Isolated Natural Killer Cells Research Articles

Cytokine Response of Natural Killer Cells to Hepatitis B Virus Infection Depends on Monocyte Co-Stimulation.

Hepatitis B virus (HBV) is a major driver of chronic hepatic inflammation, which regularly leads to liver cirrhosis or hepatocellular carcinoma. Immediate innate immune cell response is crucial for the rapid clearance of the infection. Here, natural killer (NK) cells play a pivotal role in direct cytotoxicity and the secretion of antiviral cytokines as well as regulatory function. The aim of this study was to further elucidate NK cell responses triggered by an HBV infection. Therefore, we optimized HBV in vitro models that reliably stimulate NK cells using hepatocyte-like HepG2 cells expressing the Na+-taurocholate co-transporting polypeptide (NTCP) and HepaRG cells. Immune cells were acquired from healthy platelet donors. Initially, HepG2-NTCP cells demonstrated higher viral replication compared to HepaRG cells. Co-cultures with immune cells revealed increased production of interferon-γ and tumor necrosis factor-α by NK cells, which was no longer evident in isolated NK cells. Likewise, the depletion of monocytes and spatial separation from target cells led to the absence of the antiviral cytokine production of NK cells. Eventually, the combined co-culture of isolated NK cells and monocytes led to a sufficient cytokine response of NK cells, which was also apparent when communication between the two immune cell subpopulations was restricted to soluble factors. In summary, our study demonstrates antiviral cytokine production by NK cells in response to HBV+ HepG2-NTCP cells, which is dependent on monocyte bystander activation.

Investigation into the restoration of TRPM3 ion channel activity in post-COVID-19 condition: a potential pharmacotherapeutic target.

Recently, we reported that post COVID-19 condition patients also have Transient Receptor Potential Melastatin 3 (TRPM3) ion channel dysfunction, a potential biomarker reported in natural killer (NK) cells from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) patients. As there is no universal treatment for post COVID-19 condition, knowledge of ME/CFS may provide advances to investigate therapeutic targets. Naltrexone hydrochloride (NTX) has been demonstrated to be beneficial as a pharmacological intervention for ME/CFS patients and experimental investigations have shown NTX restored TRPM3 function in NK cells. This research aimed to: i) validate impaired TRPM3 ion channel function in post COVID-19 condition patients compared with ME/CFS; and ii) investigate NTX effects on TRPM3 ion channel activity in post COVID-19 condition patients. Whole-cell patch-clamp was performed to characterize TRPM3 ion channel activity in freshly isolated NK cells of post COVID-19 condition (N = 9; 40.56 ± 11.26 years), ME/CFS (N = 9; 39.33 ± 9.80 years) and healthy controls (HC) (N = 9; 45.22 ± 9.67 years). NTX effects were assessed on post COVID-19 condition (N = 9; 40.56 ± 11.26 years) and HC (N = 7; 45.43 ± 10.50 years) where NK cells were incubated for 24 hours in two protocols: treated with 200 µM NTX, or non-treated; TRPM3 channel function was assessed with patch-clamp protocol. This investigation confirmed impaired TRPM3 ion channel function in NK cells from post COVID-19 condition and ME/CFS patients. Importantly, PregS-induced TRPM3 currents were significantly restored in NTX-treated NK cells from post COVID-19 condition compared with HC. Furthermore, the sensitivity of NK cells to ononetin was not significantly different between post COVID-19 condition and HC after treatment with NTX. Our findings provide further evidence identifying similarities of TRPM3 ion channel dysfunction between ME/CFS and post COVID-19 condition patients. This study also reports, for the first time, TRPM3 ion channel activity was restored in NK cells isolated from post COVID-19 condition patients after in vitro treatment with NTX. The TRPM3 restoration consequently may re-establish TRPM3-dependent calcium (Ca2+) influx. This investigation proposes NTX as a potential therapeutic intervention and TRPM3 as a treatment biomarker for post COVID-19 condition.

CTLA-4 Blockade of Natural Killer Cells Increases Cytotoxicity against Acute Lymphoid Leukaemia Cells Neda.

There is interest in using cytotoxic T lymphocyte antigen-4 (CTLA-4) immunotherapy to treat blood cancers. Unfortunately, patients with acute lymphoblastic leukaemia (ALL) frequently exhibit resistance to treatment and natural killer (NK) cell exhaustion. This study aims to increase the cytotoxic potency of natural killer cells by using CTLA-4 to block the Nalm-6 leukaemia cell line. In this experimental study, NK cells were purified from the peripheral blood mononuclear cells (PBMCs) of 10 healthy people and assessed by flow cytometry for purity and viability. The purified cells were activated overnight at 37°C and 5% CO2 with interleukin-15 (IL-15, 10 ng/ml) followed by evaluation of expressions of CTLA-4, activating and inhibitory receptors, and the release of interferon gamma (IFN-γ) and granzyme B (GZM B). CTLA-4 expression on NK cells from recurrent ALL patients was also evaluated. Finally, the cytotoxic activity of NK cells was assessed after the CTLA-4 blockade. The purity of the isolated cells was 96.58 ± 2.57%. Isolated NK cells activated with IL-15 resulted in significantly higher CTLA-4 expression (8.75%, P<0.05). Similarly, CTLA-4 expression on the surface of NK cells from patients with ALL was higher (7.46%) compared to healthy individuals (1.46%, P<0.05). IL-15 reduced NKG2A expression (P<0.01), and increased expressions of NKP30 (P<0.05) and NKP46 (P<0.01). The activated NK cells released more IFN-γ (P<0.5) and GZM B (P<0.01) compared to unactivated NK cells. Blockade of CTLA-4 enhanced the NK cell killing potential against Nalm-6 cells (56.3%, P<0.05); however, IFN-γ and GZM B levels were not statistically different between the blocked and non-blocked groups. Our findings suggest that CTLA-4 blockage of Nalm-6 cells causes an increase in antitumour activity of NK cells against these cells. Our study also provides evidence for the potential of cancer immunotherapy treatment using blocking anti-CTLA-4 mAbs.

Functional integration of natural killer cells in a microfluidically perfused liver on-a-chip model

ObjectiveThe liver acts as an innate immunity-dominant organ and natural killer (NK) cells, are the main lymphocyte population in the human liver. NK cells are in close interaction with other immune cells, acting as the first line of defense against pathogens, infections, and injury.A previously developed, three-dimensional, perfused liver-on-a-chip comprised of human cells was used to integrate NK cells, representing pivotal immune cells during liver injury and regeneration. The objective of this study was to integrate functional NK cells in an in vitro model of the human liver and assess utilization of the model for NK cell-dependent studies of liver inflammation.ResultsNK cells from human blood and liver specimen were isolated by Percoll separation with subsequent magnetic cell separation (MACS), yielding highly purified blood and liver derived NK cells. After stimulation with toll-like-receptor (TLR) agonists (lipopolysaccharides, Pam3CSK4), isolated NK cells showed increased interferon (IFN)-gamma secretion. To study the role of NK cells in a complex hepatic environment, these cells were integrated in the vascular compartment of a microfluidically supported liver-on-a-chip model in close interaction with endothelial and resident macrophages. Successful, functional integration of NK cells was verified by immunofluorescence staining (NKp46), flow cytometry analysis and TLR agonist-dependent secretion of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha. Lastly, we observed that inflammatory activation of NK cells in the liver-on-a-chip led to a loss of vascular barrier integrity. Overall, our data shows the first successful, functional integration of NK cells in a liver-on-a-chip model that can be utilized to investigate NK cell-dependent effects on liver inflammation in vitro.

TRAIL-conjugated liposomes that bind natural killer cells to induce colorectal cancer cell apoptosis.

Natural killer (NK) cell functionality is a strong indicator of favorable prognosis in cancer patients, making NK cells an appealing therapeutic target to prevent lymph node dissemination. We engineered liposomes that are conjugated with anti-CD335 antibodies for NK cell targeting, and the apoptotic ligand TRAIL to kill cancer cells. Liposomes were made using a thin film hydration method followed by extrusion to approximately 100 nm in diameter and conjugation of proteins via thiol-maleimide click chemistry. TRAIL/anti-CD335 liposomes successfully bound to isolated NK cells. Once piggybacked to the surface of NK cells, these "Super Natural Killer Cells" were able to more effectively kill oxaliplatin-resistant SW620 cells and metastatic COLO205 colorectal cancer cells via TRAIL-mediated apoptosis compared to NK cells alone. Importantly, Super NK cells were more effective under physiological levels of fluid shear stress found in the lymphatics. Liposome biodistribution after intravenous administration confirmed the sustained presence of liposomes within the spleen and tumor draining mesenteric lymph nodes for at least 4 days. These results demonstrate the enhanced apoptotic effects of NK cells armored with liposomal TRAIL against clinically relevant colorectal cancer cells, providing the groundwork for in vivo treatment studies in mouse models of colorectal cancer metastasis.

Integrative Magneto-Microfluidic Separation of Immune Cells Facilitates Clinical Functional Assays.

Accurately analyzing the functional activities of natural killer (NK) cells in clinical diagnosis remains challenging due to their coupling with other immune effectors. To address this, an integrated immune cell separator is required, which necessitates a streamlined sample preparation workflow including immunological cell isolation, removal of excess red blood cells (RBCs), and buffer exchange for downstream analysis. Here, a self-powered integrated magneto-microfluidic cell separation (SMS) chip is presented, which outputs high-purity target immune cells by simply inputting whole blood. The SMS chip intensifies the magnetic field gradient using an iron sphere-filled inlet reservoir for high-performance immuno-magnetic cell selection and separates target cells size-selectively using a microfluidic lattice for RBC removal and buffer exchange. In addition, the chip incorporates self-powered microfluidic pumping through a degassed polydimethylsiloxane chip, enabling the rapid isolation of NK cells at the place of blood collection within 40min. This chip is used to isolate NK cells from whole blood samples of hepatocellular cancer patients and healthy volunteers and examined their functional activities to identify potential abnormalities in NK cell function. The SMS chip is simple to use, rapid to sort, and requires small blood volumes, thus facilitating the use of immune cell subtypes for cell-based diagnosis.

Fully Automated Continuous Centrifugal Microfluidics Isolates Natural Killer Cells with High Performance and Minimal Stress.

Natural killer (NK) cells are a part of the innate immune system, providing the first line of defense against cancer cells and pathogens at an early stage. Hence, they are attracting attention as a valuable resource for allogeneic cell immunotherapy. However, NK cells exist with limited proportion in blood, and obtaining sufficient clinical-grade NK cells with highly viable and minimal stress is critical for successful immune cell therapy. Conventional purification methods via immunoaffinity or density gradient centrifugation had several limitations in yield, purity, and cellular stress, which might cause an increased risk for graft versus host disease and reduced efficacy due to NK cell malfunction, exhaustion, and apoptosis. Moreover, reducing the variations of isolation performance caused by the manual process is another unmet need for uniform quality of the living drug. Here, an automated system using an NK disc (NKD) based on continuous centrifugal microfluidics (CCM) technology was developed to isolate NK cells from whole blood with high yield, purity, reproducibility, and low stress. The CCM technology, which operates fluidic manipulation under disc rotation, enabled precise extraction of the ultra-thin target fluid layer generated by blood centrifugation. Compared to the conventional manual method, the CCM-NKD isolated NK cells with higher yield (recovery rate) and purity, while maintaining better reproducibility. Furthermore, since the CCM-NKD maintained substantially milder centrifugation conditions (120 ×g for 10 min) compared to the conventional approach (1200 ×g for 20 min), it showed reduced cellular stress and increased antioxidant capacity in the isolated NK cells. Based on the results, the CCM-NKD is expected to be a useful tool to provide highly intact and viable cell weapons for successful immune cell therapy.

Activated NK cells reprogram MDSCs via NKG2D-NKG2DL and IFN-γ to modulate antitumor T-cell response after cryo-thermal therapy

BackgroundMyeloid‐derived suppressor cells (MDSCs) can potently inhibit T-cell activity, promote growth and metastasis of tumor and contribute to resistance to immunotherapy. Targeting MDSCs to alleviate their protumor functions and immunosuppressive...

SOT101 induces NK cell cytotoxicity and potentiates antibody-dependent cell cytotoxicity and anti-tumor activity.

SOT101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SOT101 among other immune cells specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion or activation of the regulatory T cell compartment. In this study, we showed that SOT101 induced expression of cytotoxic receptors NKp30, DNAM-1 and NKG2D on human NK cells. SOT101 stimulated dose-dependent proliferation and the relative expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+, and these displayed an enhanced cytotoxicity in vitro. Using human PBMCs and isolated NK cells, we showed that SOT101 added concomitantly or used for immune cell pre-stimulation potentiated clinically approved monoclonal antibodies Cetuximab, Daratumumab and Obinutuzumab in killing of tumor cells in vitro. The anti-tumor efficacy of SOT101 in combination with Daratumumab was assessed in a solid multiple myeloma xenograft in CB17 SCID mouse model testing several combination schedules of administration in the early and late therapeutic setting of established tumors in vivo. SOT101 and Daratumumab monotherapies decreased with various efficacy tumor growth in vivo in dependence on the advancement of the tumor development. The combination of both drugs showed the strongest anti-tumor efficacy. Specifically, the sequencing of both drugs did not matter in the early therapeutic setting where a complete tumor regression was observed in all animals. In the late therapeutic treatment of established tumors Daratumumab followed by SOT101 administration or a concomitant administration of both drugs showed a significant anti-tumor efficacy over the respective monotherapies. These results suggest that SOT101 might significantly augment the anti-tumor activity of therapeutic antibodies by increasing NK cell-mediated activity in patients. These results support the evaluation of SOT101 in combination with Daratumumab in clinical studies and present a rationale for an optimal clinical dosing schedule selection.

Structure of the human NK cell NKR-P1:LLT1 receptor:ligand complex reveals clustering in the immune synapse

Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

Transient receptor potential melastatin 3 dysfunction in post COVID-19 condition and myalgic encephalomyelitis/chronic fatigue syndrome patients

BackgroundMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a severe multisystemic condition associated with post-infectious onset, impaired natural killer (NK) cell cytotoxicity and impaired ion channel function, namely Transient Receptor Potential Melastatin 3 (TRPM3). Long-term effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has resulted in neurocognitive, immunological, gastrointestinal, and cardiovascular manifestations recently recognised as post coronavirus disease 2019 (COVID-19) condition. The symptomatology of ME/CFS overlaps significantly with post COVID-19; therefore, this research aimed to investigate TRPM3 ion channel function in post COVID-19 condition patients.MethodsWhole-cell patch-clamp technique was used to measure TRPM3 ion channel activity in isolated NK cells of N = 5 ME/CFS patients, N = 5 post COVID-19 patients, and N = 5 healthy controls (HC). The TRPM3 agonist, pregnenolone sulfate (PregS) was used to activate TRPM3 function, while ononetin was used as a TRPM3 antagonist.ResultsAs reported in previous research, PregS-induced TRPM3 currents were significantly reduced in ME/CFS patients compared with HC (p = 0.0048). PregS-induced TRPM3 amplitude was significantly reduced in post COVID-19 condition compared with HC (p = 0.0039). Importantly, no significant difference was reported in ME/CFS patients compared with post COVID-19 condition as PregS-induced TRPM3 currents of post COVID-19 condition patients were similar of ME/CFS patients currents (p > 0.9999). Isolated NK cells from post COVID-19 condition and ME/CFS patients were resistant to ononetin and differed significantly with HC (p < 0.0001).ConclusionThe results of this investigation suggest that post COVID-19 condition patients may have impaired TRPM3 ion channel function and provide further evidence regarding the similarities between post COVID-19 condition and ME/CFS. Impaired TRPM3 channel activity in post COVID-19 condition patients suggest impaired ion mobilisation which may consequently impede cell function resulting in chronic post-infectious symptoms. Further investigation into TRPM3 function may elucidate the pathomechanism, provide a diagnostic and therapeutic target for post COVID-19 condition patients and commonalities with ME/CFS patients.

P53 missense mutant G242A subverts natural killer cells in sheltering mouse breast cancer cells against immune rejection

Cancer cells acquire immunoediting ability to evade immune surveillance and thus escape eradication. It is widely known that mutant proteins encoded from tumor suppressor TP53 exhibit gain-of-function in cancer cells, thereby promoting progression; however, how mutant p53 contributes to the sheltering of cancer cells from host anticancer immunity remains unclear. Herein, we report that murine p53 missense mutation G242A (corresponding to human G245A) suppresses the activation of host natural killer (NK) cells, thereby enabling breast cancer cells to avoid immune assault. We found that serial injection of EMT6 breast cancer cells that carry wild-type (wt) Trp53, like normal fibroblasts, promoted NK activity in mice, while SVTneg2 cells carrying Trp53 G242A+/+ mutation decreased NK cell numbers and increased CD8+ T lymphocyte numbers in spleen. Innate immunity based on NK cells and CD8 T cells was reduced in p53 mutant-carrying transgenic mice (Trp53 R172H/+, corresponding to human R175H/+). Further, upon co-culture with isolated NK cells, EMT6 cells substantively activated NK cells and proliferation thereof, increasing interferon-gamma (IFN-γ) production; however, SVTneg2 cells suppressed NK cell activation. Further mechanistic study elucidated that p53 can modulate expression by cancer cells of Mult-1 and H60a, which are activating and inhibitory ligands for NKG2D receptors of NK cells, respectively, to enhance immune surveillance against cancer. Our findings demonstrate that wt p53 is requisite for NK cell-based immune recognition and elimination of cancerous cells, and perhaps more importantly, that p53 missense mutant presence in cancer cells impairs NK cell-attributable responses, thus veiling cancerous cells from host immunity and enabling cancer progression.

Impaired TRPM3-dependent calcium influx and restoration using Naltrexone in natural killer cells of myalgic encephalomyelitis/chronic fatigue syndrome patients

BackgroundMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious disorder of unknown aetiology. While the pathomechanism of ME/CFS remains elusive, reduced natural killer (NK) cell cytotoxic function is a consistent immunological feature. NK cell effector functions rely on long-term sustained calcium (Ca2+) influx. In recent years evidence of transient receptor potential melastatin 3 (TRPM3) dysfunction supports the hypothesis that ME/CFS is potentially an ion channel disorder. Specifically, reports of single nucleotide polymorphisms, low surface expression and impaired function of TRPM3 have been reported in NK cells of ME/CFS patients. It has been reported that mu (µ)-opioid receptor (µOR) agonists, known collectively as opioids, inhibit TRPM3. Naltrexone hydrochloride (NTX), a µOR antagonist, negates the inhibitory action of µOR on TRPM3 function. Importantly, it has recently been reported that NTX restores impaired TRPM3 function in NK cells of ME/CFS patients.MethodsLive cell immunofluorescent imaging was used to measure TRPM3-dependent Ca2+ influx in NK cells isolated from n = 10 ME/CFS patients and n = 10 age- and sex-matched healthy controls (HC) following modulation with TRPM3-agonist, pregnenolone sulfate (PregS) and TRPM3-antaognist, ononetin. The effect of overnight (24 h) NTX in vitro treatment on TRPM3-dependent Ca2+ influx was determined.ResultsThe amplitude (p < 0.0001) and half-time of Ca2+ response (p < 0.0001) was significantly reduced at baseline in NK cells of ME/CFS patients compared with HC. Overnight treatment of NK cells with NTX significantly improved TRPM3-dependent Ca2+ influx in ME/CFS patients. Specifically, there was no significance between HC and ME/CFS patients for half-time response, and the amplitude of Ca2+ influx was significantly increased in ME/CFS patients (p < 0.0001).ConclusionTRPM3-dependent Ca2+ influx was restored in ME/CFS patients following overnight treatment of isolated NK cells with NTX in vitro. Collectively, these findings validate that TRPM3 loss of function results in altered Ca2+ influx supporting the growing evidence that ME/CFS is a TRP ion channel disorder and that NTX provides a potential therapeutic intervention for ME/CFS.

Hypoxia-inducible factor-1 alpha expression is induced by IL-2 via the PI3K/mTOR pathway in hypoxic NK cells and supports effector functions in NKL cells and ex vivo expanded NK cells

Natural killer (NK) cells are cytotoxic innate lymphocytes that are specialized to kill tumor cells. NK cells are responsive to the primary cytokine IL-2 in the tumor microenvironment (TME), to activate its effector functions against tumors. Despite their inherent ability to kill tumor cells, dysfunctional NK cells observed within advanced solid tumors are associated with poor patient survival. Hypoxia in the TME is a major contributor to immune evasion in solid tumors that could contribute to impaired NK cell function. HIF-1α is a nodal regulator of hypoxia in driving the adaptive cellular responses to changes in oxygen concentrations. Whether HIF-1α is expressed in hypoxic NK cells in the context of IL-2 and whether its expression regulates NK cell effector function are unclear. Here, we report that freshly isolated NK cells from human peripheral blood in hypoxia could not stabilize HIF-1α protein coincident with impaired anti-tumor cytotoxicity. However, ex vivo expansion of these cells restored HIF-1α levels in hypoxia to promote antitumor cytotoxic functions. Similarly, the human NK cell line NKL expressed HIF-1α upon IL-2 stimulation in hypoxia and exhibited improved anti-tumor cytotoxicity and IFN-γ secretion. We found that ex vivo expanded human NK cells and NKL cells required the concerted activation of PI3K/mTOR pathway initiated by IL-2 signaling in combination with hypoxia for HIF-1α stabilization. These findings highlight that HIF-1α stabilization in hypoxia maximizes NK cell effector function and raises the prospect of NK cells as ideal therapeutic candidates for solid tumors.

Tim-3 and PD-1 blocking cannot restore the functional properties of natural killer cells in early clinical stages of chronic lymphocytic leukemia: An in vitro study.

Programmed death-1 (PD-1) and T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) are two major immune checkpoint receptors expressed on immune cells and their expression is related to the exhaustion phenotype. In the present in vitro study, blocking of PD-1 and Tim-3 molecules was performed on isolated natural killer (NK) cells from patients with chronic lymphocytic leukemia (CLL) to restore their functional properties. NK cells fraction was positively isolated from fresh peripheral blood of 18 CLL patients, treated with anti-PD-1 and anti-Tim-3 blocking monoclonal antibodies and co-cultured with K562 target cells to evaluate their apoptosis induction by Annexin V-PI method. Blocked NK cells were also incubated with anti-CD107a antibody to assess their degranulation properties by flow cytometry. The level of secreted tumor node factor-alpha (TNF-α) and interferon-gamma (IFN-γ) by NK cells was also measured by ELISA. Our results showed similar functional properties in terms of degranulation and apoptosis of K562 target cells by isolated NK cells from CLL patients in PD-1/Tim-3 blocked and control groups. It was also shown that blocking of PD-1 and Tim-3 could not improve the production of pro-inflammatory TNF-α and IFN-γ cytokines by isolated NK cells from CLL patients. Altogether, our results indicated that pretreatment of NK cells with anti-PD-1 and anti-Tim-3 blocking antibodies in CLL patients at early clinical stages cannot improve their functional properties. Besides many other malignancies, the application of checkpoint inhibitors in CLL needs more investigations and complementary studies.

The Direct Influence of Cytomegalovirus Lysate on the Natural Killer Cell Receptor Repertoire.

Natural killer (NK) cells are essential for controlling certain viral infections, including cytomegalovirus (CMV). In particular, the importance of NK cells in the context of CMV infection is underscored by the adaptive capabilities of these cells. Evidence suggests that some viruses can directly interfere with NK cell compartments and their activation and lead to shape-shifting the NK cell receptor repertoire. Still, it remains unknown whether the CMV can interact with NK cells without intermediaries. Here, we examined whether the direct effects of CMV lysate alter phenotypical properties of NK cells. To investigate this issue, NK cells were isolated from the blood of CMV seropositive healthy donors by negative magnetic separation. Isolated NK cells were cultured in the presence of CMV lysate and analyzed for the expression of NKG2A, NKG2C, and CD57 by FACS caliber. The results showed that NKG2C expression is significantly upregulated in the presence of CMV lysate compared to without stimulated group (mean increase, 6.65 %; 95% CI, 0.2582 to 13.02; p=0.043; R square: 0.38). Likewise, results have shown a significant decrease in the frequency of NKG2A+CD57- NK cell subsets (p=0.005; 95% CI, -13.49 to -3.151; R square: 0.5957) in the stimulated group compared to without stimulated ones. According to these results, CMV may drive a direct influence on NK cell receptor repertoire, including the expansion of NK cells expressing NKG2C receptor, which is needed for further studies.

IMMU-42. OPTIMIZING IMMUNOTHERAPY FOR GLIOMA USING CYTOKINE-ACTIVATED NATURAL KILLER CELLS

Abstract INTRODUCTION Glioblastoma multiforme (GBM) is the most common primary central nervous system malignancy associated with a 12-15 month survival after surgery and radio-chemotherapy. Utilizing adoptive cellular immunotherapy using natural killer (NK) cells has developed over the past two decades for a variety of hematologic malignancies. This approach in solid malignancies is limited by questions of cell dose versus tumor burden, insufficient tumor infiltration, and a tumor microenvironment that suppresses NK cell function. METHODS We isolated NK cells from healthy volunteers and activated them using IL-2, -15, -12, -18, then perform cytotoxic assays in the presence of glioma stem cells. We also tested the efficacy of the NK cells with intracranial delivery in a pre-clinical murine model of glioma. We tested various concentrations of IL-2 and IL-15 on the cytokine culture platform. RESULTS In this study, we demonstrate human NK cells, activated using a cytokine cocktail of interleukins-2, -15, -12, and -18, exert strong cytotoxic events against glioma cell lines. To further examine the efficacy of activated NK cells in vitro, we utilized intracranially xenografted glioma lines and demonstrated a survival benefit with tumor bed injections of these cytokine-activated NK cells (p=0.0089). We were able to confirm that NK cells cultured with low doses (200u IL2; 50ng/ml IL15) of both cytokines are just as effective as higher doses. This is important, as in vivoexhaustion of NK cells stimulated with high doses of either cytokine has been well validated. We also found that low-dose irradiation (4Gy) of glioma cells prior to co-culture with cytokine-activated NK cells promoted increased targeted glioma cell killing within 4 hours(32% cell killing). CONCLUSIONS These findings suggest that in a clinical study, injection of cytokine-activated NK cells into the glioblastoma tumor bed could be used as adjuvant treatment following either stereotactic radiation or surgical resection.

ADCC against MICA/B Is Mediated against Differentiated Oral and Pancreatic and Not Stem-Like/Poorly Differentiated Tumors by the NK Cells; Loss in Cancer Patients due to Down-Modulation of CD16 Receptor.

Simple SummaryNatural Killer cells are known to eliminate tumors directly or via antibody dependent cellular cytotoxicity. The complete modes and mechanisms of such killings are yet to be delineated. It is also unclear at what stages of tumor differentiation NK cells are capable of mediating the two modes of tumor killing. In this report we provide evidence that NK cells mediate killing of both stem-like/poorly differentiated tumors and well-differentiated tumors via direct cytotoxicity and antibody dependent cellular cytotoxicity, respectively. By using antibodies to MICA/B, EGFR and PDL1 surface receptors expressed on well-differentiated but not on stem-like/poorly differentiated tumors we demonstrate significant NK cell mediated antibody dependent cellular cytotoxicity in the absence of direct killing. In addition, our results suggested the possibility of CD16 receptors mediating both direct cytotoxicity and antibody dependent cellular cytotoxicity, resulting in the competitive use of these receptors in either direct killing or antibody dependent cellular cytotoxicity.Tumor cells are known to upregulate major histocompatibility complex-class I chain related proteins A and B (MICA/B) expression under stress conditions or due to radiation exposure. However, it is not clear whether there are specific stages of cellular maturation in which these ligands are upregulated or whether the natural killer (NK) cells differentially target these tumors in direct cytotoxicity or antibody-dependent cell cytotoxicity (ADCC). We used freshly isolated primary and osteoclast (OCs)-expanded NK cells to determine the degree of direct cytotoxicity or of ADCC using anti-MICA/B monoclonal antibodies (mAbs) against oral stem-like/poorly-differentiated oral squamous cancer stem cells (OSCSCs) and Mia PaCa-2 (MP2) pancreatic tumors as well as their well-differentiated counterparts: namely, oral squamous carcinoma cells (OSCCs) and pancreatic PL12 tumors. By using phenotypic and functional analysis, we demonstrated that OSCSCs and MP2 tumors were primary targets of direct cytotoxicity by freshly isolated NK cells and not by ADCC mediated by anti-MICA/B mAbs, which was likely due to the lower surface expression of MICA/B. However, the inverse was seen when their MICA/B-expressing differentiated counterparts, OSCCs and PL12 tumors, were used in direct cytotoxicity and ADCC, in which there was lower direct cytotoxicity but higher ADCC mediated by the NK cells. Differentiation of the OSCSCs and MP2 tumors by NK cell-supernatants abolished the direct killing of these tumors by the NK cells while enhancing NK cell-mediated ADCC due to the increased expression of MICA/B on the surface of these tumors. We further report that both direct killing and ADCC against MICA/B expressing tumors were significantly diminished by cancer patients’ NK cells. Surprisingly, OC-expanded NK cells, unlike primary interleukin-2 (IL-2) activated NK cells, were found to kill OSCCs and PL12 tumors, and under these conditions, we did not observe significant ADCC using anti-MICA/B mAbs, even though the tumors expressed a higher surface expression of MICA/B. In addition, differentiated tumor cells also expressed higher levels of surface epidermal growth factor receptor (EGFR) and programmed death-ligand 1(PDL1) and were more susceptible to NK cell-mediated ADCC in the presence of anti-EGFR and anti-PDL1 mAbs compared to their stem-like/poorly differentiated counterparts. Overall, these results suggested the possibility of CD16 receptors mediating both direct cytotoxicity and ADCC, resulting in the competitive use of these receptors in either direct killing or ADCC, depending on the differentiation status of tumor cells and the stage of maturation and activation of NK cells.

Selectively and Efficiently Expanding Natural Killer Cells in Vitro

Natural killer (NK) cells are the main innate immune cells, and have powerful antitumor activity in vitro, but NK cells based immunotherapy does not often get satisfactory results in clinical practice due to insufficient number and poor activity of NK cells.. Therefore, how to improve expansion efficiency and killing activity of NK cells in vitro is critical. Here we report an in vitro culture system culturing peripheral blood mononuclear cells (PBMC) with irradiated LCL as feeder cells in 10%FBS/RPMI1640 culture medium in the presence of 100U/ml IL-2 and 5ng/ml IL-15 for 14 days could selectively expand NK cells. With this culture system, the purity of NK cells increased from 11.5%± 7.01% to 75.3±10.9% (P&amp;lt; 0.01), and the expansion efficiency was about 625±63 times than the original PBMC. Compared with the fresh NK cells isolated from PBMC, there was no significant change in the expression of inhibitory receptors on the surface of expanded NK cells, while the expression of activating receptors was significantly increased (p&amp;lt;0.05). The cytotoxicity of expanded NK cells against leukemia cell line K562 was significantly higher than that of freshly isolated NK cells (72± 5.6% vs 8.7±2.3%, E: T=1, P&amp;lt;0.01), expanded NK cells also showed stronger cytotoxicity to LCL (22±3.6%, E: T=1) and primary leukemia cells (42±5.2%, E:T=1). In summary, we could selectively expand NK cells with this in vitro culture system, the expanded NK cells gained higher expression of NCRs and NKG2D receptors, and displayed greater cytotoxicity against tumor cells, therefore could be readily applied to anti-tumor treatment in clinical treatment. Figure Disclosures No relevant conflicts of interest to declare.

Flexible ZnO-mAb nanoplatforms for selective peripheral blood mononuclear cell immobilization

Cancer is the second cause of death worldwide. This devastating disease requires specific, fast, and affordable solutions to mitigate and reverse this trend. A step towards cancer-fighting lies in the isolation of natural killer (NK) cells, a set of innate immune cells, that can either be used as biomarkers of tumorigenesis or, after autologous transplantation, to fight aggressive metastatic cells. In order to specifically isolate NK cells (which express the surface NKp30 receptor) from peripheral blood mononuclear cells, a ZnO immunoaffinity-based platform was developed by electrodeposition of the metal oxide on a flexible indium tin oxide (ITO)-coated polyethylene terephthalate (PET) substrate. The resulting crystalline and well-aligned ZnO nanorods (NRs) proved their efficiency in immobilizing monoclonal anti-human NKp30 antibodies (mAb), obviating the need for additional procedures for mAb immobilization. The presence of NK cells on the peripheral blood mononuclear cell (PBMCs) fraction was evaluated by the response to their natural ligand (B7-H6) using an acridine orange (AO)-based assay. The successful selection of NK cells from PBMCs by our nanoplatform was assessed by the photoluminescent properties of AO. This easy and straightforward ZnO-mAb nanoplatform paves the way for the design of biosensors for clinic diagnosis, and, due to its inherent biocompatibility, for the initial selection of NK cells for autotransplantation immunotherapies.