Increase In Natural Killer Cell Activity Research Articles

The Consumption of Lacticaseibacillus rhamnosus HDB1258 Changes Human Gut Microbiota and Induces Immune Enhancement Through NK Cell Activation.

The gut microbiota can play an important role in enhancing the host's complex immune system. In this regard, many studies indicate that probiotics consumption has a beneficial impact on alterations in the composition of the gut microbiota. Our previous study demonstrated that the oral administration of Lacticaseibacillus rhamnosus HDB1258 (HDB1258) enhances immune cell activity and alters the composition of gut microbiota in C57BL/6 mice, thereby showing its potential as a novel immunostimulatory ingredient. Therefore, this clinical trial assessed the effects of HDB1258 on human natural killer (NK) cell activity and changes in gut microbiota. It also investigated the correlation between gut microbiota and NK cell activity following HDB1258 supplementation. Participants (n = 71) were randomized into placebo and HDB1258 groups, and NK cell activity and gut microbiota were investigated at baseline (week 0) and endline (week 8). The present study showed that HDB1258 significantly increased NK cell activity and resulted in positive regulatory effects on the gut microbial balance in subjects compared to the placebo group. HDB1258 affected the gut microbial balance by inducing the growth of beneficial bacteria such as Lactococcus and Sutterella. Especially, the changes in Escherichia-Shigella composition were negatively correlated with the changes in NK cell activity after HDB1258 consumption. There was also a positive correlation between the NK cell activity in the HDB1258 group and the composition of Prevotella 9 and Adlercreutzia. These findings suggest that HDB1258 may improve the host's intestinal environment by regulating gut bacteria related to immune response and promote NK cell activation. This study was registered at clinical research information service (CRIS: KCT0008204).

Long-term dynamics of natural killer cells in response to SARS-CoV-2 vaccination: Persistently enhanced activity postvaccination.

Natural Killer (NK) cells play a significant role in the early defense against virus infections and cancer. Recent studies have demonstrated the involvement of NK cells in both the induction and effector phases of vaccine-induced immunity in various contexts. However, their role in shaping immune responses following SARS-CoV-2 vaccination remains poorly understood. To address this matter, we conducted a comprehensive analysis of NK cell phenotype and function in SARS-CoV-2 unexposed individuals who received the BNT162b2 vaccine. We employed a longitudinal study design and utilized a panel of 53 15-mer overlapping peptides covering the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein to assess NK cell function at 0 and 20 days following the first vaccine, and 30 and 240 days following booster. Additionally, we evaluated the levels of total IgG anti-Spike antibodies and their potential neutralizing ability. Our findings revealed an increased NK cell activity upon re-exposure to RBD when combined with IL12 and IL18 several months after booster. Concurrently, we observed that the frequencies of NKG2A + NK cells declined over the course of the follow-up period, while NKG2C increased only in CMV positive subjects. The finding that NK cell functions are inducible 9 months after vaccination upon re-exposure to RBD and cytokines, sheds light on the role of NK cells in contributing to SARS-CoV-2 vaccine-induced immune protection and pave the way to further studies in the field.

Decreased LDHB expression in breast tumor cells causes NK cell activation and promotes tumor progression.

Abnormal metabolism is the underlying reason for breast cancer progression. Decreased lactate dehydrogenase B (LDHB) has been detected in breast cancer but the function of LDHB remains unknown. Western blot was used to analyze LDHB expression in breast cancer cells. The impact of LDHB on tumor cell migration and invasion was determined using Transwell assays, wound healing assays, and a mouse lung metastasis model. Subcutaneous tumor formation, a natural killer (NK) cell cytotoxicity assay, and flow cytometry evaluated NK cell activation. Immunofluorescence and quantitative real-time PCR detected NK cell activation markers. Kaplan-Meier analysis evaluated the effect of immune cell infiltration on prognosis. Single-sample gene set enrichment analysis determined NK cell activation scores. A support vector machine predicted the role of LDHB in NK cell activation. In this study we showed that LDHB inhibits the breast cancer cell metastasis and orchestrates metabolic reprogramming within tumor cells. Our results revealed that LDHB-mediated lactic acid clearance in breast cancer cells triggers NK cell activation within the tumor microenvironment. Our findings, which were confirmed in a murine model, demonstrated that LDHB in tumor cells promotes NK cell activation and ultimately results in the eradication of malignant cells. Clinically, our study further validated that LDHB affects immune cell infiltration and function. Specifically, its expression has been linked to enhanced NK cell-mediated cytotoxicity and improved patient survival. Furthermore, we identified LDHB expression in tumors as an important predictor of NK cell activation, with strong predictive ability in some cancers. Our results suggest that LDHB is a promising target for activating the tumor immune microenvironment in breast cancer, where LDHB-associated lactic acid clearance leads to increased NK cell activity. This study highlights the critical role of LDHB in regulating immune responses and its potential as a therapeutic target for breast cancer.

Efbalropendekin Alfa enhances human natural killer cell cytotoxicity against tumor cell lines in vitro.

IL-15 has shown preclinical activity by enhancing the functional maturation of natural killer (NK) cells. Clinical evaluation of the potential anticancer activity of most cytokines, including IL-15, has been limited by low tolerability and rapid in vivo clearance. Efbalropendekin Alfa (XmAb24306) is a soluble IL15/IL15-receptor alpha heterodimer complex fused to a half-life extended Fc domain (IL15/IL15Rα-Fc), engineered with mutations to reduce IL-15 affinity for CD122. Reduced affinity drives lower potency, leading to prolonged pharmacodynamic response in cynomolgus monkeys. We show that in vitro, human NK cells treated with XmAb24306 demonstrate enhanced cytotoxicity against various tumor cell lines. XmAb24306-treated NK cells also exhibit enhanced killing of 3D colorectal cancer spheroids. Daratumumab (dara), a monoclonal antibody (mAb) that targets CD38 results in antibody-dependent cellular cytotoxicity (ADCC) of both multiple myeloma (MM) cells and NK cells. Addition of XmAb24306 increases dara-mediated NK cell ADCC against various MM cell lines in vitro. Because NK cells express CD38, XmAb24306 increases dara-mediated NK cell fratricide, but overall does not negatively impact the ADCC activity against a MM cell line likely due to increased NK cell activity of the surviving cells. These data show that XmAb24306 increases direct and ADCC-mediated human NK cell cytotoxicity in vitro.

Gut-microbiota prompt activation of natural killer cell on alcoholic liver disease

ABSTRACT The liver is rich in innate immune cells, such as natural killer (NK) cells, natural killer T cells, and Kupffer cells associated with the gut microbiome. These immune cells are dysfunctional owing to alcohol consumption. However, there is insufficient data on the association between immune cells and gut microbiome in alcoholic liver disease (ALD). Therefore, the purpose of this study was to evaluate the effects of probiotic strains on NK cells in ALD patients. In total, 125 human blood samples [control (n = 22), alcoholic hepatitis (n = 43), and alcoholic cirrhosis (n = 60]) were collected for flow cytometric analysis. C57BL/6J mice were divided into four groups (normal, EtOH-fed, and 2 EtOH+strain groups [Phocaeicola dorei and Lactobacillus helveticus]). Lymphocytes isolated from mouse livers were analyzed using flow cytometry. The frequency of NK cells increased in patients with alcoholic hepatitis and decreased in patients with alcoholic cirrhosis. The expression of NKp46, an NK cell-activating receptor, was decreased in patients with alcoholic hepatitis and increased in patients with alcoholic cirrhosis compared to that in the control group. The number of cytotoxic CD56dimCD16+ NK cells was significantly reduced in patients with alcoholic cirrhosis. We tested the effect of oral administration P. dorei and L. helveticus in EtOH-fed mice. P. dorei and L. helveticus improved liver inflammation and intestinal barrier damage caused by EtOH supply and increased NK cell activity. Therefore, these observations suggest that the gut microbiome may ameliorate ALD by regulating immune cells.

Inhibiting N-glycan processing increases the antibody binding affinity and effector function of human natural killer cells.

Novel approaches are required to improve the efficacy of immunotherapies and increase the proportion of patients who experience a benefit. Antibody-dependent cell-mediated cytotoxicity (ADCC) contributes to the efficacy of many monoclonal antibodies therapies. Natural killer (NK) cells mediate ADCC, though responses are highly variable and depend on prior treatment as well as other factors. Thus, strategies to increase NK cell activity are expected to improve multiple therapies. Both cytokine treatment and NK cell receptor engineering are being explored to increase ADCC. Post-translational modifications, including glycosylation, are widely recognized as mediators of cellular processes but minimally explored as an alternative strategy to increase ADCC. We evaluated the impact of treatment with kifunensine, an inhibitor of asparagine-linked (N-)glycan processing, on ADCC using primary and cultured human NK cells. We also probed affinity using binding assays and CD16a structure with nuclear magnetic resonance spectroscopy. Treating primary human NK cells and cultured YTS-CD16a cells with kifunensine doubled ADCC in a CD16a-dependent manner. Kifunensine treatment also increased the antibody-binding affinity of CD16a on the NK cell surface. Structural interrogation identified a single CD16a region, proximal to the N162 glycan and the antibody-binding interface, perturbed by the N-glycan composition. The observed increase in NK cell activity following kifunensine treatment synergized with afucosylated antibodies, further increasing ADCC by an additional 33%. These results demonstrate native N-glycan processing is an important factor that limits NK cell ADCC. Furthermore, optimal antibody and CD16a glycoforms are defined that provide the greatest ADCC activity.

Euonymus alatus (Thunb.) Siebold leaf extract enhanced immunostimulatory effects in a cyclophosphamide-induced immunosuppressed rat model.

Euonymus alatus (Thunb.) Siebold (EA) is a medicinal plant used in some Asian countries to treat various diseases, including cancer, hyperglycemia, diabetes, urticaria, dysmenorrhea, and arthritis. Owing to the wide range of pharmacological applications of EA, various roles of EA are being studied. We evaluated the immune-enhancing effect of EA treatment in a cyclophosphamide (Cy)-induced immunosuppressed rat model. We analyzed the immune enhancement effect of EA on macrophages by western blotting. In addition, cell viability and natural killer (NK) cell activity were analyzed in splenocytes following EA treatment. For in vivo studies, analysis of weekly body weight, spleen weight, immune cell count, cytokine levels, and spleen histological findings was performed following EA administration in Cy-induced immunocompromised rats. EA significantly increased cell viability and phospho-nuclear factor-kappa B and phospho-extracellular signal-regulated kinase protein levels in the macrophages. EA significantly increased NK cell activity in splenocytes compared with the control group. In Cy-induced immunosuppressed rats, EA administration increased spleen tissue weight and the contents of leukocytes, lymphocytes, granulocytes, intermediate cells, and plasma cytokines (tumor necrosis factor-α and interferon-γ). In addition, improvement in the damaged spleen tissue was observed. These findings confirm that EA exerts an immune-enhancing effect, thereby suggesting its potential as an immunostimulatory agent or functional food.

IFN-β activates cytotoxic function of human natural killer cells toward IL-27 and poly(I:C) stimulated PC3 and DU145 cells

Natural killer (NK) cell phenotype and function are altered in patients with prostate cancer, and increased NK cell activity is associated with a better prognosis in patients with disease. For patients with advanced stage prostate cancer, immunotherapies are a promising approach when standard treatment options have been exhausted. With the rapid emergence of NK cell-based therapies, it is important to understand the mechanisms by which NK cells can be triggered to kill cancer cells that have developed immune-evasive strategies. Altering the cytokine profiles of advanced prostate cancer cells may be an area to explore when considering ways in which NK cell activation can be modulated. We have previously demonstrated that combining the cytokine, IL-27, with TLR3 agonist, poly(I:C), changes cytokine secretion in the advanced prostate cancer models, PC3 and DU145 cells. Herein, we extend our previous work to study the effect of primary human NK cells on prostate cancer cell death in an in vitro co-culture model. Stimulating PC3 and DU145 cells with IL-27 and poly(I:C) induced IFN-β secretion, which was required for activation of primary human NK cells to kill these stimulated prostate cancer cells. PC3 cells were more sensitized to NK cell-mediated killing when compared to DU145 cells, which was attributed to differential levels of IFN-β produced in response to stimulation with IL-27 and poly(I:C). IFN-β increased granzyme B secretion and membrane-bound TRAIL expression by co-cultured NK cells. We further demonstrated that these NK cells killed PC3 cells in a partially TRAIL-dependent manner. This work provides mechanistic insight into how the cytotoxic function of NK cells can be improved to target cancer cells.

Abstract 2912: High-throughput screening and single-cell transcriptomics identify synergies between oncology drugs and natural killer cell immunotherapy

Abstract Background & Objective: Natural killer (NK) cell-based immunotherapies are emerging therapeutic approaches that aim to target malignant cells resistant to conventional treatments and T cell-based immunotherapies. Existing oncology drugs may enhance the anti-cancer function of NK cells, although large-scale high-throughput testing of NK cell-drug combinations has not been previously attempted. Our objective was to evaluate the potential of approved and investigational oncology drugs to synergize with NK cell-based immunotherapy in acute myeloid leukemia (AML), with the aim to discover both potentiating and inhibiting compounds. Methods: A high-throughput drug sensitivity and resistance screen including 528 oncology drugs was combined with expanded NK cells to evaluate their cytotoxicity against luciferase-expressing AML cells (MOLM-14). The most prominent drugs were investigated further with single-cell RNA sequencing to reveal underlying biological mechanisms. Results: Overall, 191 drugs had an inhibitory effect on cytotoxicity, 294 had no effect, and 43 had an enhancing effect. The most notable NK cell cytotoxicity-potentiating drugs were pevonedistat (NEDD8 inhibitor), daporinad (NAMPT inhibitor), and JAK inhibitors. These drugs also induced distinct changes in NK and target cell transcriptional profiles. When combined with NK cells, pevonedistat and daporinad induced an upregulation of HLA-E and CXCL10 in target cells. In addition, MYC and FLT3 proto-oncogenes were downregulated in target cells when combining NK cells with pevonedistat. Moreover, in NK cells, daporinad induced dysregulated expression of genes related to NK cell effector functions such as TYROBP, TXNIP and KLRC1. Treatment with JAK inhibitors downregulated MHC I expression on target cells, providing a putative mechanism for increased NK cell activity. Validation of key hits is ongoing in additional AML cell lines. Conclusion: We discovered novel drugs with both potentiating and inhibiting effects on NK cell cytotoxicity against AML cells in vitro. Our results demonstrate the potential of using a high-throughput framework for studying NK cell-drug interactions against malignant cells, while aiming to improve treatment for AML and other malignancies through combination immunotherapies. Citation Format: Jonas O. Bouhlal, Petra Nygren, Essi Laajala, Aleksandr Ianevski, Jay Klievink, Hanna Lähteenmäki, Khalid Saeed, Dean Lee, Tero Aittokallio, Olli Dufva, Satu Mustjoki. High-throughput screening and single-cell transcriptomics identify synergies between oncology drugs and natural killer cell immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2912.

Arabinogalactan G1-4A isolated from Tinospora cordifolia induces PKC/mTOR mediated direct activation of natural killer cells and through dendritic cell cross-talk

BackgroundTinospora cordifolia polysaccharide G1–4A activates antigen-presenting cells, but its effect on natural killer (NK) cells is not known. The objective of this study is to assess the effect of G1–4A on NK cells; direct effects as well as through dendritic cell (DC) cross-talk. MethodsNK cell phenotype and function were assessed in spleen cells treated in vitro with G1–4A or isolated from mice administered with G1–4A. Following treatment with G1–4A in vitro or in cells isolated from G1–4A treated mice (in vivo), activated NK cell phenotype was characterized as CD3−NKp46+CD69+ cells by flow cytometry; NK cell function was evaluated by IFN-γ secretion (ELISA) and cytotoxicity assay (calcein release by target cells in effector: target cells co-culture assay). ResultsBoth in vitro as well as in vivoG1–4A treatment increased phenotypic and functional activation of NK cells. So, we wanted to determine if this was through NK-DC crosstalk or direct activation of NK cells. There was increased NK cell activation following co-culture with bone marrow derived DC matured withG1–4A in vitro or splenic DC isolated from G1–4A administered mice indicating crosstalk. G1–4A also increased activation of NK cells in (a) CD11c depleted splenic cells that was contact dependent and (b) purified NKp46+ cells that was abrogated by PKC/mTOR inhibitors indicating direct effects on NK cells. ConclusionIn summary, treatment with G1–4A results in phenotypic and functional activation of NK cells directly as well as through NK-DC cross talk and has the potential to be used as an immunotherapeutic agent.

Eubacterium rectale Improves the Efficacy of Anti-PD1 Immunotherapy in Melanoma via l-Serine-Mediated NK Cell Activation.

Natural killer (NK) cells, as key immune cells, play essential roles in tumor cell immune escape and immunotherapy. Accumulating evidence has demonstrated that the gut microbiota community affects the efficacy of anti-PD1 immunotherapy and that remodeling the gut microbiota is a promising strategy to enhance anti-PD1 immunotherapy responsiveness in advanced melanoma patients; however, the details of the mechanism remain elusive. In this study, we found that Eubacterium rectale was significantly enriched in melanoma patients who responded to anti-PD1 immunotherapy and that a high E. rectale abundance was related to longer survival in melanoma patients. Furthermore, administration of E. rectale remarkably improved the efficacy of anti-PD1 therapy and increased the overall survival of tumor-bearing mice; moreover, application of E. rectale led to a significant accumulation of NK cells in the tumor microenvironment. Interestingly, conditioned medium isolated from an E. rectale culture system dramatically enhanced NK cell function. Gas chromatography-mass spectrometry/ultrahigh performance liquid chromatography-tandem mass spectrometry-based metabolomic analysis showed that l-serine production was significantly decreased in the E. rectale group; moreover, administration of an l-serine synthesis inhibitor dramatically increased NK cell activation, which enhanced anti-PD1 immunotherapy effects. Mechanistically, supplementation with l-serine or application of an l-serine synthesis inhibitor affected NK cell activation through Fos/Fosl. In summary, our findings reveal the role of bacteria-modulated serine metabolic signaling in NK cell activation and provide a novel therapeutic strategy to improve the efficacy of anti-PD1 immunotherapy in melanoma.

Effect of orally administered heat-treated Lactobacillus plantarum LM1004 on the innate immune system: A randomized, placebo-controlled, double-blind study

• Supplementation with heat-treated Lactobacillus plantarum LM1004 (HT-LM1004) for eight weeks activated the innate immunity in healthy adults. • Natural killer cells were enhanced with HT-LM1004 compared to the control group. • Serum level of interleukin-12 by dietary intake of HT-LM1004 was elevated from baseline. • Treatment of HT-LM1004 was safe and well tolerated. Heat-treated Lactobacillus plantarum LM1004 (HT-LM1004) isolated from Korean kimchi and further processed has been reported to exert the stimulatory activities on the innate immune system in our previous in vitro and in vivo studies. The aim of the present study was to examine the effect of HT-LM1004 on changes in natural killer (NK) cell activity, the serum concentrations of cytokines and safety parameters in healthy adults aged 19–75 years. In the present randomized, placebo-controlled, double-blind trial, one hundred subjects consumed a capsule containing 2 × 10 10 cells of HT-LM1004 or placebo once per day over eight weeks. Significant increases in NK cell activity (%) were observed in the treatment group after eight weeks of HT-LM1004 ingestion compared to the control group ( p = 0.036). This study also demonstrated a consistent trend of enhanced serum level of interleukin (IL)-12 by dietary intake of HT-LM1004 without any side effects.

HLA-B∗46 associates with rapid HIV disease progression in Asian cohorts and prominent differences in NK cell phenotype

Human leukocyte antigen (HLA) alleles have been linked to HIV disease progression and attributed to differences in cytotoxic T lymphocyte (CTL) epitope representation. These findings are largely based on treatment-naive individuals of European and African ancestry. We assessed HLA associations with HIV-1 outcomes in 1,318 individuals from Thailand and found HLA-B∗46:01 (B∗46) associated with accelerated disease in three independent cohorts. B∗46 had no detectable effect on HIV-specific Tcell responses, but this allele is unusual in containing an HLA-C epitope that binds inhibitory receptors on natural killer (NK) cells. Unbiased transcriptomic screens showed increased NK cell activation in people with HIV, without B∗46, and simultaneous single-cell profiling of surface proteins and transcriptomes revealed a NK cell subset primed for increased responses in the absence of B∗46. These findings support a role for NK cells in HIV pathogenesis, revealed by the unique properties of the B∗46 allele common only in Asia.

Biodegradable nanoparticle-induced sting pathway activation for the treatment of cancer.

e14552 Background: Recent advances in the field of cancer immunology have highlighted the importance of the immune system for eliminating tumors. Numerous studies have shown that tumor-infiltrating immune cells such as antigen-presenting cells (APCs), T cells, and natural killer (NK) cells play critical roles in tumor control. However, the inflammatory anti-tumor immune response is counteracted by the induction of immune regulatory mechanisms within the tumor microenvironment (TME). These findings have led to the development of immune-targeted therapies, which are aimed at activating anti-tumor immune signaling pathways and enhancing anti-tumor immune function. While immunotherapies, have revolutionized the treatment of several solid tumors and leukemias, at best response rates remain low at 25%-30%, and a portion of patients eventually develop resistance to therapy leading to disease progression and mortality. Methods: We have previously reported the potent effects of 3-4 daily intravenous infusions of immune modifying poly(lactic- co-glycolic acid) (PLGA) nanoparticles, ONP-302, free from drugs or other bioactive agents, for the amelioration of acute inflammatory diseases by targeting myeloid cells. The present studies describe a novel use for ONP-302 nanoparticles, employing an altered dosing scheme to reprogram myeloid cells resulting in significant enhancement of tumor immunity. The efficacy of ONP-302 nanoparticles at inducing an anti-tumor immune response was evaluated using syngeneic mouse tumor models. Results: ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased NK cell activation via an IL-15-dependent mechanism. Additionally, ONP-302 treatment increased PD-1/PD-L1 expression in the tumor microenvironment, thereby allowing for functionality of anti-PD-1 for treatment in the B16.F10 melanoma tumor model which is normally unresponsive to monotherapy with anti-PD-1. Conclusions: These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells via activation of the STING/IL-15/NK cell mechanism, as well as increasing anti-PD-1 response rates.

SARS-CoV-2 peptides bind to NKG2D and increase NK cell activity

Immune dysregulation is commonly observed in patients with coronavirus disease 2019 (COVID-19). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces severe lung inflammation and innate immune cell dysregulation. However, the precise interaction between SARS-CoV-2 and the innate immune system is currently unknown. To understand the interaction between SARS-CoV-2 and natural killer (NK) cells, several SARS-CoV-2 S protein peptides capable of binding to the NKG2D receptor were screened by in silico analysis. Among them, two peptides, cov1 and cov2, bound to NK cells and NKG2D receptors. These cov peptides increased NK cytotoxicity toward lung cancer cells, stimulated interferon gamma (IFN-γ) production by NK cells, and likely mediated these responses through the phosphorylation of Vav1, a key downstream-signaling molecule of NKG2D and NK activation genes. The direct interaction between SARS-CoV-2 and NK cells is a novel finding, and modulation of this interaction has potential clinical application as a therapeutic target for COVID-19.

IPSC-Derived Natural Killer Cells and Macrophages Synergistically Kill Acute Myeloid Leukemia Blasts

Despite many advances, the treatment of acute myeloid leukemia (AML) remains challenging, and few patients are cured by therapies other than allogeneic hematopoietic cell transplant. Treatment with natural killer (NK) cells from allogeneic donors is a promising therapy that can achieve remissions in 30-50% of AML patients. To generate an improved cell-based therapy for AML, our group has produced NK cells from induced pluripotent stem cells (iPSCs). iPSC-derived NK cells effectively kill AML cells, but may benefit from additional modifications or combination with other therapies to durably cure AML. Based on studies that demonstrate that targeting the CD47 pathway on macrophages and NK cells improves anti-tumor activity and is an effective treatment for patients with AML, we investigated the combination of iPSC-derived NK cells with iPSC-derived macrophages with and without CD47 blockade for the treatment of AML.AML clinical trials combining anti-CD47 monoclonal antibodies (mAb) with chemotherapy have demonstrated an anti-tumor effect primarily thought to be mediated though a macrophage immune-checkpoint blockade mechanism. To determine if addition of iPSC-derived macrophages can improve the cytotoxicity of NK cells against AML blasts, we co-cultured iPSC-NK cells and MOLM13 or MV-4-11 AML cells with iPSC-macrophages in a standard cytotoxicity assay. Similar results were found for cytotoxicity tests against both AML cell lines (Figure panels A and B). While macrophages alone did not kill AML blasts, the addition of iPSC-macrophages to iPSC-NK cells significantly improved killing of the AML cells by 50% (p<0.01). Addition of an anti-CD47 mAb (B6H12) further increased killing of these AML cells by the iPSC-NK cell + iPSC-macrophage combination treatment by an additional 23% (p<0.01). Intriguingly, the addition of just the anti-CD47 mAb to the iPSC-NK cells also significantly increased anti-AML activity, although this increased killing was consistently lower than what was seen with addition of iPSC-macrophages combined with anti-CD47 and iPSC-NK cells. Addition of the CD47 mAb to iPSC-macrophages without NK cells did not result in increased anti-AML cytotoxicity. We also demonstrated that blockade of SIRPα (the receptor on NK cells for CD47) significantly increased NK cell killing of AML blasts by 16% (p<0.05). Furthermore, the combination of iPSC-NK cells + iPSC-macrophages + SIRPα mAb led to a 37% increase in cytotoxicity compared to iPSC-NK cells + iPSC-macrophages alone (p<0.01).To confirm that addition of anti-CD47 or anti-SIRPα antibodies increased NK cell activation via loss of the inhibitory CD47-SIRPα interaction between NK cells and AML blasts and not by another mechanism, we tested the effect of adding both CD47 mAb and anti-SIRPα antibodies. Compared to addition of either anti-CD47 or anti-SIRPα mAb alone, the combination induced no additional increase in anti-AML activity by the NK cells. These results suggest that the CD47-SIRPα interaction between AML and NK cells is an important inhibitory immune-checkpoint on NK cells. To control for the effect of blocking SIRPα on macrophages, we tested the addition of the SIRPα mAb to iPSC-macrophages. This combination did not improve on the lack of cytotoxicity exhibited by macrophages alone. We also evaluated if ADCC mediated by the anti-CD47 mAb binding AML blasts could account for the increase in cytotoxicity and found that blockade of Fc-receptors on NK cells does not diminish the increase in cytotoxicity seen with addition of the mAb, excluding a role for ADCC.To investigate if these findings with AML could be extended to other hematologic malignancies, we tested the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against the RPMI-8226 multiple myeloma cell line (Figure C). Here, we again demonstrate that CD47 blockade combined with iPSC-macrophages leads to increased NK cell-mediated anti-myeloma activity. In vivo studies testing the combination of iPSC-NK cells, iPSC-macrophages and CD47 mAb against human AML and myeloma cells in mouse xenograft-models are ongoing. Together our results indicate that blocking the CD47-SIRPα interaction between NK cells and tumor cells consistently mediates improved anti-tumor activity. Furthermore, iPSC-derived NK cells and macrophages provide an important, standardized, “off-the-shelf” cell therapy approach that can be translated into novel clinical therapies. [Display omitted] DisclosuresKaufman: Shoreline Biosciences: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qihan Biotech: Consultancy, Current holder of stock options in a privately-held company; VisiCELL Medical: Consultancy, Current holder of stock options in a privately-held company.

Human IL-15 Inhibits NK Cells Specific for Human NK-92 Cells

Recent studies have indicated that natural killer (NK) cells present in peripheral blood mononuclear cells (PBMCs) might be responsible for the somewhat poor outcome of clinical trials conducted with the NK cell line NK-92, as well as chimeric antigen receptor-modified NK-92 cells against leukemias and lymphomas. These NK cells and how their cytotoxic profiles can be altered by some common gamma chain receptor-dependent cytokines or by removal of CD4+ cells have been addressed herein. A time-resolved fluorometric assay using 2.2':6'.2"-terpyridine-6.6"-dicarboxylic acid-labeled NK-92 or K562 as target cells was used for measuring the cytotoxic activity of cytokine-treated PBMCs and purified NK cells. Pre-incubation with 25 ng/ml interleukin 12 (IL-12), IL-15 or IL-21 for 72 h increased NK cell activity against K562 cells by more than 90% (1:25 target:effector ratio), whereas the corresponding NK cell activity against NK-92 cells was reduced by 15.9±0.1% by IL-12 and 50.6±2.9% by IL-15 compared to cells treated with medium alone. IL-7, on the other hand, increased NK activity against K562 to a much smaller extent (10.4±0.4%) and inhibited NK-92 cell lysis by 15.2±0.3%. Interestingly, similar amounts of IL-2 potentiated NK cell activity against both K562 and NK-92 cells by 50.9±0.5% and 14.3±0.9%, respectively. Purification of NK cells with magnetic beads demonstrated that NK cells indeed were responsible for the observed cytotoxic activity against both NK-92 cells (58.5±9.10%, 1:100 target:effector ratio) and K562 cells (81.6±9.57%, 1:100 target:effector ratio). Elimination of CD4+ cells from PBMCs did not alter the NK activity profile. This study highlights a problem that might arise with immune-based NK-92 and chimeric antigen receptor-transduced NK-92 cell therapies and pinpoints the need for evaluating new NK-like cell lines.

Adaptive Natural Killer Cell Functional Recovery in Hepatitis C Virus Cured Patients.

Current evidence suggests that dysfunctional natural killer (NK) cell responses during hepatitis C virus (HCV) infection can be restored after viral eradication with direct acting antivirals (DAAs). However, the fate of the recently described adaptive NK cell population, endowed with increased ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC), during HCV infection is poorly defined, while no study has explored the effects of DAAs on this NK subset. We performed multicolor flow cytometry to investigate CD57+ FcεRIγneg adaptive and FcεRIγpos conventional NK cell phenotype and function before and after DAA treatment in 59 patients chronically infected with HCV, 39 with advanced liver fibrosis, and 20 with mild-moderate liver fibrosis. Moreover, bulk NK cell phenotype and function were analyzed after cytokine activation following contact with K562 target cells. The proportion of FcεRIγneg NK cells in patients with HCV was associated with increased HCV load at baseline, and it was significantly reduced after treatment. Patients with an advanced fibrosis stage displayed increased NK cell activation and exhaustion markers that normalized after therapy. Of note, adaptive NK cells from patients with HCV were characterized by increased programmed death receptor 1 expression and reduced ADCC activity at baseline. DAA treatment restored ADCC ability and reduced programmed death receptor 1 expression. HCV profoundly affects the frequency, phenotype, and function of adaptive NK cells. DAA therapy restores a normal adaptive NK phenotype and enhances interferon-gamma production by this cell subset.

Efficacy and safety of CAEC (Canavalia gladiata arctium lappa extract complex) on immune function enhancement: An 8 week, randomised, double-blind, placebo-controlled clinical trial

• CAEC enhanced immune function by stimulating NK cell activity and IL-10 expression. • CAEC significantly increased NK cell activity at E:T = 25:1 and 50:1 after 8 weeks. • CAEC significantly increased the level of IL-10 after 8 weeks. • CAEC will be able to serve as a functional food for stimulating immunity. The aim of this study was to evaluate the efficacy and safety of CAEC (Canavalia gladiata arctium lappa extract complex) in adults with white blood cells between 3 and 8 × 10 3 cells/µL by assessing natural killer (NK) cell activity and immune-related biomarkers. Overall, 100 participants (CAEC group = 50, placebo group = 50) were randomised, and administered CAEC or placebo once daily for 8 weeks, with an evaluation visit performed every 4 weeks. NK cell activity, the primary outcome, significantly increased in CAEC when compared with the placebo at effector-to-target (E:T) ratios of 25:1 and 50:1 after 8 weeks. Compared with the placebo group, IL-10 demonstrated significant differences at week 8 in the CAEC group. Our findings demonstrate that CAEC is safe and enhances immune function by stimulating NK cell activity through IL-10 expression. Hence, we anticipate that CAEC can be developed as a functional food for stimulating immunity.

Low-dose influenza vaccine Grippol Quadrivalent with adjuvant Polyoxidonium induces a T helper-2 mediated humoral immune response and increases NK cell activity

The influenza vaccine Grippol® Quadrivalent (GQ) is a new vaccine, containing the adjuvant Polyoxidonium® and recombinant hemagglutinins from 4 strains of the influenza virus in amount of 5–6 μg of each hemagglutinin per human dose. These doses of antigens are about 3 times less than the standard dose recommended by WHO. We sought to characterize the immune response to the GQ vaccine and to determine the contribution of the adjuvant in this response. BALB/c mice were vaccinated with GQ or with adjuvant-free antigen mixtures (AGs). Then, the antibody response, the number of memory T cells in the spleen, and the functional properties of splenocytes were determined. The vaccine GQ has been shown to induce antibodies to all 4 influenza hemagglutinins. The vaccination with GQ caused a strong increase in the AG-induced proliferation and production of Th2 cytokines ex vivo. These effects were equal to effect achieved by standard dose of antigens. Vaccination also caused the accumulation of CD4+ large lymphocytes with the phenotype of central and effector memory T cells in the spleen. The GQ vaccine enhanced the cytolytic activity of natural killer (NK) cells, whereas the adjuvant-free mixture of AGs in lowered and standard doses did not affect NK activity. We did not find a noticeable response of Th1 and CD8+ T cells to vaccination. In vitro, the GQ vaccine stimulated the maturation of human monocyte-derived dendritic cells (DCs) enhancing the expression of HLA-DR, CD80, CD83, CD86 and ICOSL molecules. Polyoxidonium without AGs also induced expression of ICOSL, which plays an important role in T-dependent humoral immune response. In summary, the low-dose influenza vaccine GQ with Polyoxidonium adjuvant is immunogenic, induces a Th2-polarized T-cell response and CD4+ memory T cells maturation, activates the production of antibodies to influenza hemagglutinins, and increases the activity of NK cells.