Search

Background: High levels of the polyunsaturated fatty acid arachidonic acid (AA) within the ovarian carcinoma (OC) microenvironment correlate with reduced relapse-free survival. Furthermore, OC progression is tied to compromised immunosurveillance, partially attributed to the impairment of natural killer (NK) cells. However, potential connections between AA and NK cell dysfunction in OC have not been studied. Methods: We employed a combination of phosphoproteomics, transcriptional profiling and biological assays to investigate AA's impact on NK cell functions. Results: AA (i) disrupts interleukin-2/15-mediated expression of pro-inflammatory genes by inhibiting STAT1-dependent signaling, (ii) hampers signaling by cytotoxicity receptors through disruption of their surface expression, (iii) diminishes phosphorylation of NKG2D-induced protein kinases, including ERK1/2, LYN, MSK1/2 and STAT1, and (iv) alters reactive oxygen species production by transcriptionally upregulating detoxification. These modifications lead to a cessation of NK cell proliferation and a reduction in cytotoxicity. Conclusion: Our findings highlight significant AA-induced alterations in the signaling network that regulates NK cell activity. As low expression of several NK cell receptors correlates with shorter OC patient survival, these findings suggest a functional linkage between AA, NK cell dysfunction and OC progression. Plain English summary: High levels of the fatty acid arachidonic acid (AA) in the ovarian cancer environment are associated with shorter survival. Ovarian cancer progression is linked to weakened immune defenses, partly due to malfunctioning natural killer (NK) cells. We used various scientific methods to study how AA affects NK cell function. Our results show that AA significantly changes the signaling processes that control NK cell activity. Specifically, AA (i) disrupts the expression of pro-inflammatory genes, (ii) impairs the function of receptors that activate killing mechanisms by preventing them from appearing on the cell surface, (iii) reduces the activity of intracellular signaling proteins that are activated by these receptors and (iv) changes the production of reactive oxygen species. These changes stop NK cells from multiplying and reduce their ability to kill cancer cells, suggesting that AA contributes to NK cell dysfunction and cancer progression. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cell therapy represents an attractive immunotherapy approach against recurrent epithelial ovarian cancer (EOC), as EOC is sensitive to NK cellmediated cytotoxicity. However, NK cell antitumor activity is dampened by suppressive factors in EOC patient ascites. Here, we integrated functional assays, soluble factor analysis, high-dimensional flow cytometry cellular component data and clinical parameters of advanced EOC patients to study the mechanisms of ascites-induced inhibition of NK cells. Using a suppression assay, we found that ascites from EOC patients strongly inhibits peripheral blood-derived NK cells and CD34+ progenitor-derived NK cells, albeit the latter were more resistant. Interestingly, we found that higher ascites-induced NK cell inhibition correlated with reduced progression-free and overall survival in EOC patients. Furthermore, we identified transforming growth factor (TGF)-b1 to correlate with ascites-induced NK cell dysfunction and reduced patient survival. In functional assays, we showed that proliferation and anti-tumor reactivity of CD34+ progenitor-derived NK cells are significantly affected by TGF-b1 exposure. Moreover, inhibition of TGF-b1 signaling with galunisertib partly restored NK cell functionality in some donors. For the cellular components, we showed that the secretome is associated with a different composition of CD45+ cells between ascites of EOC and benign reference samples with higher proportions of macrophages in the EOC patient samples. Furthermore, we revealed that higher TGF-b1 levels are associated with the presence of M2-like macrophages, B cell populations and T-regulatory cells in EOC patient ascites. These findings reveal that targeting TGF-b1 signaling could increase NK cell immune responses in high-grade EOC patients. [ABSTRACT FROM AUTHOR]

The serine/threonine/tyrosine kinase 1 (STYK1) is a receptor protein-tyrosine kinase (RPTK)-like molecule that is detected in several human organs. STYK1 plays an important role in promoting tumorigenesis and metastasis in various cancers. By analyzing the expression of RTKs in immune cells in the database of 2013 Immunological Genome Project, we found that STYK1 was principally expressed in NK cells. In order to investigate the function of STYK1, we used CRISPR/Cas9 technology to generate STYK1-deleted mice, we found STYK1 deletion mice have normal number, development, and function of NK cells in spleen and bone marrow in tumor-free resting state. To examine the tumor surveillance of STYK1 in vivo, we utilized a variety of tumor models, including NK cell-specific target cell (ß2M and RMA-S) clearance experiments in vivo, subcutaneous and intravenous injection of B16F10 melanoma model, and the spontaneous breast cancer model MMTV-PyMT. Surprisingly, we discovered that deletion of the oncogenic STYK1 promoted the four-model tumor progression, and we observed a reduction of NK cell accumulation in the tumor tissues of STYK1 deletion mice compared to WT mice. In order to study the mechanism of STYK1 in NK, RNA sequence of STYK1−/− and WT NK have unveiled a disparity in the signaling pathways linked to migration and adhesion in STYK1−/− NK cells. Further analysis of chemokine receptors associated with NK cell migration revealed that STYK1-deficient NK cells exhibited a significant reduction in CCR2 expression. The STYK1 expression was negatively associated with tumor progression in glioma patients. Overall, our study found the expression of STYK1 in NK cell mediates NK cell anti-tumor response through regulating CCR2 and infiltrating into tumor tissue. [ABSTRACT FROM AUTHOR]

Cannabidiol (CBD), the major non-psychoactive component of cannabis, exhibits anti-inflammatory properties, but less is known about the immunomodulatory potential of CBD on activated natural killer (NK) cells and/or their targets. Many tumor cells present heat shock protein 70 (Hsp70) on their cell surface in a tumor-specific manner and although a membrane Hsp70 (mHsp70) positive phenotype serves as a target for Hsp70-activated NK cells, a high mHsp70 expression is associated with tumor aggressiveness. This study investigated the immuno-modulatory potential of CBD on NK cells stimulated with TKD Hsp70 peptide and IL-2 (TKD+IL-2) and also on HCT116 p53wt and HCT116 p53-/- colorectal cancer cells exhibiting high and low basal levels of mHsp70 expression. Apart from an increase in the density of NTB-A and a reduced expression of LAMP-1, the expression of all other activatory NK cell receptors including NKp30, NKG2D and CD69 which are significantly up-regulated after stimulation with TKD+IL-2 remained unaffected after a co-treatment with CBD. However, the release of major pro-inflammatory cytokines by NK cells such as interferon-γ (IFN-γ) and the effector molecule granzyme B (GrzB) was significantly reduced upon CBD treatment. With respect to the tumor target cells, CBD significantly reduced the elevated expression of mHsp70 but had no effect on the low basal mHsp70 expression. Expression of other NK cell ligands such as MICA and MICB remained unaffected, and the NK cell ligands ULBP and B7-H6 were not expressed on these target cells. Consistent with the reduced mHsp70 expression, treatment of both effector and target cells with CBD reduced the killing of high mHsp70 expressing tumor cells by TKD+IL-2+CBD pre-treated NK cells but had no effect on the killing of low mHsp70 expressing tumor cells. Concomitantly, CBD treatment reduced the TKD+IL-2 induced increased release of IFN-γ, IL-4, TNF-α and GrzB, but CBD had no effect on the release of IFN-α when NK cells were co-incubated with tumor target cells. Cannabidiol (CBD) may potentially diminish the anti-tumor effectiveness of TKD+IL-2 activated natural killer (NK) cells. • Cannabidiol(CBD), as a potential antitumor agent, the immunomodulatory effects on activated NK cells is remain unclear. • Many tumor cells present heat shock protein 70 (Hsp70) on their cell surface and a membrane Hsp70 (mHsp70) positive phenotype serves as a target for Hsp70-activated NK cells. • Cannabidiol (CBD) may potentially diminish the anti-tumor effectiveness of TKD+IL-2 activated natural killer (NK) cells. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to investigate the impact of surgical intervention on peripheral blood T lymphocyte subsets and natural killer (NK) cell activity in pediatric patients with obstructive sleep apnea hypopnea syndrome (OSAHS). Methods: A total of 36 OSAHS children, 32 children with tonsillar hypertrophy, and 30 healthy children were enrolled. Clinical data and polysomnography (PSG) results were collected. Peripheral blood samples were analyzed for T lymphocyte subsets, NK cells, and cytokine levels including Th1 (IFN-γ, IL-2, TNF-α), Th2 (IL-4, IL-10), and Th17 (IL-17). Results: At baseline, OSAHS children exhibited lower LSaO2 levels and higher AHI values compared to healthy children. They also showed decreased percentages of CD3 + T cells, CD4 + T cells, NK cells, and elevated CD8 + T cells and CD4+/CD8 + ratio. Levels of IFN-γ, IL-2, TNF-α, IL-4, and IL-17 were significantly lower in OSAHS children. Post-surgery improvements were observed in LSaO2, AHI, and immune markers at 3 months and 6 months. Pearson's correlation analysis revealed significant associations between LSaO2, AHI, and peripheral blood immune parameters at baseline and 6 months post-surgery. Conclusion: Surgical intervention in pediatric OSAHS influences peripheral blood T lymphocyte subsets and NK cell activity. Early intervention and monitoring of immune function are crucial for the recovery and healthy development of affected children. [ABSTRACT FROM AUTHOR]

Background: The relationship between chronic hepatitis B (CHB) infection and natural killer (NK) cell dysfunction is well-established, but the specific role of HBV viral antigens in driving NK cell impairment in patients with CHB remains unclear. This study investigates the modulatory effects of hepatitis B virus subviral particles (HBVsvp, a representative model for HBsAg) on the phenotypic regulation (activating and inhibitory receptors), cytokine production and cytotoxic potential of peripheral blood mononuclear cell-derived natural killer cells (PBMCs-derived NK cell), which contributes to NK cell dysfunction in CHB infection, potentially serving as an effective HBV immune evasion strategy by the virus. Methods: NK cells were isolated from peripheral blood of patients with CHB (n=5) and healthy individuals (n=5), stimulated with HBVsvp. Subsequent flow cytometric characterization involved assessing changes in activating (NKp46 and NKG2D) and inhibitory (CD94) receptors expression, quantifying TNF-α and IFN-δ cytokine secretion, and evaluating the cytotoxic response against HepG2.2.15 cells with subsequent HBVsvp quantification. Results: In CHB patients, in vitro exposure of PBMCs-derived NK cell with HBVsvp (represent HBsAg model) significantly reduced NK cell-activating receptors expression (P = 0.022), increased expression of CD94 + NK cells (p = 0.029), accompanied with a reduced TNF-α-IFN-γ cytokine levels, and impaired cytotoxic capacity (evidenced by increased cell proliferation and elevated HBVsvp levels in co-cultures with HepG2.2.15 cells in a time-dependent), relative to healthy donors. Conclusion: These findings suggest that HBVsvp may induce dysfunctional NK cell responses characterized by phenotypic imbalance with subsequent reduction in cytokine and cytotoxic levels, indicating HBVsvp immunosuppressive effect that compromises antiviral defense in CHB patients. These data enhance our understanding of NK cell interactions with HBsAg and highlight the potential for targeting CD94 inhibitory receptors to restore NK cell function as an immunotherapeutic approach. Further clinical research is needed to validate these observations and establish their utility as reliable biomarkers. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is the most common type of primary malignant brain tumor and has a dismal overall survival rate. To date, no GBM therapy has yielded successful results in survival for patients beyond baseline surgical resection, radiation, and chemotherapy. Immunotherapy has taken the oncology world by storm in recent years and there has been movement from researchers to implement the immunotherapy revolution into GBM treatment. Natural killer (NK) cell-based immunotherapies are a rising candidate to treat GBM from multiple therapeutic vantage points: monoclonal antibody therapy targeting tumor-associated antigens (TAAs), immune checkpoint inhibitors, CAR-NK cell therapy, Bi-specific killer cell engagers (BiKEs), and more. NK therapies often focus on tumor antigens for targeting. Here, we reviewed some common targets analyzed in the fight for GBM immunotherapy relevant to NK cells: EGFR, HER2, CD155, and IL-13Rα2. We further propose investigating the Lectin-like Transcript 1 (LLT1) and cell surface proliferating cell nuclear antigen (csPCNA) as targets for NK cell-based immunotherapy. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) cell therapies utilize CARs to redirect immune cells towards cancer cells expressing specific antigens like human epidermal growth factor receptor 2 (HER2). Despite their potential, CAR T cell therapies exhibit variable response rates and adverse effects in some patients. Non-invasive molecular imaging can aid in predicting patient outcomes by tracking infused cells post-administration. CAR-T cells are typically autologous, increasing manufacturing complexity and costs. An alternative approach involves developing CAR natural killer (CAR-NK) cells as an off-the-shelf allogeneic product. In this study, we engineered HER2-targeted CAR-NK cells co-expressing the positron emission tomography (PET) reporter gene human sodium-iodide symporter (NIS) and assessed their therapeutic efficacy and PET imaging capability in a HER2 ovarian cancer mouse model. NK-92 cells were genetically modified to express a HER2-targeted CAR, the bioluminescence imaging reporter Antares, and NIS. HER2-expressing ovarian cancer cells were engineered to express the bioluminescence reporter Firefly luciferase (Fluc). Co-culture experiments demonstrated significantly enhanced cytotoxicity of CAR-NK cells compared to naive NK cells. In vivo studies involving mice with Fluc-expressing tumors revealed that those treated with CAR-NK cells exhibited reduced tumor burden and prolonged survival compared to controls. Longitudinal bioluminescence imaging demonstrated stable signals from CAR-NK cells over time. PET imaging using the NIS-targeted tracer 18F-tetrafluoroborate ([18F]TFB) showed significantly higher PET signals in mice treated with NIS-expressing CAR-NK cells. Overall, our study showcases the therapeutic potential of HER2-targeted CAR-NK cells in an aggressive ovarian cancer model and underscores the feasibility of using human-derived PET reporter gene imaging to monitor these cells non-invasively in patients. [ABSTRACT FROM AUTHOR]

Abstract Background High levels of the polyunsaturated fatty acid arachidonic acid (AA) within the ovarian carcinoma (OC) microenvironment correlate with reduced relapse-free survival. Furthermore, OC progression is tied to compromised immunosurveillance, partially attributed to the impairment of natural killer (NK) cells. However, potential connections between AA and NK cell dysfunction in OC have not been studied. Methods We employed a combination of phosphoproteomics, transcriptional profiling and biological assays to investigate AA’s impact on NK cell functions. Results AA (i) disrupts interleukin-2/15-mediated expression of pro-inflammatory genes by inhibiting STAT1-dependent signaling, (ii) hampers signaling by cytotoxicity receptors through disruption of their surface expression, (iii) diminishes phosphorylation of NKG2D-induced protein kinases, including ERK1/2, LYN, MSK1/2 and STAT1, and (iv) alters reactive oxygen species production by transcriptionally upregulating detoxification. These modifications lead to a cessation of NK cell proliferation and a reduction in cytotoxicity. Conclusion Our findings highlight significant AA-induced alterations in the signaling network that regulates NK cell activity. As low expression of several NK cell receptors correlates with shorter OC patient survival, these findings suggest a functional linkage between AA, NK cell dysfunction and OC progression.

Abstract The serine/threonine/tyrosine kinase 1 (STYK1) is a receptor protein-tyrosine kinase (RPTK)-like molecule that is detected in several human organs. STYK1 plays an important role in promoting tumorigenesis and metastasis in various cancers. By analyzing the expression of RTKs in immune cells in the database of 2013 Immunological Genome Project, we found that STYK1 was principally expressed in NK cells. In order to investigate the function of STYK1, we used CRISPR/Cas9 technology to generate STYK1-deleted mice, we found STYK1 deletion mice have normal number, development, and function of NK cells in spleen and bone marrow in tumor-free resting state. To examine the tumor surveillance of STYK1 in vivo, we utilized a variety of tumor models, including NK cell-specific target cell (ß2M and RMA-S) clearance experiments in vivo, subcutaneous and intravenous injection of B16F10 melanoma model, and the spontaneous breast cancer model MMTV-PyMT. Surprisingly, we discovered that deletion of the oncogenic STYK1 promoted the four-model tumor progression, and we observed a reduction of NK cell accumulation in the tumor tissues of STYK1 deletion mice compared to WT mice. In order to study the mechanism of STYK1 in NK, RNA sequence of STYK1−/− and WT NK have unveiled a disparity in the signaling pathways linked to migration and adhesion in STYK1−/− NK cells. Further analysis of chemokine receptors associated with NK cell migration revealed that STYK1-deficient NK cells exhibited a significant reduction in CCR2 expression. The STYK1 expression was negatively associated with tumor progression in glioma patients. Overall, our study found the expression of STYK1 in NK cell mediates NK cell anti-tumor response through regulating CCR2 and infiltrating into tumor tissue.

Glioblastoma (GBM) poses a significant challenge in oncology and stands as the most aggressive form of brain cancer. A primary contributor to its relentless nature is the stem‐like cancer cells, called glioblastoma stem cells (GSCs). GSCs have the capacity for self‐renewal and tumorigenesis, leading to frequent GBM recurrences and complicating treatment modalities. While natural killer (NK) cells exhibit potential in targeting and eliminating stem‐like cancer cells, their efficacy within the GBM microenvironment is limited due to constrained infiltration and function. To address this limitation, novel investigations focusing on boosting NK cell activity against GSCs are imperative. This study presents two streamlined image‐based assays assessing NK cell migration and cytotoxicity towards GSCs. It details protocols and explores the strengths and limitations of these methods. These assays could aid in identifying novel targets to enhance NK cell activity towards GSCs, facilitating the development of NK cell‐based immunotherapy for improved GBM treatment.

The lymph nodes are vital to enable adaptive immune responses to infection. Natural killer (NK) cells are cytotoxic lymphocytes that directly kill cancer cells and modulate the activation of other immune cells during anti-tumour immune response. NK cells in the lymph nodes are involved in the regulation of T-cell and B-cell populations and the clearance of viral infections. In solid tumours, lymph nodes are a frequent site of metastasis and immune cell priming, whilst in haematological malignancies, tumour cells can proliferate in the lymph nodes. Thus, lymph nodes are an important site in anti-tumour immunity and therapy resistance. It is therefore crucial to identify strategies to increase recruitment and overcome suppression of NK cells in the lymph node microenvironment to improve tumour clearance. In this review, we summarise the literature interrogating NK cell phenotype and function in the lymph nodes in the context of infection and cancer and evaluate both current and potential strategies to mobilise and activate NK cells within the lymph nodes of cancer patients. [ABSTRACT FROM AUTHOR]

β-(1,3/1,4)-glucan is a major component of cereal grains, such as oats and barley. In this study, we investigated the effects of cooked waxy barley, which contains β-(1,3/1,4)-glucan, on upper respiratory tract physical symptoms and mood status by performing a randomized, parallel-group, comparative trial. The primary outcome was assessed using the Wisconsin Upper Respiratory Symptom Survey-21 and Profile of Mood States second edition. Twenty-seven healthy Japanese adult participants were supplemented with 100 g of cooked waxy barley (containing 1.8 g of β-glucan) or 100 g of cooked white rice daily for 8 weeks. Participants receiving cooked waxy barley reported a reduction in cumulative days of sneezing (p < 0.05) and feeling tired (p < 0.0001) compared with the control group. After the intervention period, there were significantly less severe nasal symptoms, such as runny nose, plugged nose, and sneezing (p < 0.05), and a significantly greater reduction of the Tension-Anguish score (p < 0.05) in the barley group than in the control group. This study suggests that supplementation of cooked waxy barley containing β-(1,3/1,4)-glucan prevents or alleviates nasal upper respiratory tract symptoms and improves mood status. The findings of this study should be confirmed by double-blind trials with a larger number of participants. [ABSTRACT FROM AUTHOR]

Natural killer cell‐derived extracellular vesicles (NK‐EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK‐EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine‐based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK‐EVs against leukaemia K562 cells (suspension model) and breast cancer MDA‐MB‐231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin‐based cell viability potency assay reliably and reproducibly measured a dose‐response of NK‐EVs' cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra‐run and inter‐run variability. The assay signal showed specificity and selectivity of NK‐EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5‐hour treatment with NK‐EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA‐MB‐231 cells per test with a consistent range. Importantly, NK‐EVs' dose‐response for cytotoxicity showed a strong correlation (|ρ| ∼ 0.8) with the levels of known cytotoxic factors associated with the NK‐EVs' corona (FasL, GNLY, GzmB, PFN and IFN‐γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK‐EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA‐binding reporter assay and confluence assay), our results support this highly sensitive resazurin‐based viability potency assay as a high‐throughput and quantitative method for assessing NK‐EVs' cytotoxicity against both suspension and adherent cancer models for evaluating NK‐EVs' biotherapeutics. [ABSTRACT FROM AUTHOR]

Simple Summary: The ADCC bioassays used to assess mAb-induced ADCC require continued development/refinement to meet regulatory requirements. We used trastuzumab and a lactate de-hydrogenase (LDH)-based ADCC bioassay as a model to define critical parameters of the ADCC bioassay. We find that a 4 to 24 h recovery cultivation is required to restore peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cell activity toward ADCC when using cryopreserved PBMCs. Several bioassay parameters, including preparation of effector cells, E/T ratio, target cell selection, bioassay media components, and treatment time can also influence the data quality of the ADCC activity. The mechanisms of mAb-induced ADCC have been well established. However, the ADCC bioassays used to quantify mAb-induced ADCC require continued development/refinement to properly assess and compare the potency of newly developed therapeutic mAbs and biosimilars to meet regulatory requirements. We used trastuzumab and a lactate dehydrogenase (LDH)-based ADCC bioassay as a model to define critical parameters of the ADCC bioassay, describing how several bioassay parameters, including preparation of effector cells, E/T ratio, target cell selection, bioassay media components, and treatment time can influence the data quality of the ADCC activity. We confirm that a 4 to 24 h recovery cultivation is required to restore peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cell activity toward ADCC when using cryopreserved PBMCs. Furthermore, we delineated the cellular mechanisms underlying the restored ADCC activity following the recovery cultivation. We observed that CD69, an early marker of NK cell activation, was upregulated and a new subset CD56dim/CD16dim population was dramatically increased in the recovered NK cells, which led to an increase in expression and secretion of perforin, granzyme B, and cytokine production. This study provides comprehensive technical insights into ADCC bioassay optimization to inform trastuzumab biosimilar development. The knowledge gained from this study can also be leveraged to guide bioassay development for therapeutic mAbs with ADCC as the primary mechanism of action. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) poses a significant challenge in oncology and stands as the most aggressive form of brain cancer. A primary contributor to its relentless nature is the stem‐like cancer cells, called glioblastoma stem cells (GSCs). GSCs have the capacity for self‐renewal and tumorigenesis, leading to frequent GBM recurrences and complicating treatment modalities. While natural killer (NK) cells exhibit potential in targeting and eliminating stem‐like cancer cells, their efficacy within the GBM microenvironment is limited due to constrained infiltration and function. To address this limitation, novel investigations focusing on boosting NK cell activity against GSCs are imperative. This study presents two streamlined image‐based assays assessing NK cell migration and cytotoxicity towards GSCs. It details protocols and explores the strengths and limitations of these methods. These assays could aid in identifying novel targets to enhance NK cell activity towards GSCs, facilitating the development of NK cell‐based immunotherapy for improved GBM treatment. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor‐ (CAR‐)modified T cells have been successfully used to treat blood cancer. With the improved research on anti‐tumour adoptive cell therapy, researchers have focused on immune cells other than T lymphocytes. Natural killer (NK) cells have received widespread attention as barriers to natural immunity. Compared to T lymphocyte‐related adoptive cell therapy, the use of NK cells to treat tumours does not cause graft‐versus‐host disease, significantly improving immunity. Moreover, NK cells have more sources than T cells, and the related modified cells are less expensive. NK cells function through several pathways in anti‐tumour mechanisms. Currently, many anti‐tumour clinical trials have used NK cell‐related adoptive cell therapies. In this review, we have summarized the recent progress in NK cell‐related adoptive cellular immunotherapy for tumour treatment and propose the current challenges faced by CAR‐NK cell therapy. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cell therapy represents an attractive immunotherapy approach against recurrent epithelial ovarian cancer (EOC), as EOC is sensitive to NK cell-mediated cytotoxicity. However, NK cell antitumor activity is dampened by suppressive factors in EOC patient ascites. Here, we integrated functional assays, soluble factor analysis, high-dimensional flow cytometry cellular component data and clinical parameters of advanced EOC patients to study the mechanisms of ascites-induced inhibition of NK cells. Using a suppression assay, we found that ascites from EOC patients strongly inhibits peripheral blood-derived NK cells and CD34+ progenitor-derived NK cells, albeit the latter were more resistant. Interestingly, we found that higher ascites-induced NK cell inhibition correlated with reduced progression-free and overall survival in EOC patients. Furthermore, we identified transforming growth factor (TGF)-β1 to correlate with ascites-induced NK cell dysfunction and reduced patient survival. In functional assays, we showed that proliferation and anti-tumor reactivity of CD34+ progenitor-derived NK cells are significantly affected by TGF-β1 exposure. Moreover, inhibition of TGF-β1 signaling with galunisertib partly restored NK cell functionality in some donors. For the cellular components, we showed that the secretome is associated with a different composition of CD45+ cells between ascites of EOC and benign reference samples with higher proportions of macrophages in the EOC patient samples. Furthermore, we revealed that higher TGF-β1 levels are associated with the presence of M2-like macrophages, B cell populations and T-regulatory cells in EOC patient ascites. These findings reveal that targeting TGF-β1 signaling could increase NK cell immune responses in high-grade EOC patients.

BackgroundThe relationship between chronic hepatitis B (CHB) infection and natural killer (NK) cell dysfunction is well-established, but the specific role of HBV viral antigens in driving NK cell impairment in patients with CHB remains unclear. This study investigates the modulatory effects of hepatitis B virus subviral particles (HBVsvp, a representative model for HBsAg) on the phenotypic regulation (activating and inhibitory receptors), cytokine production and cytotoxic potential of peripheral blood mononuclear cell-derived natural killer cells (PBMCs-derived NK cell), which contributes to NK cell dysfunction in CHB infection, potentially serving as an effective HBV immune evasion strategy by the virus.MethodsNK cells were isolated from peripheral blood of patients with CHB (n=5) and healthy individuals (n=5), stimulated with HBVsvp. Subsequent flow cytometric characterization involved assessing changes in activating (NKp46 and NKG2D) and inhibitory (CD94) receptors expression, quantifying TNF-α and IFN- γ cytokine secretion, and evaluating the cytotoxic response against HepG2.2.15 cells with subsequent HBVsvp quantification.ResultsIn CHB patients, in vitro exposure of PBMCs-derived NK cell with HBVsvp (represent HBsAg model) significantly reduced NK cell-activating receptors expression (P = 0.022), increased expression of CD94 + NK cells (p = 0.029), accompanied with a reduced TNF-α - IFN-γ cytokine levels, and impaired cytotoxic capacity (evidenced by increased cell proliferation and elevated HBVsvp levels in co-cultures with HepG2.2.15 cells in a time-dependent), relative to healthy donors.ConclusionThese findings suggest that HBVsvp may induce dysfunctional NK cell responses characterized by phenotypic imbalance with subsequent reduction in cytokine and cytotoxic levels, indicating HBVsvp immunosuppressive effect that compromises antiviral defense in CHB patients. These data enhance our understanding of NK cell interactions with HBsAg and highlight the potential for targeting CD94 inhibitory receptors to restore NK cell function as an immunotherapeutic approach. Further clinical research is needed to validate these observations and establish their utility as reliable biomarkers.

The transactivator of transcription (Tat) is a HIV regulatory protein which promotes viral replication and chemotaxis. HIV-1 shows extensive genetic diversity, HIV-1 subtype C being the most dominant subtype in the world. Our hypothesis is the frequency of CSF CD3+CD56+ and CD3-CD56dim is reduced in HIV-1C compared to HIV-1B due to the Tat C30S31 substitution in HIV-1C. 34 CSF and paired blood samples (PWH, n = 20; PWoH, n = 14) were studied. In PWH, the percentage of CD3+CD56+ was higher in CSF than in blood (p

Abstract Background Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy and the most frequently acute leukemia of stem cell precursors and the myeloid derivatives in adult. Longitudinal studies have indicated the therapeutic landscape and drug resistance for patients with AML are still intractable, which largely attribute to the deficiency of detailed information upon the pathogenesis. Methods In this study, we compared the cellular phenotype of resident NK cells (rAML-NKs, rHD-NKs) and expanded NK cells (eAML-NKs, eHD-NKs) from bone marrow of AML patients (AML) and healthy donors (HD). Then, we took advantage of the co-culture strategy for the evaluation of the in vitro cytotoxicity of NK cells upon diverse tumor cell lines (e.g., K562, Nalm6, U937). With the aid of RNA-sequencing (RNA-SEQ) and bioinformatics analyses (e.g., GOBP analysis, KEGG analysis, GSEA, volcano plot), we verified the similarities and differences of the omics features between eAML-NKs and eHD-NKs. Results Herein, we verified the sharp decline in the content of total resident NK cells (CD3−CD56+) in rAML-NKs compared to rHD-NKs. Differ from the expanded eHD-NKs, eAML-NKs revealed decline in diverse NK cell subsets (NKG2D+, CD25+, NKp44+, NKp46+) and alterations in cellular vitality but conservations in cytotoxicity. According to transcriptomic analysis, AML-NKs and HD-NKs showed multifaceted distinctions in gene expression profiling and genetic variations. Conclusions Collectively, our data revealed the variations in the cytobiological and transcriptomic features between AML-NKs and HD-NKs in bone marrow environment. Our findings would benefit the further development of novel biomarkers for AML diagnosis and NK cell-based cytotherapy in future.

Background: Natural killer (NK) cells, CD3− lymphocytes, are critical players in cancer immune surveillance. This study aimed to assess two types of CD3− NK cell classifications (subsets), that is, convectional subsets (based on CD56 and CD16 expression) and new subsets (based on CD56, CD27, and CD11b expression), and their functional molecules in the peripheral blood of patients with breast cancer (BC) in comparison with healthy donors (HDs). Methods: Thirty untreated females with BC and 20 age‐matched healthy women were enrolled. Peripheral blood samples were collected and directly incubated with fluorochrome‐conjugated antibodies against CD3, CD56, CD16, CD27, CD11b, CD96, NKG2C, NKG2D, NKp44, CXCR3, perforin, and granzyme B. Red blood cells were then lysed using lysing solution, and the stained cells were acquired on four‐color flow cytometer. Result: Our results indicated 15% of lymphocytes in peripheral blood of patients with BC and HDs had NK cells phenotype. However, the frequency of total NK cells (CD3−CD56+), and NK subsets (based on conventional and new classifications) was not significantly different between patients and HDs. We observed mean fluorescent intensity (MFI) of CXCR3 in total NK cells (p =.02) and the conventional cytotoxic (CD3−CD56dim CD16+) NK cells (p =.03) were significantly elevated in the patients with BC compared to HDs. Despite this, the MFI of granzyme B expression in conventional regulatory (CD3−CD56brightCD16−/+) NK cells and CD3−CD56−CD16+ NK cells (p =.03 and p =.004, respectively) in the patients was lower than healthy subjects. Conclusion: The higher expression of chemokine receptor CXCR3 on total NK cells in patients with BC may be associated with increased chemotaxis‐related NK cell infiltration. However, lower expression of granzyme B in conventional regulatory NK cells and CD3−CD56−CD16+ NK cells in the patients compared to HDs suggests reduced cytotoxic activity of the NK cells in BC. These results might demonstrate accumulating NK subsets with a dysfunctional phenotype in the peripheral blood of patients with BC. [ABSTRACT FROM AUTHOR]

Background: Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy and the most frequently acute leukemia of stem cell precursors and the myeloid derivatives in adult. Longitudinal studies have indicated the therapeutic landscape and drug resistance for patients with AML are still intractable, which largely attribute to the deficiency of detailed information upon the pathogenesis. Methods: In this study, we compared the cellular phenotype of resident NK cells (rAML-NKs, rHD-NKs) and expanded NK cells (eAML-NKs, eHD-NKs) from bone marrow of AML patients (AML) and healthy donors (HD). Then, we took advantage of the co-culture strategy for the evaluation of the in vitro cytotoxicity of NK cells upon diverse tumor cell lines (e.g., K562, Nalm6, U937). With the aid of RNA-sequencing (RNA-SEQ) and bioinformatics analyses (e.g., GOBP analysis, KEGG analysis, GSEA, volcano plot), we verified the similarities and differences of the omics features between eAML-NKs and eHD-NKs. Results: Herein, we verified the sharp decline in the content of total resident NK cells (CD3−CD56+) in rAML-NKs compared to rHD-NKs. Differ from the expanded eHD-NKs, eAML-NKs revealed decline in diverse NK cell subsets (NKG2D+, CD25+, NKp44+, NKp46+) and alterations in cellular vitality but conservations in cytotoxicity. According to transcriptomic analysis, AML-NKs and HD-NKs showed multifaceted distinctions in gene expression profiling and genetic variations. Conclusions: Collectively, our data revealed the variations in the cytobiological and transcriptomic features between AML-NKs and HD-NKs in bone marrow environment. Our findings would benefit the further development of novel biomarkers for AML diagnosis and NK cell-based cytotherapy in future. [ABSTRACT FROM AUTHOR]

Immunotherapy based on adoptive transfer of natural killer (NK) cells is a promising strategy for circumventing the limitations of cancer treatments. However, components of the immunosuppressive tumor microenvironment (TME), such as transforming growth factor-beta (TGF-β), compromise the therapeutic efficacy of NK cells significantly. To address these limitations, we developed a novel method of engineering NK cells for adaptive transfer. The method is based on nanogels that serve two functions: (1) they overcome the TGF-β-mediated stress environment of the TME, and (2) they enhance the direct anti-tumor activity of NK cells. Previously, we demonstrated that cationic compounds such as 25 K branched polyethylenimine (25 K bPEI) prime NK cells, putting them in a 'ready-to-fight' state. Based on these findings, we designed nanogels that have two primary characteristics: (1) they encapsulate galunisertib (Gal), which is used clinically to inhibit TGF-β receptor activity, thereby blocking TGF-β signaling; and (2) they provide cells with a surface coating of 25 K bPEI. When grown in culture medium containing TGF-β, nanogel-treated NK cells demonstrated greater migration ability, degranulation activity, and cytotoxicity towards cancer cells than untreated NK cells. Additionally, the in vivo efficacy of nanogel-treated NK cells against PC-3 xenografts was significantly greater than that of Chem_NK cells primed by 25 K bPEI alone. These findings suggest that Gal-loaded 25 K bPEI-coated nanogels exert anti-tumor effects via chemical priming, as well suppressing the effects of TGF-β on NK cells. We also expect 25 K bPEI-based nanogels to have great potential to overcome the suppressive effects of the TME through their NK cell-priming activity and delivery of the desired chemicals. [Display omitted] [ABSTRACT FROM AUTHOR]

Opinion statement: Monoclonal antibody (mAb) therapy is now considered a main component of cancer therapy in Australia. Although traditionally thought of as pure signalling inhibitors, a large proponent of these medications function through antibody-dependent cell-mediated cytotoxicity (ADCC). Currently, most protocols and institutional guidelines for ADCC-mediated mAbs promote the use of corticosteroids as premedication: this is implemented to reduce infusion-related reactions (IRRs) and antiemesis prophylaxis and combat concurrently administered chemotherapy-related syndromes. Concerningly, the inhibitory effects of ADCC by corticosteroids are well documented; henceforth, it is possible the current standard of care is misaligned to the literature surrounding ADCC. Subsequently, clinicians' decisions to act in contrast to this literature may be reducing the efficacy of mAbs. The literature suggests that the redundant use of corticosteroids should be cautioned against when used in conjunction with ADCC-mediated mAbs—this is due to the consequent reduction in anti-tumour activity. Owing to the fact IRRs typically occur upon initial infusion, the authors advocate for individual clinicians and institutional protocols to considering augmenting their practice to corticosteroid premedication at the first dose only, unless clinically indicated. Additionally, product information (PI) and consumer medicine information (CMI) documents distributed by Australian and international regulatory agencies should consider disclosing the risk of concurrent steroids with these medications. Moreover, the authors suggest considering alternative medications for the management of side effects. [ABSTRACT FROM AUTHOR]

Abstract Natural killer cell‐derived extracellular vesicles (NK‐EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK‐EVs' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine‐based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK‐EVs against leukaemia K562 cells (suspension model) and breast cancer MDA‐MB‐231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin‐based cell viability potency assay reliably and reproducibly measured a dose‐response of NK‐EVs’ cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra‐run and inter‐run variability. The assay signal showed specificity and selectivity of NK‐EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5‐hour treatment with NK‐EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA‐MB‐231 cells per test with a consistent range. Importantly, NK‐EVs’ dose‐response for cytotoxicity showed a strong correlation (|ρ| ∼ 0.8) with the levels of known cytotoxic factors associated with the NK‐EVs’ corona (FasL, GNLY, GzmB, PFN and IFN‐γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK‐EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA‐binding reporter assay and confluence assay), our results support this highly sensitive resazurin‐based viability potency assay as a high‐throughput and quantitative method for assessing NK‐EVs’ cytotoxicity against both suspension and adherent cancer models for evaluating NK‐EVs’ biotherapeutics.

Abstract The metastasis of melanoma cells to regional lymph nodes and distant sites is an important contributor to cancer-related morbidity and mortality among patients with melanoma. This intricate process entails dynamic interactions involving tumor cells, cellular constituents, and non-cellular elements within the microenvironment. Moreover, both microenvironmental and systemic factors regulate the metastatic progression. Central to immunosurveillance for tumor cells are natural killer (NK) cells, prominent effectors of the innate immune system with potent antitumor and antimetastatic capabilities. Recognizing their pivotal role, contemporary immunotherapeutic strategies are actively integrating NK cells to combat metastatic tumors. Thus, a meticulous exploration of the interplay between metastatic melanoma and NK cells along the metastatic cascade is important. Given the critical involvement of NK cells within the melanoma tumor microenvironment, this comprehensive review illuminates the intricate relationship between components of the melanoma tumor microenvironment and NK cells, delineating their multifaceted roles. By shedding light on these critical aspects, this review advocates for a deeper understanding of NK cell dynamics within the melanoma context, driving forward transformative strategies to combat this cancer.

Natural killer (NK) cells are innate lymphocytes that can rapidly mount a response to their targets by employing diverse mechanisms. Due to their functional attributes, NK cells have been implicated in anti-viral and anti-tumour immune responses. Although traditionally known to mount non-specific, rapid immune responses, in recent years, the notion of memory NK cells with adaptive features has gained more recognition. Memory NK cells emerge in response to different stimuli, such as viral antigens and specific cytokine combinations. They form distinct populations, accompanied by transcriptional, epigenetic and metabolic reprogramming, resulting in unique phenotypic and functional attributes. Several clinical trials are testing the efficacy of memory NK cells due to their enhanced functionality, bioenergetic profile and persistence in vivo. The therapeutic potential of NK cells is being harnessed in viral infections, with wider applications in the cancer field. In this review, we summarise the current state of research on the generation of memory NK cells, along with their clinical applications in viral infection and cancer.

Glioblastoma multiforme (GBM) is the most common type of primary malignant brain tumor and has a dismal overall survival rate. To date, no GBM therapy has yielded successful results in survival for patients beyond baseline surgical resection, radiation, and chemotherapy. Immunotherapy has taken the oncology world by storm in recent years and there has been movement from researchers to implement the immunotherapy revolution into GBM treatment. Natural killer (NK) cell-based immunotherapies are a rising candidate to treat GBM from multiple therapeutic vantage points: monoclonal antibody therapy targeting tumor-associated antigens (TAAs), immune checkpoint inhibitors, CAR-NK cell therapy, Bi-specific killer cell engagers (BiKEs), and more. NK therapies often focus on tumor antigens for targeting. Here, we reviewed some common targets analyzed in the fight for GBM immunotherapy relevant to NK cells: EGFR, HER2, CD155, and IL-13Rα2. We further propose investigating the Lectin-like Transcript 1 (LLT1) and cell surface proliferating cell nuclear antigen (csPCNA) as targets for NK cell-based immunotherapy.

Simple Summary: Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate of only 11.6%, partially due to limited therapeutic options. Immunotherapy-based approaches, such as immune checkpoint inhibitors, have proven ineffective, in part due to the inability of cytotoxic, effector immune cells to sufficiently infiltrate tumors. Thus, understanding how the PDAC tumor microenvironment (TME) regulates the accumulation of immune cells is critical to improving immunotherapy-based approaches. Chemokines are small molecules that function as chemotactic factors which regulate the migration, infiltration, and accumulation of immune cells. Here, we comprehensively assess the structural and functional role of chemokines, examine the effects of chemokines that are present in the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME), specifically those produced by cancer cells and stromal components, and evaluate their impact on immune cell trafficking, both in promoting and suppressing anti-tumor responses. We further explore the impact of chemokines on patient outcomes in PDAC and their role in the context of immunotherapy treatments, and review clinical trials that have targeted chemokine receptors and ligands in the treatment of PDAC. Lastly, we highlight potential strategies that can be utilized to harness chemokines in order to increase cytotoxic immune cell infiltration and the anti-tumor effects of immunotherapy. [ABSTRACT FROM AUTHOR]

The metastasis of melanoma cells to regional lymph nodes and distant sites is an important contributor to cancer-related morbidity and mortality among patients with melanoma. This intricate process entails dynamic interactions involving tumor cells, cellular constituents, and non-cellular elements within the microenvironment. Moreover, both microenvironmental and systemic factors regulate the metastatic progression. Central to immunosurveillance for tumor cells are natural killer (NK) cells, prominent effectors of the innate immune system with potent antitumor and antimetastatic capabilities. Recognizing their pivotal role, contemporary immunotherapeutic strategies are actively integrating NK cells to combat metastatic tumors. Thus, a meticulous exploration of the interplay between metastatic melanoma and NK cells along the metastatic cascade is important. Given the critical involvement of NK cells within the melanoma tumor microenvironment, this comprehensive review illuminates the intricate relationship between components of the melanoma tumor microenvironment and NK cells, delineating their multifaceted roles. By shedding light on these critical aspects, this review advocates for a deeper understanding of NK cell dynamics within the melanoma context, driving forward transformative strategies to combat this cancer. [ABSTRACT FROM AUTHOR]

Natural killer cell‐derived extracellular vesicles (NK‐EVs) have shown promising potential as biotherapeutics for cancer due to their unique attributes as cytotoxic nanovesicles against cancer cells and immune‐modulatory activity towards immune cells. However, a biomanufacturing workflow is needed to produce clinical‐grade NK‐EVs for pre‐clinical and clinical applications. This study established a novel biomanufacturing workflow using a closed‐loop hollow‐fibre bioreactor to continuously produce NK‐EVs from the clinically relevant NK92‐MI cell line under serum‐free, Xeno‐free and feeder‐free conditions following GMP‐compliant conditions. The NK92 cells grown in the bioreactor for three continuous production lots resulted in large quantities of both NK cell and NK‐EV biotherapeutics at the end of each production lot (over 109 viable cells and 1013 EVs), while retaining their cytotoxic payload (granzyme B and perforin), pro‐inflammatory cytokine (interferon‐gamma) content and cytotoxicity against the human leukemic cell line K562 with limited off‐target toxicity against healthy human fibroblast cells. This scalable biomanufacturing workflow has the potential to facilitate the clinical translation of adoptive NK cell‐based and NK‐EV‐based immunotherapies for cancer with GMP considerations. [ABSTRACT FROM AUTHOR]

Abstract Background Natural killer (NK) cells, CD3− lymphocytes, are critical players in cancer immune surveillance. This study aimed to assess two types of CD3− NK cell classifications (subsets), that is, convectional subsets (based on CD56 and CD16 expression) and new subsets (based on CD56, CD27, and CD11b expression), and their functional molecules in the peripheral blood of patients with breast cancer (BC) in comparison with healthy donors (HDs). Methods Thirty untreated females with BC and 20 age‐matched healthy women were enrolled. Peripheral blood samples were collected and directly incubated with fluorochrome‐conjugated antibodies against CD3, CD56, CD16, CD27, CD11b, CD96, NKG2C, NKG2D, NKp44, CXCR3, perforin, and granzyme B. Red blood cells were then lysed using lysing solution, and the stained cells were acquired on four‐color flow cytometer. Result Our results indicated 15% of lymphocytes in peripheral blood of patients with BC and HDs had NK cells phenotype. However, the frequency of total NK cells (CD3−CD56+), and NK subsets (based on conventional and new classifications) was not significantly different between patients and HDs. We observed mean fluorescent intensity (MFI) of CXCR3 in total NK cells (p = .02) and the conventional cytotoxic (CD3−CD56dim CD16+) NK cells (p = .03) were significantly elevated in the patients with BC compared to HDs. Despite this, the MFI of granzyme B expression in conventional regulatory (CD3−CD56brightCD16−/+) NK cells and CD3−CD56−CD16+ NK cells (p = .03 and p = .004, respectively) in the patients was lower than healthy subjects. Conclusion The higher expression of chemokine receptor CXCR3 on total NK cells in patients with BC may be associated with increased chemotaxis‐related NK cell infiltration. However, lower expression of granzyme B in conventional regulatory NK cells and CD3−CD56−CD16+ NK cells in the patients compared to HDs suggests reduced cytotoxic activity of the NK cells in BC. These results might demonstrate accumulating NK subsets with a dysfunctional phenotype in the peripheral blood of patients with BC.

Abstract Background Natural killer (NK) cells hold great promise in treating diverse hematopoietic and solid tumors. Despite their availability from peripheral blood and cord blood, stem cell-derived NK cells offer an 'off-the-shelf' solution. Hematopoietic stem and progenitor cells (HSPCs) derived from cord blood pose no risk to the newborn or mother and are virtually ideal sources for NK cell differentiation. Methods We developed a modified protocol to differentiate HSPCs to NK cells under serum-free conditions using defined factors. The HSPC-derived NK (HSC-NK) cells could be expanded in a K562 feeder cell-dependent manner. Furthermore, using lentivirus transduction, chimeric antigen receptor (CAR)-modified HSPCs could be differentiated into NK cells, leading to the establishment of CAR-NK cells. Results The efficiency of NK cell differentiation from HSPCs was increased through the simple modulation of osmotic pressure by the addition of sodium chloride or glucose. Furthermore, the hyperosmosis-primed HSC-NK cells exhibited enhanced proliferation capacity and maintained normal functional characteristics, including transcriptome and antitumor efficacy. The optimized protocol yielded approximately 1.8 million NK cells from a single CD34-positive cell within a 28-day cycle, which signifies more than a ten-fold increase in efficiency relative to the conventional methods. This optimized protocol was also suitable for generating CAR-NK cells with high yields compared to standard conditions. Conclusions The results of this study establish high osmotic pressure as a simple yet powerful adjustment that significantly enhances the efficiency and functionality of HSC-NK cells, including CAR-NK cells. This optimized protocol could lead to cost-effective, high-yield NK cell therapies, potentially revolutionizing cancer immunotherapy strategies.

The lymph nodes are vital to enable adaptive immune responses to infection. Natural killer (NK) cells are cytotoxic lymphocytes that directly kill cancer cells and modulate the activation of other immune cells during anti-tumour immune response. NK cells in the lymph nodes are involved in the regulation of T-cell and B-cell populations and the clearance of viral infections. In solid tumours, lymph nodes are a frequent site of metastasis and immune cell priming, whilst in haematological malignancies, tumour cells can proliferate in the lymph nodes. Thus, lymph nodes are an important site in anti-tumour immunity and therapy resistance. It is therefore crucial to identify strategies to increase recruitment and overcome suppression of NK cells in the lymph node microenvironment to improve tumour clearance. In this review, we summarise the literature interrogating NK cell phenotype and function in the lymph nodes in the context of infection and cancer and evaluate both current and potential strategies to mobilise and activate NK cells within the lymph nodes of cancer patients.

Abstract Natural killer cell‐derived extracellular vesicles (NK‐EVs) have shown promising potential as biotherapeutics for cancer due to their unique attributes as cytotoxic nanovesicles against cancer cells and immune‐modulatory activity towards immune cells. However, a biomanufacturing workflow is needed to produce clinical‐grade NK‐EVs for pre‐clinical and clinical applications. This study established a novel biomanufacturing workflow using a closed‐loop hollow‐fibre bioreactor to continuously produce NK‐EVs from the clinically relevant NK92‐MI cell line under serum‐free, Xeno‐free and feeder‐free conditions following GMP‐compliant conditions. The NK92 cells grown in the bioreactor for three continuous production lots resulted in large quantities of both NK cell and NK‐EV biotherapeutics at the end of each production lot (over 109 viable cells and 1013 EVs), while retaining their cytotoxic payload (granzyme B and perforin), pro‐inflammatory cytokine (interferon‐gamma) content and cytotoxicity against the human leukemic cell line K562 with limited off‐target toxicity against healthy human fibroblast cells. This scalable biomanufacturing workflow has the potential to facilitate the clinical translation of adoptive NK cell‐based and NK‐EV‐based immunotherapies for cancer with GMP considerations.

Given the increasing incidence of pancreatic cancer and the low survival rate, the exploration of the complex tumor microenvironment and the development of novel treatment options is urgent. NK cells, known for their cytotoxic abilities and modulation of other immune cells, are vital in recognizing and killing cancer cells. However, hypoxic conditions in the tumor microenvironment have been found to impair NK cell functionality and contribute to tumor immune escape. Therefore, we aimed to uncover the mechanism through which hypoxia mediates the immune escape of pancreatic cancer cells, focusing on the influence of miR- 1275/AXIN2 on NK cells. Using a combination of GEO dataset screening, Tumor Immune Estimation Resource 2.0 immunoscore screening, and the Cancer Genome Atlas data, we identified a correlation between miR-1275 and NK cells. The down-regulation of miR-1275 was associated with decreased NK cell activity and survival in patients with pancreatic cancer. Pathway analysis further linked miR-1275 expression with the hypoxic HIF1A pathway. In vitro experiments were conducted using the NK-92 cell, revealing that hypoxia significantly reduced miR- 1275 expression and correspondingly decreased the cell-killing ability of NK cells. Upregulation of miR-1275 increased perforin, IFN-g and TNF-a expression levels and enhanced NK cell cytotoxicity. Additionally, miR-1275 was found to bind to and inhibit AXIN2 expression, which when overexpressed, partially alleviated the promotive effect of upregulated miR-1275 on NK-92 cell killing ability. In conclusion, this research underscores the critical role of the miR-1275/AXIN2 axis in hypoxia-mediated immune escape in pancreatic cancer, thus opening new potential avenues for treatment strategies. [ABSTRACT FROM AUTHOR]

Simple Summary: Adoptive cell therapy (ACT) is an innovative approach to combat cancer and infectious diseases using specialised immune cells, such as chimeric antigen receptor T-cells (CAR-Ts), tumour-infiltrating lymphocytes (TILs) and virus-specific T-cells (VSTs). Such therapies are manufactured individually for each patient and can be negatively affected by poor cell quality, often impaired by prior treatments, age, and complex manufacturing. To overcome this, this field is assessing the potential of creating cell therapies from "fit" donors to provide off-the-shelf treatment options. Induced pluripotent cells (iPSCs) have renewable characteristics and offer a solution towards off-the-shelf therapy. iPSCs can be used as an unlimited source to derive different immune cells, including natural killer (NK) cells and T-cells. iPSCs can be further genetically modified and used create different ACTs. In this review, we describe the methodologies for generating such cell therapies from iPSCs and discuss the current advances and challenges with a focus on CAR-T/NK-, TIL- and VST-based therapies. Adoptive cell therapy (ACT) has transformed the treatment landscape for cancer and infectious disease through the investigational use of chimeric antigen receptor T-cells (CAR-Ts), tumour-infiltrating lymphocytes (TILs) and viral-specific T-cells (VSTs). Whilst these represent breakthrough treatments, there are subsets of patients who fail to respond to autologous ACT products. This is frequently due to impaired patient T-cell function or "fitness" as a consequence of prior treatments and age, and can be exacerbated by complex manufacturing protocols. Further, the manufacture of autologous, patient-specific products is time-consuming, expensive and non-standardised. Induced pluripotent stem cells (iPSCs) as an allogeneic alternative to patient-specific products can potentially overcome the issues outlined above. iPSC technology provides an unlimited source of rejuvenated iPSC-derived T-cells (T-iPSCs) or natural killer (NK) cells (NK-iPSCs), and in the context of the growing field of allogeneic ACT, iPSCs have enormous potential as a platform for generating off-the-shelf, standardised, "fit" therapeutics for patients. In this review, we evaluate current and future applications of iPSC technology in the CAR-T/NK, TIL and VST space. We discuss current and next-generation iPSC manufacturing protocols, and report on current iPSC-based adoptive therapy clinical trials to elucidate the potential of this technology as the future of ACT. [ABSTRACT FROM AUTHOR]

Summary: Adoptive cellular therapy using chimeric antigen receptor (CAR) T cells has led to a paradigm shift in the treatment of various hematologic malignancies. However, the broad application of this approach for myeloid malignancies and solid cancers has been limited by the paucity and heterogeneity of target antigen expression, and lack of bona fide tumor‐specific antigens that can be targeted without cross‐reactivity against normal tissues. This may lead to unwanted on‐target off‐tumor toxicities that could undermine the desired antitumor effect. Recent advances in synthetic biology and genetic engineering have enabled reprogramming of immune effector cells to enhance their selectivity toward tumors, thus mitigating on‐target off‐tumor adverse effects. In this review, we outline the current strategies being explored to improve CAR selectivity toward tumor cells with a focus on natural killer (NK) cells, and the progress made in translating these strategies to the clinic. [ABSTRACT FROM AUTHOR]

Endometrial receptivity is largely determined by the immunophenotype of endometrium, especially uterine NK-cells (uNK). Immune component is directly involved in the formation of favourable microenvironment for the blastocyst implantation and placenta formation, but the way it changes during the maturation of endometrial tissue in healthy fertile women is still underexplored. The endometrium was collected from 47 healthy oocyte donors after controlled ovarian stimulation: 23 women on the day of oocytes retrieval (OR) and 24 women on the term of implantation window (IW). The OR group was analysed, published previously and used as a comparison group to show the dynamic of changes. Isolated endometrial lymphocytes and peripheral blood samples were stained with monoclonal antibodies and analysed according to the three-color flow cytometry protocol. The proportion of NK-cells (CD3−CD56+) in endometrium grew significantly in the implantation window compared to the oocytes retrieval day. NK-cells acquired a more differentiated phenotype from the day of OR until IW: the expression of CD8 and CD158a significantly increased, while the expression of HLA-DR significantly decreased. Significant correlations between peripheral blood and endometrial NK-cells were found in CD8 expression during OR and IW, CD335(p46)neg and CD335(p46)++ subsets during IW term. Immunophenotype of receptive endometrium forms due to the accumulation of uNK-cells, which actively proliferate, become mature, differentiative, and ready to meet the embryo. Endometrial immunophenotype is peculiar and specific but not autonomic and isolated. Differentiation (CD8 on NK-cells), and activity (p46 on NK-cells) of peripheral blood lymphocytes is reflected in endometrial lymphocytes profile, and therefore the research of peripheral blood immunophenotype is relevant. • Endometrial immunophenotype forms the receptivity and conditions for the implantation. • Endometrium matures with changes in the number and phenotype of uNK-cells. • On implantation window term, NK-cells actively proliferate and become differentiative. • Endometrial immunophenotype is specific but not autonomic and isolated. [ABSTRACT FROM AUTHOR]

Background: Natural killer (NK) cells hold great promise in treating diverse hematopoietic and solid tumors. Despite their availability from peripheral blood and cord blood, stem cell-derived NK cells offer an 'off-the-shelf' solution. Hematopoietic stem and progenitor cells (HSPCs) derived from cord blood pose no risk to the newborn or mother and are virtually ideal sources for NK cell differentiation. Methods: We developed a modified protocol to differentiate HSPCs to NK cells under serum-free conditions using defined factors. The HSPC-derived NK (HSC-NK) cells could be expanded in a K562 feeder cell-dependent manner. Furthermore, using lentivirus transduction, chimeric antigen receptor (CAR)-modified HSPCs could be differentiated into NK cells, leading to the establishment of CAR-NK cells. Results: The efficiency of NK cell differentiation from HSPCs was increased through the simple modulation of osmotic pressure by the addition of sodium chloride or glucose. Furthermore, the hyperosmosis-primed HSC-NK cells exhibited enhanced proliferation capacity and maintained normal functional characteristics, including transcriptome and antitumor efficacy. The optimized protocol yielded approximately 1.8 million NK cells from a single CD34-positive cell within a 28-day cycle, which signifies more than a ten-fold increase in efficiency relative to the conventional methods. This optimized protocol was also suitable for generating CAR-NK cells with high yields compared to standard conditions. Conclusions: The results of this study establish high osmotic pressure as a simple yet powerful adjustment that significantly enhances the efficiency and functionality of HSC-NK cells, including CAR-NK cells. This optimized protocol could lead to cost-effective, high-yield NK cell therapies, potentially revolutionizing cancer immunotherapy strategies. [ABSTRACT FROM AUTHOR]

Purpose: Natural killer (NK) cells play a critical role in cancer immunosurveillance and hold promise as both therapies and prognostic markers in advanced disease. We explore factors that may influence NK cell concentration in the peripheral blood of women with breast cancer in Côte d'Ivoire compared to healthy controls and implications for future research in our context. Methods: In this cross-sectional case-control study, blood samples were taken from 30 women diagnosed with breast cancer within 6 months of diagnosis and fifteen healthy women at University Teaching Hospital [Centre Hospitalier Universitaire (CHU)] Treichville in Abidjan, Côte d'Ivoire, from March to September 2018. The blood draw could take place at any time following diagnosis and through treatment. Demographic and clinical data were collected. NK cells were isolated, stained, analysed and counted using the flow cytometer at the Department of Immunology at CHU of Cocody. All p-values were two-sided. Results: Mean age among 30 women with breast cancer was 49 years old compared to 45 years old for 15 controls (p = 0.41). Among 30 women with breast cancer, 4 (13.3%) had Stage 2 disease, 14 (46.7 %) at Stage 3, and 12 (40%) at Stage 4. Fourteen (46.7%) had breast cancer that was hormone receptor-positive (HR+) HER2-negative, 10 (33.3%) had triple-negative cancer, three (10.0%) had HR+HER2+ disease, and three (10.0%) HRHER2+ cancer. NK cell concentration was not associated with cancer diagnosis, age, cancer stage, subtype, or type of treatment patients received (p > 0.05). Conclusion: Although we did not find an association between NK cell concentration, cancer characteristics or treatment, our results be limited by the small sample size and timing of blood draw. Our next steps include a larger study to explore circulating NK cells prior to any treatment and NK cell infiltration within breast cancer tumour and correlating this with response to treatment and prognosis. [ABSTRACT FROM AUTHOR]

Allogeneic natural killer (NK) cell-based immunotherapy is a promising, well-tolerated adjuvant therapeutic approach for acute myeloid leukemia (AML). For reproducible NK cell immunotherapy, a homogenous, pure and scalable NK cell product is preferred. Therefore, we developed a good manufacturing practice (GMP)-compliant, cytokine-based ex vivo manufacturing process for generating NK cells from CD34+ hematopoietic stem and progenitor cells (HSPC). This manufacturing process combines amongst others IL15 and IL12 and the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1) to generate a consistent and active NK cell product that fits the requirements for NK cell immunotherapy well. The cell culture protocol was first optimized to generate NK cells with required expansion and differentiation capacity in GMP-compliant closed system cell culture bags. In addition, phenotype, antitumor potency, proliferative and metabolic capacity were evaluated to characterize the HSPC-NK product. Subsequently, seven batches were manufactured for qualification of the process. All seven runs demonstrated consistent results for proliferation, differentiation and antitumor potency, and preliminary specifications for the investigational medicinal product for early clinical phase trials were set. This GMP-compliant manufacturing process for HSPC-NK cells (named RNK001 cells) is used to produce NK cell batches applied in the clinical trial 'Infusion of ex vivo-generated allogeneic natural killer cells in combination with subcutaneous IL2 in patients with acute myeloid leukemia' approved by the Dutch Ethics Committee (EudraCT 2019-001929-27). [ABSTRACT FROM AUTHOR]

Spore-forming probiotic bacteria, including Bacillus coagulans, are resilient and produce a variety of beneficial metabolites. We evaluated the immune-modulating effects of the novel probiotic strain Bacillus coagulans JBI-YZ6.3, where the germinated spores, metabolite fraction, and cell wall fraction were tested in parallel using human peripheral blood mononuclear cell cultures under both normal and lipopolysaccharide-induced inflamed culture conditions. The expression of CD25 and CD69 activation markers was evaluated via flow cytometry. Supernatants were tested for cytokines, interferons, chemokines, and growth factors using Luminex arrays. The germinated spores were highly immunogenic; both the cell wall and metabolite fractions contributed significantly. Under normal culture conditions, increased levels of immune activation were observed as increased expressions of CD25 and CD69 relative to natural killer cells, suggesting an increased ability to attack virus-infected target cells. On monocytes, a complex effect was observed, where the expression of CD25 increased under normal conditions but decreased under inflamed conditions. This, in combination with increased interleukin-10 (IL-10) and decreased monocyte chemoattractant protein-1 (MCP-1) production under inflamed conditions, points to anti-inflammatory effects. The production of the stem cell-related growth factor granulocyte colony-stimulating Factor (G-CSF) was enhanced. Further research is warranted to characterize the composition of the postbiotic metabolite fraction and document the characteristics of immunomodulating agents secreted by this probiotic strain. [ABSTRACT FROM AUTHOR]

BackgroundCoronavirus Disease 2019 (Covid-19) continues to challenge the limits of our knowledge and our healthcare system. Here we sought to define the host immune response, a.k.a, the "cytokine storm" that has been implicated in fatal COVID-19 using an AI-based approach.MethodOver 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a 'seed' gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. An AI-based approach was used to explore the utility of the signature in navigating the uncharted territory of Covid-19, setting therapeutic goals, and finding therapeutic solutions.FindingsThe 166-gene signature was surprisingly conserved across all viral pandemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViP and severe-ViP signatures, respectively. The ViP signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determine severity/fatality. Precise therapeutic goals could be formulated; these goals were met in high-dose SARS-CoV-2-challenged hamsters using either neutralizing antibodies that abrogate SARS-CoV-2•ACE2 engagement or a directly acting antiviral agent, EIDD-2801. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine prognosticated disease severity.InterpretationThe ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.FundingThis work was supported by the National Institutes for Health (NIH) [grants CA151673 and GM138385 (to DS) and AI141630 (to P.G), DK107585-05S1 (SD) and AI155696 (to P.G, D.S and S.D), U19-AI142742 (to S.C, cchiCooperative Centers for Human Immunology)]; Research Grants Program Office (RGPO) from the University of California Office of the President (UCOP) (R00RG2628 & R00RG2642 to P.G, D.S and S.D); the UC San Diego Sanford Stem Cell Clinical Center (to P.G, D.S and S.D); LJI Institutional Funds (to S.C); the VA San Diego Healthcare System Institutional funds (to L.C.A). GDK was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists.One sentence summaryThe host immune response in COVID-19.

Simple Summary: In this study, we provide a basal and longitudinal evaluation of immune cells in advanced non-small-cell lung cancer (NSCLC) patients undergoing PD-1 or PD-L1 blockade. We aimed to explore if any data could be predictive of better outcomes and long-term survival and to detect eventual connections among immune cell subsets and sarcopenia, another known risk factor for progression disease (PD). We found that natural killer (NK) cell basal levels are higher in patients with disease control (DC) compared to PD patients; higher NK cell basal levels predict a longer overall survival (OS); lower NK values represent a risk factor for PD; and after three months of immune check-point inhibitors (ICIs) treatment, NK cells (and the subclass CD56bright) significantly increase in DC patients. Interestingly, sarcopenic patients show lower NK cell values at basal levels. Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of tumors. Natural killer (NK) cells can play an important role in cancer immune surveillance. The aim of this prospective observational study was to analyze peripheral blood mononuclear cells (PBMCs) in patients with advanced non-small-cell lung cancer (NSCLC) receiving ICIs in order to identify predictive factors for better survival outcomes. Methods: Forty-seven stage IV NSCLC patients were enrolled. Patients underwent baseline (T0) and longitudinal (T1) evaluations after ICIs. Peripheral immune blood cell counts were analyzed using flow cytometry. Results: Basal levels of CD3−CD56+ NK cells were higher in patients with controlled disease (DC) compared to progression disease (PD) patients (127 cells/µL vs. 27.8 cells/µL, p < 0.001). Lower NK cell values were independent prognostic factors for shorter overall survival (OS) (HR 0.992; 95% CI 0.987–0.997, p < 0.001) and progression-free survival (PFS) (HR 0.988; 95% CI 0.981–0.994, p < 0.001). During the longitudinal evaluation, CD3−CD56+ NK cells (138.1 cells/µL vs. 127 cells/µL, p = 0.025) and CD56bright NK cells (27.4 cells/µL vs. 18.1 cells/µL, p = 0.034) significantly increased in the DC group. Finally, lower values of CD3−CD56+ NK cells (28.3 cells/µL vs. 114.6 cells/µL, p = 0.004) and CD56dim NK cells (13.2 cells/µL vs. 89.4 cells/µL, p < 0.001) were found in sarcopenic patients compared to patients without sarcopenia. Conclusions: Peripheral NK cells could represent a non-invasive and useful tool to predict ICI therapy response in NSCLC patients, and the association of low NK cell levels with sarcopenia deserves even more attention in clinical evaluation. [ABSTRACT FROM AUTHOR]

Background: Natural killer (NK) cells are classified as innate immune cells which can directly recognize and kill tumor cells without antigen sensitization. NK cell-based adoptive immunotherapy for blood malignancies has attracted more attention in recent years. Objective: To analyze different NK cell subsets in the peripheral blood and bone marrow (BM) of patients with multiple myeloma (MM). Methods: Using flow cytometry we analyzed: (i) the distribution of distinct NK cell subpopulations (i.e. CD16low CD56low, CD16pos CD56high, CD16neg CD56high, CD16high CD56low, CD16neg CD56low, CD16low CD56low CD38pos) in the BM from MM patients at distinct disease stages. (ii) the expression of NKG2D, DNAM-1 and NKp30, and (iii) the expression of CD107a in CD16low CD56low CD38pos and CD16low CD56low CD38neg NK cells subsets. Results: CD16low CD56low CD38pos was the dominant subset in BM from patients with MM at the CR stage with a decreased expression of NKp30. CD16low CD56low CD38pos subset showed a higher proportion of CD107a expression compared to CD16low CD56low CD38neg cells. In vitro experiments indicated that the CD16low CD56low CD38pos NK cell subset possesses more cytotoxicity than CD16low CD56low CD38neg NK cells. Conclusion: Our data suggest that CD16low CD56low CD38pos NK cells may reflect as an effector population with the potential therapeutic target in patients with MM. This group of cells may be useful for adoptive immunotherapy in MM in the future.

Abstract Cellular immunotherapy harnesses the body's own immune system to fight cancer by using engineered T cells, macrophages, or natural killer (NK) cells. Compared to chimeric antigen receptor T (CAR‐T) cells that are commonly used to treat hematological malignancies, CAR‐NK cells have shown remarkable therapeutic effectiveness while exhibiting enhanced safety, reduced risk of graft‐versus‐host disease, fewer side effects, and amplified antitumor efficacy. Preclinical trials have unveiled the high potential of adoptive CAR‐NK cell therapy to curtail or even eliminate both hematological malignancies and solid tumors in animal models. We brought forth herein the design principle of CAR‐NK cells, highlighted the latest progress in the preclinical testing and clinical trials of CAR‐NK cells, briefly delved into discussed major roadblocks in CAR‐NK therapy, and discussed potential solutions to surmount these challenges. Given the accelerated progress in both basic and translational studies on immune cell engineering, CAR‐NK cell therapy promises to become a serious contender and important addition to the next‐generation cell‐based immunotherapy.