Naive Natural Killer Cells Research Articles

NK Cell-derived Exosomes From NK Cells Previously Exposed to Neuroblastoma Cells Augment the Antitumor Activity of Cytokine-activated NK Cells.

Immune cell-derived exosomes can increase immunity against tumors. In contrast, tumor-derived exosomes can reduce the immunity and can change the tumor microenvironment to further develop and provide metastasis. These effects take place by an alteration in the innate and adaptive immune cell functions. In this experiment, we studied the natural killer (NK) cells' effectiveness on tumor cells after expansion and thereafter incubated it with exosomes. The exosomes were derived from 2 populations of NK cells: (1) naive NK cells and, (2) NK cells previously exposed to neuroblastoma (NB) cells. Moreover, we have studied the NB-derived exosomes on NK cell function. The molecular load of the characterized exosomes (by means of nanoparticle-tracking analysis, flow cytometry, scanning electron microscopy, and western blot) from NK cells exposed to the NB cell revealed their expression of natural killer cell receptors in addition to CD56, NKG2D, and KIR2DL2 receptors. These exosomes were used to treat NK cells and thereafter administered to NB tumor cells both in vitro and in vivo. Our results showed some kind of NK cells' education by the exosomes. This education from NK cells previously exposed to NB cell-derived exosomes caused efficient and greater cytotoxicity against NB tumors, but NB-derived exosomes act as tumor promoters by providing a tumor supporting niche. Hence, this method of preparing the exosomes has a dramatic effect on activation of anti-NK cells against NB cells.

Tumor-priming converts NK cells to memory-like NK cells

ABSTRACT Fascinating earlier evidence suggests an intrinsic capacity of human natural killer (NK) cells to acquire adaptive immune features in the context of cytomegalovirus (CMV) infection or pro-inflammatory cytokine stimulation. Since the role of memory NK cells in cancer has so far remained elusive and adoptive NK cell transfer in relapsing pediatric acute B cell precursor leukemia (BCP-ALL) patients awaits improvement, we asked the question whether tumor-priming could promote the generation of memory NK cells with enhanced graft-vs.-leukemia (GvL) reactivity. Here, we provide substantial evidence that priming of naive human NK cells with pediatric acute B cell leukemia or acute myeloid leukemia specimens induces a functional conversion to tumor-induced memory-like (TIML)-NK cells displaying a heightened tumor-specific cytotoxicity and enhanced perforin synthesis. Cell cycles analyses reveal that tumor-priming sustainably alters the balance between NK cell activation and apoptosis in favor of survival. In addition, gene expression patterns differ between TIML- and cytokine-induced memory-like (CIML)-NK cells with the magnitude of regulated genes being distinctly higher in TIML-NK cells. As such, the tumor-induced conversion of NK cells triggers the emergence of a so far unacknowledged NK cell differentiation stage that might promote GvL effects in the context of adoptive cell transfer.

Cytomegalovirus generates long-lived antigen-specific NK cells with diminished bystander activation to heterologous infection.

Natural killer (NK) cells play a key role in the host response to cytomegalovirus (CMV) and can mediate an enhanced response to secondary challenge with CMV. We assessed the ability of mouse CMV (MCMV)-induced memory Ly49H(+) NK cells to respond to challenges with influenza, an acute viral infection localized to the lung, and Listeria monocytogenes, a systemic bacterial infection. MCMV-memory NK cells did not display enhanced activation or proliferation after infection with influenza or Listeria, as compared with naive Ly49H(+) or Ly49H(-) NK cells. Memory NK cells also showed impaired activation compared with naive cells when challenged with a mutant MCMV lacking m157, highlighting their antigen-specific response. Ex vivo, MCMV-memory NK cells displayed reduced phosphorylation of STAT4 and STAT1 in response to stimulation by IL-12 and type I interferon (IFN), respectively, and IFN-γ production was reduced in response to IL-12 + IL-18 compared with naive NK cells. However, costimulation of MCMV-memory NK cells with IL-12 and m157 antigen rescues their impaired response compared with cytokines alone. These findings reveal that MCMV-primed memory NK cells are diminished in their response to cytokine-driven bystander responses to heterologous infections as they become specialized and antigen-specific for the control of MCMV upon rechallenge.

Cross-talk between CD8+ T cells and natural killers: the role of mitochondrial Aa2+ transport

Unraveling complex interactions between immune cells is a key to the development of new strategies for immunotherapy. In the present study, we investigated functional outcome of bidirectional interaction between activated CD8+T and naive natural killer (NK) cells. We found that phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Io)-stimulated CD8+T cells form multiple intercellular contacts with naive NK lymphocytes. Co-culture of activated T cells with naive NK cells results in the selective down-regulation of CD25 molecule in T cells while elevating CD25 and CD69 expression on naive NK cells. Further, CD8+T and NK cells cross-regulate mitochondrial homeostasis including calcium transport. This effect is dependent on both cytokines and intercellular contacts, and partially involves natural killer group 2 member D (NKG2D) receptor activation. Data also suggest that activated CD8+T cells might directly transfer mitochondria and activation molecules such as CD25 and CD69 to naive NK cells. Alterations in phosporylation status of multiple signaling proteins during CD8+T/NK interaction suggest a functional remodeling whereby NK cells shift activated CD8+T cells towards T central-memory (TCM) phenotype and activated CD8+T lymphocytes alter naive state of NK cells towards effector/regulatory phenotype. Inhibition of mitochondrial Ca2+ uptake (mCU) or Na+/Ca2+ exchanger (mNCE) with Ru360 and {type:entrez-protein,attrs:{text:CGP37157,term_id:875406365,term_text:CGP37157}}CGP37157 respectively mimicked observed alterations in CD8+ and NK cells upon their interaction. These data suggest a potential role of mitochondrial Ca2+ homeostasis in the acquisition of mixed activation/regulatory phenotype by NK cells during T cell-NK cell interaction. We believe that understanding the mechanisms of CD8+-NK interplay will help to develop new approaches for cellular immunotherapy.

Exposure to particular matter increases susceptibility to respiratory Staphylococcus aureus infection in rats via reducing pulmonary natural killer cells.

Epidemiological studies have shown a correlation between exposure to fine particular matter (PM2.5) and increased respiratory infection, but the mechanisms have remained poorly defined. By using an experimental system we evaluated the effect of PM2.5 exposure on susceptibility to subsequent pulmonary Staphylococcus aureus (S. aureus) infection and its potential mechanisms. Rats were intratracheally instilled with a single dose of PM2.5 sample or PBS followed by an intratracheal inoculation with bacteria S. aureus at 24h after PM2.5 exposure. The rats were examined at 24h post infection. We found that exposure of rats to PM2.5 significantly increased inflammatory cells and levels of IL-6 and TNF-α in bronchoalveolar lavage fluids (BALF). Prior PM2.5 exposure markedly increased the susceptibility of rats to subsequent S. aureus infection. The mechanistic studies showed that alveolar macrophages (AMs) from PM2.5-experienced lungs had depressed phagocytosis of S. aureus, and prior PM2.5 exposure significantly decreased the natural killer (NK) cells recruited into the airways following subsequent S. aureus infection. Further, adoptive transfer of naive NK cells to the lung of prior PM2.5-exposed rats restored PM2.5-impaired antibacterial host defense. The presence of NK cells markedly enhanced the ability of AMs to phagocytose S. aureus ex vivo. Thus, our study identifies PM2.5-impaired NK cell response in the lung to be a novel critical mechanism for PM2.5-mediated susceptibility to S. aureus bacterial infection, which provides a potential mechanism to explain the epidemiological findings that associate ambient air pollution and increased lung bacterial infections. Our findings also suggest that enhancing pulmonary NK cells may be considered for future therapeutic approaches to clinically antibiotic-resistant S. aureus infection in the lung.

Way to go to exploit NK cells' versatile talents for cancer immunotherapy.

Way to go to exploit NK cells' versatile talents for cancer immunotherapy.

A LysM and SH3-domain containing region of the Listeria monocytogenes p60 protein stimulates accessory cells to promote activation of host NK cells.

Listeria monocytogenes (Lm) infection induces rapid and robust activation of host natural killer (NK) cells. Here we define a region of the abundantly secreted Lm endopeptidase, p60, that potently but indirectly stimulates NK cell activation in vitro and in vivo. Lm expression of p60 resulted in increased IFNγ production by naïve NK cells co-cultured with treated dendritic cells (DCs). Moreover, recombinant p60 protein stimulated activation of naive NK cells when co-cultured with TLR or cytokine primed DCs in the absence of Lm. Intact p60 protein weakly digested bacterial peptidoglycan (PGN), but neither muropeptide recognition by RIP2 nor the catalytic activity of p60 was required for NK cell activation. Rather, the immune stimulating activity mapped to an N-terminal region of p60, termed L1S. Treatment of DCs with a recombinant L1S polypeptide stimulated them to activate naïve NK cells in a cell culture model. Further, L1S treatment activated NK cells in vivo and increased host resistance to infection with Francisella tularensis live vaccine strain (LVS). These studies demonstrate an immune stimulating function for a bacterial LysM domain-containing polypeptide and suggest that recombinant versions of L1S or other p60 derivatives can be used to promote NK cell activation in therapeutic contexts.

Fetal and adult multipotent mesenchymal stromal cells are killed by different pathways

Multipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC), can be isolated from adult and fetal tissues. Recently, there has been considerable interest in MSC because they have features favorable for transplantation, namely their multipotency and non-immunogenic properties. We analyzed how human MSC derived from first-trimester fetal liver and adult bone marrow interact with naive and activated innate natural killer (NK) cells. NK cell function was studied by measuring killing of MSC, as well as degranulation (CD107a) induced by MSC. To assess the importance of NK cell killing, expression of surface epitopes was analyzed by flow cytometry on MSC before and after stimulation with interferon (IFN)γ. Fetal and adult MSC express several ligands to activating NK cell receptors as well as low levels of HLA class I, with large inter-individual variation. Naive peripheral blood NK cells did not lyse fetal or adult MSC, whereas interleukin (IL)2 activated allogeneic as well as autologous NK cells did. Pre-incubation of MSC with IFN-γ increased their levels of HLA class I, protecting them from NK cell recognition. Fetal and adult MSC were preferably killed via the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) pathways, respectively. Blocking NKG2D reduced NK cell degranulation in both fetal and adult MSC. Fetal and adult MSC differ in their interactions with NK cells. Both fetal and adult MSC are susceptible to lysis by activated NK cells, which may have implications for the use of MSC in cell therapy.

Versatility in NK cell memory

Versatility in NK cell memory

Critical role for the chemokine receptor CXCR6 in NK cell–mediated antigen-specific memory of haptens and viruses

Hepatic natural killer (NK) cells mediate antigen (Ag)-specific contact hypersensitivity (CHS) in T-cell and B-cell deficient mice. We now report that hepatic, but not splenic or naïve NK cells also develop specific memory to vaccines containing Ags from influenza, vesicular stomatitis virus (VSV) or human immunodeficiency virus-1 (HIV). Adoptive transfer of virus-sensitized NK cells to naïve recipients enhanced the animals' survival upon lethal challenge with the sensitizing virus, but not a different virus. NK cell memory to haptens and viruses depended upon CXCR6, a chemokine receptor on hepatic NK cells that was required for memory NK cell persistence but not for Ag recognition. Hence, hepatic NK-cells can develop adaptive immunity to structurally diverse Ags, an activity that requires NK-cell-expressed CXCR6.

IFN‐γ Production and Degranulation are Differentially Regulated in Response to Stimulation in Murine Natural Killer Cells

Activation of natural killer (NK) cells is induced via receptors like NKG2D, NKR-P1C and NKp46. This activation is balanced by interactions with inhibitory receptors. NK cell activation can lead to cytotoxicity mediated via polarized exocytosis of secretory lysosomes (degranulation) and interferon (IFN)-gamma production. We studied cell surface mobilization of a molecule present in secretory lysosomes, CD107a (LAMP-1), to monitor the relationship between degranulation of NK cells and their production of IFN-gamma at the single cell level. A comparison of responses in naive mouse NK cells and NK cells pre-activated with the type I interferon-inducer tilorone demonstrated a dramatic influence of pre-activation, allowing potent degranulation and IFN-gamma responses to NKG2D mediated stimulation that were not observed with naive NK cells. Degranulation and IFN-gamma production were performed by overlapping NK cell populations with generally higher frequencies of degranulating than IFN-gamma producing NK cells. An NK cell subset analysis based on expression of Mac-1 and CD27 revealed that immature NK cells (Mac-1(lo) CD27(hi)) are preferentially degranulating, Mac-1(hi) CD27(hi) cells perform both effector functions efficiently, while the most mature (Mac-1(hi) CD27(lo)) NK cells display reduced degranulation but with maintained IFN-gamma production.

NKG2D splice variants: a reexamination of adaptor molecule associations

NKG2D is a homodimeric C-type lectin-related receptor expressed on natural killer (NK) cells and T cells. In mice, alternative deoxyribonucleic acid (DNA) splicing generates two isoforms of NKG2D that differ in the length of their cytoplasmic domains. Their ability to induce cellular activation is mediated via association with two membrane-bound, signaling adaptor molecules, DAP10 and DAP12. It has been reported that the long form of NKG2D associates exclusively with DAP10, whereas the short variant can interact with either adaptor. The short isoform was reported to be almost undetectable in naive NK cells. Using two distinct cell types, we demonstrate that like the short isoform, the long variant of NKG2D also associates not only with DAP10 but also with DAP12. Using reporter cells (70Z/3), we demonstrate that DAP12 can compete equally with DAP10 for association with both variants of NKG2D when DAP10 and DAP12 are coexpressed. Cross-linking either isoform of NKG2D induces a calcium flux when associated exclusively with DAP10 or DAP12. Moreover, using quantitative polymerase chain reaction (PCR), we also show that the short isoform of NKG2D is expressed in naive NK cells. Our data suggest that signaling via mouse NKG2D isoforms is more complex than originally presented.

Interaction of heat shock protein 70 peptide with NK cells involves the NK receptor CD94.

Full-length Hsp70 protein (Hsp70) and the C-terminal domain of Hsp70 (Hsp70C) both stimulate the cytolytic activity of naive natural killer (NK) cells against Hsp70-positive tumor target cells. Here, we describe the characterization of Hsp70-NK cell interaction with binding studies using the human NK cell line YT. Binding of recombinant Hsp70 protein (Hsp70) and the C-terminal domain of Hsp70 (Hsp70C) to YT cells is demonstrated by immunofluorescence studies. A phenotypic characterization revealed that none of the recently described HSP-receptors (alpha2-macroglobulin receptor CD91, Toll-like receptors 2, 4, 9, CD14) are expressed on YT cells. Only the C-type lectin receptor CD94 is commonly expressed by YT cells and Hsp70 reactive NK cells. A correlation of the cell density-dependent, variable CD94 expression and the binding capacity of Hsp70 was detected. Furthermore, Hsp70 binding could be completely abrogated by preincubation of YT cells with a CD94-specific antibody. Competition assays using either unlabeled Hsp70 protein or an unrelated protein (GST) in 20-fold excess and binding studies with escalating doses of Hsp70 protein provide evidence for a specific and concentration-dependent interaction of Hsp70 with YT cells. In addition to Hsp70 and Hsp70C, a 14-mer Hsp70 peptide termed TKD is known to exhibit comparable stimulatory properties on NK cells. Similar to full-length Hsp70 protein (10 microg/ml-50 microg/ml), a specific binding of this peptide to YT cells was observed at 4 degrees C, at equivalent concentrations (2.0 microg/ml-8.0 microg/ml). Following a 30 min incubation period at 37 degrees C, membrane-bound Hsp70 protein and Hsp70 peptide TKD were completely taken up into the cytoplasm.

Regulation of CD40L expression on natural killer cells by interleukin-12 and interferon gamma: its role in the elicitation of an effective antitumor immune response.

Effector cell functions are regulated by a number of positive signals for the mediation of antitumor immunity. The CD40 and CD40 ligand (CD40L) interaction has been implicated in the generation of effective cell-mediated and humoral immune responses, where cytokines have been shown to play a significant role in the expression of these molecules. Our earlier studies have shown that spontaneous regression of a rat histiocytoma transplanted s.c. is mediated by CD8 + CD3- NK cells. The CD40-CD40L mediation during tumor regression was of interest. Tumor-transplanted animals showed enhanced expression of CD40L on natural killer (NK) and T cells, when compared to cells from normal animals. CD40 expression on AK-5 tumor cells was also induced after s.c. transplantation. Administration of anti-(interleukin-12) (anti-IL-12) and anti-(interferon gamma) (anti-IFNgamma) antibodies in tumor-bearing animals showed down-regulation of the expression of CD40L on NK and T cells with simultaneous inhibition of cytotoxic acitivity of NK cells, cytokine release and the production of antitumor antibody. Naive NK cells, when co-cultured with fixed AK-5 cells, were induced to express CD40L. CD40L expression modulated the immune response exerted by NK cells, in part by the activation of nuclear factor kB (NF-kB). Furthermore, the signaling via CD40L through the use of anti-CD40L antibody promoted the in vitro activation of cytotoxic as well as NF-kappaB binding activity in NK cells from tumor-transplanted animals. These observations demonstrate that the expression of CD40L by the effector cells is regulated by IL-12 and IFNgamma, and could effectively modulate the NK-cell-mediated immune response during the regression of AK-5 tumor.

Alpha-gal-independent dual recognition and activation of xenogeneic endothelial cells and human naïve natural killer cells.

Interaction between vascularized xenograft and host immune system is thought to occur via Galactose alpha (1,3) Galactose (Gala 1,3 gal) structures decorating the xenograft. We raised anti-Gala 1,3 gal-BSA polyclonal antibodies in baboons and investigated effect(s) of these antibodies as well as soluble Gala 1,3 gal-BSA on human naive natural killer (NK) cell interactions with porcine aortic endothelial cells. We demonstrate that human naive (unstimulated) NK cells recognize xenogeneic endothelial cells under conditions where binding to the Gala 1,3 gal structures is minimized by the presence of blocking anti-Gala 1,3 gal IgG or soluble Gala 1-3 gal and in the absence of xenoreactive natural antibodies and complement. After xenogeneic encounter both endothelial cells and human NK cells are activated. Endothelial cell activation is rapid and is manifested initially by an intraendothelial calcium transient and subsequently by expression of P-selectin and vascular endothelial cell adhesion molecule-1 on the xenoendothelium surface. NK cell activation is manifested by increased expression of perforin and increased cytotoxicity towards the xenoendothelium. Neither recognition nor activation of the xenoendothelium was affected by the introduction of either anti-Gala 1,3 gal IgG or soluble Gala 1-3 gal. Our data provide evidence that innate immune cells, such as NK cells, recognize and activate xenoendothelial cells independently of Gala 1-3 gal structures and raise the possibility of novel interactive sites on both human naive NK cells and discordant xenogeneic endothelium.

Interferon-γ–induced membrane PAF-receptor expression confers tumor cell susceptibility to NK perforin-dependent lysis

Perforin is known to display a membranolytic activity on tumor cells. Nevertheless, perforin release during natural killer (NK)–cell activation is not sufficient to induce membrane target-cell damage. On the basis of the ability of perforin to interact with phospholipids containing a choline phosphate headgroup, we identify the platelet-activating factor (PAF) and its membrane receptor as crucial components in tumor cell killing activity of human resting NK cells. We demonstrate for the first time that upon activation, naive NK cells release the choline phosphate–containing lysolipid PAF, which binds to perforin and acts as an agonist on perforin-induced membrane damage. PAF is known to incorporate cell membranes using a specific receptor. Here we show that interferon-γ (IFN–γ) secreted from activated NK cells ends in PAF-receptor expression on perforin-sensitive K562 cells but not on perforin-resistant Daudi cells. In order to prove the capacity of PAF to interact simultaneously with its membrane PAF receptor and with perforin, we successfully co-purified the 3 components in the presence of bridging PAF molecules. The functional activity of this complex was further examined. The aim was to determine whether membrane PAF-receptor expression on tumor cells, driven to express this receptor, could render them sensitive to the perforin lytic pathway. The results confirmed that transfection of the PAF-receptor complementary DNA into major histocompatibility complex class I and Fas-receptor negative tumor cells restored susceptibility to naive NK cells and perforin attack. Failure of IFN-γ to induce membrane PAF receptor constitutes the first described mechanism for tumor cells to resist the perforin lytic pathway.

Interferon-γ–induced membrane PAF-receptor expression confers tumor cell susceptibility to NK perforin-dependent lysis

AbstractPerforin is known to display a membranolytic activity on tumor cells. Nevertheless, perforin release during natural killer (NK)–cell activation is not sufficient to induce membrane target-cell damage. On the basis of the ability of perforin to interact with phospholipids containing a choline phosphate headgroup, we identify the platelet-activating factor (PAF) and its membrane receptor as crucial components in tumor cell killing activity of human resting NK cells. We demonstrate for the first time that upon activation, naive NK cells release the choline phosphate–containing lysolipid PAF, which binds to perforin and acts as an agonist on perforin-induced membrane damage. PAF is known to incorporate cell membranes using a specific receptor. Here we show that interferon-γ (IFN–γ) secreted from activated NK cells ends in PAF-receptor expression on perforin-sensitive K562 cells but not on perforin-resistant Daudi cells. In order to prove the capacity of PAF to interact simultaneously with its membrane PAF receptor and with perforin, we successfully co-purified the 3 components in the presence of bridging PAF molecules. The functional activity of this complex was further examined. The aim was to determine whether membrane PAF-receptor expression on tumor cells, driven to express this receptor, could render them sensitive to the perforin lytic pathway. The results confirmed that transfection of the PAF-receptor complementary DNA into major histocompatibility complex class I and Fas-receptor negative tumor cells restored susceptibility to naive NK cells and perforin attack. Failure of IFN-γ to induce membrane PAF receptor constitutes the first described mechanism for tumor cells to resist the perforin lytic pathway.

Preferential interaction of a novel tumor surface protein (p38.5) with naive natural killer cells.

A receptor-ligand interaction exclusive to natural killer (NK) cell-mediated recognition and triggering of tumor cell destruction has not yet been identified. In contrast, molecules that are involved in cellular adhesion and regulation of NK cytolysis have been well studied. In this report, a novel tumor surface protein is identified that exhibits characteristics of a recognition structure for naive NK cells. A tagged ligand-cell adsorption technique revealed a 38.5-kD plasma membrane protein (p38.5) from a prototypical NK-susceptible cell line (K562) that preferentially bound to NK cells (CD3(-)CD5(-)CD16(+)) relative to T lymphocytes (CD3(+)CD5(+) CD16(-)). The molecule was purified to apparent homogeneity for further characterization. An amino acid sequence of an 11-mer internal peptide of p38.5 did not exhibit homology to known proteins. Affinity-purified antibody generated against this peptide (anti-p38.5) reacted with a single protein of 38.5 kD on Western blots of whole cell extracts of K562. Flow cytometry and immunoprecipitation studies of surface-labeled tumor cells demonstrated expression of p38.5 on NK-susceptible tumor cell lines (K562, MOLT-4, Jurkat), whereas p38.5 was not detected on NK-resistant tumor cell lines (A549, Raji, MDA-MB-231). Significantly, p38.5 loss variants derived from wild-type Jurkat and Molt-4 cell lines exhibited decreased susceptibility to NK cell-mediated lysis demonstrating a strong association between cell surface expression of p38.5 and cytotoxicity. Purified p38.5 retained preferential binding to NK cells and inhibited NK activity in a dose-dependent manner, thereby providing direct evidence of a role in the lytic process. Binding studies identified a 70-kD membrane protein from NK cells as a possible receptor for the p38.5 tumor ligand. Consistent with cellular adsorption studies, the 70-kD, p38.5 binding protein was not detected on T lymphocytes. Based on studies demonstrating selective binding of p38.5 to NK cells, lack of expression on NK-resistant tumor cell lines and ability of the purified molecule to block cytolysis, we conclude that p38.5 may serve as a recognition/triggering ligand for naive human NK cells.

Activation of natural killer cells by heat-killed Listeria monocytogenes requires additional signals from lymphoid cells

Regulatory and protective functions have been attributed to murine natural killer (NK) cells in a number of infectious diseases including listeriosis. We have developed an in vitro model to study parameters underlying the activation of naive NK cells using heat-killed Listeria monocytogenes (HKL) as stimulator. Independent from expression of the cell surface marker NK1.1, NK cells lysed YAC-1 cells after in vitro stimulation with HKL or HKL + Interleukin (IL)-2, but not medium or IL-2 alone. In contrast, NK cells from severely immunocompromised SCID or RAG-1 − − mutant mice failed to respond to HKL alone, but required exogenous IL-2. Using single-gene-disruption mutant mice, we show that NK-cell activation can be supported by either T-cell receptor (TCR)αβ cells, TCRγδ cells, MHC class I or MHC class II gene products. We conclude from these data that recognition of listerial components alone is insufficient for activation of naive NK cells, and that additional costimulatory signals are necessary. These can be provided by various lymphoid cells and appear to be cytokines.

A novel ligand in lymphocyte-mediated cytotoxicity: expression of the beta subunit of H+ transporting ATP synthase on the surface of tumor cell lines.

Extracellular adenosine triphosphate (eATP) has been suggested to play a role in lymphocyte-induced tumor destruction. We now provide evidence that a protein responsible for ATP synthesis in mitochondria may also play a physiologic role in major histocompatibility complex-independent, lymphocyte-mediated cytotoxicity. A 51.5-kD protein (p51.5) bearing structural and immunologic characteristics of the beta subunit of H+ transporting ATP synthase (E.C. 3.6.1.34, beta-H+ATPase, published molecular mass of 51.6 kD) was detected on the plasma membrane of three different human tumor cell lines studied. NH2-terminal amino acid sequence analysis of purified p51.5 from K562 tumor cells revealed 100% homology of 16 residues identified in the first 21 positions to the known sequence of human mitochondrial beta-H+ ATPase. Antibody directed against a 21-mer peptide in the ATP binding region of beta-H+ ATPase (anti-beta) reacted with only one band on Western blots of whole tumor extracts and tumor membrane extracts suggesting that the antiserum reacts with a single species of protein. Anti-beta reacted with the cell membranes of tumor cells as determined by fluorescence-activated flow cytometry and immunoprecipitated a 51.5-kD protein from surface-labeled neoplastic cells (but not human erythrocytes and lymphocytes). Purified p51.5 bound to human lymphocytes and inhibited natural killer (NK) cell-mediated cytotoxicity. Furthermore, anti-beta treatment of the K562 and A549 tumor cell lines inhibited NK (by > 95%) and interleukin 2-activated killer (LAK) cell (by 75%) cytotoxicity, respectively. Soluble p51.5 upon binding to lymphocytes retained its reactivity to anti-beta suggesting that the ATP binding domain and the lymphocyte-receptor binding domain reside in distinct regions of the ligand. These results suggest that beta-H+ ATPase or a nearly identical molecule is an important ligand in the effector phase (rather than the recognition phase) of a cytolytic pathway used by naive NK and LAK cells.