Siglec-7 is an indicator of natural killer cell function in acute myeloid leukemia.

Immune dysfunction is an established risk factor in acute myeloid leukemia (AML). The cytotoxicity of natural killer (NK) cells is greatly impaired in AML, and the profile of NK cell receptors is markedly altered in AML; however, this is not yet well characterized. In this study, we found the downregulation of Siglec-7 could be utilized as a potential marker of NK cell dysfunction in AML. The absolute numbers and percentages of NK cells were declined in the peripheral blood of patients with AML, and the levels of activating receptors NKG2D, NKp46, and NKp30 were reduced in NK cells from patients with AML compared with healthy controls. In contrast, the levels of inhibitory receptors TIM-3, ILT-4, ILT-5, and PD-1 were increased in NK cells from patients with AML. Of note, the level of Siglec-7 in NK cells from patients with AML was significantly lower than that in NK cells from healthy controls, and Siglec-7+ NK cells displayed higher levels of activating receptors and stronger cytotoxicity when compared with Siglec-7- NK cells. Our data indicate that decreased Siglec-7 level may predict NK cell dysfunction in AML, and NK cells may be promising targets of immunotherapy for AML.

Novel insight from the first lung transplant of a COVID-19 patient.

BACKGROUND: To reveal detailed histopathological changes, virus distributions, immunologic properties and multi-omic features caused by SARS-CoV-2 in the explanted lungs from the world's first successful lung transplantation of a COVID-19 patient. MATERIALS AND METHODS: A total of 36 samples were collected from the lungs. Histopathological features and virus distribution were observed by optical microscope and transmission electron microscope (TEM). Immune cells were detected by flow cytometry and immunohistochemistry. Transcriptome and proteome approaches were used to investigate main biological processes involved in COVID-19-associated pulmonary fibrosis. RESULTS: The histopathological changes of the lung tissues were characterized by extensive pulmonary interstitial fibrosis and haemorrhage. Viral particles were observed in the cytoplasm of macrophages. CD3+ CD4- T cells, neutrophils, NK cells, γ/δ T cells and monocytes, but not B cells, were abundant in the lungs. Higher levels of proinflammatory cytokines iNOS, IL-1ß and IL-6 were in the area of mild fibrosis. Multi-omics analyses revealed a total of 126 out of 20,356 significant different transcription and 114 out of 8,493 protein expression in lung samples with mild and severe fibrosis, most of which were related to fibrosis and inflammation. CONCLUSIONS: Our results provide novel insight that the significant neutrophil/ CD3+ CD4- T cell/ macrophage activation leads to cytokine storm and severe fibrosis in the lungs of COVID-19 patient and may contribute to a better understanding of COVID-19 pathogenesis.

Fas Ligand localizes to intraluminal vesicles within NK cell cytolytic granules and is enriched at the immune synapse.

INTRODUCTION: T cell and NK cell cytotoxicity can be mediated via the perforin/granzyme system and Fas Ligand (FasL, CD178). FasL is synthesized as a type II transmembrane protein that binds its cognate receptor Fas (CD95). Membrane-bound FasL is expressed on the plasma membrane of activated lymphocytes and is the main form of FasL with cytotoxic activity, but whether FasL is delivered to the immune synapse along with granzyme and perforin-containing granules is unclear. METHODS: We stably expressed FasL-fluorescent fusion proteins into human NK cells and examined the localization of FasL relative to other intracellular markers by confocal and immunoelectron microscopy, and examined the trafficking of FasL during formation of immune synapses with HLA-deficient B cells. RESULTS: FasL co-localized with CD63 more strongly than perforin or Lamp1+ in cytolytic granules. Electron microscopy revealed that FasL is enriched on intraluminal vesicles (ILVs) adjacent to the dense-core within cytolytic granules. In NK cells forming immune synapses with HLA-deficient B cells, a portion of FasL-containing granules re-localize toward the immune synapse, while a distinct pool of FasL remains at the distal pole of the cell. CONCLUSIONS: Localization of FasL to intra-luminal vesicles within cytolytic granules facilitates FasL trafficking to immune synapses and cytotoxic function in NK cells.

Leptin affects filopodia and cofilin in NK-92 cells in a dose- and time-dependent manner.

Hyperleptinemia, associated with obesity, is related with immune dysfunction and carcinogenesis. Natural Killer (NK) cells, a major component of the innate immune system are mediators of anti-tumor immunity and the most actively migrating cells among leukocytes. Actin rearrangement, promoted by cofilin plays a central role in cellular migration. Leptin affects the phosphorylation-dependent activity of cofilin and thus actin remodeling. We used human NK-92 cells to explore the in vitro effects of leptin on co-localization of cofilin and F-actin and on morphological changes in NK cells. NK-92 cells were incubated with different leptin concentrations (10 and 100 ng/mL) for 30 min and 24 h and immunocytochemically stained. Results demonstrate a dose- and time-dependent influence of leptin on cellular morphology. Utilizing confocal microscopy, we observed that the co-localization of cofilin-1 and F-actin was slightly influenced by leptin. In summary, the present study demonstrates an impact of a physiological leptin stimulation on the filopodia length, and a time-dependent effect on the co-localization of cofilin and F-actin in NK-92 cells.

Exposure to silver nanoparticles affects viability and function of natural killer cells, mostly via the release of ions.

Natural killer (NK) cells play a crucial role in linking innate and adaptive immune responses, especially during viral infections and tumor surveillance. They have two major effector functions: the killing of stressed/abnormal cells and the release of cytokines. Their activity is regulated via inhibitory and activating surface receptors. At the same time that the production and use of engineered nanoparticles is steadily increasing, the risk for exposure to silver nanoparticles (AgNPs) from consumer products or biomedical applications is growing. Given this, we assessed the effects of 20-nm big AgNPs on NK cells, which represent an important part of the immune system. Our study involved overnight exposure of human blood NK cells to different concentrations of AgNPs, and silver (Ag) ion controls, and analyzing them for viability, surface receptor expression, intracellular markers, cytokine release, and killing potential. Exposure to AgNPs, but not to Ag ion controls, reduced the viability and the cytotoxic potential after polyriboinosinic-polyribocytidylic acid stimulation of NK cells and increased the expression of the inhibitory receptor CD159a. Exposure to AgNPs and Ag ion controls reduced the expression of the activating receptors CD335 and of CD16 and increased the expression of the activating receptor CD314. Overall, exposure to AgNPs changes NK cells' function and phenotype and may present a risk for modulating human immune responses, which should be further investigated.

Advances in Molecular Imaging Strategies for In Vivo Tracking of Immune Cells.

Tracking of immune cells in vivo is a crucial tool for development and optimization of cell-based therapy. Techniques for tracking immune cells have been applied widely for understanding the intrinsic behavior of immune cells and include non-radiation-based techniques such as optical imaging and magnetic resonance imaging (MRI), radiation-based techniques such as computerized tomography (CT), and nuclear imaging including single photon emission computerized tomography (SPECT) and positron emission tomography (PET). Each modality has its own strengths and limitations. To overcome the limitations of each modality, multimodal imaging techniques involving two or more imaging modalities are actively applied. Multimodal techniques allow integration of the strengths of individual modalities. In this review, we discuss the strengths and limitations of currently available preclinical in vivo immune cell tracking techniques and summarize the value of immune cell tracking in the development and optimization of immune cell therapy for various diseases.

FoxO1-mediated autophagy is required for NK cell development and innate immunity.

Natural killer (NK) cells exert a crucial role in early immune responses as a major innate effector component. However, the underlying mechanisms of NK cell development remain largely elusive. Here we show that robust autophagy appears in the stage of immature NK cells (iNKs), which is required for NK cell development. Autophagy defects result in damaged mitochondria and accumulation of reactive oxygen species (ROS) that leads to apoptosis of NK cells. Autophagy protects NK cell viability during development through removal of damaged mitochondria and intracellular ROS. Phosphorylated Forkhead box O (FoxO)1 is located to the cytoplasm of iNKs and interacts with Atg7, leading to induction of autophagy. FoxO1 deficiency or an inactive FoxO1(AAA) mutant abrogates autophagy initiation in iNKs and impairs NK cell development and viral clearance. Therefore we conclude that FoxO1-mediated autophagy is required for NK cell development and NK cell-induced innate immunity.

Agonist antibody that induces human malignant cells to kill one another.

An attractive, but as yet generally unrealized, approach to cancer therapy concerns discovering agents that change the state of differentiation of the cancer cells. Recently, we discovered a phenomenon that we call "receptor pleiotropism" in which agonist antibodies against known receptors induce cell fates that are very different from those induced by the natural agonist to the same receptor. Here, we show that one can take advantage of this phenomenon to convert acute myeloblastic leukemic cells into natural killer cells. Upon induction with the antibody, these leukemic cells enter into a differentiation cascade in which as many as 80% of the starting leukemic cells can be differentiated. The antibody-induced killer cells make large amounts of perforin, IFN-γ, and granzyme B and attack and kill other members of the leukemic cell population. Importantly, induction of killer cells is confined to transformed cells, in that normal bone marrow cells are not induced to form killer cells. Thus, it seems possible to use agonist antibodies to change the differentiation state of cancer cells into those that attack and kill other members of the malignant clone from which they originate.

Ultrastructure alteration of decidual natural killer cells in women with unexplained recurrent miscarriage: a possible association with impaired decidual vascular remodelling.

This study aimed to evaluate the extent of remodelling of intra-decidual segments of the spiral arteries in human deciduas between the 6th and 10th gestational weeks in women with unexplained recurrent miscarriages (RM) in comparison to gestational-matched controls. A possible association with the number, immunoexpressive behaviour and ultrastructural changes of decidual natural killer cells (dNKCs) was investigated. Decidual biopsies were obtained from RM cases (n = 40) and women with no history of spontaneous miscarriage and at least one live birth at term (n = 30). Staining was performed using PAS, anti-CD34 and anti-CD56 antibodies, using an avidin-biotin-peroxides technique. Analysis by means of light and transmission electron microscopy was employed. To determine the extent of remodelling of decidual vessels, a quantitative score was analysed using histological criteria of vascular transformation and then related to the number of CD56(+) dNKCs. In RM, dNKCs were distributed among decidual cells and around the vessels. They possessed numerous polyploidic protrusions on cell membranes crossing from one cell to another. The cells became more irregular and exhibited heterogeneous electron-dense granules in their cytoplasm compared to controls. The non-remodelling score and number of dNKCs were significantly increased in RM group (p < 0.001). The number of dNKCs was significantly correlated with the scores in both control (r = 0.491; p = 0.006) and RM (r = 0.852; p < 0.001) groups. It appears that dNKCs play a key role in impaired decidual artery remodelling that may be involved with early RM. This may be due to increased numbers of cells or impaired cellular interactions resulting from alterations to the ultrastructure.

NK cells kill mycobacteria directly by releasing perforin and granulysin.

Although the mechanisms underlying the cytotoxic effect of NK cells on tumor cells and intracellular bacteria have been studied extensively, it remains unclear how these cells kill extracellular bacterial pathogens. In this study, we examine how human NK cells kill Mycobacterium kansasii and M.tb. The underlying mechanism is contact dependent and requires two cytolytic proteins: perforin and granulysin. Mycobacteria induce enhanced expression of the cytolytic proteins via activation of the NKG2D/NCR cell-surface receptors and intracellular signaling pathways involving ERK, JNK, and p38 MAPKs. These results suggest that NK cells use similar cellular mechanisms to kill both bacterial pathogens and target host cells. This report reveals a novel role for NK cells, perforin, and granulysin in killing mycobacteria and highlights a potential alternative defense mechanism that the immune system can use against mycobacterial infection.

Visualization of the immunological synapse by dual color time-gated stimulated emission depletion (STED) nanoscopy.

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in <80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Gap junction intercellular communications regulate NK cell activation and modulate NK cytotoxic capacity.

Gap junctions (GJs) mediate intercellular communication between adjacent cells. Previously, we showed that connexin 43 (Cx43), the main GJ protein in the immune system, mediates Ag transfer between human dendritic cells (DCs) and is recruited to the immunological synapse during T cell priming. This crosstalk contributed to T cell activation, intracellular Ca(2+) responses, and cytokine release. However, the role of GJs in NK cell activation by DCs and NK cell-mediated cytotoxicity against tumor cells remains unknown. In this study, we found polarization of Cx43 at the NK/DC and NK/tumor cell-contact sites, accompanied by the formation of functional GJs between NK/DCs and NK/tumor cells, respectively. Cx43-GJ-mediated intercellular communication (GJIC) between human NK and DCs was bidirectional. Blockage of Cx43-GJIC inhibited NK cell activation, though it affected neither the phenotype nor the function of DCs. Cx43 knockdown or inhibition using mimetic peptides greatly reduced CD69 and CD25 expression and IFN-γ release by DC-stimulated NK cells. Moreover, blocking Cx43 strongly inhibited the NK cell-mediated tumor cell lysis associated with inhibition of granzyme B activity and Ca(2+) influx. Our data identify a novel and active role for Cx43-GJIC in human NK cell activation and antitumor effector functions that may be important for the design of new immune therapeutic strategies.

Rapid reuptake of granzyme B leads to emperitosis: an apoptotic cell-in-cell death of immune killer cells inside tumor cells.

A cell-in-cell process refers to the invasion of one living cell into another homotypic or heterotypic cell. Different from non-apoptotic death processes of internalized cells termed entosis or cannibalism, we previously reported an apoptotic cell-in-cell death occurring during heterotypic cell-in-cell formation. In this study, we further demonstrated that the apoptotic cell-in-cell death occurred only in internalized immune killer cells expressing granzyme B (GzmB). Vacuole wrapping around the internalized cells inside the target cells was the common hallmark during the early stage of all cell-in-cell processes, which resulted in the accumulation of reactive oxygen species and subsequent mitochondrial injury of encapsulated killer or non-cytotoxic immune cells. However, internalized killer cells mediated rapid bubbling of the vacuoles with the subsequent degranulation of GzmB inside the vacuole of the target cells and underwent the reuptake of GzmB by killer cells themselves. The confinement of GzmB inside the vacuole surpassed the lysosome-mediated cell death occurring in heterotypic or homotypic entosis processes, resulting in a GzmB-triggered caspase-dependent apoptotic cell-in-cell death of internalized killer cells. On the contrary, internalized killer cells from GzmB-deficient mice underwent a typical non-apoptotic entotic cell-in-cell death similar to that of non-cytotoxic immune cells or tumor cells. Our results thus demonstrated the critical involvement of immune cells with cytotoxic property in apoptotic cell-in-cell death, which we termed as emperitosis taken from emperipolesis and apoptosis. Whereas entosis or cannibalism may serve as a feed-on mechanism to exacerbate and nourish tumor cells, emperitosis of immune killer cells inside tumor cells may serve as an in-cell danger sensation model to prevent the killing of target cells from inside, implying a unique mechanism for tumor cells to escape from immune surveillance.

Arf-like GTPase Arl8b regulates lytic granule polarization and natural killer cell-mediated cytotoxicity.

Natural killer (NK) lymphocytes contain lysosome-related organelles (LROs), known as lytic granules, which upon formation of immune synapse with the target cell, polarize toward the immune synapse to deliver their contents to the target cell membrane. Here, we identify a small GTP-binding protein, ADP-ribosylation factor-like 8b (Arl8b), as a critical factor required for NK cell-mediated cytotoxicity. Our findings indicate that Arl8b drives the polarization of lytic granules and microtubule-organizing centers (MTOCs) toward the immune synapse between effector NK lymphocytes and target cells. Using a glutathione S-transferase pull-down approach, we identify kinesin family member 5B (KIF5B; the heavy chain of kinesin-1) as an interaction partner of Arl8b from NK cell lysates. Previous studies showed that interaction between kinesin-1 and Arl8b is mediated by SifA and kinesin-interacting protein (SKIP) and the tripartite complex drives the anterograde movement of lysosomes. Silencing of both KIF5B and SKIP in NK cells, similar to Arl8b, led to failure of MTOC-lytic granule polarization to the immune synapse, suggesting that Arl8b and kinesin-1 together control this critical step in NK cell cytotoxicity.

Effects of formaldehyde exposure on human NK cells in vitro.

Natural killer (NK) cells play a pivotal role in human immunologic surveillance. Formaldehyde (FA), a ubiquitous environmental contaminant, has been classified as a carcinogen to humans. Although it is known that immune cells are sensitive to FA, so far little is known about how it's affecting the activity of human NK cells. To probe it, the primary human NK cells were treated with different concentrations of FA (3200, 1600, 800, 400, 200, 100, 50, and 0 µM) in vitro. The morphology, viability, apoptosis, cytotoxicity (killing tumor cell activity) and cytokine and cytolytic proteins secretion of NK cells were evaluated respectively. Our results reveal that FA could induce NK cells death obviously in a concentration-dependent manner. With the decreased concentrations of FA from 3200 µM to 800 µM, accordingly, the viability of NK cells increased from 65. 2 ± 12.1% to 78.48 ± 10.3% (p<0.05), and the cytotoxicity of NK cells recovered from 29.2 ± 8.5% to 63.9 ± 5.9% (p<0.05). The secretion of perforin was affected significantly by FA, whereas the secretion of IFN-γ and granzyme-B altered slightly. It is concluded that human NK cell is sensitive to FA, 800 µM may be a critical concentration of FA inhibiting the activity of human NK cell.

Membrane-type 6 matrix metalloproteinase regulates the activation-induced downmodulation of CD16 in human primary NK cells.

CD16 (FcγRIIIa), the low-affinity receptor for IgG, expressed by the majority of human NK cells, is a potent activating receptor that facilitates Ab-dependent cell-mediated cytotoxicity (ADCC). ADCC dysfunction has been linked to cancer progression and poor prognosis for chronic infections, such as HIV; thus, understanding how CD16 expression is regulated by NK cells has clinical relevance. Importantly, CD16 cell-surface expression is downmodulated following NK cell activation and, in particular, exposure to stimulatory cytokines (IL-2 or IL-15), likely owing to the action of matrix metalloproteinases (MMPs). In this article, we identify membrane-type 6 (MT6) MMP (also known as MMP25) as a proteinase responsible for CD16 downmodulation. IL-2-induced upregulation of MT6/MMP25 cell-surface expression correlates with CD16 downmodulation. MT6/MMP25, sequestered in intracellular compartments in unstimulated NK cells, translocates to the cell surface after stimulation; moreover, it polarizes to the effector-target cell interface of the CD16-mediated immunological synapse. siRNA-mediated disruption of MT6/MMP25 expression enhances the ADCC capacity of NK cells, emphasizing the important functional role of MT6/MMP25 in the regulation of ADCC activity. Thus, this study uncovers a previously unknown role of MT6/MMP25 in human NK cells, and suggests that inhibition of MT6/MMP25 activity could improve ADCC efficacy of therapeutically administered NK cells that require IL-2 for culture and expansion.

Natural killer cells: frequency, phenotype and function in healthy cats.

Natural killer (NK) cells play a central role in innate immunity and have been shown to influence adaptive immune responses as well. This study aimed to provide a general NK cell quantification and phenotyping in several compartments of healthy cats and assess their functional properties. The results indicated that NK numbers, both absolute and relative, and phenotype mostly correspond with those found in bovine, ovine, human and murine immunology. However, there were also distinct differences, especially with regard to the expression of the integrin CD11b and the selectin CD62L (between 10 and 30% of feline NK cells stain positive for these markers) and the relative frequencies in lymph nodes (6.7%), which stand central in NK cell development. Caution should be taken when extrapolating findings on NK cell properties over species, notwithstanding the generally accepted evolutionary conservation of NK cells and their subtypes. It was also shown that K562 cells, the 'golden' target cell line for NK functionality tests did not work for feline cells. The feline kidney cell line CRFK proved to be very responsive to NK- and NKT-mediated lysis and therefore, represents an ideal alternative target. This study is a good reference for NK cell numbers, both absolute and relative, phenotype and function in several anatomical compartments of healthy cats and for cat-specific cytotoxic assays involving both NK and NKT cells.

Functional distinction of rat liver natural killer cells from spleen natural killer cells under normal and acidic conditions in vitro.

BACKGROUND: The microenvironment within solid tumors has often been shown to exhibit an acidic extracellular pH. Although the morphologic and functional differences in natural killer (NK) cells of the liver and spleen have been reported previously under physiological conditions, the difference under acidic conditions is still unclear. This study was to investigate the differences in the morphological and functional characteristics between rat liver and spleen NK cells under normal and acidic conditions in vitro. METHODS: Liver and spleen NK cells were isolated and purified from Sprague-Dawley rats by density gradient centrifugation and the Dynabeads(®) FlowComp(TM) Flexi system, and stimulated for 4 days with or without IL-2 or treated with low pH or control for different times. Morphology was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cell death and proliferation assays were performed by flow cytometry, IFN-gamma production was tested by ELISA, and cytotoxic activity was evaluated by lactate dehydrogenase (LDH) release assay. RESULTS: Liver NK cells had significantly higher levels of cytotoxic activity than spleen NK cells under normal and acidic conditions, and the maximum difference was observed at pH 5.6. Further analysis revealed that the cytotoxic activity of NK cells was correlated with morphology, cell death, proliferative activity and IFN-gamma production. By TEM, liver NK cells contained a greater number of electron-dense granules per cell at pH 5.6. Moreover, a modest elevation of cell death and reduction of proliferation of liver NK cells occurred within a range of 5.6-7.2. Interestingly, an acidic extracellular pH only marginally, and not significantly, suppressed IFN-gamma production by liver NK cells. CONCLUSION: The sharp morphological and functional differences shown by the two types of NK cells in vitro indicate that liver NK cells are unexpectedly resistant to pH shock.

HLA-G inhibition of NK-cell cytolytic function is uncoupled from tumor cell lipid raft reorganization.

HLA-G is a non-classical HLA class I molecule with tolerogenic properties and restricted tissue distribution. The expression of HLA-G can be induced by tumors thus providing an efficient way to escape the anti-tumoral immune response. Although lipid rafts regulate diverse immunological mechanisms their relationship with HLA-G remains controversial. Our results show that HLA-G-mediated inhibition of both the interaction between NK and tumor cells, and of intracellular calcium flux in NK cells conjugated to their target cells were independent of lipid raft integrity. In addition, cytotoxicity assays indicated that HLA-G continued to efficiently inhibit NK-cell cytolytic function in several different tumor cells independently of lipid raft integrity. Confocal microscopy with 3D reconstruction combined with biochemical analysis showed that HLA-G was mainly localized outside the lipid rafts of tumor cells after cross-linking with specific antibody and remained excluded from lipid rafts during interaction with the ILT2 inhibitory receptor of NK cells. This study indicates that the inhibitory function of HLA-G is uncoupled from lipid raft organization, further distinguishing HLA-G from classical HLA molecules and providing novel information in the understanding of tumor immune escape mechanism mediated through HLA-G.

IQGAP1 involvement in MTOC and granule polarization in NK-cell cytotoxicity.

Natural killer (NK) cells form a region of tight contact called the NK immunological synapse (NKIS) with their target cells. This is a dynamic region serving as a platform for targeted signaling and exocytotic events. We previously identified IQGAP1 as a cytoskeletal component of the NK-like cell line YTS. The present study was undertaken to determine the role of IQGAP1 in the function of NK cells. Silencing of IQGAP1 expression resulted in almost complete loss of the cytotoxic activity of YTS cells. Loss of IQGAP1 did not prevent conjugate formation with target cells but it did result in a failure to reorient the microtubule organizing centre to the immune synapse. Significantly, IQGAP1 expression was required for the perigranular accumulation of an F-actin network. IQGAP1 was shown to undergo marked rearrangements during synapse maturation in effector target conjugates of YTS or primary NK cells. These results suggest previously undescribed role(s) for IQGAP1 in regulating multiple aspects of cytoskeletal organization and granule polarization in NK cells.