CD94 functions as a natural killer cell inhibitory receptor for different HLA class I alleles: identification of the inhibitory form of CD94 by the use of novel monoclonal antibodies

CD94 molecules have been suggested to function as inhibitory natural killer cell (NK) receptors involved in the recognition of HLA-B alleles sharing the Bw6 supertypic specificity. In this study, we show that CD94 molecules may play a more general role: they are also involved in the recognition of other HLA class I molecules, including HLA-C and at least some HLA-A alleles. The inhibitory effect mediated by CD94 molecules on NK cytolytic activity is lower in magnitude than that of bona fide inhibitory receptors such as p58 or p70. Distinct from the other human NK receptors involved in HLA class I recognition, CD94 is expressed on virtually all NK cells. In addition, it has been shown to be functionally heterogeneous since, in different clones, CD94 mediated either cell triggering or inhibition. Although NK cells expressing inhibitory CD94 molecules are usually characterized by a CD94^bright phenotype, there is no precise correlation between fluorescence intensity and inhibitory or activating function. Here, we describe two novel monoclonal antibodies (mAb) which selectively recognize inhibitory CD94 molecules and bind to a subset (variable in size among different donors) of CD94^bright cells. The use of these mAb allows the direct assessment of NK cells expressing inhibitory CD94 receptors both at the population and at the clonal level.     

Cloning of NKG2-F,a new member of the NKG2 family of human natural killer cell receptor genes

The NKG2 family of genes encodes at least four different type II transmembrane molecules (NKG2-A, NKG2-B, NKG2-C and NKG2-E) which contain a C-lectin domain. These proteins have been shown to be covalently associated with CD94, another C-type lectin member. The heterodimers are involved in natural killer cell-mediated recognition of different HLA-allotypes. Here we describe the cloning of a new NKG2-related gene, termed NKG2-F, localized 25 kb from NKG2-A as well as its relationship with the previously described NKG2-D cDNA. Despite the similarities with the other NKG2 genes, NKG2-F encodes a putative protein which does not contain any lectin domain. However, a conserved 24-amino acid sequence, present in all members of the NKG2 family, suggests that NKG2-F is also able to form heterodimers with CD94.     

Balance between activating NKG2D,DNAM‐1, NKp44 and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and synovial hyperplasia leading to progressive joint destruction. Fibroblast‐like synoviocytes (FLS) are central components of the aggressive, tumour‐like synovial structure termed pannus, which invades the joint space and cartilage. A distinct natural killer (NK) cell subset expressing the inhibitory CD94/NKG2A receptor is present in RA synovial fluid. Little is known about possible cellular interactions between RA‐FLS and NK cells. We used cultured RA‐FLS and the human NK cell line Nishi, of which the latter expresses an NK receptor repertoire similar to that of NK cells in RA synovial fluid, as an in vitro model system of RA‐FLS/NK cell cross‐talk. We show that RA‐FLS express numerous ligands for both activating and inhibitory NK cell receptors, and stimulate degranulation of Nishi cells. We found that NKG2D, DNAM‐1, NKp46 and NKp44 are the key activating receptors involved in Nishi cell degranulation towards RA‐FLS. Moreover, blockade of the interaction between CD94/NKG2A and its ligand HLA‐E expressed on RA‐FLS further enhanced Nishi cell degranulation in co‐culture with RA‐FLS. Using cultured RA‐FLS and the human NK cell line Nishi as an in vitro model system of RA‐FLS/NK cell cross‐talk, our results suggest that cell‐mediated cytotoxicity of RA‐FLS may be one mechanism by which NK cells influence local joint inflammation in RA.     

NKG2-C is a receptor on human natural killer cells that recognizes structures on K562 target cells

NKG2-C is a member of the recently discovered NKG2 family of genes and proteins, which are preferentially expressed on human natural killer (NK) cells. These potential NK cell receptors belong to a larger class of type II transmembrane proteins with a C-type lectin domain. We show here that NKG2-C is expressed as a 36-kDa glycoprotein by translation in vitro, recombinant expression and immunoprecipitation from a human NK cell clone. Further, a recombinant soluble NKG2-C-receptor binds specifically to K562 cells, which are target cells for NK cell killing, and to RPMI 8866 cells, which are feeder cells for NK cells; several other hematopoietic cell lines tested do not show any binding. The binding structures on the surface of K562 cells disappear, concomitant with a loss in susceptibility to killing when the cells are induced to differentiate with phorbol ester and Ca²⁺ ionophore. Our data suggest the presence of specific target molecules for NKG2-C on K562 cells, since overall glycosylation, Lewis X and Lewis Y structures, as well as the mucin-like CD43 molecule, do not change following induction of the cells. We propose that NKG2-C mediates a specific interaction of NK cells and their target cells with functional importance for NK cell killing.     

Cloning of a mouse homolog of CD94 extends the family of C-type lectins on murine natural killer cells

Two families of major histocompatibility complex (MHC) class I-specific receptors are found on natural killer (NK) cells: immunoglobulin-like receptors and C-type lectin receptors. In mice, the latter category is represented by the Ly49 family of receptors, whereas in humans, NK cells express the distantly related CD94, which forms MHC class I-specific heterodimers with NKG2 family members. Humans also express the MHC class I-specific p50/p58/p70 family of immunoglobulin-like receptors, but these have not been identified in mice. Hence, there is no known instance of an MHC class I-specific receptor that is expressed by both human and murine NK cells. Here we report the cloning of CD94 from the CB.17 and C57BL/6 strains of mice. Mouse CD94 is 54 % identical and 66 % similar to human CD94, and is also a member of the C-type lectin superfamily. Mouse CD94 is expressed efficiently on the cell surface of cells transiently transfected with the corresponding cDNA, but surface CD94 was unable to mediate detectable binding to MHC class I-expressing ConA blasts. Notably, mouse CD94, like human CD94, has a very short cytoplasmic tail, suggesting the existence of partner chains that may play a role in ligand binding and signaling. Like many other C-type lectins expressed by NK cells, mouse CD94 maps to the NK complex on distal chromosome 6, synteneic to human CD94. We also demonstrate that mouse CD94 is highly expressed specifically by mouse NK cells, raising the possibility that mice, like humans, express multiple families of MHC class I-specific receptors on their NK cells. Murine homologs of human NKG2 family members have not yet been identified, but we report here the existence of a murine NKG2D-like sequence that also maps to the murine NK complex near CD94 and Ly49 family members.     

MS患者CD94/NKG2A-bright和CD94/NKG2A-dim NK细胞亚群对IFN-β的差异性反应

目的： 分析多发性硬化（MS）进展型患者不同表型NK细胞亚群对临床主要治疗方法的反应性差异.方法： 分离患者外周血中的NK细胞, 以流式细胞术根据表面抑制性受体CD94/NKG2A表达情况分为两个亚群CD94/NKG2A-bright和CD94/NKG2A-dim.分别加入IFN-β, 测定两个亚群表面CD94/NKG2A变化及细胞增殖, 同时检测两种亚群分泌IL-10和TGF-β情况.结果： CD94/NKG2A阳性表达的NK细胞占25.5%, 其中CD94/NKG2A-bright和CD94/NKG2A-dim分别占其中的23.6%和76.4%.加入IFN-β, CD94/NKG2A-bright组增殖率明显低于CD94/NKG2A-dim组, CD94/NKG2A表达峰度变化不大.CD94/NKG2A-dim组中CD94/NKG2A表达显著增加.两个亚群分泌的IL-10和TGF-β与未刺激组相比, 均有明显差异.CD94/NKG2A-bright和CD94/NKG2A-dim组间亦有明显差异.结论： IFN-β通过诱导NK细胞CD94/NKG2A表达在非特异免疫系统中抑制NK细胞; 同时刺激IL-10 和TGF-β分泌进一步发挥对免疫系统的抑制.CD94/NKG2A-bright和CD94/NKG2A-dim对IFN-β反应有差异性.     

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.     

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.     

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.     

The CD94/NKG2C killer lectin-like receptor constitutes an alternative activation pathway for a subset of CD8+ T cells

The CD94/NKG2C killer lectin-like receptor (KLR) specific for HLA-E is coupled to the KARAP/DAP12 adapter in a subset of NK cells, triggering their effector functions. We have studied the distribution and function of this KLR in T lymphocytes. Like other NK cell receptors (NKR), CD94/NKG2C was predominantly expressed by a CD8(+) T cell subset, though TCRgammadelta(+) NKG2C(+) and rare CD4(+) NKG2C(+) cells were also detected in some individuals. Coculture with the 721.221 HLA class I-deficient lymphoma cell line transfected with HLA-E (.221-AEH) induced IL-2Ralpha expression in CD94/NKG2C+ NK cells and a minor subset of CD94/NKG2C(+) T cells, promoting their proliferation; moreover, a similar response was triggered upon selective engagement of CD94/NKG2C with a specific mAb. CD8(+) TCRalphabeta CD94/NKG2C(+) T cell clones, that displayed different combinations of KIR and CD85j receptors, expressed KARAP/DAP12 which was co-precipitated by an anti-CD94 mAb. Specific engagement of the KLR triggered cytotoxicity and cytokine production in CD94/NKG2C(+) T cell clones, inducing as well IL-2Ralpha expression and a proliferative response. Altogether these results support that CD94/NKG2C may constitute an alternative T cell activation pathway capable of driving the expansion and triggering the effector functions of a CTL subset.     

Specific engagement of the CD94/NKG2-A killer inhibitory receptor by the HLA-E class Ib molecule induces SHP-1 phosphatase recruitment to tyrosine-phosphorylated NKG2-A: evidence for receptor function in heterologous transfectants

It has been recently demonstrated that the CD94/NKG2-A killer inhibitory receptor (KIR) specifically recognizes the HLA-E class Ib molecule. Moreover, the apparent CD94-mediated specific recognition of different HLA class Ia allotypes, transfected into the HLA-defective cell line 721.221, indeed depends on their selective ability to concomitantly stabilize the surface expression of endogenous HLA-E molecules, which confer protection against CD94/NKG2-A⁺ effector cells. In the present study, we show that a selective engagement of the CD94/NKG2-A inhibitory receptor with a specific monoclonal antibody (mAb) (Z199) was sufficient to induce tyrosine phosphorylation of the NKG2-A subunit and SHP-1 recruitment. These early biochemical events, commonly related to negative signaling pathways, were also detected upon the specific interaction of NK cells with an HLA-E⁺ 721.221 transfectant (.221-AEH), and were prevented by pre-incubation of .221-AEH with an anti-HLA class I mAb. Furthermore, mAb cross-linking of the CD94/NKG2-A receptor, segregated from other NK-associated molecules by transfection into a rat basophilic leukemia cell line (RBL-2H3), promoted tyrosine phosphorylation of NKG2-A and co-precipitation of SHP-1, together with an inhibition of secretory events triggered via FcϵRI. Remarkably, interaction of CD94/NKG2-A⁺ RBL cells with the HLA-E⁺ .221-AEH transfectant specifically induced a detectable association of SHP-1 with NKG2-A, constituting a more formal evidence for the receptor-HLA class I interaction.     

Expression of killer cell inhibitory receptors on human uterine natural killer cells

The establishment of the human placenta in early pregnancy is characterized by the presence of large numbers of natural killer (NK) cells within the maternal decidua in close proximity to the fetally-derived invading extravillous trophoblast which expresses at least two HLA class I molecules, HLA-G and HLA-C. These NK cells have an unusual phenotype, CD56^bright CD16, distinguishing them from adult peripheral blood NK cells. They may control key events in trophoblast migration and therefore placentation. Human NK cells in peripheral blood express receptors for polymorphic HLA class I molecules. This family of receptors, known as killer cell inhibitory receptors (KIR), are expressed on overlapping subsets of NK cells to give an NK cell repertoire which differs between individuals. Using a panel of monoclonal antibodies to several members of the KIR family and analysis by flow cytometry, we have found that KIR are expressed by decidual NK cells. There is variation in both the percentage of cells expressing a particular receptor and the density of receptor expression between decidual NK cells from different individuals. Comparison of NK cells from decidua and peripheral blood of the same individual showed that NK cells from these two different locations express different repertoires of KIR. Receptors are present in individuals who do not possess the relevant class I ligand, raising the possibility that these NK receptors may be involved in recognition of the allogeneic fetus by the mother at the implantation site.     

The inhibitory NK cell receptor CD94/NKG2A and the activating receptor CD94/NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues

The human non-classical MHC class I molecule HLA-E is a ligand for both an inhibitory NK cell receptor (CD94/NKG2A) and an activating receptor (CD94/NKG2C). To identify HLA-E surface recognized by both receptors, especially to determine if both receptors recognize the same epitope, we made a series of individually Ala-substituted HLA-E proteins and analyzed their binding to CD94/NKG2A orCD94/NKG2C. Eight HLA-E mutations that significantly impaired HLA-E binding to CD94/NKG2A are all found in the top of alpha1/alpha2 domain of HLA-E. These results suggest that CD94/NKG2A binds a HLA-E surface equivalent to a NKG2D binding site on MICA. Of the eight mutations that impaired HLA-E binding to CD94/NKG2A, six significantly impaired HLA-E binding to CD94/NKG2C suggesting that CD94/NKG2C also binds a similar surface of HLA-E. Unexpectedly, the two HLA-E mutations (D69A and H155A) selectively abrogated HLA-E binding to CD94/NKG2A, not largely affected CD94/NKG2C. These results indicate that a mostly shared, but partly distinct set of HLA-E residues is discriminated by the two receptors.     

人自然杀伤细胞抑制性受体P58.1基因的克隆与鉴定

目的 克隆及鉴定P58.1基因。方法 采用RT-PCR技术,从正常人外周血单个核细胞中扩增P58.1基因全长cDNA,经酶切后构建重组克隆载体,测序鉴定。结果 RT-PCR获得预期的扩增产物P58.1全长cDNA,成功构建pSPORT1-P58.1重组克隆载体,酶切、酶谱分析与预期结果相符。DNA测序结果与GenBank登记的人P58.1cDNA全长碱基序列一致。结论 pSPORT1-58.1重组克隆载体构建成功;P58.1基因序列与文献报道一致,为下一步构建表达载体和基因转染等工作打下良好的基础。     

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.     

Association of CD94/NKG2A, CD94/NKG2C, and its ligand HLA-E polymorphisms with Behcet's disease

Inhibitory CD94/NKG2A and activating CD94/NKG2C receptors are expressed on natural killer, CD4, and CD8 T cells and recognize human leukocyte antigen (HLA)-E, resulting in the modulation of cytotoxic activity and cytokine production. An imbalance in cytotoxic activity and cytokine production has been implicated in Behcet's disease (BD). The results of this study showed that the NKG2A c.-4258*C, c.338-90*G, and CD94 c.-134*T alleles (P= 0.015, OR = 0.8; P < 0.0001, OR = 0.5; and P= 0.034, OR = 0.8, respectively) were associated with decreased risk and that NKG2A c.284-67_-62del, c.1077*C, and the activating receptor, NKG2C c.305*T were not associated with 345 patients with BD. But a significant difference in NKG2C c.305*T was detected among BD patients with ocular lesions and arthritis (P < 0.0001, OR = 2.1 and P= 0.0001, OR = 1.8, respectively). We already showed in our previous research that HLA-E*0101 also appears to contribute to a reduction in risk through the inhibitory CD94/NKG2A-mediated immune response. This result led us to the analyses of the combined risk of the HLA-E and the NKG2A for BD. Individuals harboring HLA-E*0101, NKG2A c.-4258*C, and c.338-90*G evidenced a reduced risk of BD compared with healthy controls (21.1% vs 40.1%, P < 0.0001, OR = 0.4). By way of contrast, individuals without the HLA-E*0101, NKG2A c.-4258*C, and c.338-90*G alleles evidenced a twofold increased risk of BD (P= 0.014, OR = 2.0). Individuals without HLA-E*0101, NKG2A c.-4258*G/*G, and c.338-90*G evidenced a 4.8-fold increase in BD risk (P= 0.0002, OR = 4.8). Although the effects of these single nucleotide polymorphisms (SNPs) remain unclear, our results indicate that the SNPs of the inhibitory receptor CD94/NKG2A and its haplotypes, as well as its ligand HLA-E, are associated with BD immune systems.     

The ILT2(LIR1) and CD94/NKG2A NK cell receptors respectively recognize HLA-G1 and HLA-E molecules co-expressed on target cells

Previous studies on NK recognition of HLA-G1 employed as targets 721.221 transfectants (.221-G1) that unknowingly co-expressed the HLA-E molecule, subsequently found to be a major ligand for the CD94/NKG2 receptors. In the present study we re-evaluated the relative role played by CD94/NKG2 and ILT2(LIR1) molecules in recognition of HLA-G1 by NK clones. We employed as targets .221-G1 cells and a surface HLA-E-negative transfectant, .221-G1(E_neg), generated by site-directed mutagenesis of the HLA-G1 leader sequence. The antagonistic effects of receptor- (i.e. CD94/NKG2A, ILT2) and ligand-specific mAb (i.e. HLA-G, HLA-E) were assessed. In addition, binding of an ILT2-Ig fusion protein to the .221-AEH, expressing only HLA-E, and the .221-G1(E_neg) transfectants was analyzed. Our data demonstrate that NK recognition of cells expressing HLA-G1 involves at least two non-overlapping receptor-ligand systems: the CD94/NKG2 interaction with HLA-E, and the engagement of the ILT2(LIR1) receptor by HLA-G1 molecules.