Clonal analysis of NK cell development from bone marrow progenitors in vitro: orderly acquisition of receptor gene expression

In the mouse, two families of MHC class I-specific receptors, namely Ly49 and CD94/NKG2, have been identified on NK cells. Individual NK cells can express several Ly49 molecules as well as members of the CD94/NKG2 family. The expression of multiple receptors with different specificities for MHC class I is thus thought to generate NK cells with diverse recognition patterns. To delineate the mechanism by which NK cells begin to express different patterns of Ly49 and CD94/NKG2 molecules, we developed a clonal assay in which NK1.1(-), IL-2/ IL-15 receptor beta+ NK precursors generated by culture of multipotential Lin(-), c-kit+ progenitors in IL-7, stem cell factor and flt3 ligand are induced to differentiate into NK1.1+ , Ly49+ NK cells. Examination of the clonal populations thus generated revealed heterogeneity in the pattern of Ly49 and CD94/NKG2 gene expression. In addition, a distinct kinetic pattern of expression was observed. CD94, NKG2A, NKG2C and Ly49B were expressed first followed by Ly49G, then Ly49C and I and finally, Ly49A, D, E and F. The data suggest a stochastic but ordered acquisition of class I receptors on NK cells in which developing NK cells become capable of expressing distinct receptors at different times but show no absolute prerequisite to express the receptors that are acquired early in NK development for the expression of those that are acquired later.     

Ly49 and CD94/NKG2: developmentally regulated expression and evolution

Summary: Murine natural killer (NK) cells express two families of MHC class I-specific receptors, namely the Ly49 family and CD94/NKG2 heterodimers. Stochastic co-expression of these receptors generates diverse receptor repertoires in adult NK-cell populations, whereas fetal NK cells have much more limited receptor diversity as they mostly express CD94/NKG2A but not Ly49. These receptors are also expressed on CD8⁺ T cells and NK1.1⁺ T cells and regulate their functions, but their expression pattern on NK cells is significantly different from those on T cells. Thus, expression of Ly49 and CD94/NKG2 is developmentally regulated. NK cells acquire the Ly49 family of receptors in an orderly manner as they differentiate from bone marrow progenitors in vitro . Similarly, acquisition of CD94 and NKG2 by NK cells as they differentiate from embryonic stem cells is also orderly. To gain insight into the mechanisms regulating Ly49 expression, potential regulatory regions of several Ly49 genes have been examined. Ly49 genes with different expression patterns have remarkably similar sequences in the putative regulatory regions. Finally, a functional Ly49 gene has been identified in baboon, and primate comparisons suggest that functional extinction of the Ly49 gene in the human lineage seems to have been a relatively recent event.
This research was supported by the National Cancer Institute of Canada and the Medical Research Council of Canada with core support from the BC Cancer Agency. KM and BW were supported by studentships from the University of British Columbia and the National Science and Engineering Research Council of Canada. RL is a recipient of a Leukemia Research Fund of Canada fellowship. SL was supported by the Deutsche Forschungsgemeinschaft.     

Computational modeling of human natural killer cell development suggests a selection process regulating coexpression of KIR with CD94/NKG2A

Natural killer cells fail to lyse target cells expressing sufficient levels of self MHC class I molecules, providing one mechanism to secure self tolerance. Inhibition of lysis is mediated by inhibitory receptors expressed by NK cells, such as the murine Ly49 receptors, human KIR receptors and CD94/NKG2A, expressed by both species. To ensure that most, if not all, NK cells express at least one inhibitory receptor for self MHC class I, selection processes have been postulated for murine NK cells regulating the number and identity of inhibitory receptors expressed by each cell. The presence of similar selection processes in human NK cells has not been demonstrated. In previous studies using mathematical modeling we have shown that, in the Ly49 system, the sequential model (in which gene expression and selection operate simultaneously) is most likely to explain the observed expression frequencies. We also predicted the parameters (such as receptor-ligand binding affinity levels) under which the models fit with the observed frequencies. This study aims to evaluate whether these models may be valid in the human system. Our data suggest that if selection operates during human NK cell development, it affects the co-expression of CD94/NKG2A and KIR rather than KIR expression alone, and is more likely to be governed by the two-step selection model.     

Ly49E expression points toward overlapping,but distinct,natural killer (NK) cell differentiation kinetics and potential of fetal versus adult lymphoid progenitors

Using a new antibody, we found previously that contrary to adult natural killer (NK) cells, fetal NK cells have a unique phenotype, as they exclusively express Ly49E. This can be explained by an intrinsic different NK differentiation potential of fetal versus adult lymphoid progenitors, by immaturity of fetal NK cells or by instability of Ly49E expression. Here, we show that adult progenitor cells were still capable of differentiating into Ly49E-expressing NK cells but at a much lower frequency. Surprisingly, Ly49E expression in vitro did not require stromal cells. Kinetic analysis in vivo showed that Ly49E was expressed early, together with CD94/NKG2 and Ly49G2, followed by Ly49C, and finally Ly49D. Transfer of sorted Ly49E-positive fetal NK cells showed stable Ly49E expression, and later, part of these cells up-regulated other Ly49 members. These data indicate that although there are intrinsic differences, there is no strict fetal and adult wave of NK cell differentiation.     

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.     

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.     

The balancing act: inhibitory Ly49 regulate NKG2D-mediated NK cell functions

NK cells use NKG2D receptor to recognize 'induced-self'. In apparent violation of the 'missing-self' hypothesis, NK cells stimulated through NKG2D can lyse target cells despite normal expression levels of MHC class I molecules. Although, 'overriding' of the inhibitory by the activating signals had been postulated the precise role of inhibitory Ly49 receptors on NKG2D-mediated activation has only started emerging. We propose that NKG2D-mediated activation is a function of 'altering the balance' in the signaling strength between the activating NKG2D and inhibiting Ly49 receptors. Balance in the signaling strength depends on the expression levels of activating ligands on the target cells. Qualitative and quantitative variations of MHC class I molecules expressed on the target cells also plays a major role in determining this 'altered-balance'. Consequently, the nature of Ly49 receptors expressed on specific NK subsets determines the level of NKG2D-mediated NK cell activation. These observations provide a firm basis of 'altered-balance' in NK signaling and describe an active interplay between inhibitory Ly49 and activating NKG2D receptors.     

Natural killer cell receptors for major histocompatibility complex class I and related molecules in cytomegalovirus infection

Downmodulation of major histocompatibility complex (MHC) class I molecules by cytomegalovirus (CMV) impairs the engagement of specific leucocyte-inhibitory receptors, rendering infected cells vulnerable to natural killer (NK) cells. Members of the murine Ly49 and human KIR families, CD85j (ILT2 or leucocyte Ig-like receptor-1), as well as the CD94/NKG2A-inhibitory killer lectin-like receptor (KLR) fulfil this surveillance role. On the other hand, NK-activating receptors specific to ligands expressed on virus-infected cells may overcome the control by inhibitory receptors. In this regard, NKG2D and Ly49H lectin-like molecules trigger NK-cell functions recognizing, respectively class I-related stress-inducible molecules and the m157 murine CMV glycoprotein. Among a variety of immune evasion strategies, CMV promotes the synthesis of class I surrogates and selectively preserves the expression of some class I molecules in infected cells; moreover, CMV interferes with the expression of ligands for NKG2D. We herein review these aspects of the host-pathogen interaction, discussing a number of open issues.     

Structure of the Ly49 family of natural killer (NK) cell receptors and their interaction with MHC class I molecules

Natural killer (NK) cells are an essential component of the innate immunity toward tumors and virally infected cells. The function of NK cells is regulated by a precise balance between inhibitory and activating signals. These signals are mediated by NK cell receptors that bind either classical MHC class I molecules or their structural relatives such as MICA, ULBP, RAE-1, and H-60. Two separate families of NK cell receptors have been identified: the immunoglobulin-like family (KIR, LIR) and C-type lectin-like family (Ly49, NKG2D, and CD94/NKG2). Here we summarize the structure of Ly49 C-type lectin-like proteins hitherto solved (Ly49A, Ly49C and Ly49I) and their interaction with MHC class I molecules as determined by the co-crystal structure of Ly49A/H-2Dd and Ly49C/H-2Kb.     

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.     

Persistent expression of CD94/NKG2 receptors by virus-specific CD8 T cells is initiated by TCR-mediated signals

Subsets of CD8 T cells express receptors that are critical in regulating the activity of NK cells. To characterize the expression of these receptors on CD8 T cells we made use of transgenic mice that express a H-2Kb restricted TCR specific for the immunodominant epitope located within the HSV-1 glycoprotein B (gB). Few naive gB-specific T cells express Ly49 or CD94/NKG2 receptors. Following acute infection of C57BL/6 mice with either HSV-1 or a recombinant influenza virus that encodes the gB determinant, gB-specific T cells showed a dramatic upregulation of CD94/NKG2 receptors. Moreover, gB-specific CD8 T cells that expressed CD94/NKG2 receptors were also found to express another NK receptor, KLRG1. We established that while Ag-stimulated gB-specific CD8 T cells primarily express inhibitory isoforms of CD94/NKG2 receptors, these cells remain capable of producing gammaIFN upon peptide stimulation. While peak CD94/NKG2 expression on gB-specific cells was reached 2-3 days following infection, it remained elevated beyond 60 days post-infection with either HSV-1 or a gB-expressing recombinant influenza virus. The data imply that the prolonged expression was not due to persistence of replicating virus and suggest that while recognition of the cognate Ag is necessary to trigger expression of CD94/NKG2 receptors, it is not required for their continued expression on memory T cells.     

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.     

The Qa-1b molecule binds to a large subpopulation of murine NK cells

Recent studies on human NK cells have demonstrated that the NK cell CD94/NKG2 receptors bind to the nonclassical MHC class I molecule HLA-E. A functional CD94/NKG2 complex has not yet been identified in rodents, but cDNA encoding rat and mouse CD94 and NKG2 have recently been cloned, suggesting that CD94/NKG2 receptors may exist in species other than man. The mouse nonclassical MHC class I molecule Qa-1 shares several features with HLA-E. This suggests that Qa-1 may be similarly recognized by murine NK cells. To study the ability of Qa-1 to bind to murine NK cells, we have produced a soluble tetrameric form of Qa-1^b . In the present study, we demonstrate that Qa-1^b tetramers distinctly bind to a large subset of fresh or IL-2-activated NK1.1⁺ /CD3⁻ splenocytes independently of the expression of Ly49 inhibitory receptors. Binding occurs whether NK cells have evolved in an MHC class I-expressing or in an MHC class I-deficient environment. Our data suggest the existence of a Qa-1-recognizing structure on a large subpopulation of murine NK cells that may be similar to the human CD94/NKG2 heterodimeric complex.     

Mouse CD146/MCAM is a marker of natural killer cell maturation

CD146/melanoma cell adhesion molecule is an adhesion molecule expressed by endothelial cells and by a small fraction of activated T and B lymphocytes in humans. In order to analyze the pattern of CD146 expression in mouse leukocytes at steady-state conditions, we generated a set of novel rat anti-mouse CD146 monoclonal antibodies. CD146 expression was undetectable on monocytes, dendritic cells, T cells or B cells, but was expressed on about 30% of neutrophils and 60% of NK cells. Within murine lymphocytes, CD146 was defined as a novel NK-specific surface molecule. An increased percentage of CD146+ cells was found in the most mature CD27(-)CD11b+ NK cell subpopulation, which also displays higher expression of Ly49C/I, Ly49D and KLRG1 and lower expression of NKG2A/C/E molecules. CD146+ NK cells were found to be less cytotoxic and produce less IFN-gamma than CD146(-) NK cells upon stimulation with target cells or activating antibodies. These findings define CD146 as a marker of mouse NK cell maturation that may be used as an alternative to the combined use of CD27 and CD11b staining to detect final stages of NK cell maturation.     

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.     

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.     

Structural basis of MHC class I recognition by natural killer cell receptors

Summary: Natural killer (NK)-cell function is regulated by NK receptors that recognize MHC class I (MHC-I) molecules on target cells. Two structurally distinct families of NK receptors have been identified, the immunoglobulin-like family (killer cell immunoglobulin-like receptors (KIRs), leukocyte immunoglobulin-like receptors (LIRs)) and the C-type lectin-like family (Ly49, CD94/NKG2A, NKG2D, CD69). Recently, the three-dimensional structures of several NK receptors were determined, in free form or bound to MHC-I. These include those of unbound KIRs, NKG2D, CD69, LIR-1 and the CD94 subunit of the CD94/NKG2A heterodimer. Together, these structures define the basic molecular architecture of both the immunoglobulin-like and C-type lectin-like families of NK receptors. In addition, crystal structures have been reported for the complex between Ly49A and H-2D^d, and for KIR2DL2 bound to HLA-Cw3. The complex structures provide a framework for understanding MHC-I recognition by NK receptors from both families and reveal striking differences in the nature of this recognition, despite the receptors' functional similarity.
This research was supported, in part, by National Institutes of Health grants R01 AI47900 and R37 36900 (RAM) and a fellowship from the Cancer Research Institute (MWS). We are grateful to DW Wolan and IA Wilson for providing coordinates of NKG2D prior to publication, and to members of our laboratories for encouragement.     

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.