Is the tumor cell side of the immunological synapse a polarized secretory domain?

The formation of a lytic immunological synapse (IS) is crucial for cytotoxic lymphocytes to accurately target and effectively eliminate malignant cells. While significant attention has been focused on the lymphocyte side of the IS, particularly its role as a secretory domain for lytic granules, the cancer cell side of the IS has remained relatively underexplored. Recent findings have revealed that cancer cells can rapidly polarize their actin cytoskeleton toward the IS upon interaction with natural killer (NK) cells, thereby evading NK cell-mediated cytotoxicity. In this Brief Research Report, we present preliminary findings suggesting that actin cytoskeleton remodeling at the cancer cell side of the IS is associated with the targeted secretion of small extracellular vesicles towards the interacting NK cell. We observed that multivesicular bodies (MVBs) preferentially accumulate in the synaptic region in cancer cells exhibiting synaptic accumulation of F-actin, compared to those lacking actin cytoskeleton remodeling. Extracellular immunofluorescence staining revealed increased surface exposure of CD63 at the cancer cell side of the IS, suggestive of the fusion of MVBs with the plasma membrane. This hypothesis was supported by a pH-sensitive probe demonstrating dynamic trafficking of CD63 to the extracellular region of the IS. Collectively, our data support the notion that cancer cells can engage in targeted secretion of extracellular vesicles in response to NK cell attack, underscoring the need for further research into the potential role of this process in facilitating cancer cell immune evasion.

Increased peritoneal TGF-ß1 is associated with ascites-induced NK-cell dysfunction and reduced survival in high-grade epithelial ovarian cancer.

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)-ß1 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-ß1 exposure. Moreover, inhibition of TGF-ß1 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-ß1 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-ß1 signaling could increase NK cell immune responses in high-grade EOC patients.

STYK1 mediates NK cell anti-tumor response through regulating CCR2 and trafficking.

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.

Peripheral NK cell phenotypic alteration and dysfunctional state post hepatitis B subviral particles stimulation in CHB patients: evading immune surveillance.

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- γ 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.

Prospective Molecular Targets for Natural Killer Cell Immunotherapy against Glioblastoma Multiforme.

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.

Proliferation capability of natural killer cells upon cytokines stimulation correlated negatively with serum lactate dehydrogenase level in coronary artery disease patients.

Background: Natural killer (NK) cells are proposed to participate in coronary artery disease (CAD) development. However, little is known about how CAD patients' NK cells respond to different stimulatory factors in terms of proliferation capability. Methods and results: Twenty-nine CAD patients' peripheral blood NK cells were isolated and individually treated with IL-2, IL-12, IL-15, IL-18, IL-21, cortisone acetate, hydrocortisone, or ascorbic acid for 36 hours, followed by cell cycle analysis using flow cytometry. The ratio of S and G2/M phase cell number to total cell number was defined as a proliferation index (PrI) and used for proliferative capability indication. The results showed that these eight factors resulted in different life cycle changes in the 29 NK cell samples. Remarkably, 28 out of 29 NK cell samples showed an obvious increase in PrI upon ascorbic acid treatment. The serum lactate dehydrogenase (LDH) level of the 29 CAD patients was measured. The results showed a negative correlation between serum LDH level and the CAD patients' NK cell PrI upon stimulation of interleukins, but not the non-interleukin stimulators. Consistently, a retrospective analysis of 46 CAD patients and 32 healthy donors showed that the circulating NK cell number negatively correlated with the serum LDH level in CAD patients. Unexpectedly, addition of LDH to NK cells significantly enhanced the production of IFN-γ, IL-10 and TNF-α, suggesting a strong regulatory role on NK cell's function. Conclusion: Ascorbic acid could promote the proliferation of the CAD patients' NK cells; LDH serum level may function as an indicator for NK cell proliferation capability and an immune-regulatory factor.

NK Cells in the Lymph Nodes and Their Role in Anti-Tumour Immunity.

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.

iPSC-derived NK cells expressing high-affinity IgG Fc receptor fusion CD64/16A to mediate flexible, multi-tumor antigen targeting for lymphoma.

Introduction: NK cells can mediate tumor cell killing by natural cytotoxicity and by antibody-dependent cell-mediated cytotoxicity (ADCC), an anti-tumor mechanism mediated through the IgG Fc receptor CD16A (FcγRIIIA). CD16A polymorphisms conferring increased affinity for IgG positively correlate with clinical outcomes during monoclonal antibody therapy for lymphoma, linking increased binding affinity with increased therapeutic potential via ADCC. We have previously reported on the FcγR fusion CD64/16A consisting of the extracellular region of CD64 (FcγRI), a high-affinity Fc receptor normally expressed by myeloid cells, and the transmembrane/cytoplasmic regions of CD16A, to create a highly potent and novel activating fusion receptor. Here, we evaluate the therapeutic potential of engineered induced pluripotent stem cell (iPSC)-derived NK (iNK) cells expressing CD64/16A as an "off-the-shelf", antibody-armed cellular therapy product with multi-antigen targeting potential. Methods: iNK cells were generated from iPSCs engineered to express CD64/16A and an interleukin (IL)-15/IL-15Rα fusion (IL-15RF) protein for cytokine independence. iNK cells and peripheral blood NK cells were expanded using irradiated K562-mbIL21-41BBL feeder cells to examine in in vitro and in vivo assays using the Raji lymphoma cell line. ADCC was evaluated in real-time by IncuCyte assays and using a xenograft mouse model with high circulating levels of human IgG. Results: Our data show that CD64/16A expressing iNK cells can mediate potent anti-tumor activity against human B cell lymphoma. In particular, (i) under suboptimal conditions, including low antibody concentrations and low effector-to-target ratios, iNK-CD64/16A cells mediate ADCC, (ii) iNK-CD64/16A cells can be pre-loaded with tumor-targeting antibodies (arming) to elicit ADCC, (iii) armed iNK-CD64/16A cells can be repurposed with additional antibodies to target new tumor antigens, and (iv) cryopreserved, armed iNK-CD64/16A are capable of sustained ADCC in a tumor xenograft model under saturating levels of human IgG. Discussion: iNK-CD64/16A cells allow for a flexible use of antibodies (antibody arming and antibody targeting), and an "off-the-shelf" platform for multi-antigen recognition to overcome limitations of adoptive cell therapies expressing fixed antigen receptors leading to cancer relapse due to antigen escape variants.

The immunomodulatory effects of cannabidiol on Hsp70-activated NK cells and tumor target cells.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: Cannabidiol (CBD) may potentially diminish the anti-tumor effectiveness of TKD+IL-2 activated natural killer (NK) cells.

Distorted frequency and functionality of natural killer cells in pemphigus vulgaris: A potential therapeutic target.

Pemphigus vulgaris (PV) is a rare autoimmune disorder where autoantibodies target the desmosomal proteins resulting in blistering of oral mucosa and skin. While the pathogenesis of PV is mainly mediated by the adaptive immune system, key players of innate immunity are also emerging. This study outlines the phenotypic as well as functional attributes of NK cells in PV. Through in-depth analysis using flow cytometry we identified an increase in the frequency of CD56+ CD3- NK cells and their subtypes in periphery. Along with this there is an increased frequency of IFNγ+ CD56bright CD16dim NK cells. mRNA expression of sorted NK cells for differentially expressed genes, particularly key transcription factors such as T-bet and EOMES, as well as surface receptors like NKG2D and KIR2D, and the cytokine IFNγ, displayed significant upregulation. A significant activation of NK cells was seen in the disease state. The levels of perforin and IFNγ were significantly elevated in the culture supernatants of patients. Additionally, a significantly higher cytotoxicity of NK cells in PV was observed. In lesioned tissues of PV, NK related markers were significantly increased. Lastly, we observed NK cells using confocal microscopy in the tissue biopsies of patients which showed significant infiltration of CD56+ CD3- NK cells at the lesional sites. This study aimed to shed light on the pivotal role of NK cells in the immunopathology of PV, offering a thorough understanding of their behaviour and changes in expression which might help in contributing to the development of novel therapeutics.

The impact of surgical intervention on peripheral blood T lymphocyte subsets and natural killer cell activity in pediatric obstructive sleep apnea hypopnea syndrome (OSAHS).

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.

Define Critical Parameters of Trastuzumab-Mediated ADCC Assays via Assay Optimization Processes, Focusing on the Impact of Cryopreserved Effector Cells on Assay Performance.

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.

Effects of Chronic Barley Consumption on Upper Respiratory Tract Symptoms in Japanese Healthy Adults: A Randomized, Parallel-Group, Controlled Trial.

ß-(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 ß-(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 ß-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 ß-(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.

Evaluation of resazurin phenoxazine dye as a highly sensitive cell viability potency assay for natural killer cell-derived extracellular vesicle-based cancer biotherapeutics.

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.

Natural killer cell-related anti-tumour adoptive cell immunotherapy.

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.

Imaging CAR-NK cells targeted to HER2 ovarian cancer with human sodium-iodide symporter-based positron emission tomography.

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.

S309-CAR-NK cells bind the Omicron variants in vitro and reduce SARS-CoV-2 viral loads in humanized ACE2-NSG mice.

Recent progress on chimeric antigen receptor (CAR)-NK cells has shown promising results in treating CD19-positive lymphoid tumors with minimal toxicities [including graft versus host disease (GvHD) and cytokine release syndrome (CRS) in clinical trials. Nevertheless, the use of CAR-NK cells in combating viral infections has not yet been fully explored. Previous studies have shown that CAR-NK cells expressing S309 single-chain fragment variable (scFv), hereinafter S309-CAR-NK cells, can bind to SARS-CoV-2 wildtype pseudotyped virus (PV) and effectively kill cells expressing wild-type spike protein in vitro. In this study, we further demonstrate that the S309-CAR-NK cells can bind to different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants in vitro. We also show that S309-CAR-NK cells reduce virus loads in the NOD/SCID gamma (NSG) mice expressing the human angiotensin-converting enzyme 2 (hACE2) receptor challenged with SARS-CoV-2 wild-type (strain USA/WA1/2020). Our study demonstrates the potential use of S309-CAR-NK cells for inhibiting infection by SARS-CoV-2 and for the potential treatment of COVID-19 patients unresponsive to otherwise currently available therapeutics. IMPORTANCE: Chimeric antigen receptor (CAR)-NK cells can be "off-the-shelf" products that treat various diseases, including cancer, infections, and autoimmune diseases. In this study, we engineered natural killer (NK) cells to express S309 single-chain fragment variable (scFv), to target the Spike protein of SARS-CoV-2, hereinafter S309-CAR-NK cells. Our study shows that S309-CAR-NK cells are effective against different SARS-CoV-2 variants, including the B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants. The S309-CAR-NK cells can (i) directly bind to SARS-CoV-2 pseudotyped virus (PV), (ii) competitively bind to SARS-CoV-2 PV with 293T cells expressing the human angiotensin-converting enzyme 2 (hACE2) receptor (293T-hACE2 cells), (iii) specifically target and lyse A549 cells expressing the spike protein, and (iv) significantly reduce the viral loads of SARS-CoV-2 wild-type (strain USA/WA1/2020) in the lungs of NOD/SCID gamma (NSG) mice expressing hACE2 (hACE2-NSG mice). Altogether, the current study demonstrates the potential use of S309-CAR-NK immunotherapy as an alternative treatment for COVID-19 patients.

Chimeric antigen receptor-natural killer cell therapy: current advancements and strategies to overcome challenges.

Chimeric antigen receptor-natural killer (CAR-NK) cell therapy is a novel immunotherapy targeting cancer cells via the generation of chimeric antigen receptors on NK cells which recognize specific cancer antigens. CAR-NK cell therapy is gaining attention nowadays owing to the ability of CAR-NK cells to release potent cytotoxicity against cancer cells without side effects such as cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GvHD). CAR-NK cells do not require antigen priming, thus enabling them to be used as "off-the-shelf" therapy. Nonetheless, CAR-NK cell therapy still possesses several challenges in eliminating cancer cells which reside in hypoxic and immunosuppressive tumor microenvironment. Therefore, this review is envisioned to explore the current advancements and limitations of CAR-NK cell therapy as well as discuss strategies to overcome the challenges faced by CAR-NK cell therapy. This review also aims to dissect the current status of clinical trials on CAR-NK cells and future recommendations for improving the effectiveness and safety of CAR-NK cell therapy.

Impact of a Liver Immune Status Index among Living Liver Transplant Recipients with Hepatocellular Carcinoma.

Introduction: Hepatocellular carcinoma (HCC) is a major global health challenge, being the fifth most prevalent neoplasm and the third leading cause of cancer-related deaths worldwide. Liver transplantation offers a potentially curative approach for HCC, yet the risk of recurrence posttransplantation remains a significant concern. This study investigates the influence of a liver immune status index (LISI) on the prognosis of patients undergoing living-donor liver transplantation for HCC. Methods: In a single-center study spanning from 2001 to 2020, 113 patients undergoing living-donor liver transplantation for HCC were analyzed. LISI was calculated for each donor liver using body mass index, serum albumin levels, and the fibrosis-4 index. This study assessed the impact of donor LISI on short-term recurrence rates and survival, with special attention to its correlation with the antitumor activity of natural killer (NK) cells in the liver. Results: The patients were divided into two grades (high donor LISI, >-1.23 [n = 43]; and low donor LISI, ≤-1.23 [n = 70]). After propensity matching to adjust the background of recipient factors, the survival rates at 1 and 3 years were 92.6% and 88.9% and 81.5% and 70.4% in the low and high donor LISI groups, respectively (p = 0.11). The 1- and 3-year recurrence-free survival were 88.9% and 85.2% and 74.1% and 55.1% in the low and high donor LISI groups, respectively (p = 0.02). Conclusions: This study underscores the potential of an LISI as a noninvasive biomarker for assessing liver NK cell antitumor capacity, with implications for living-donor liver transplantation for HCC. Donor LISI emerges as a significant predictor of early recurrence risk following living-donor liver transplantation for HCC, highlighting the role of the liver antitumor activity of liver NK cells in managing liver malignancies.