Rocaglamide enhances NK cell-mediated killing of non-small cell lung cancer cells by inhibiting autophagy

Targeting macroautophagy/autophagy is a novel strategy in cancer immunotherapy. In the present study, we showed that the natural product rocaglamide (RocA) enhanced natural killer (NK) cell-mediated lysis of non-small cell lung cancer (NSCLC) cells in vitro and tumor regression in vivo. Moreover, this effect was not related to the NK cell recognition of target cells or expressions of death receptors. Instead, RocA inhibited autophagy and restored the level of NK cell-derived GZMB (granzyme B) in NSCLC cells, therefore increasing their susceptibility to NK cell-mediated killing. In addition, we further identified that the target of RocA was ULK1 (unc-51 like autophagy activating kinase 1) that is required for autophagy initiation. Using firefly luciferase containing the 5´ untranslated region of ULK1, we found that RocA inhibited the protein translation of ULK1 in a sequence-specific manner. Taken together, RocA could block autophagic immune resistance to NK cell-mediated killing, and our data suggested that RocA was a promising therapeutic candidate in NK cell-based cancer immunotherapy.     

Reduced release and binding of perforin at the immunological synapse underlies the age-related decline in natural killer cell cytotoxicity

Physiological aging is accompanied by a marked reduction in natural killer (NK) cell cytotoxicity (NKCC) at the single cell level, but the underlying mechanisms are unknown. To address this issue, we isolated NK cells from healthy young (≤ 35 years) and old (≤ 60 years) subjects and examined the effect of age on events fundamental to the process of NKCC. Simultaneous assessment of NKCC and NK cell–target cell conjugate formation revealed a marked age‐associated decline in NK cell killing but comparable conjugate formation, indicating a post‐target cell binding defect was responsible for impaired NKCC. Despite a reduction in the proportion of NK cells expressing the activatory receptor NKp46, NK cells from old donors were not hyporesponsive to stimulation, as no age‐associated difference was observed in the expression of the early activation marker CD69 following target cell coculture. Furthermore, intracellular levels of the key cytotoxic effector molecules perforin and granzyme B, and the fusion of secretory lysosomes with the NK cell membrane were also similar between the two groups. However, when we examined the binding of the pore‐forming protein perforin to the surface of its target cell, an event that correlated strongly with target cell lysis, we found the percentage of perforin positive target cells was lower following coculture with NK cells from old subjects. Underlying this reduction in binding was an age‐associated impairment in perforin secretion, which was associated with defective polarization of lytic granules towards the immunological synapse. We propose that reduced perforin secretion underlies the reduction in NKCC that accompanies physiological aging.     

Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34(+) cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-gamma production. However, NK cell cytotoxicity and IFN-gamma production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-gamma Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-gamma production plays an important role in Fas-mediated cytotoxicity.     

Perforin is recaptured by natural killer cells following target cells stimulation for cytotoxicity

When encountering target cells, NK (natural killer) cells exocytose Pfn (perforin) and granzyme B to kill challengers. We previously reported that granzyme B is recycled and reused by NK cells via clathrin-dependent endocytosis. However, whether Pfn, a main secretory vesicle content, indispensible to granzyme B killing, undergoes endocytosis remains unknown. We demonstrate that Pfn is recaptured by early endosomes of NK cells via a clathrin-dependent endocytosis after target cell stimulation. Inhibition of clathrin-dependent endocytosis significantly attenuated the cytotoxicity of NK cells. The data suggest that the recovery of Pfn contributes to the cytotoxicity of NK cells. The assay of endocytosis of lytic molecule presents a particular focus for exploring the mechanism of abnormal cytotoxicity of NK cells.     

IFN increases class I MHC antigen expression on adenovirus-infected human cells without inducing resistance to natural killer cell killing.

It has been previously shown that unstimulated NK cells cannot preferentially lyse adenovirus serotypes 2 and 5-infected human cells. In this study, the ability of IFN to promote the selective NK cell-mediated lysis of adenovirus-infected human cells was determined. The relationship between target cell susceptibility to NK cell-mediated killing and class I Ag expression was also analyzed through the use of adenovirus serotype 2 and 5 mutants that do not make the adenovirus early region 3 19-kDa class I binding protein. IFN induced the selective lysis of adenovirus serotype 2 and 5-infected human cells by activating NK cells (IFN-alpha) and protecting uninfected, but not adenovirus-infected cells, from NK cell-mediated lysis (IFN-gamma). IFN-gamma increased the expression of class I Ag on the surface of cells infected with the adenovirus early region 3 deletion mutants, dl327 or dl801, to a level equal to or greater than that expressed on uninfected cells. Despite the increased expression of class I Ag, IFN-gamma could not protect these adenovirus-infected cells from NK cell-mediated lysis. Thus, dl327 or dl801 infection prevented IFN-gamma's induction of cytolytic resistance to NK cell-mediated killing but left IFN-gamma's induction of class I Ag intact. Surface class I Ag levels were substantially higher on IFN-gamma-treated, dl327-, and dl801-infected cells in comparison to cells infected with wild type adenovirus serotype 5. Again, higher target cell levels of class I Ag did not correlate with increased resistance to NK cell-mediated lysis because there was equivalent NK cell-mediated killing of IFN-gamma-treated adenovirus serotype 5-, dl327-, or dl801-infected cells. Thus, IFN-gamma only protects uninfected cells from NK cell-mediated killing, irrespective of target class I Ag levels, and thereby concentrates NK lytic activity on just adenovirus-infected cells. These data demonstrate that IFN-gamma's ability to protect target cells from NK cell-mediated cytolysis is unrelated to IFN-gamma's induction of surface class I MHC Ag.     

Myxoma Virus Infection Promotes NK Lysis of Malignant Gliomas In Vitro and In Vivo

Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas.     

The human cytomegalovirus protein UL16 mediates increased resistance to natural killer cell cytotoxicity through resistance to cytolytic proteins

Several reports have shown that human cytomegalovirus (HCMV)-infected cells are resistant to NK lysis. These studies have focused on receptor-ligand interactions, and different HCMV proteins have been indicated to mediate inhibitory NK signals. Here, we report that the HCMV protein UL16 is of major importance for the ability of HCMV-infected cells to resist NK cell-mediated cytotoxicity. Fibroblasts infected with the UL16 deletion mutant HCMV strain exhibited a 70% increased sensitivity to NK killing at 7 days postinfection compared to AD169-infected cells. Interestingly, HCMV-infected cells did not appear to engage inhibitory molecules on NK cells, since the levels of granzyme B were not reduced in supernatants obtained from NK cell cocultures with infected target cells compared to uninfected target cells. Furthermore, HCMV-infected cells, but not cells infected with the UL16 deletion mutant HCMV strain, exhibited a significantly increased resistance to the action of cytolytic proteins, including perforin, granzyme B, streptolysin O, and porcine NK lysin. In addition, fluorescence-activated cell sorting for UL16-positive transfected cells resulted in protection levels of 90% compared to control cells carrying the green fluorescent protein vector. Thus, the UL16 protein mediates an increased protection against the action of cytolytic proteins released by activated NK cells, possibly by a membrane-stabilizing mechanisms, rather than by delivering negative signals to NK cells.     

Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells

We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH–GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20°C for 2 h and then released by warming up to 37°C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH–GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.     

Organelle proteomics: identification of the exocytic machinery associated with the natural killer cell secretory lysosome

Natural killer (NK) cells and cytotoxic T lymphocytes eliminate virally infected and transformed cells. Target cell killing is mediated by the regulated exocytosis of secretory lysosomes, which deliver perforin and proapoptotic granzymes to the infected or transformed cell. Yet despite the central role that secretory lysosome exocytosis plays in the immune response to viruses and tumors, little is known about the molecular machinery that regulates the docking and fusion of this organelle with the plasma membrane. To identify potential components of this exocytic machinery we used proteomics to define the protein composition of the NK cell secretory lysosome membrane. Secretory lysosomes were isolated from the NK cell line YTS by subcellular fractionation, integral membrane proteins and membrane-associated proteins were enriched using Triton X-114 and separated by SDS-PAGE, and tryptic peptides were identified by LC ESI-MS/MS. In total 221 proteins were identified unambiguously in the secretory lysosome membrane fraction of which 61% were predicted to be either integral membrane proteins or membrane-associated proteins. A significant proportion of the proteins identified play a role in vesicular trafficking, including members of both the Rab GTPase and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and protein families. These proteins include Rab27a and the SNARE vesicle-associated membrane protein-7, both of which were enriched in the secretory lysosome fraction and represent potential components of the machinery that regulates the exocytosis of this organelle in NK cells.     

Role of P-glycoprotein in human natural killer-like cell line-mediated cytotoxicity

Natural killer (NK) cells express the highest amount of P-glycoprotein (Pgp), a product of the multidrug resistance (MDR) 1 gene, among lymphoid cells, and our previous studies demonstrated that Pgp is required for NK cell-mediated cytotoxicity. In this study we examined the role of Pgp in NK cell-mediated cytotoxicity using a human NK-like cell line, i.e., YTN cells and two MDR reversing agents, nicardipine and its structural analog, AHC-93. These two agents inhibited the Pgp function (rhodamine-123 excretion) as well as cell-mediated cytotoxicity, confirming that Pgp is critical for NK cell-mediated cytotoxicity. As revealed by video-rate ultraviolet laser-scanning confocal microscopy, AHC-93 did not inhibit the increase in the intracellular calcium concentration upon binding to target cells, whereas nicardipine did, as reported previously. These two reagents relocated acridine orange dye from lysosomes to the cytoplasm at concentrations similar to those required for the inhibition of cell-mediated cytotoxicity. These results suggest that Pgp is directly or indirectly involved in pH regulation in lysosomes, but not in calcium homeostasis.     

Role for the Rac1 exchange factor Vav in the signaling pathways leading to NK cell cytotoxicity

Here we investigate the activation of and a possible role for the hematopoietic Rac1 exchange factor, Vav, in the signaling mechanisms leading to NK cell-mediated cytotoxicity. Our data show that direct contact of NK cells with a panel of sensitive tumor targets leads to a rapid and transient tyrosine phosphorylation of Vav and to its association with tyrosine-phosphorylated Syk. Vav tyrosine phosphorylation is also observed following the activation of NK cells through the low-affinity Fc receptor for IgG (Fc gamma RIII). In addition, we demonstrate that both direct and Ab-mediated NK cell binding to target cells result in the activation of nucleotide exchange on endogenous Rac1. Furthermore, Vav antisense oligodeoxynucleotide treatment leads to an impairment of NK cytotoxicity, with Fc gamma RIII-mediated killing being more sensitive to the abrogation of Vav expression. These results provide new insight into the signaling pathways leading to cytotoxic effector function and define a role for Vav in the activation of NK cell-mediated killing.     

Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H

Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (NK) cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co-culture of YT cells, a human NK cell line, and GFP-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process.     

Decay-accelerating factor expression on either effector or target cells inhibits cytotoxicity by human natural killer cells.

Previous studies have shown that freshly isolated CD16+ NK cells are deficient in the expression of decay-accelerating factor (DAF), or CD55, a membrane regulator of C3 activation. In this study we investigated the significance, for NK cell-mediated lysis, of DAF expression on the target and effector cells. The effect of DAF expression on the susceptibility of NK cell targets was investigated by several means: first, DAF- cell lines were cloned from K562; second, the cloned DAF- cells were reconstituted with exogenous purified DAF; and third, anti-DAF F(ab')2 was used to block DAF function on the DAF+ K562 cells. Consistently, the presence of DAF in the target cell membrane, either naturally occurring or experimentally incorporated, afforded the target cell protection against lysis, and this protection could be blocked with anti-DAF. Similarly, targets for antibody-dependent cell-mediated cytotoxicity with exogenous DAF incorporated in their plasma membrane became less sensitive to antibody-dependent cell-mediated cytotoxicity by NK cells compared with the same target cells without incorporated DAF in their membranes. DAF incorporated in the plasma membranes of the effector NK cells made the NK cells less effective at killing K562 targets. The known function of DAF is to regulate C3 activation, and we were able to demonstrate that the isolated NK cell is capable of releasing C3. It is also possible that the participation of DAF in NK cell function represents a new, noncomplement-dependent function for DAF.     

IFN-gamma production dominates the early human natural killer cell response to Coxsackievirus infection

Coxsackieviruses (CV) are important human pathogens that have been implicated in the pathogenesis of several diseases, including myocarditis and pancreatitis. How the human immune system recognizes and controls CV infections is not well understood. Studies in mice suggest that natural killer (NK) cells play a critical role in viral clearance and host survival, but the mechanism(s) by which human NK cells may contribute to the host anti-CV defence has not been investigated. Here we show that CVB3 infection markedly reduces HLA class I cell surface expression but does not increase the expression of the activating NK cell receptor ligands MICA/B and ULBP1-3 on human cells. We also demonstrate that the lowered target cell HLA class I surface expression does not correlate with an increased susceptibility to NK cell-mediated killing. However, NK cells responded with a robust production of interferon γ (IFN-γ) when peripheral blood mononuclear cells were cocultured with infected cells. In summary, this study shows that CVB3 interferes with the expression of NK cell receptor ligands on infected cells and indicates that IFN-γ production, rather than cytotoxicity, marks the early human NK cell response to CVB3 infection.     

Phosphoinositide breakdown and evidence for protein kinase C involvement during human NK killing

Conjugation between human NK cells and susceptible target cells (K562 and Jurkat) leads to breakdown of inositol lipids in the effector cells but not when conjugated with resistant target cells. Extracellular Ca2+ is required for this activation. Sphingosine inhibits NK killing in both normal and IL-2-activated NK cells. Phorbol esters, TPA, and PDBU enhanced NK killing at low concentrations, where 4-alpha-PDIDE did not. The diacylglycerol derivative OAG increased NK cell killing and activated PKC from human lymphocytes. These results strongly suggest that phosphoinositide breakdown and activation of PKC is involved in NK killing.