectively expressed by NK cells. The expression is strain dependent, with high expression in DA and low in PVG NK cells, correlating with the expression of rat CD94. Ly-49 genes have previously been identified in the rat, and the existence of rat NKG2 genes in addition to a CD94 orthologue suggests that NK cell populations utilize different C-type lectin receptors for MHC class I molecules in parallel.     

HLA-G1 co-expression boosts the HLA class I-mediated NK lysis inhibition

It is now acknowledged that the pattern of HLA-G expression is not restricted to extravillous cytotrophoblast cells, as several studies described HLA-G in HLA class I+ cells, such as thymic epithelial cells, cytokine-activated monocytes and some tumors. In these situations, HLA-G may provide an additional inhibitory signal to escape from NK cell-mediated cytotoxicity. Accordingly, the aim of this study was to define the behavior of HLA-G once it is co-expressed into an HLA-A, -B, -C and -E+ cell line. For this purpose, HLA-G1 cDNA was transfected into an HLA class I+ melanoma cell line which was used as a target towards freshly isolated peripheral blood NK cells. Cytotoxic experiments using either anti-HLA-G1 or anti-HLA-G1 inhibitory receptor mAb show that HLA-G1 boosts the HLA class I-mediated inhibition of polyclonal NK cells through interaction with ILT-2, which appears as the major HLA-G1 inhibitory receptor involved. Nevertheless, HLA-G1 is also able to inhibit the cytolytic activity of an ILT-2- NK clone which otherwise expresses another HLA-G1 inhibitory receptor belonging to the KIR103 gene family. In order to more precisely define the relative role exerted by HLA-G1 versus -E on polyclonal NK cells, antibody-blocking assays were carried out using either anti-HLA class I or anti-CD94/NKG2A. Results demonstrate that in the absence of HLA-G1, the naturally expressed HLA class I-mediated NK inhibition is predominantly exerted by HLA-E through binding with CD94/NKG2A. In contrast, once HLA-G1 is expressed, it becomes the major NK inhibitory ligand.     

Structure and function of the CD94 C-type lectin receptor complex involved in recognition of HLA class I molecules

Summary: A multigene family of immunoglobulin superfamily (Ig-SF) killer cell inhibitory receptors (KIRs) specifically recognize HLA class I molecules, while the interaction with H-2 products is mediated by members of the murine Ly49 C-type lectin family. A common structural feature of these receptors with inhibitory function is the presence of cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that couple them to SHP phosphatases. Strong support for the involvement of the CD94 C-type lectin receptor complex in NK cell-mediated recognition of Bw6+ HLA-B, HLA A and HLA-C alleles has been obtained. The cloned CD94 molecule covalently assembles with at least two different glyco-proteins (43 kDa and 39 kDa) to form functional receptors. NK cells inhibited upon HLA recognition express the CD94/p43 dimer, whose specificity for HLA molecules partially overlaps the Ig-SF receptor system. By contrast. NK clones bearing the homologous CD94/p39 receptor are triggered upon its ligation by CD94-specific mAbs. Remarkably, a set of Ig-SF receptors (p50) homologous to p58 KIRs also display an activating function. CD94-associated molecules belong to the NKG2 family of C-type lectins; the NKG2-A gene encodes for the p43 subunit. which contains cytoplasmic ITIMS. Expression of the different CD94 heterodimeric receptors will enable precise analysis of their putative interaction with HLA class I molecules.     

Molecular characterization of a gene in the rat homologous to human CD94

Three classes of major histocompatibility (MHC) class I binding receptors on natural killer (NK) cells have so far been described: CD94/NKG2 heterodimeric receptors and killer cell inhibitory receptors in the human, and Ly-49 homodimers in rodents. CD94, NKG2 and Ly-49 belong to the C-type lectin super-family. As yet, CD94 and NKG2 molecules have not been detected in rodents or Ly-49 in humans. It has therefore been proposed that the two receptors represent functional equivalents in these species. The present study describes the cDNA cloning of a novel rat gene encoding a protein of 179 amino acids, 54.2 % identical to human CD94. The single-copy Cd94 gene is localized to the rat NK gene complex (NKC), within 50 kb from Nkrp2, between the Nkrp1 and Ly49 gene clusters. By Northern blot analysis, we showed that rat CD94 is selectively expressed by NK cells and a small subset of T cells, similar to the human ortho-logue. This expression is strain dependent, with high expression in DA NK cells and low in PVG NK cells. Evidence is presented that this difference is not due to receptor repertoire shaping by MHC-encoded ligands, but is controlled by genetic elements residing within the NKC. The identification of a rat CD94 orthologue suggests that NK cell populations utilize two different C-type lectin receptors for MHC class I molecules in parallel.     

Reciprocal age related change in natural killer cell receptors for MHC class I

Natural killer (NK) cells are essential for healthy aging. NK cell activation is controlled by MHC class I-specific CD94/NKG2 receptors and killer immunoglobulin-like receptors (KIR). To assess NK cytotoxic function in isolation from MHC receptor engagement, we measured the ability of purified NK cells to kill mouse P815 target cells in the presence of anti-CD16 mAb. CD16-mediated cytotoxicity did not change with age, indicating that NK activation and cytotoxic granule release remained functional. We then investigated MHC class I receptor expression on NK cells. There was an age related decrease in CD94 and NKG2A expression and a reciprocal age related increase in KIR expression. NKG2A expression also declined with age on CD56(+) T cells. CD94/NKG2A receptor function was proportional to expression, indicating that NK cell inhibitory signaling pathways were intact. NKG2A and KIR expression were complementary, suggesting that CD94/NKG2A function could substitute for inhibitory KIR function during polyclonal NK cell development in both young and elderly adults. The distinct roles of CD94/NKG2A and KIR receptors suggest that shifting MHC class I receptor expression patterns reflect age related changes in NK cell and CD56(+) T cell turnover and function in vivo.     

HLA-G recognition by human natural killer cells. Involvement of CD94 both as inhibitory and as activating receptor complex

The lack of classical human histocompatibility leukocyte antigen (HLA) molecules in human placenta prevents the recognition and lysis by maternal T lymphocytes but poses the problem of susceptibility to natural killer (NK) cell-mediated lysis. The nonclassical HLA class I molecule HLA-G may mediate protection from NK cells. NK cells are known to express a number of HLA class I-specific inhibitory receptors. These include members of the immunoglobulin (Ig) superfamily (p58, p70, p140), characterized by a defined allele specificity, and CD94/NKG2A with a broad specificity for different HLA class I molecules. We analyzed a series of NK cell clones derived from normal peripheral blood expressing different NK receptors (NKR). Clones were analyzed for their cytolytic activity against the HLA class I-negative 221 cell line either untransfected or transfected with HLA-G (221/G) or other informative alleles, as control. All clones expressing CD94/NKG2A [as identified by the Z199 monoclonal antibody (mAb)] displayed a markedly reduced cytolytic activity against 221/G. Moreover, mAb directed to the CD94/NKG2A complex completely restored target cell lysis. Among NKG2A-negative NK clones, different functional patterns could be detected. Clones expressing inhibitory receptors belonging to the Ig superfamily lysed 221/G target cells with equal or higher efficiency than untransfected 221 cells. These data indicated that p58, p70 and p140 do not function as HLA-G-specific inhibitory NKR, and that HLA-G-specific activating NKR also exist. Further analysis indicated that in these clones (characterized by the CD94⁺/NKG2A^? phenotype) mAb specific for CD94, but not for the other NKR, reversed the activating effect. Infrequent clones were also isolated that, in spite of the lack of CD94/NKG2A, displayed HLA-G specificity, thus suggesting the existence of a different, still unknown NKR.