Natural Killer Cell Cytotoxicity Research Articles

A 5-ALA mediated photodynamic therapy increases natural killer cytotoxicity against oral squamous cell carcinoma cell lines.

Oral squamous cell carcinoma (OSCC) constitutes over 90% of oral cancers, known for its aggressiveness and poor prognosis. Photodynamic therapy (PDT) has emerged as a promising adjuvant therapy and is linked to immunogenic cell death, activating innate and adaptive anti-tumor responses. Natural Killer (NK) cells, key players in malignant cell elimination, have not been extensively studied in PDT. This study evaluates whether PDT increases OSCC cell lines' susceptibility to NK cell cytotoxicity. PDT, using 5-aminolevulinic acid (5-ALA) and LED irradiation, was applied to Ca1 and Luc4 cell lines. Results showed a dose-dependent viability decrease post-PDT. Gene expression analysis revealed upregulation of NK cell-activating ligands (ULBP1-4, MICA/B) and decreased MHC class I expression in Ca1, suggesting increased NK cell susceptibility. Enhanced NK cell cytotoxicity was confirmed in Ca1 but not in Luc4 cells. These findings indicate that PDT may enhance NK cell-mediated cytotoxicity in OSCC, offering potential for improved treatment strategies.

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.

Regulation of the PD-1/PD-L1 Axis and NK Cell Dysfunction by Exosomal miR-552-5p in Gastric Cancer

ObjectiveNK cells play a vital role in tumor immune resistance. Various factors affect NK cell activity. While NK cell dysfunction has been observed in numerous malignancies, the underlying mechanisms in gastric cancer remain unclear.MethodFlow cytometry was used to identify the phenotypic distribution and expression of activated receptors on NK cells. ELISA was used to determine the expression of cytokines. We examined the expression of NK cell-related genes and explored their association with survival and prognosis. Additionally, we conducted PCR detection of miR-552-5p expression levels in plasma exosomes of patients and investigated its correlation with phenotypic distribution and activated receptors. We used flow cytometry and ELISA to verify the role of miR-552-5p in NK cell dysfunction. Furthermore, we investigated the potential role of PD-1/PD-L1 in regulating NK cell dysfunction in patients’ cells.ResultsWe observed a significant decrease in the percentage of NKG2D and NKp30 and IFN-γ and TNF-α in patients than in healthy volunteers. Patients with low levels of CD56, CD16, NKG2D, and NKP46 exhibited poorer survival prognoses. Moreover, increased expression levels of plasma exosomal miR-552-5p in patients were negatively associated with NK cell phenotypic distribution and activated receptor expression. MiR-552-5p downregulated the secretion of perforin, granzyme, and IFN-γ as well as the expression of NKp30, NKp46, and NKG2D. Additionally, it suppressed the cytotoxicity of NK cells. The inhibitory effect of miR-552-5p, on NK cell function was reversed when anti-PD-L1 antibodies were used.ConclusionExosomal miR-552-5p targets the PD-1/PD-L1 axis, leading to impaired NK cell function.

Acute natural killer cells response to a continuous moderate intensity and a work-matched high intensity interval exercise session in metastatic cancer patients treated with chemotherapy

BackgroundIt has been suggested that the acute natural killer (NK) cell response to aerobic exercise might contribute to the tumor suppressor effect of regular exercise observed in preclinical studies. Moreover, because this response is modulated by exercise intensity, high-intensity intervals exercise (HIIE) might represent an interesting therapeutic approach in cancer patients. However, this immune response remains unstudied in cancer patients currently undergoing chemotherapy. ObjectiveTo characterize the acute NK cell response following a moderate-intensity continuous aerobic exercise session (MOD), and a HIIE session in metastatic cancer patients treated with chemotherapy. MethodsTwelve cancer patients (45–65 years old) underwent a MOD and a duration and work-matched HIIE trial, in a block-randomized order. Peripheral blood mononuclear cells (PBMC) were isolated before, after and 1h after each trial. NK cell subsets were enumerated using flow cytometry and complete blood counts. The surface expression of the cytotoxic NK cell (cNK; CD56dimCD16+) subset was evaluated for its expression of the differentiation markers CD57 and CD158a, the activating receptor NKG2D, the immune checkpoints TIM-3 and PD-1, and the chemokine receptors CXCR3, CXCR4 and CCR2. ResultscNK cell blood counts increased immediately following MOD (p < 0.001) and decreased back to pre-exercise values 1 h after exercise cessation (p < 0.001). The most responsive cNK cell subsets were expressing CD57, CD158a, NKG2D, TIM-3 and CXCR3. The HIIE trial elicited a similar biphasic response, without any difference between trials (all p ≥ 0.38). However, significant changes in the MFI values of CXCR4 and NKG2D were observed in the cNK cell subset following HIIE (all p ≤ 0.038), but not MOD. ConclusionIn metastatic cancer patients undergoing chemotherapy, both MOD and HIIE can elicit an acute mobilisation and egress of NK cells exhibiting phenotypic characteristics associated with high cytotoxicity and tumor homing. Future longitudinal trials are needed to determine if combining aerobic exercise training and chemotherapy will translate towards favorable immune and clinical outcomes.

TLR Agonists Modify NK Cell Activation and Increase Its Cytotoxicity in Acute Lymphoblastic Leukemia.

Natural killer (NK) cells play a crucial role in innate immunity, particularly in combating infections and tumors. However, in hematological cancers, NK cells often exhibit impaired functions. Therefore, it is very important to activate its endosomal Toll-like receptors (TLRs) as a potential strategy to restore its antitumor activity. We stimulated NK cells from the peripheral blood mononuclear cells from children with acute lymphoblastic leukemia and NK cells isolated, and the NK cells were stimulated with specific TLR ligands (Poly I:C, Imiquimod, R848, and ODN2006) and we evaluated changes in IFN-γ, CD107a, NKG2D, NKp44 expression, Granzyme B secretion, cytokine/chemokine release, and cytotoxic activity. Results revealed that Poly I:C and Imiquimod enhanced the activation of both immunoregulatory and cytotoxic NK cells, increasing IFN-γ, CD107a, NKG2D, and NKp44 expression. R848 activated immunoregulatory NK cells, while ODN2006 boosted CD107a, NKp44, NKG2D, and IFN-γ secretion in cytotoxic NK cells. R848 also increased the secretion of seven cytokines/chemokines. Importantly, R848 and ODN 2006 significantly improved cytotoxicity against leukemic cells. Overall, TLR stimulation enhances NK cell activation, suggesting TLR8 (R848) and TLR9 (ODN 2006) ligands as promising candidates for antitumor immunotherapy.

Comprehensive mapping of immune perturbations associated with secondary hemophagocytic lymphohistiocytosis.

Secondary hemophagocytic lymphohistiocytosis (sHLH) is a hyperinflammatory syndrome characterized by immune disorders. It is imperative to elucidate the immunophenotypic panorama and the interactions among these cells in patients. Human peripheral blood mononuclear cells were collected from healthy donors and sHLH patients and tested using multicolor flow cytometry. We used FlowSOM to explore and visualize the immunophenotypic characteristics of sHLH. By demonstrating the phenotypes of immune cells, we discovered that sHLH patients had significantly higher levels of CD56+ monocytes, higher levels of myeloid-derived suppressor cells, low-density neutrophil-to-T cell ratio, and higher heterogeneous T cell activation than healthy donors. However, natural killer cell cytotoxicity and function were impaired. We then assessed the correlations among 30 immune cell types and evaluated metabolic analysis. Our findings demonstrated polymorphonuclear myeloid-derived suppressor cells, CD56+ monocytes, and neutrophil-to-T cell ratio were elevated abnormally in sHLH patients, which may indicate an association with immune overactivation and inflammatory response. We are expected to confirm that they are involved in the occurrence of the disease through further in-depth research.

Dual roles of CD11b+CD33+HLA-DR-/lowCD14- myeloid-derived suppressor cells with a granulocytic morphology following allogeneic hematopoietic stem cell transplantation: from inflammation promoters to immune suppressors within 90 days.

Granulocytic myeloid-derived suppressor cells (G-MDSCs) show fast recovery following allogeneic hematopoietic stem cell transplantation (allo-HSCT) constituting the major part of peripheral blood in the early phase. Although G-MDSCs mediate immune suppression through multiple mechanisms, they may also promote inflammation under specific conditions. G-MDSCs were isolated from 82 patients following allo-HSCT within 90 days after allo-HSCT, and their interactions with autologous CD3+ T-cells were examined. T-cell proliferation was assessed by flow cytometry following CFSE staining, while differentiation and interferon-γ secretion were characterized using chemokine receptor profiling and ELISpot assays, respectively. NK cell cytotoxicity was evaluated through co-culture with K562 cells. An aGVHD xenogeneic model in humanized mice was employed to study the in vivo effects of human leukocytes. Furthermore, transcriptional alterations in G-MDSCs were analyzed via RNA sequencing to investigate functional transitions. G-MDSCs promoted inflammation in the early-stage, by facilitating cytokine secretion and proliferation of T cells, as well as their differentiation into pro-inflammatory T helper subsets. At day 28, patients with a higher number of G-MDSCs exhibited an increased risk of developing grades II-IV aGvHD. Besides, adoptive transfer of G-MDSCs from patients at day 28 into humanized mice exacerbated aGvHD. However, at day 90, G-MDSCs led to immunosuppression, characterized by upregulated expression of indoleamine 2,3-dioxygenase gene and interleukin-10 secretion, coupled with the inhibition of T cell proliferation. Furthermore, transcriptional analysis of G-MDSCs at day 28 and day 90 revealed that 1445 genes were differentially expressed. These genes were associated with various pathways, revealing the molecular signatures of early post-transplant differentiation in G-MDSCs. In addition, genes linked to the endoplasmic reticulum stress were upregulated in patients without aGvHD. The acquisition of immunosuppressive function by G-MDSCs may depend on the activation of CXCL2 and DERL1 genes. Our findings revealed the alteration in the immune characteristics of G-MDSCs within the first 90 days post-allo-HSCT. Moreover, the quantity of G-MDSCs at day 28 may serve as a predictive indicator for the development of aGvHD.

Boosting the anti-tumor activity of natural killer cells by caripe 8 – A Carapichea ipecacuanha isolated cyclotide

Cyclotides are head-to-tail cyclized peptides with a unique cystine-knot motif. Their structure provides exceptional resistance against enzymatic, chemical, or thermal degradation compared to other peptides. Peptide-based therapeutics promise high specificity, selectivity and lower immunogenicity, making them safer alternatives to small molecules or large biologicals. Cyclotides were researched due to their anti-cancer properties by inducing apoptosis in tumor cells in the past, but the impact of cyclotides on cytotoxic immune cells was poorly studied. Natural Killer (NK) cells are cytotoxic innate lymphoid cells and play an important role in the defense against infected, stressed and transformed cells. NK cells do not need prior sensitization and act in an antigen independent manner, holding promising potential in the field of immunotherapy. To investigate the effect of immunomodulatory cyclotides on NK cells, we evaluated several peptide-enriched plant extracts on NK cell mediated cytotoxicity. We observed that the extract samples derived from Carapichea ipecacuanha (Brot.) L. Andersson augments the killing potential of mouse NK cells against different tumor targets in vitro. Subsequent isolation of cyclotides from C. ipecacuanha extracts led to the identification of a primary candidate that enhances cytotoxicity of both mouse and human NK cells. The augmented killing is facilitated by the increased degranulation capacity of NK cells. In addition, we noted a direct toxic effect of caripe 8 on tumor cells, suggesting a dual therapeutic potential in cancer treatment. This study offers novel insights how natural peptides can influence NK cell cytotoxicity. These pre-clinical findings hold significant promise for advancing current immunotherapeutic approaches.

The Novel Fusion Protein Melittin-MIL-2 Exhibits Strong Antitumor Immune Effect in Lung Adenocarcinoma Cell A549.

In previous studies, we developed a novel fusion protein named "melittin-MIL-2" which exhibited more anti-tumor activity. However, it remains unclear whether melittin-MIL-2 possesses antitumor immune effect on lung adenocarcinoma. In this study, the immune effect and mechanism of melittin-MIL-2 inhibiting the growth and invasion of lung adenocarcinoma will be investigated, in order to provide novel perspectives for the immunotherapy of lung cancer. The results indicated that melittin-MIL-2 promoted T cell proliferation, enhanced NK cell cytotoxicity, and boosted IFN-γ secretion in PBMCs. After melittin-MIL-2 stimulation, perforin expression and LAK/NK-like killing activities of human PBMCs and NK cells were significantly enhanced. Melittin-MIL-2 is capable of hampering the development and proliferation of lung adenocarcinoma cell A549. ICAM-1 and Fas expression in A549 cells exposed to melittin-MIL-2 rose significantly. The expression levels of TLR8 and VEGF in A549 cells decreased significantly after melittin-MIL-2 stimulation. Invivo, melittin-MIL-2 substantially impeded the growth of lung adenocarcinoma and formed an immune-stimulating microenvironment locally in tumor tissues. In conclusion, the novel fusion protein melittin-MIL-2 exhibits strong anti-tumor immune effect in lung adenocarcinoma cell A549 via activating the LFA-1/ICAM-1 and Fas/FasL pathways to enhance cytolytic activity, upregulating the secretion of IFN-γ and perforin, and boosting LAK/NK-like killing activities. Immuno-effector cells and their secreted cytokines can form immune stimulation microenvironment locally in lung adenocarcinoma Lewis mice tissue.

LINC00665 promotes the progression and immune evasion of lung cancer by facilitating the translation of TCF7 protein through dependence on IRES

ObjectiveTo investigate the influence of LINC00665 on the development and immune evasion of lung cancer.MethodsTumor tissues and corresponding adjacent tissues were collected from 84 lung cancer patients, categorized into non-metastatic (n = 58) and metastatic (n = 26) groups. LINC00665 expression in lung cancer and metastatic lung cancer tissues was assessed via qRT-PCR. Pearson correlation analysis was conducted to examine the correlation between LINC00665 and immune-modulating cytokines (TGF-β, IL-10, IL-1β, IFN-γ, IL-2, TNF-α). A549 and H1299 cells, with relatively high LINC00665 expression, were used for in vitro studies. Cells were transfected with LINC00665-targeting shRNA, and changes in proliferation, apoptosis, migration, invasion, and NK cell cytotoxicity were assessed. Downstream molecular mechanisms of LINC00665 were investigated using GEO database analysis, highlighting the association with HHLA2. LINC00665’s role in promoting HHLA2 expression via binding with TCF7 was explored. In low LINC00665-expressing A549/H1299 cells, overexpression of HHLA2 was performed to evaluate effects on malignant behavior and NK cell sensitivity. A xenograft model was established for in vivo validation through tumor volume and weight measurements, Ki-67 immunoreactivity analysis, and flow cytometry analysis of CD107a + NK cells.ResultsLINC00665, TCF7 mRNA, and HHLA2 mRNA expression levels were significantly higher in lung cancer tissues than adjacent tissues, with non-metastatic lung cancer showing higher expression than metastatic lung cancer. In metastatic lung cancer, LINC00665 positively correlated with immune-suppressive cytokines (TGF-β, IL-10, IL-1β) and negatively correlated with anti-tumor cytokines (IFN-γ, IL-2, TNF-α). LINC00665 knockdown significantly inhibited lung cancer cell growth and metastasis, promoting sensitivity to NK cells. Further analysis revealed that LINC00665 recruits transcription factor TCF7 to upregulate HHLA2 expression in lung cancer cells, thereby facilitating lung cancer development and immune escape.ConclusionLINC00665, through recruitment of TCF7 and upregulation of HHLA2, inhibits NK cell cytotoxicity, promoting the development and immune evasion of lung cancer.

Deep Learning-Based Automated Analysis of NK Cell Cytotoxicity in Single Cancer Cell Arrays

The cytotoxicity assay of immune cells based on live cell imaging offers comprehensive information at the single cell-level information, but the data acquisition and analysis are labor-intensive. To overcome this limitation, we previously developed single cancer cell arrays that immobilize cancer cells in microwells as single cell arrays, thus allow high-throughput data acquisition. In this study, we utilize deep learning to automatically analyze NK cell cytotoxicity in the context of single cancer cell arrays. Defined cancer cell position and the separation of NK cells and cancer cells along distinct optical planes facilitate segmentation and classification by deep learning. Various deep learning models are evaluated to determine the most appropriate model. The results of the deep learning-based automated data analysis are consistent with those of the previous manual analysis. The integration of the microwell platform and deep learning would present new opportunities for the analysis of cell–cell interactions.

Lung Cancer Organoid System to Evaluate the Cytotoxicity of Natural Killer Cells.

Natural killer (NK) cells are gaining growing attention due to their promise for immunotherapy. A fast and accurate system is needed to test NK cell biology and their therapeutic application. Here, we report a lung cancer organoid-based system to evaluate NK cells' cytotoxicity. We first established the lung cancer organoids on top of Matrigel, which allows the co-culture with NK cells. When co-cultured, NK cells moved close to and inside the lung cancer organoids. When we analyzed by flow cytometry, co-culture of NK cells induced a significantly higher ratio of cell death of lung cancer organoids, suggesting that lung cancer organoids can be employed to test the cytotoxicity of NK cells. Finally, the pretreatment of NK cells with A83-01, a TGFβ inhibitor, significantly enhanced the cell death of lung cancer organoids by NK cells, indicating that lung cancer organoid-based system faithfully recapitulates cell line-based system in evaluating the in vitro cytotoxicity of NK cells. These data represent that cancer organoid-based NK cell co-culture system is a reliable platform for studying NK cell biology and evaluating their cytotoxicity for screening for NK cell immunotherapy.

In vivo AAV-SB-CRISPR screens of tumor-infiltrating primary NK cells identify genetic checkpoints of CAR-NK therapy.

Natural killer (NK) cells have clinical potential against cancer; however, multiple limitations hinder the success of NK cell therapy. Here, we performed unbiased functional mapping of tumor-infiltrating NK (TINK) cells using in vivo adeno-associated virus (AAV)-SB (Sleeping Beauty)-CRISPR (clustered regularly interspaced short palindromic repeats) screens in four solid tumor mouse models. In parallel, we characterized single-cell transcriptomic landscapes of TINK cells, which identified previously unexplored subpopulations of NK cells and differentially expressed TINK genes. As a convergent hit, CALHM2-knockout (KO) NK cells showed enhanced cytotoxicity and tumor infiltration in mouse primary NK cells and human chimeric antigen receptor (CAR)-NK cells. CALHM2 mRNA reversed the CALHM2-KO phenotype. CALHM2 KO in human primary NK cells enhanced their cytotoxicity, degranulation and cytokine production. Transcriptomics profiling revealed CALHM2-KO-altered genes and pathways in both baseline and stimulated conditions. In a solid tumor model resistant to unmodified CAR-NK cells, CALHM2-KO CAR-NK cells showed potent in vivo antitumor efficacy. These data identify endogenous genetic checkpoints that naturally limit NK cell function and demonstrate the use of CALHM2 KO for engineering enhanced NK cell-based immunotherapies.

Alginate-gelatine hydrogel microspheres protect NK cell proliferation and cytotoxicity under hypoxic conditions

Aims This study aimed to encapsulate natural killer (NK) cells in a hydrogel to sustain their function within the hypoxic tumour microenvironments. Methods An alginate-gelatine hydrogel was generated via electrospray technology. Hydrogel biocompatibility was assessed through cell counting kit-8 and Live/Dead assays to ascertain cell. Moreover, we analysed lactate dehydrogenase assays to evaluate the cytotoxicity against tumours and utilised RT-qPCR to analyse cytokine gene level. Results Alginate and gelatine formed hydrogels with diameters ranging from 489.2 ± 23.0 μm, and the encapsulation efficiency was 34.07 ± 1.76%. Encapsulated NK cells exhibited robust proliferation and tumour-killing capabilities under normoxia and hypoxia. Furthermore, encapsulation provided a protective shield against cell viability under hypoxia. Importantly, tumour-killing cytotoxicity through cytokines upregulation such as granzyme B and interferon-gamma was preserved under hypoxia. Conclusion The encapsulation of NK cells not only safeguards their viability but also reinforces anticancer capacity, countering the inhibition of activation induced by hypoxia.

NK cells with adhesion defects and reduced cytotoxic functions are associated with a poor prognosis in multiple myeloma

AbstractThe promising results obtained with immunotherapeutic approaches for multiple myeloma (MM) call for a better stratification of patients based on immune components. The most pressing being cytotoxic lymphocytes such as natural killer (NK) cells that are mandatory for MM surveillance and therapy. Here, we performed a single-cell RNA sequencing analysis of NK cells from 10 patients with MM and 10 age/sex–matched healthy donors that revealed important transcriptomic changes in the NK cell landscape affecting both the bone marrow (BM) and peripheral blood compartment. The frequency of mature cytotoxic CD56dim NK cell subsets was reduced in patients with MM at the advantage of late-stage NK cell subsets expressing NF-κB and interferon-I inflammatory signatures. These NK cell subsets accumulating in patients with MM were characterized by low CD16 and CD226 expression and poor cytotoxic functions. MM CD16/CD226Lo NK cells also had adhesion defects with reduced lymphocyte function-associated antigen 1 (LFA-1) integrin activation and actin polymerization that may account for their limited effector functions in vitro. Finally, analysis of BM-infiltrating NK cells in a retrospective cohort of 177 patients with MM from the Intergroupe Francophone du Myélome (IFM) 2009 trial demonstrated that a high frequency of NK cells and their low CD16 and CD226 expression were associated with a shorter overall survival. Thus, CD16/CD226Lo NK cells with reduced effector functions accumulate along MM development and negatively affect patients’ clinical outcomes. Given the growing interest in harnessing NK cells to treat myeloma, this improved knowledge around MM–associated NK cell dysfunction will stimulate the development of more efficient immunotherapeutic drugs against MM.

CEACAM1-engineered MSCs have a broad spectrum of immunomodulatory functions and therapeutic potential via cell-to-cell interaction

Mesenchymal stem cells (MSCs) have garnered attention for their regenerative and immunomodulatory capabilities in clinical trials for various diseases. However, the effectiveness of MSC-based therapies, especially for conditions like graft-versus-host disease (GvHD), remains uncertain. The cytokine interferon (IFN)-γ has been known to enhance the immunosuppressive properties of MSCs through cell-to-cell interactions and soluble factors. In this study, we observed that IFN-γ-treated MSCs upregulated the expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), associated with immune evasion through the inhibition of natural killer (NK) cell cytotoxicity. To co-opt this immunomodulatory function, we generated MSCs overexpressing CEACAM1 and found that CEACAM1-engineered MSCs significantly reduced NK cell activation and cytotoxicity via cell-to-cell interaction, independent of NKG2D ligand regulation. Furthermore, CEACAM1-engineered MSCs effectively inhibited the proliferation and activation of T cells along with the inflammatory responses of monocytes. In a humanized GvHD mouse model, CEACAM1-MSCs, particularly CEACAM1-4S-MSCs, demonstrated therapeutic potential by improving survival and alleviating symptoms. These findings suggest that CEACAM1 expression on MSCs contributes to MSC-mediated regulation of immune responses and that CEACAM1-engineered MSC could have therapeutic potential in conditions involving immune dysregulation.

CD155 blockade enhances allogeneic natural killer cell-mediated antitumor response against osteosarcoma.

Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect but has been ineffective for solid tumors like osteosarcoma (OS). OS expresses CD155 which interacts strongly with inhibitory receptors TIGIT and CD96 but also binds to activating receptor DNAM-1 on natural killer (NK) cells. CD155 has never been targeted after alloBMT. Combining adoptively transferred allogeneic NK (alloNK) cells with CD155 blockade after alloBMT may enhance a GVT effect against OS. Murine NK cells were activated and expanded ex vivo with soluble IL-15/IL-15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT and alloNK cell infusion with anti-CD155 either before or after tumor induction, with select groups receiving anti-DNAM-1 pretreated alloNK cells. Tumor growth, GVHD and survival were monitored, and differential gene expression of lung tissue was assessed by RNA microarray. AlloNK cells exhibited superior cytotoxicity against CD155-expressing OS compared to synNK cells, and this activity was enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1. In vivo, CD155 blockade with alloNK infusion increased survival when treating OS that relapsed after alloBMT. No benefit was seen for treating established OS before alloBMT. Treatment with combination CD155 and anti-DNAM-1 pretreated alloNK ameliorated survival and tumor control benefits seen with CD155 blockade alone. RNA microarray showed mice treated with alloNK and CD155 blockade had increased expression of cytotoxicity genes and the NKG2D ligand H60a, whereas mice treated with anti-DNAM-1 pretreated alloNK cells resulted in upregulation of NK cell inhibitory receptor genes. Whereas blocking DNAM-1 on alloNK abrogated cytotoxicity, blocking NKG2D had no effect, implying DNAM-1:CD155 engagement drives alloNK activation against OS. These results demonstrate the safety and efficacy of infusing alloNK cells with CD155 blockade to mount a GVT effect against OS and show benefits are in part through DNAM-1. Defining the hierarchy of receptors that govern alloNK responses is critical to translating alloNK cell infusions and immune checkpoint inhibition for solid tumors treated with alloBMT. Allogeneic bone marrow transplant (alloBMT) has yet to show efficacy in treating solid tumors, such as osteosarcoma (OS). CD155 is expressed on OS and interacts with natural killer (NK) cell receptors, such as activating receptor DNAM-1 and inhibitory receptors TIGIT and CD96 and has a dominant inhibitory effect on NK cell activity. Targeting CD155 interactions on allogeneic NK cells could enhance anti-OS responses, but this has not been tested after alloBMT. CD155 blockade enhances allogeneic natural killer cell-mediated cytotoxicity against OS and improved event-free survival after alloBMT in an in vivo mouse model of metastatic pulmonary OS. Addition of DNAM-1 blockade abrogated CD155 blockade-enhanced allogeneic NK cell antitumor responses. These results demonstrate efficacy of allogeneic NK cells combined with CD155 blockade to mount an antitumor response against CD155-expressing OS. Translation of combination adoptive NK cell and CD155 axis modulation offers a platform for alloBMT treatment approaches for pediatric patients with relapsed and refractory solid tumors.

Asciminib stands out as the superior tyrosine kinase inhibitor to combine with anti-CD20 monoclonal antibodies for the treatment of CD20+ Philadelphia-positive B-cell precursor acute lymphoblastic leukemia in preclinical models.

Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia (Ph+ BCPALL) is a high-risk acute lymphoblastic leukemia subtype characterized by the presence of BCR::ABL1 fusion gene. Tyrosine kinase inhibitors (TKIs) combined with chemotherapy are established as the first-line treatment. Additionally, rituximab (RTX), an anti-CD20 monoclonal antibody (mAb) is administered in adult BCP-ALL patients with ≥20% of CD20+ blasts. In this study, we observed a marked prevalence of CD20 expression in patients diagnosed with Ph+ BCP-ALL, indicating a potential widespread clinical application of RTX in combination with TKIs. Consequently, we examined the influence of TKIs on the antitumor effectiveness of anti-CD20 mAbs by evaluating CD20 surface levels and conducting in vitro functional assays. All tested TKIs were found to uniformly downregulate CD20 on leukemic cells, diminishing the efficacy of RTX-mediated complement-dependent cytotoxicity. Interestingly, these TKIs displayed varied effects on NK cell-mediated antibody-dependent cytotoxicity and macrophage phagocytic function. While asciminib demonstrated no inhibition of effector cell functions, dasatinib notably suppressed the anti-CD20-mAb-mediated NK cell cytotoxicity and macrophage phagocytosis of BCP-ALL cells. Dasatinib and ponatinib also decreased NK cell degranulation in vitro. Importantly, oral administration of dasatinib, but not asciminib, compromised NK cell activity within patients' blood, determined by ex vivo degranulation assay. Our results indicate that asciminib might be preferred over other TKIs for combination therapy with anti-CD20 mAbs.

Enhanced treatment of breast cancer brain metastases with oncolytic virus expressing anti-CD47 antibody and temozolomide

Limited therapeutic options are available for patients with breast cancer brain metastases (BCBM), and thus there is an urgent need for novel treatment approaches. We previously engineered an effective oncolytic herpes simplex virus-1 (oHSV) expressing a full-length anti-CD47 monoclonal antibody (mAb) with a human IgG1 scaffold (OV-αCD47-G1) that was used to treat both ovarian cancer and glioblastoma. Here, we demonstrate that the combination of OV-αCD47-G1 and temozolomide (TMZ) improve outcomes in preclinical models of BCBM. The combination of TMZ with OV-αCD47-G1 synergistically increased macrophage phagocytosis against breast tumor cells and led to greater activation of NK cell cytotoxicity. Additionally, the combination of OV-αCD47-G1 with TMZ significantly prolonged the survival of tumor-bearing mice when compared to TMZ or OV-αCD47-G1 alone. Combination treatment with the mouse counterpart of OV-αCD47-G1, termed OV-A4-IgG2b, also enhanced mouse macrophage phagocytosis, NK cell cytotoxicity, and survival in an immunocompetent model of mice bearing BCBM compared to TMZ or OV-A4-IgG2b alone. Collectively, these results suggest that OV-αCD47-G1 combined with TMZ should be explored in patients with BCBM.

Tethering of hexokinase 2 to mitochondria promotes resistance of liver cancer cells to natural killer cell cytotoxicity.

Hexokinases (HKs) control the first step of glucose catabolism. A switch of expression from liver HK (glucokinase, GCK) to the tumor isoenzyme HK2 is observed in hepatocellular carcinoma progression. Our prior work revealed that HK isoenzyme switch in hepatocytes not only regulates hepatic metabolic functions but also modulates innate immunity and sensitivity to Natural Killer (NK) cell cytotoxicity. This study investigates the impact of HK2 expression and its mitochondrial binding on the resistance of human liver cancer cells to NK-cell-induced cytolysis. We have shown that HK2 expression induces resistance to NK cell cytotoxicity in a process requiring mitochondrial binding of HK2. Neither HK2 nor GCK expression affects target cells' ability to activate NK cells. In contrast, mitochondrial binding of HK2 reduces effector caspase 3/7 activity both at baseline and upon NK-cell activation. Furthermore, HK2 tethering to mitochondria enhances their resistance to cytochrome c release triggered by tBID. These findings indicate that HK2 mitochondrial binding in liver cancer cells is an intrinsic resistance factor to cytolysis and an escape mechanism from immune surveillance.