Live cell imaging of highly activated natural killer cells against human hepatocellular carcinoma in vivo

Background aimsTracking administered natural killer (NK) cells in vivo is critical for developing an effective NK cell-based immunotherapy against human hepatocellular carcinoma (HCC). Here the authors established a new molecular imaging using ex vivo-activated NK cells and investigated real-time biodistribution of administered NK cells during HCC progression.MethodsEx vivo-expanded NK cells from healthy donors were labeled with a near-infrared lipophilic cytoplasmic dye, and their proliferation, surface receptor expression and cytotoxicity activity were evaluated. Human HCC HepG2 cells were implanted into the livers of NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ (NSG) mice. The authors administered 1,1’-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-labeled NK cells intravenously to non-tumor-bearing and intrahepatic HCC tumor-bearing NSG mice. Fluorescent imaging was performed using a fluorescence-labeled organism bioimaging instrument. Single cell suspensions from the resected organs were analyzed using flow cytometry.ResultsThe fluorescent DiR dye was nontoxic and did not affect the proliferation or surface receptor expression levels of the NK cells, even at high doses. The administered DiR-labeled NK cells immediately migrated to the lungs of the non-tumor-bearing NSG mice, with increased NK cell signals evident in the liver and spleen after 4 h. NK cells migrated to the intrahepatic tumor-bearing livers of both early- and late-stage HCC mice within 1 h of injection. In early-stage intrahepatic tumor-bearing mice, the fluorescence signal increased in the liver until 48 h post-injection and decreased 7 days after NK injection. In late-stage HCC, the NK cell fluorescence signal was the highest in the liver for 7 days after NK injection and persisted for 14 days. The purity of long-term persistent CD45⁺CD56⁺CD3⁻ NK cells was highest in early- and late-stage HepG2-bearing liver compared with normal liver 2 weeks after NK injection, whereas highest purity was still observed in the lungs of non-tumor-bearing mice. In addition, Ki-67 expression was detected in migrated human NK cells in the liver and lung up to 72 h after administration. With HepG2 tumor progression, NK cells reduced the expression of NKp30 and NKG2D.ConclusionsAdministered NK cells were successfully tracked in vivo by labeling the NK cells with near-infrared DiR dye. Highly expanded, activated NK cells migrated rapidly to the tumor-bearing liver, where they persisted for 14 days after administration, with high purity of CD45⁺CD56⁺CD3⁻ NK cells. Liver biodistribution and persistence of administered NK cells showed significantly different accumulation patterns during HCC progression.     

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

Background aimsMultipotent 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.MethodsWe 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.ResultsFetal 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.ConclusionsFetal 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.     

Treatment with 4-Methylpyrazole Modulated Stellate Cells and Natural Killer Cells and Ameliorated Liver Fibrosis in Mice

MethodsLiver fibrosis was induced by intraperitoneal injections of carbon tetrachloride (CCl₄) or bile duct ligation (BDL) for two weeks. To inhibit ADH3-mediated retinol metabolism, 10 μg 4-methylpyrazole (4-MP)/g of body weight was administered to mice treated with CCl₄ or subjected to BDL. The mice were sacrificed at week 2 to evaluate the regression of liver fibrosis. Liver sections were stained for collagen and α-smooth muscle actin (α-SMA). In addition, HSCs and NK cells were isolated from control and treated mice livers for molecular and immunological studies.ResultsTreatment with 4-MP attenuated CCl₄- and BDL-induced liver fibrosis in mice, without any adverse effects. HSCs from 4-MP treated mice depicted decreased levels of retinoic acids and increased retinol content than HSCs from control mice. In addition, the expression of α-SMA, transforming growth factor-β1 (TGF-β1), and type I collagen α1 was significantly reduced in the HSCs of 4-MP treated mice compared to the HSCs from control mice. Furthermore, inhibition of retinol metabolism by 4-MP increased interferon-γ production in NK cells, resulting in increased apoptosis of activated HSCs.ConclusionsBased on our data, we conclude that inhibition of retinol metabolism by 4-MP ameliorates liver fibrosis in mice through activation of NK cells and suppression of HSCs. Therefore, retinol and its metabolizing enzyme, ADH3, might be potential targets for therapeutic intervention of liver fibrosis.     

Miltefosine enhances infectivity of a miltefosine-resistant Leishmania infantum strain by attenuating its innate immune recognition

BackgroundMiltefosine (MIL) is currently the only oral drug available to treat visceral leishmaniasis but its use as first-line monotherapy has been compromised by an increasing treatment failure. Despite the scarce number of resistant clinical isolates, MIL-resistance by mutations in a single aminophospholipid transporter gene can easily be selected in a laboratory environment. These mutations result in a reduced survival in the mammalian host, which can partially be restored by exposure to MIL, suggesting a kind of drug-dependency.Methodology/Principal findingsTo enable a combined study of the infection dynamics and underlying immunological events for differential in vivo survival, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of MIL-resistant (MIL-R) and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results in C57Bl/6 and BALB/c mice show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites. In addition, we demonstrated that MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production.Conclusions/SignificanceDifferential induction of innate immune responses in the liver was found to underlie the attenuated phenotype of a MIL-R parasite and its peculiar feature of drug-dependency. The impact of MIL on hepatic NK and NKT activation and IFN-γ production following recognition of a MIL-R strain indicates that this mechanism may sustain infections with resistant parasites and contribute to treatment failure.     

Differential effects of mycophenolate mofetil and cyclosporine A on peripheral blood and cord blood natural killer cells activated with interleukin-2

Background aimsGraft-versus-host disease remains a major cause of death after hematopoietic stem cell transplantation. Cyclosporine (CsA) and mycophenolate mofetil (MMF) have been successfully used alone or in combination as prophylaxis for graft-versus-host disease. Although the effects of these drugs on T cells have been studied, little is known about the effects of both drugs on natural killer (NK) cells. We examined if the sensitivity of umbilical cord blood (CB) NK cells to MMF and/or CsA differs from their adult counterparts.MethodsAn approach that was based on flow cytometry and real-time polymerase chain reaction was used to assess the effects of MMF, CsA and the combination of both drugs on the viability, activation, proliferation and cytotoxicity of peripheral blood (PB) and CB NK cells after culture with interleukin-2.ResultsMMF alone or together with CsA induced cell death of CB NK cells but not of PB NK cells. MMF and CsA had differential effects on NK cell activation but significantly reduced proliferation of CB NK cells. MMF reduced perforin expression by PB NK cells, whereas CsA alone or together with MMF drastically decreased degranulation of CB and PB NK cells. However, neither affected cytokine secretion by PB and CB NK cells.ConclusionsThis study showed that CB NK cells were more sensitive to MMF and CsA than were PB NK cells. MMF and CsA had significant effects on NK cells that could jeopardize the beneficial effects of NK cells after hematopoietic stem cell transplantation.     

Analysis of ex vivo expanded and activated clinical-grade human NK cells after cryopreservation

Background aimsSeveral methods to expand and activate (EA) NK cells ex vivo have been developed for the treatment of relapsed or refractory cancers. Infusion of fresh NK cells is generally preferred to the infusion of cryopreserved/thawed (C/T) NK cells because of concern that cryopreservation diminishes NK cell activity. However, there has been little head-to-head comparison of the functionality of fresh versus C/T NK cell products.MethodsWe evaluated activity of fresh and C/T EA NK cells generated by interleukin (IL)-15, IL-2 and CD137L expansion.ResultsAnalysis of C/T NK cell products demonstrated decreased recovery of viable CD56⁺ cells, but the proportion of NK cells in the C/T EA NK cell product did not decrease compared with the fresh EA NK cell product. Fresh and C/T EA NK cells demonstrated increased granzyme B compared with NK cells pre-expansion, but only fresh EA NK cells showed increased NKG2D. Compared with fresh EA NK cells, cytotoxic ability of C/T EA NK cells was reduced, but C/T EA NK cells remained potently cytotoxic against tumor cells via both antibody-independent and antibody-dependent mechanisms within 4 h post-thaw. Fresh EA NK cells generated high levels of gamma interferon (IFN-γ), which was abrogated by JAK1/JAK2 inhibition with ruxolitinib, but C/T EA NK cells showed lower IFN-γ unaffected by JAK1/JAK2 inhibition.DiscussionUsage of C/T EA NK cells may be an option to provide serial “boost” NK cell infusions from a single apheresis to maximize NK cell persistence and potentially improve NK-induced responses to refractory cancer.     

Intraperitoneal delivery of human natural killer cells for treatment of ovarian cancer in a mouse xenograft model

Background aimsThere is an urgent need for novel therapeutic strategies for relapsed ovarian cancer. Dramatic clinical anti-tumor effects have been observed with interleukin (IL)-2 activated natural killer (NK) cells; however, intravenous delivery of NK cells in patients with ovarian cancer has not been successful in ameliorating disease. We investigated in vivo engraftment of intraperitoneally (IP) delivered NK cells in an ovarian cancer xenograft model to determine if delivery mode can affect tumor cell killing and circumvent lack of NK cell expansion.MethodsAn ovarian cancer xenograft mouse model was established to evaluate efficacy of IP-delivered NK cells. Tumor burden was monitored by bioluminescent imaging of luciferase-expressing ovarian cancer cells. NK cell persistence, tumor burden and NK cell trafficking were evaluated. Transplanted NK cells were evaluated by flow cytometry and cytotoxicity assays.ResultsIP delivery of human NK cells plus cytokines led to high levels of circulating NK and was effective in clearing intraperitoneal ovarian cancer burden in xenografted mice. NK cells remained within the peritoneal cavity 54 days after injection and had markers of maturation. Additionally, surviving NK cells were able to kill ovarian cancer cells at a rate similar to pre-infusion levels, supporting that in vivo functionality of human NK cells can be maintained after IP infusion.ConclusionsIP delivery of NK cells leads to stable engraftment and antitumor response in an ovarian cancer xenograft model. These data support further pre-clinical and clinical evaluation of IP delivery of allogeneic NK cells in ovarian cancer.     

Natural killer cells have a synergistic anti-tumor effect in combination with chemoradiotherapy against head and neck cancer

BackgroundThe use of natural killer (NK) cells is a promising approach in the field of cancer immunotherapy; however, combination treatments are required to enhance the effects of NK cell immunotherapy. In this study, we assessed the potential of irradiation and cisplatin as a chemoradiotherapy (CRT) regimen to augment the effects of NK cell immunotherapy in head and neck squamous cell carcinoma (HNSCC).MethodsNK cells were expanded using our recently established K562-OX40 ligand and membrane-bound interleukin (IL)-18 and IL-21 feeder cells in the presence of IL-2/IL-15 from peripheral blood of healthy donors.ResultsThe results showed an increase in the purity of NK cells and expression of activation markers such as NKG2D and lymphocyte function–associated antigen 1 during the expansion process, which is positively correlated to the NK cell infiltration and overall survival in patients with HNSCC. CRT induced NK cell activation ligand (ULBP2) and adhesion molecules (ICAM-1, -2 and -3) on HNSCC, leading to enhanced cytotoxicity of NK cells against HNSCC.ConclusionsOur findings suggest that the NK cells have a potent anti-tumor effect in combination with CRT against HNSCC.     

Clinical-grade,large-scale,feeder-free expansion of highly active human natural killer cells for adoptive immunotherapy using an automated bioreactor

Background aimsNatural killer (NK) cell-based adoptive immunotherapy is a promising approach for the treatment of cancer. Ex vivo expansion and activation of NK cells under good manufacturing practice (GMP) conditions are crucial for facilitating large clinical trials. The goal of this study was to optimize a large-scale, feeder-free, closed system for efficient NK cell expansion.MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors and myeloma patients were cultured for 21 days using flasks, cell culture bags and bioreactors. Final products from different expansions were evaluated comparatively for phenotype and functionality.ResultsSignificant NK cell expansions were obtained in all systems. The bioreactor yielded a final product rich in NK cells (mean 38%) ensuring that a clinically relevant cell dose was reached (mean 9.8 × 10⁹ NK cells). Moreover, we observed that NK cells expanded in the bioreactor displayed significantly higher cytotoxic capacity. It was possible to attribute this partially to a higher expression level of NKp44 compared with NK cells expanded in flasks.ConclusionsThese results demonstrate that large amounts of highly active NK cells for adoptive immunotherapy can be produced in a closed, automated, large-scale bioreactor under feeder-free current GMP conditions, facilitating clinical trials for the use of these cells.     

Fc-engineered anti-CD33 monoclonal antibody potentiates cytotoxicity of membrane-bound interleukin-21 expanded natural killer cells in acute myeloid leukemia

BackgroundQualitative and quantitative defects in natural killer (NK) cells have been noted in patients with acute myeloid leukemia (AML), providing rationale for infusion of donor-derived NK cells. We previously showed that decitabine enhances expression of NKG2D ligands in AML with additive cytotoxicity when NK cells and Fc (fragment crystallizable region)-engineered CD33 monoclonal antibody (CD33mAb) was used. We conducted a phase 1 study evaluating decitabine and haploidentical NK cells in relapsed AML. Using patient samples from this study, we evaluated whether ex vivo donor-derived expanded NK cells with or without CD33mAb was effective in decitabine-treated AML.MethodsBone marrow aspirates were collected from patients at pre- and post-NK cell infusion. NK cells from healthy donors were expanded for 14 days using irradiated K562 feeder cells displaying membrane-bound IL-21 (mbIL-21). Patient samples were used to test in vitro activity of mbIL-21 NK cells ± CD33m Ab-dependent cellular cytotoxicity (ADCC) and AML patient derived xenograft (PDX) mice were developed to test in vivo activity.ResultsUpon incubation with primary AML blasts, mbIL-21 NK cells showed variable donor-dependent intra-cellular interferon-γ production, which increased with CD33mAb-coated AML. ADCC assays revealed mbIL-21 NK cells effectively lysed primary AML blasts with higher activity on CD33mAb-coated AML. Importantly, CD33mAb-dependent enhanced cytotoxicity by mbIL-21 NK cells was maintained in AML cells from patients even 24 days post-decitabine treatment. In vivo infusion of mbIL-21 NK cells in AML PDX mice, treated with CD33mAb, reduced the tumor burden.DiscussionThese data show the therapeutic utility of mbIL-21 NK cells that can be further potentiated by addition of CD33mAb in AML.     

Concurrent transposon engineering and CRISPR/Cas9 genome editing of primary CLL-1 chimeric antigen receptor–natural killer cells

BackgroundNatural killer (NK) cell genome editing promises to enhance the innate and alloreactive anti-tumor potential of NK cell adoptive transfer. DNA transposons are versatile non-viral gene vectors now being adapted to primary NK cells, representing important tools for research and clinical product development.Aims and MethodsWe set out to generate donor-derived, primary chimeric antigen receptor (CAR)-NK cells by combining the TcBuster transposon system with Epstein–Barr virus–transformed lymphoblastoid feeder cell-mediated activation and expansion.ResultsThis approach allowed for clinically relevant NK-cell expansion capability and CAR expression, which was further enhanced by immunomagnetic selection based on binding to the CAR target protein.The resulting CAR-NK cells targeting the myeloid associated antigen CLL-1 efficiently targeted CLL-1–positive AML cell lines and primary AML populations, including a population enriched for leukemia stem cells. Subsequently, concurrent delivery of CRISPR/Cas9 cargo was applied to knockout the NK cell cytokine checkpoint cytokine-inducible SH2-containing protein (CIS, product of the CISH gene), resulting in enhanced cytotoxicity and an altered NK cell phenotype.ConclusionsThis report contributes a promising application of transposon engineering to donor-derived NK cells and emphasizes the importance of feeder mediated NK cell activation and expansion to current protocols.     

Chronic HCV Infection Affects the NK Cell Phenotype in the Blood More than in the Liver

Although epidemiological and functional studies have implicated NK cells in protection and early clearance of HCV, the mechanism by which they may contribute to viral control is poorly understood, particularly at the site of infection, the liver. We hypothesized that a unique immunophenotypic/functional NK cell signature exists in the liver that may provide insights into the contribution of NK cells to viral control. Intrahepatic and blood NK cells were profiled from chronically infected HCV-positive and HCV-negative individuals. Baseline expression of activating and inhibitory receptors was assessed, as well as functional responses following stimulation through classic NK cell pathways. Independent of HCV infection, the liver was enriched for the immunoregulatory CD56^bright NK cell population, which produced less IFNγ and CD107a but comparable levels of MIP1β, and was immunophenotypically distinct from their blood counterparts. This profile was mostly unaltered in chronic HCV infection, though different expression levels of NKp46 and NKG2D were associated with different grades of fibrosis. In contrast to the liver, chronic HCV infection associated with an enrichment of CD161^lowperforin^high NK cells in the blood correlated with increased AST and 2B4 expression. However, the association of relatively discrete changes in the NK cell phenotype in the liver with the fibrosis stage nevertheless suggests an important role for the NK response. Overall these data suggest that tissue localization has a more pervasive effect on NK cells than the presence of chronic viral infection, during which these cells might be mostly attuned to limiting immunopathology. It will be important to characterize NK cells during early HCV infection, when they should have a critical role in limiting infection.     

Natural Killer Cell Receptors and Cytotoxic Activity in Phosphomannomutase 2 Deficiency (PMM2-CDG)

BackgroundPMM2-CDG is the most common N-glycosylation defect and shows an increased risk of recurrent and/or severe, sometimes fatal, infections in early life. We hypothesized that natural killer (NK) cells, as important mediators of the immune response against microbial pathogens and regulators of adaptive immunity, might be affected in this genetic disorder.ObjectiveTo evaluate possible defects on PMM2-CDG NK peripheral blood cell number, killing activity and expression of membrane receptors.MethodsWe studied fresh and activated NK cells from twelve PMM2-CDG cells. The number and expression of lymphoid surface receptors were studied by flow cytometry. The NK responsiveness (frequency of degranulated NK cells) and killing activity against K562 target cells was determined in the NK cytotoxicity assay.ResultsWe found an increase of blood NK cells in three patients with a severe phenotype. Two of them, who had suffered from moderate/severe viral infections during their first year of life, also had reduced T lymphocyte numbers. Patient activated NK cells showed increased expression of CD54 adhesion molecule and NKG2D and NKp46 activating receptors. NKp46 and 2B4 expression was inversely correlated with the expression of NKG2D in activated PMM2-CDG cells. Maximal NK activity against K562 target cells was similar in control and PMM2-CDG cells. Interestingly, the NK cell responsiveness was higher in patient cells. NKG2D and specially CD54 increased surface expression significantly correlated with the increased NK cell cytolytic activity according to the modulation of the killer activity by expression of triggering receptors and adhesion molecules.ConclusionsOur results indicate that hypoglycosylation in PMM2-CDG altered NK cell reactivity against target cells and the expression of CD54 and NKG2D, NKp46 and 2B4 activating receptors during NK cell activation. This suggests a defective control of NK cell killing activity and the overall anti-viral immune response in PMM2-CDG patients. The present work improves our understanding of the immunological functions in PMM2-CDG and possibly in other CDG-I types.     

Migration of iron-labeled KHYG-1 natural killer cells to subcutaneous tumors in nude mice, as detected by magnetic resonance imaging

Background aimsA novel cell line of cytotoxic natural killer (NK) cells, KHYG-1, was examined in vivo for immunotherapy against prostate cancer. The feasibility of using magnetic resonance imaging (MRI) tracking to monitor the fate of injected NK cells following intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c.) administration was assessed.MethodsPC-3M human prostate cancer cells were injected s.c. into the flank of nude mice (day 0). KHYG-1 NK cells were labeled with an iron oxide contrast agent and injected s.c., i.v. or i.p. on day 8. Mice were imaged by MRI on days 7, 9 and 12. Tumor sections were examined with fluorescence microscopy and immunohistologic staining for NK cells.ResultsNK cells were detected in the tumors by histology after all three administration routes. NK cells and fluorescence from the iron label were co-localized. Signal loss was seen in the areas around the tumors and between the tumor lobes in the s.c. group.ConclusionsWe are the first to label this cell line of NK cells with an iron oxide contrast agent. Accumulation of NK cells was visualized by MRI after s.c. injection but not after i.v. and i.p. injection.     

Effect of exposure to interleukin-21 at various time points on human natural killer cell culture

Background aimsInterleukin-21 (IL-21) can enhance the effector function of natural killer (NK) cells but also limits their proliferation when continuously combined with IL-2/IL-15. Paradoxically, membrane-bound (mb)-IL-21 has been shown to improve human NK cell proliferation when cultured with IL-2/mb-IL-15. To clarify the role of IL-21, we investigated the effect of the timing of IL-21 addition to NK cell culture.MethodsIL-2/IL-15–activated NK cells were additionally treated with IL-21 according to the following schedules; (i) control (without IL-21); (ii) first week (day 0 to day 7); (iii) intermittent (the first 3 days of each week for 7 weeks); (iv) after 1 week (day 8 to day 14); and (v) continuous (day 0 to day 49). The expression of NK receptors, granzyme B, perforin, CD107a, interferon-γ, telomere length and NK cell death were measured by flow cytometry.ResultsCompared with the control (2004.2-fold; n = 10 healthy donors) and intermittent groups (2063.9-fold), a strong proliferative response of the NK cells on day 42 was identified in the “first week” group (3743.8-fold) (P < 0.05). NK cells treated with IL-21 in the “first week” group showed cytotoxicity similar to that in control cells. On day 28, there was a significant increase in cytotoxicity of “first week” NK cells that received IL-21 treatment for an additional 2 days compared with the “first week” NK cells (P < 0.05).ConclusionsThese data suggest that controlling temporal exposure of IL-21 during NK cell proliferation can be a critical consideration to improve the yields and cytotoxicity of NK cells.     

Age-related variation in the proportion and activity of murine liver natural killer cells and their cytotoxicity against regenerating hepatocytes

We investigated the distribution of liver NK cells in mice of various ages and their cytotoxicity against regenerating hepatocytes. Liver NK cells were identified by asialo GM1 antibody in mononuclear cell suspension from the liver, whereas NK activity was assayed against YAC-1 target cells. Mononuclear cells in the liver consisted of more than 25% NK cells with potent NK activity in C3H/He mice, 8 wk of age. The strain-specific distribution (C3H/He greater than C57BL/6 greater than DBA/2) of liver NK cells was the same as those in the spleen and blood. The proportion of liver NK cells and the level of NK activity in C3H/He mice were further demonstrated to vary depending on age, in that both the proportion and the function were generated at 4 wk of age, reached a maximum between the 6th and 8th wk, and then rapidly decreased around the 9th wk. The appearance of an increased number of NK cells in the liver seemed to coincide with the slowing of the rapid increase of murine liver weight. We then investigated whether liver NK cells mediated their cytotoxicity against regenerating hepatocytes. Both specific 51Cr-release assay and single cell cytotoxicity assay demonstrated that liver NK cells were significantly cytotoxic against regenerating hepatocytes in partially hepatectomized liver, but to a lesser extent against normal hepatocytes in resting liver. Morphologic study revealed that normal liver predominantly consisted of hepatocytes with binuclei (greater than 60%) but that regenerating liver mainly consisted of hepatocytes with a single nucleus (greater than 70%). One-nucleus hepatocytes were more susceptible to the cytotoxicity of liver NK cells. A comparative study of restoration kinetics of the liver weight and the number of liver NK cells after partial hepatectomy also showed a unique relationship. These results raise the possibility that liver NK cells might be responsible for regulating hepatocyte growth.     

Irradiated chimeric antigen receptor engineered NK-92MI cells show effective cytotoxicity against CD19+ malignancy in a mouse model

Background aimsAnti-CD19 chimeric antigen receptor (CAR)-modified T cells have shown dramatic cytotoxicity against B-cell malignancies. Currently, autologous T cells are conventionally used to manufacture CAR T cells. Low quality or insufficient quantity of autologous T cells may lead to failure of CAR T preparations. Moreover, CAR T preparation usually takes 1–2 weeks, which is too long for patients with rapid disease progression to successfully infuse CAR T cells. Thus, the development of a ready-to-use CAR immunotherapy strategy is needed. NK-92, a natural killer (NK) cell line derived from an NK lymphoma patient, has been gradually applied as a CAR-modified effector cell. To avoid the potential development of secondary NK lymphoma in patients, large doses of radiation are used to treat NK-92 cells before clinical application, which ensures the safety but reduces the cytotoxicity of NK-92 cells. Therefore, it is crucial to explore a suitable radiation dose that ensures short life span and good cytotoxicity of CAR NK-92 cells.MethodsNK-92MI, a modified IL-2-independent NK-92 cell line, was used to establish an anti-CD19 CAR NK. The suitable radiation dose of CAR NK was then explored in vitro and validated in vivo, and the specific cytotoxicity of irradiated and unirradiated CAR NK against CD19⁺ malignant cells was assessed.ResultsCAR NK exhibited specific cytotoxicity against CD19⁺ malignant cells. Irradiation ensured a short life span of CAR NK in vitro and in vivo. Encouragingly, irradiated CAR NK displayed an anti-CD19⁺ malignancy capacity similar to that of unirradiated CAR NK.ConclusionsFive Gy is a suitable radiation dose to ensure the safety and effectiveness of CD19 CAR NK-92MI cells.     

Transient blockade of TBK1/IKKε allows efficient transduction of primary human natural killer cells with vesicular stomatitis virus G-pseudotyped lentiviral vectors

Background aimsVesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors (LVs) are widely used to reliably generate genetically modified, clinical-grade T-cell products. However, the results of genetically modifying natural killer (NK) cells with VSV-G LVs have been variable. The authors explored whether inhibition of the IKK-related protein kinases TBK1 and IKKε, key signaling molecules of the endosomal TLR4 pathway, which is activated by VSV-G, would enable the reliable transduction of NK cells by VSV-G LVs.MethodsThe authors activated NK cells from peripheral blood mononuclear cells using standard procedures and transduced them with VSV-G LVs encoding a marker gene (yellow fluorescent protein [YFP]) or functional genes (chimeric antigen receptors [CARs], co-stimulatory molecules) in the presence of three TBK1/IKKε inhibitors (MRT67307, BX-795, amlexanox). NK cell transduction was evaluated by flow cytometry and/or western blot and the functionality of expressed CARs was evaluated in vitro.ResultsBlocking TBK1/IKKε during transduction of NK cells enabled their efficient transduction by VSV-G LVs as judged by YFPexpression of 40–50%, with half maximal effective concentrations of 1.1 µM (MRT67307), 5 µM (BX-795) and 24.8 µM (amlexanox). Focusing on MRT67307, the authors successfully generated NK cells expressing CD19-CARs or HER2-CARs with an inducible co-stimulatory molecule. CAR NK cells exhibited increased cytolytic activity and ability to produce cytokines in comparison to untreated controls, confirming CAR functionality.ConclusionsThe authors demonstrate that inhibition of TBK1/IKKε enables the reliable generation of genetically modified NK cells using VSV-G LVs. The authors’ protocol can be readily adapted to generate clinical-grade NK cells and thus has the potential to facilitate the clinical evaluation of genetically modified NK cell-based therapeutics in the future.     

The cajanine derivative LJ101019C regulates the proliferation and enhances the activity of NK cells via Kv1.3 channel-driven activation of the AKT/mTOR pathway

BackgroundNatural killer (NK) cells play important roles in immune responses and have been wildly used in immunotherapy. Nevertheless, some limitations remain. It is urgent to explore novel and safe strategies to enhance NK cell activity.PurposeThe aim of this study was to investigate the immuno-stimulatory effects and to reveal the molecular mechanism of LJ101019C, a derivative of a natural small-molecule compounds cajanine, on NK cells.MethodsCell proliferation was examined by CCK8 assay, then we used the cytotoxicity detection kit to detect the cytotoxicity of NK cells. The change of cell cycle, intracellular reactive oxygen species (ROS) level and mitochondrial mass were evaluated by FACS and Operetta high-content image analysis, respectively. Furthermore, the IFN-γ secretion of NK cells were measured by ELISA. The Kv1.3 protein expression and function were detected by western blot and patch-clamp technique, respectively. The role of Kv1.3 in AKT/mTOR pathway activation was determined by western blot.ResultsThe results showed that LJ101019C at relatively low concentrations (0.05–0.1 µM) significantly increased the proliferation of NK cells. And 1 µM LJ101019C could elevate the proportion of NK cells in the S-phase of the cell cycle (*p < 0.1). Furthermore, the cytotoxic effects of NK cells targeting MIA PaCa-2 cells were significantly enhanced by 0.1 and 1 µM LJ101019C, and were associated with the enhanced secretion of IFN-γ by NK cells (*p < 0.1; **p < 0.05). 0.1 and 1 µM LJ101019C increased intracellular levels of ROS (**p < 0.05), and 0.1 µM LJ101019C elevated mitochondrial mass (*p < 0.1). Electrophysiological recordings indicated that LJ101019C led to a remarkably increase the Kv1.3 current density. Moreover, western blot results indicated that LJ101019C elevated Kv1.3 protein expression and activated AKT/mTOR signaling via increasing the expression of Kv1.3 in NK cells.ConclusionLJ101019C increases the proliferation and the cytotoxicity of NK cells at relatively low concentrations. The mechanism is the activation of AKT/mTOR signaling pathway driven by up-regulation of Kv1.3 in NK cells. These suggest LJ101019C is a promising candidate for improving the efficacy of NK cell-based immunotherapies.