Natural Killer Cell Clones Research Articles

Common cytological and cytogenetic features of Epstein-Barr virus (EBV)-positive natural killer (NK) cells and cell lines derived from patients with nasal T/NK-cell lymphomas, chronic active EBV infection and hydroa vacciniforme-like eruptions.

In this study, we describe the cytological and cytogenetic features of six Epstein-Barr virus (EBV)-infected natural killer (NK) cell clones. Three cell clones, SNK-1, -3 and -6, were derived from patients with nasal T/NK-cell lymphomas; two cell clones, SNK-5 and -10, were isolated from patients with chronic active EBV infection (CAEBV); and the other cell clone, SNK-11, was from a patient with hydroa vacciniforme (HV)-like eruptions. An analysis of the number of EBV-terminal repeats showed that the SNK cell clones had monoclonal EBV genomes identical to the original EBV-infected cells of the respective patients, and SNK cells had the type II latency of EBV infection, suggesting that not only the cell clones isolated from nasal T/NK-cell lymphomas but also those isolated from CAEBV and HV-like eruptions had been transformed by EBV to a certain degree. Cytogenetic analysis detected deletions in chromosome 6q in five out of the six SNK cell clones, while 6q was not deleted in four control cell lines of T-cell lineage. This suggested that a 6q deletion is a characteristic feature of EBV-positive NK cells, which proliferated in the diseased individuals. The results showed that EBV-positive NK cells in malignant and non-malignant lymphoproliferative diseases shared common cytological and cytogenetic features.

Soluble HLA class I induces NK cell apoptosis upon the engagement of killer-activating HLA class I receptors through FasL-Fas interaction

The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I-Cw4 or -Cw3 from untransfected or -Cw4 or -Cw3 alleles transfected HLA-A(-), B(-), C(-), G(-), E(+), F(+) 721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR- or anti-KIR-specific monoclonal antibodies (mAbs). After sHLA-I-activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I-mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor-mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H(+)-adenosine triphosphatase (H(+)-ATPase) of granules, inhibited activating receptor-induced NK cell cytolysis, suggesting that activating receptor-mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-gamma (IFN-gamma) was detectable in culture supernatant of activating receptor(+) NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor-mediated IFN-gamma production. This suggests that activating receptor-induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-gamma, an antiviral cytokine able to amplify immune responses.

Expansion of natural killer cells in patients with head and neck cancer: detection of "noninhibitory" (activating) killer Ig-like receptors on circulating natural killer cells.

In a group of patients with head and neck cancers (H&NC), the expansion of the population of CD3-,CD16+ natural killer (NK) cells in the peripheral blood was studied. Cytofluorimetric analysis of the expression of killer Ig-like receptors (KIR, namely p58.1, p58.2, p58.3, p70, and p140) and CD94-NKG2a was performed. Cytolytic activities were studied using 51Cr release assay. T and NK cell cloning was performed using limiting dilution culture conditions. Cytokine production was analyzed using commercial enzyme immunoassays. Phenotypic analysis showed that the expanded populations were heterogeneous. Even in the presence of a large number of circulating NK cells, "nonspecific" cytolytic capacities were heavily reduced, whereas cytolytic capacity related to T cells was virtually normal. Unlike NK cell clones derived from healthy donors, most NK cells derived from H&NC patients expressed surface "activating" NK cell receptors (KAR) for HLA, detected by use of a redirected cytolytic assay. Analysis of the CD4+ subpopulation at the clonal level demonstrated that they had a severe proliferative defect. These experimental data indicated that H&NC patients have a polyclonal expansion of functionally deficient NK cells expressing KAR. In addition, the proliferative capacity of patients' "helper" cells was strongly inhibited, thus accounting for a severe impairment of cytolytic activity of the expanded NK cells.

NK Cell-Mediated Lysis of Autologous HCMV-Infected Skin Fibroblasts Is Highly Variable among NK Cell Clones and Polyclonal NK Cell Lines

Lysis of human cytomegalovirus (HCMV)-infected fibroblasts by autologous natural killer (NK) cells was examined in vitro. For NK cell clones, receptor expression was determined at the level of mRNA and cell-surface protein and compared to the lysis of HCMV AD169 strain-infected fibroblasts in which HLA class I was >70% downregulated. The clones ranged broadly in their ability to lyse AD169-infected fibroblasts, correlating neither with the expression of inhibitory KIR, leukocyte inhibitory receptor-1, or CD94:NKG2A receptors nor with the number of different inhibitory KIR expressed per clone. Some lines of polyclonal NK cells preferentially lysed AD169-infected cells and similarly lysed fibroblasts infected with mutant virus RV798, which lacks the genes for downregulating HLA class I. These results demonstrate that NK cell lysis of HCMV-infected autologous fibroblasts is more complex than a simple missing-self mechanism involving downregulation of HLA class I and failure to engage inhibitory self-specific KIR.

Soluble HLA class I molecules induce natural killer cell apoptosis through the engagement of CD8: evidence for a negative regulation exerted by members of the inhibitory receptor superfamily

Herein, we show that CD8(dull), CD8(intermediate), and CD8(bright) natural killer (NK) cell clones can be identified. Triggering of CD8 with its natural ligand(s), represented by soluble HLA class I (sHLA-I), isolated either from serum of healthy donors or from HLA-I(-) 721.221 lymphoblastoid cell line transfected with HLA-A2, -Cw4, and -Bw46 alleles, or HLA-G1 leads to NK cell apoptosis. The magnitude of this effect directly correlated with the level of CD8 expression. sHLA-I-induced apoptosis depends on the interaction with CD8, as it was inhibited by masking this molecule with specific monoclonal antibodies (mAbs). Moreover, sHLA-I or CD8 cross-linking with specific mAbs elicited intracellular calcium increases, Fas ligand (FasL) messenger RNA transcription, and FasL secretion, which were needed for delivering the death signal. Indeed, this apoptosis was inhibited by preincubation of NK cell clones with Fas or FasL antagonist mAbs, indicating that the Fas/FasL pathway is involved. Furthermore, members of the inhibitory receptor superfamily, such as CD94/NKG2 complex or killer inhibitory receptors, were shown to exert an inhibitory effect on sHLA-I-mediated apoptosis and secretion of FasL. These findings suggest that interaction between sHLA-I and CD8 evokes an apoptotic signal that is down-regulated by inhibitory receptor superfamily that function as survival receptors in NK cells.

Human dendritic cells activate resting natural killer (NK) cells and are recognized via the NKp30 receptor by activated NK cells.

During the innate response to many inflammatory and infectious stimuli, dendritic cells (DCs) undergo a differentiation process termed maturation. Mature DCs activate antigen-specific naive T cells. Here we show that both immature and mature DCs activate resting human natural killer (NK) cells. Within 1 wk the NK cells increase two– to fourfold in numbers, start secreting interferon (IFN)-γ, and acquire cytolytic activity against the classical NK target LCL721.221. The DC-activated NK cells then kill immature DCs efficiently, even though the latter express substantial levels of major histocompatibility complex (MHC) class I. Similar results are seen with interleukin (IL)-2–activated NK cell lines and clones, i.e., these NK cells kill and secrete IFN-γ in response to immature DCs. Mature DCs are protected from activated NK lysis, but lysis takes place if the NK inhibitory signal is blocked by a human histocompatibility leukocyte antigen (HLA)-A,B,C–specific antibody. The NK activating signal mainly involves the NKp30 natural cytotoxicity receptor, and not the NKp46 or NKp44 receptor. However, both immature and mature DCs seem to use a NKp30 independent mechanism to act as potent stimulators for resting NK cells. We suggest that DCs are able to control directly the expansion of NK cells and that the lysis of immature DCs can regulate the afferent limb of innate and adaptive immunity.

Identification, molecular cloning and functional characterization of NKp46 and NKp30 natural cytotoxicity receptors in Macaca fascicularis NK cells.

Natural killer (NK) cell recognition and function in humans is regulated by multiple cell surface receptors. The "on" signal leading to NK cell triggering is primarily mediated by natural cytotoxicity receptors (NCR). Analysis of NK cells in primate animal models is of particular relevance because NK cells may play an essential role in host defenses against infections. We analyzed Macaca fascicularis PBMC and in vitro-derived NK cell populations and clones by cytofluorometry, using a wide panel of mAb, and by cytolytic activity assays. In addition, RT-PCR strategy and transient transfections were used to isolate M. fascicularis NCR. NCR-specific mAb reactivity (anti-NKp46 and anti-NKp30) was present on M. fascicularis PBMC and on NK cell cultures. Macaque NCR were functional in both redirected killing and in mAb-mediated masking assays. Cloning of macNKp46 and macNKp30 NCR homologous genes showed a high sequence similarity (86 % and 88 %, respectively) with their human counterparts. Attempts at identifying NKp44 surface reactivity and at cloning the macaque homologue were unsuccessful. NKp46 and NKp30 NCRs, but not NKp44, are highly conserved in M. fascicularis NK cells. This suggests the possibility of a staged appearance of the NCR during phylogenesis and provides a useful tool for the study of natural immunity correlates of protection in primate SIV/SHIV infection models.

Final steps of natural killer cell maturation: a model for type 1-type 2 differentiation?

Analysis of cytokine and differentiation antigen expression in human natural killer (NK) cells revealed that interleukin 13 (IL-13) and interferon gamma (IFN-gamma) are produced at sequential stages during irreversible IL-12-induced differentiation. In human NK cell clones, polyclonal CD3-CD161+CD56- cells and peripheral lymphocytes, IL-4 induced the proliferation of both IL-13+ NK and T cells, whereas IL-12 allowed a proliferation-independent accumulation of IFN-gamma+ cells. These data disproved the NK1-NK2 hypothesis and challenge the current T helper 1 (TH1)-TH2 paradigm. We propose that the cytokine environment regulates a type 2-->0-->1 developmental progression, with IL-12 needed for terminal differentiation and IL-4 delaying this process, rather than a type 1 versus type 2 decision of a type 0 cell.

Natural killer cells lyse autologous herpes simplex virus infected targets using cytolytic mechanisms distributed clonotypically

Natural killer (NK) cells have the capability of lysing targets that have down-regulated the expression of HLA class I molecules. Herpes simplex virus (HSV) infection results in a profound reduction of HLA class I molecules on the surface of infected cells. For this reason, NK cell populations kill efficiently HSV-infected cells. The recent availability of a panel of monoclonal antibodies directed to NK receptors for HLA class I (CD158a, CD158b, anti-p70, anti-p140, and CD94) allowed an accurate dissection of the NK cell subpopulations. Using this approach, the relationship between the expression of NK cell receptors and the capability of lysing HSV-infected cell targets was analyzed at the clonal level. NK cell clones were derived from healthy donors, and cytolytic properties were assayed against HSV-infected autologous fibroblasts. NK cell clones, classified according to the expression of natural killer-cell receptors on their surface, displayed a great heterogeneity of cytolytic properties against HSV-infected cells. Nevertheless, a more accurate functional analysis demonstrated not only that HSV infection downregulated the expression of HLA-A and HLA-B and did not modify the expression of HLA-C, but also that NK cell clones expressing the “activating” form of the anti HLA-C NK cell receptor were more cytolytic than other clones. This finding suggests that two different and clonally distributed mechanisms of NK cell activation may be employed by NK cells to kill HSV-infected autologous target cells. J. Med. Virol. 62:354–363, 2000. © 2000 Wiley-Liss, Inc.

The Tyrosine Kinase Pyk-2/Raftk Regulates Natural Killer (Nk) Cell Cytotoxic Response, and Is Translocated and Activated upon Specific Target Cell Recognition and Killing

The compartmentalization of plasma membrane proteins has a key role in regulation of lymphocyte activation and development of immunity. We found that the proline-rich tyrosine kinase-2 (PYK-2/RAFTK) colocalized with the microtubule-organizing center (MTOC) at the trailing edge of migrating natural killer (NK) cells. When polyclonal NK cells bound to K562 targets, PYK-2 translocated to the area of NK-target cell interaction. The specificity of this process was assessed with NK cell clones bearing activatory or inhibitory forms of CD94/NKG2. The translocation of PYK-2, MTOC, and paxillin to the area of NK-target cell contact was regulated upon specific recognition of target cells through NK cell receptors, controlling target cell killing. Furthermore, parallel in vitro kinase assays showed that PYK-2 was activated in response to signals that specifically triggered its translocation and NK cell mediated cytotoxicity. The overexpression of both the wt and a dominant-negative mutant of PYK-2, but not ZAP-70 wt, prevented the specific translocation of the MTOC and paxillin, and blocked the cytotoxic response of NK cells. Our data indicate that subcellular compartmentalization of PYK-2 correlates with effective signal transduction. Furthermore, they also suggest an important role for PYK-2 on the assembly of the signaling complexes that regulate the cytotoxic response.

Phenotypic and functional analysis of the HLA-class I-specific inhibitory receptors of natural killer cells isolated from peripheral blood of patients undergoing bone marrow transplantation from matched unrelated donors.

Limited information is available on the natural killer cell reconstitution after bone marrow transplantation and on the possible role of these cells in graft-versus-host-disease. Blood samples were collected at different time intervals after transplantation. Lymphocytes were analyzed for informative markers by immunocytofluorimetric analysis. Natural killer cells derived from patients undergoing matched unrelated donor transplant were cloned by limiting dilution in the presence of phytohemoagglutinin and IL2. The natural killer cell clones were analyzed for cytolytic activity. In the nine patients analyzed undergoing transplantation from sibling donors, the majority of peripheral blood lymphocytes during the first 80 d after BMT were represented by T lymphocytes, while in the 15 patients undergoing matched unrelated donor transplant natural killer cells consistently outnumbered T lymphocytes. During the early phases after transplantation, most CD56+CD3- natural killer cells did not express CD16 which was expressed at later intervals. Analysis of the inhibitory receptors specific for HLA-class I molecules showed that CD94/NKG2A, specific for HLA-E, unlike normal donors, was expressed by all natural killer cells including the early appearing CD16-negative ones. Killer inhibitory receptors of the Ig superfamily were expressed late and in low percentages after transplantation and were always coexpressed with CD94/NKG2A. Natural killer-cell clones efficiently lysed the HLA-class I-negative cell lines K562 and 721-221. Natural killer-cell populations or clones isolated from patients with graft-versus-host-disease, failed to lyse donor or recipient derived phytohemoagglutinin-induced lymphoblasts. Our analysis shows that (1) recipients of matched unrelated donors transplants exhibit a high proportion of NK cells (2) all NK cells express CD94/NKG2A while the HLA-class I-specific inhibitory receptors of Ig superfamily appear at later stages and (3) donor NK cells do not lysed donor or recipient target cells.

An acidic microenvironment inhibits antitumoral non-major histocompatibility complex-restricted cytotoxicity: implications for cancer immunotherapy.

Local immunosuppression may explain the failure of an effective immune response against solid tumors. Although it is well known that the interstitial pH is significantly lower in solid tumors than in normal tissue, only a few studies in the mouse system have investigated the influence of this acidic milieu on the anti-tumoral cytotoxic response. Here the authors report the suppression of human non-major histocompatibility complex (MHC)-restricted cytotoxicity against tumor cells by an acidic extracellular pH (pHe). Unstimulated peripheral blood mononuclear cells, lymphokine-activated killer (LAK) cells, and natural killer cell clones were used as effector cells. According to pH measurements in solid tumors, representative pH values of 7.2 to 5.3 were chosen during the cytotoxic assays. Target cell lysis was measured using two nonradioactive fluorometric methods, namely two-color flow cytometry and a modified calcein-release assay, which allowed cell-mediated cytotoxicity to be measured and compared with that in adherent targets. Using K562, Daudi, or Raji as suspended target cell lines, the cytotoxic activity of unstimulated peripheral blood mononuclear cells and of LAK cells was markedly reduced by a decreasing pHe. An extracellular pH of 5.8 to 5.3 resulted in a nearly complete loss of the cytotoxic response. This pHe-dependent impairment of the killing activity could also be shown for killer cells stimulated with interleukins-7 and -12, phytohemagglutinin, or lipopolysaccharide. The lytic potential of homogeneous natural killer cell clones as effectors was also strictly influenced by the surrounding pH. The pHe dependence of the non-MHC-restricted killer cell functions against tumor cells seems to be a general phenomenon, because the cytolytic activity of LAK cells against six human adherent tumor cell lines (HeLa, HepG2, LS174T, LS174Te, MCF-7, and RT112) was also clearly reduced under acidic conditions. To initiate the killing process, adhesion molecules play an important role in recognition and binding of the target cell. However, flow cytometric analysis revealed that the expression pattern of relevant adhesion molecules was unaffected by acidic pHe. In conclusion, these data clearly indicate an inhibition of non-MHC-restricted cytotoxicity against tumor cells by an acidic pHe, which may contribute to the failure of immunosurveillance against solid tumors. Consequently, efforts to enhance the anti-tumoral cytotoxicity by immunotherapies may have limited success.

Natural killer cells lyse autologous herpes simplex virus infected targets using cytolytic mechanisms distributed clonotypically

Natural killer (NK) cells have the capability of lysing targets that have down-regulated the expression of HLA class I molecules. Herpes simplex virus (HSV) infection results in a profound reduction of HLA class I molecules on the surface of infected cells. For this reason, NK cell populations kill efficiently HSV-infected cells. The recent availability of a panel of monoclonal antibodies directed to NK receptors for HLA class I (CD158a, CD158b, anti-p70, anti-p140, and CD94) allowed an accurate dissection of the NK cell subpopulations. Using this approach, the relationship between the expression of NK cell receptors and the capability of lysing HSV-infected cell targets was analyzed at the clonal level. NK cell clones were derived from healthy donors, and cytolytic properties were assayed against HSV-infected autologous fibroblasts. NK cell clones, classified according to the expression of natural killer-cell receptors on their surface, displayed a great heterogeneity of cytolytic properties against HSV-infected cells. Nevertheless, a more accurate functional analysis demonstrated not only that HSV infection downregulated the expression of HLA-A and HLA-B and did not modify the expression of HLA-C, but also that NK cell clones expressing the "activating" form of the anti HLA-C NK cell receptor were more cytolytic than other clones. This finding suggests that two different and clonally distributed mechanisms of NK cell activation may be employed by NK cells to kill HSV-infected autologous target cells.

Role of Natural Killer Cell Alloreactivity in HLA-Mismatched Hematopoietic Stem Cell Transplantation

Because of the expression of inhibitory receptors (KIR) for major histocompatibility complex (MHC) class I allotypes, a person's natural killer (NK) cells will not recognize and will, therefore, kill cells from individuals lacking his/her KIR epitopes. This study investigated the role of NK cell alloreactivity in human HLA haplotype-mismatched hematopoietic stem cell transplantation and, specifically, the role of the three major NK specificities, ie, those for HLA-C group 1, HLA-C group 2, and HLA-Bw4 alleles. In 20 of 60 donor-recipient pairs, KIR epitope incompatibility and functional analyses of donor NK cell clones predicted donor NK cells could cause graft-versus-host (GVH)/graft-versus-leukemia (GVL) reactions. NK cell clones of donor origin were obtained from transplanted recipients and tested for lysis of recipient's cryopreserved pretransplant lymphocytes. Despite the absence of GVH disease, we detected high frequencies of NK clones which killed recipient's target cells. Lysis followed the rules of NK cell alloreactivity, being blocked only by the MHC class I KIR epitope which was missing in the recipient. The alloreactive NK clones also killed the allogeneic leukemia. Transplants from these KIR epitope incompatible donors had higher engraftment rates. Therefore, a GVL effector and engraftment facilitating mechanism, which is independent of T-cell-mediated GVH reactions, may be operational in HLA mismatched hematopoietic cell transplants.

NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis.

After culture in interleukin (IL)-2, natural killer (NK) cells acquire an increased capability of mediating non-major histocompatibility complex (MHC)-restricted tumor cell lysis. This may reflect, at least in part, the de novo expression by NK cells of triggering receptors involved in cytolysis. In this study we identified a novel 44-kD surface molecule (NKp44) that is absent in freshly isolated peripheral blood lymphocytes but is progressively expressed by all NK cells in vitro after culture in IL-2. Different from other markers of cell activation such as CD69 or VLA.2, NKp44 is absent in activated T lymphocytes or T cell clones. Since NKp44 was not detected in any of the other cell lineages analyzed, it appears as the first marker specific for activated human NK cells. Monoclonal antibody (mAb)-mediated cross-linking of NKp44 in cloned NK cells resulted in strong activation of target cell lysis in a redirected killing assay. This data indicated that NKp44 can mediate triggering of NK cell cytotoxicity. mAb-mediated masking of NKp44 resulted in partial inhibition of cytolytic activity against certain (FcgammaR-negative) NK-susceptible target cells. This inhibition was greatly increased by the simultaneous masking of p46, another recently identified NK-specific triggering surface molecule. These data strongly suggest that NKp44 functions as a triggering receptor selectively expressed by activated NK cells that, together with p46, may be involved in the process of non-MHC-restricted lysis. Finally, we show that p46 and NKp44 are coupled to the intracytoplasmic transduction machinery via the association with CD3zeta or KARAP/DAP12, respectively; these associated molecules are tyrosine phosphorylated upon NK cell stimulation.

Murine Nkg2d and Cd94 are clustered within the natural killer complex and are expressed independently in natural killer cells.

Natural killer (NK) cells express C-type lectin-like receptors, encoded in the NK gene complex, that interact with major histocompatibility complex class I and either inhibit or activate functional activity. Human NK cells express heterodimers consisting of CD94 and NKG2 family molecules, whereas murine NK cells express homodimers belonging to the Ly-49 family. The corresponding orthologues for other species, however, have not been described. In this report, we used probes derived from the expressed sequence tag database to clone C57BL/6-derived cDNAs homologous to human NKG2-D and CD94. Among normal tissues, murine NKG2-D and CD94 transcripts are highly expressed only in activated NK cells, including both Ly-49A+ and Ly-49A- subpopulations. Additionally, mNKG2-D is expressed in murine NK cell clones KY-1 and KY-2, whereas mCD94 expression is observed only in KY-1 cells but not KY-2. Last, we have finely mapped the physical location of the Cd94 (centromeric) and Nkg2d (telomeric) genes between Cd69 and the Ly49 cluster in the NK complex. Thus, these data indicate the expanding complexity of the NK complex and the corresponding repertoire of C-type lectin-like receptors on murine NK cells.

Signaling through human killer cell activating receptors triggers tyrosine phosphorylation of an associated protein complex.

Our understanding of the biology of human natural killer (NK) cells has significantly advanced in recent years upon identification of a family of NK cell-expressed genes that encode killer cell inhibitory receptors (KIR). Individual KIR can selectively bind various HLA class I allotypes and consequently transduce inhibitory signals that block NK cell lysis of ligand-bearing target cells. A distinct subset of related and linked genes express truncated versions of KIR that are otherwise highly homologous in amino acid sequence. Interestingly, these receptors appear to transmit stimulatory signals into NK cells and have been termed killer cell activating receptors (KAR). In this report, we demonstrate that recognition of HLA-Cw3 by the p50 KAR, NKAT8, can potentiate the cytotoxic response of appropriate NK cell clones. Specific cross-linking of this KAR with a monoclonal antibody resulted in intracellular calcium mobilization, protein tyrosine phosphorylation, and phosphorylation of the MAP kinases, ERK1 and ERK2. In addition, we identified a KAR-associated disulfide-linked dimer of a 13-kDa protein that was absent in the Jurkat T cell line and is predicted to participate in these activation signaling events. Upon treatment of NK cells with pervanadate, the disulfide-linked p13 and additional proteins of 25, 30, 37 and 50-95 kDa were identified as KAR-associated tyrosine phosphoproteins. Importantly, p13 was inducibly tyrosine phosphorylated upon cross-linking of NKAT8, which strongly suggests that the associated p13 provides KAR with appropriate cytoplasmic structure to couple with tyrosine kinase-mediated signaling effectors.

Functionally and Structurally Distinct NK Cell Receptor Repertoires in the Peripheral Blood of Two Human Donors

The expression of KIR and CD94:NKG2 receptors was determined for more than 100 natural killer (NK) cell clones obtained from two blood donors who differ in their HLA class I and KIR genes. More than 98% of the clones were inhibited by individual autologous class I allotypes, and every clone was inhibited by the combination of autologous allotypes. The patterns of inhibition correlate with expression of inhibitory receptors of defined specificity. One donor possesses three class I ligands for KIR, and a majority of NK cells use KIR as their inhibitory receptor; the second donor possesses only a single ligand for KIR, and a majority of NK cells use the more broadly reactive CD94:NKG2a as their inhibitory receptor. Because of these differences, the first donor has subpopulations of NK cells that kill cells of the second donor, whereas the NK cells of the second donor are universally tolerant of cells from the first donor.

Expression of the Ly49A gene in murine natural killer cell clones is predominantly but not exclusively mono-allelic.

The Ly49 family of natural killer (NK) cell receptors are major histocompatibility complex (MHC) class I-specific inhibitory receptors that are distributed to overlapping NK cell subsets. Extending earlier studies of polyclonal NK cell populations, we have employed an analysis of short-term NK cell clones from Ly49A heterozygous mice, to demonstrate that the Ly49A gene is usually expressed from one or the other allele in each Ly49A+ cell. However, we also detected a small percentage of clones that expressed both Ly49A alleles. The possibility that the colonies exhibiting bi-allelic Ly49A gene expression had been inoculated with more than one cell was ruled out by parallel analysis of clones isolated from mixtures of NK cells from Ly49A homozygous mice. The frequency of bi-allelic Ly49A+ clones suggested that the two Ly49A alleles in an NK cell are chosen for expression independently. These data are consistent with the proposal that mono-allelic Ly49A gene expression may arise as a consequence of a stochastic Ly49 gene activation mechanism. Analysis of Ly49A+ clones from MHC-different mice demonstrated that class I-deficient mice harbored a greater number of bi-allelic Ly49A+ cells than did H-2d mice, which express a Ly49A ligand. Although the numbers were insufficiently large for a clear assignment, H-2b mice may harbor an intermediate number of bi-allelic Ly49A+ NK cells. The effects of MHC expression on the prevalence of bi-allelic Ly49A+ cells suggest that an MHC-dependent education process modifies the Ly49 repertoire.

P46, a novel natural killer cell-specific surface molecule that mediates cell activation.

Limited information is available on the surface molecules that are involved in natural killer (NK) cell triggering. In this study, we selected the BAB281 monoclonal antibody (mAb) on the basis of its ability to trigger NK-mediated target cell lysis. BAB281 identified a novel NK cell-specific surface molecule of 46 kD (p46) that is expressed by all resting or activated NK cells. Importantly, unlike the NK cell antigens identified so far, the expression of p46 was strictly confined to NK cells. Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones. The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A. Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex-restricted cytolysis mediated by human NK cells.