bims-nakice 2026-05-31 papers

bims-nakice

Biomed News

on Natural killer cells

Issue of 2026–05–31
twenty papers selected by
Santosh Phuyal, Oslo Universitetssykehus

MINFLUX nanoscopy to study the NK cell immune synapse.
Assessment of NK cell cytotoxicity induced by IL-15 based immunotherapy against cancer cells.
Efficient, fratricide free non-viral engineering of CD70-targeted CAR NK cells for hematologic and solid tumor immunotherapy.
NK cell hyperactivation drives macrophage repolarization and limits M2 bias in pemphigus vulgaris.
LFA-1 interaction with GBP-130 on Plasmodium falciparum-infected red blood cells mediates NK cell activation and parasite control.
Characterization of anti-tumor activity in different NK cell subsets by multiparametric flow cytometry.
[A new nomenclature for natural killer cells].
CD49a expression defines a metabolically robust, cytokine-biased liver NK cell subset in rhesus macaques during lentivirus infection.
Characterization of natural killer (NK) cells in lung adenocarcinoma and construction of an NK risk signature based on single-cell and macromolecular RNA-seg data.
Antibody Blockade of Ly49/MHC-I interactions enhances Innate and Adaptive Immunity Against Cancer Metastasis.
Cytokine preactivation and ADCC: a potent strategy for enhancing human NK cell effector functions against 3D tumor models.
Ablation of Cbl-b in ROBO1 CAR-NK92 Cells Enhances Their Antitumor Efficacy.
Umbilical cord blood natural killer cells improve anti-GD2 antibody efficacy in neuroblastoma: from mouse to human.
Osteopontin-driven TGF-β1 signaling via integrin αvβ3-NF-κB axis impairs NK cell function in head and neck squamous cell carcinoma.
Deficient TRPM3-linked mitochondrial Ca2+ influx in natural killer cells associated with myalgic encephalomyelitis/chronic fatigue syndrome.
Triple-Negative Breast Cancer Cells Utilize IL8 and CXCL1 to Suppress NK Cells' Function and Facilitate Cancer Metastasis.
Natural killer cell immunotherapy for pancreatic cancer: Release the brakes.
Efficient NK cell transduction with VSV-G-pseudotyped lentiviral vectors.
Overexpression of SLC44A4 suppresses ferroptosis and reduces lipid peroxidation via noncanonical NF-κB signaling in a NIK-dependent manner.
Rapid assessment of immune effector cell-mediated cytotoxicity using mRNA lipid nanoparticles.

Methods Cell Biol. 2026 ;pii: S0091-679X(26)00021-X. [Epub ahead of print]207 179-202

MINFLUX nanoscopy to study the NK cell immune synapse.

Nora Ross, Daniel P Leaman, Jessica Matthias, Michael B Zwick, Emily M Mace, Scott C Henderson, Charles Daniel Murin.

  Antibody dependent cellular cytotoxicity (ADCC) is an effector function performed by natural killer (NK) cells to target and clear viral infections and cancer. ADCC is a critical feature of several antibody and cellular therapeutics as well as vaccination strategies. Using microscopy to understand the details of molecular events driving ADCC is essential to improving such therapeutics but has been limited by technologies that cannot practically provide the spatial resolution necessary to study protein function at the single molecule level in cells. In this chapter, we describe a model system using MINFLUX nanoscopy to study the molecular distribution of human FcγRIIIa (CD16a), the IgG receptor, in the NK cell immunological synapse during ADCC. The technique described here will enable further exploration of how CD16a drives NK cell ADCC and can also be applied to the study of other important protein receptors for which nanometer localization precision is needed.

Keywords:  ADCC; Antibodies; Cytotoxicity; Fc receptors; MINFLUX; NK cells

DOI:  https://doi.org/10.1016/bs.mcb.2026.01.021
Methods Cell Biol. 2026 ;pii: S0091-679X(26)00122-6. [Epub ahead of print]207 203-218

Assessment of NK cell cytotoxicity induced by IL-15 based immunotherapy against cancer cells.

Camille Rolin, Gilles Iserentant, Aubin Pitiot, Carole Seguin-Devaux.

  Natural Killer (NK) cells are at the interface of the innate and adaptive immune system. While they are naturally able to kill pathogen-infected, malignant and compromised cells, they also drive the recruitment and activation of other immune cells through the production of inflammatory cytokines and chemokines. Evaluation of NK cells cytotoxic activity is critical to determine accurately the anti-tumor potency of novel immunotherapies, in particular, when the later aim to overcome NK cells exhaustion driven by an immunosuppressive environment such as the tumor microenvironment. Here, we provide a detailed protocol for the assessment of primary NK cells cytotoxic functions against pancreatic cancer cells stimulated by a directed approach on NK cells based on the cytokine IL-15. This article describes the protocol of a flow cytometry-based assay and a calcein acetoxymethyl ester (AM) cell viability assay to quantify NK cell cytotoxic activity. Both methods are robust, fast and sensitive to be applicable in clinics and can readily be adapted for the assessment of other immunotherapies on NK cells cytotoxicity.

Keywords:  Calcein; Cytotoxicity; Flow cytometry; Immunotherapy; NK cells

DOI:  https://doi.org/10.1016/bs.mcb.2026.04.001
Mol Ther Adv. 2026 Jun 11. 34(2): 201734

Efficient, fratricide free non-viral engineering of CD70-targeted CAR NK cells for hematologic and solid tumor immunotherapy.

Jae-Woong Chang, Joshua Krueger, Timothy D Folsom, Joseph G Skeate, Erin M Stelljes, Walker S Lahr, Nicholas J Slipek, Emily J Pomeroy, Beau R Webber, Branden S Moriarity.

  Natural killer (NK) cells are a component of the innate immune system with potential safety advantages over T cells for adoptive cell therapy (ACT). However, NK cells lack the potency and persistence needed for tumor clearance. Here, we report an efficient and cost-effective non-viral method for engineering CD70 targeted CAR NK cells using a hyperactive TcBuster DNA transposon system while simultaneously knocking out CD70 via CRISPR-Cas9 to prevent fratricide. CD70 CAR NK cells engineered in this manner display improved efficacy in killing acute myeloid lymphoma (AML) cells and renal cell carcinoma (RCC) cells in vitro and in vivo. Incorporating soluble IL-15 cytokine armoring into the transposon cassette further improves survival and expansion in vivo. Our findings demonstrate a novel application of non-viral transposon-based cell engineering combined with CRISPR-Cas9 knockout to produce a clinically scalable product for ACT against multiple cancer types.

Keywords:  CRISPR-Cas9 CD70; TcBuster; acute myeloid leukemia; chimeric antigen receptor; fratricide; natural killer cells; renal cell carcinoma

DOI:  https://doi.org/10.1016/j.omta.2026.201734
Front Immunol. 2026 ;17 1774059

NK cell hyperactivation drives macrophage repolarization and limits M2 bias in pemphigus vulgaris.

Vishakha Hooda, Aishwarya Singh, Sujay Khandpur, Sudheer Arava, Taruna Madan, Alpana Sharma.

   Background: Macrophages and natural killer (NK) cells are innate immune components proven to be either pathogenic, protective, or both in autoimmune disorders. Nonetheless, the function of macrophages in the pathogenesis of pemphigus vulgaris (PV), an autoimmune skin disorder, is lacking.
Objectives: We aimed to investigate macrophages and their crosstalk with NK cells in PV, with a particular focus on how these interactions influence their functionalities.
Methods: Flow cytometry and immunofluorescence was utilised to assess macrophage profile along with expression of receptors on NK cells and macrophages. In-vitro macrophage skewing, and NK-macrophage interactions (including F-actin accumulation, activation markers, cytotoxicity, IFNγ and perforin secretion, NOS2, CD86, CD206 expression, phagocytic activity, and arginase activity were examined. NKG2D blocking and related assays were performed to elucidate its role in crosstalk.
Results: Expression of NKG2D on NK cells and MICA/B on macrophages was upregulated. CD56+ NK cells and CD68+ macrophages co-localized in lesions. A significant increase in macrophages in both the periphery and lesional skin was observed. Both M1 and M2 macrophages were upregulated in blood, whereas M1 increased and M2 decreased in skin lesions. Macrophages displayed skewing toward an M2 phenotype in vitro. Upon co-culturing, F-actin accumulation increased during interaction. The interaction led to increased activation, cytotoxicity, and IFNγ and perforin secretion in NK cells when M1 stimulation and Dsg3 autoantigen were given. In macrophages, increased expression of iNOS and CD86 and higher phagocytic activity were noted, whereas arginase activity and expression along with CD206 were downregulated. NKG2D blockade on NK cells resulted in partial but significant decrease in NK cells activity including activation, perforin, cytotoxicity and IFNγ release. The reduction were prominent in M1 state. M1 like markers NOS2 and CD86 were reduced and arginase activity non significantly improved under M2 state post blockade.
Conclusion: This study helped in comprehending functionally active NK- macrophage axis, where NK cells promote pro inflammatory M1 state, and dysregulate M2 macrophage functionality. NKG2D blockade resulted in partial reversal suggesting its critical, though not elusive, contribution indicating multifactorial crosstalk mechanism. Targeting NK cell activity or promoting M2 functionality could potentially lead to improvements in disease pathogenesis.

Keywords:  autoimmunity; macrophages NK crosstalk; monocytes; natural killer cells; pemphigus vulgaris

DOI:  https://doi.org/10.3389/fimmu.2026.1774059
Elife. 2026 May 28. pii: RP110942. [Epub ahead of print]15

LFA-1 interaction with GBP-130 on Plasmodium falciparum-infected red blood cells mediates NK cell activation and parasite control.

Osama Mukhtar, Ravi Dutt, Ashutosh Panda, Poonam Kumari, Suneet Shekhar Singh, Gourab Paul, Neha Prakash, Madiha Abbas, Md Muzahidul Islam, Priya Arora, Alma Tammour, Asif Mohmmed, Dhiraj Kumar, Pawan Malhotra.

  Natural killer (NK) cells contribute to early immunity against Plasmodium falciparum by recognizing and eliminating infected red blood cells (iRBCs), a process mediated in part by the integrin LFA-1. However, the cognate parasite ligand for LFA-1 has remained unknown. Here, we identify glycophorin binding protein-130 (PfGBP-130) as a surface-expressed ligand on iRBCs that binds the I-domain of LFA-1 (LFA-1 αI). Using an LFA-1 αI-Fc fusion protein, we demonstrate stage-specific binding to iRBCs, and LC-MS/MS analysis of immunoprecipitates of αI-Fc bound to iRBC revealed PfGBP-130 as a high-confidence interactor. Recombinant PfGBP-130 binds NK and THP-1 cells in an LFA-1-dependent manner. Co-culture assays show that PfGBP-130 promotes NK cell activation and degranulation and facilitates contact-dependent killing of iRBCs. Neutralizing antibodies against PfGBP-130 significantly impair these responses. Our findings establish PfGBP-130 as the LFA-1 ligand on iRBCs, providing new insight into NK cell-mediated immunity in malaria and identifying a potential target for host-directed interventions.

Keywords:  LFA-1; NK cells; P. falciparum; PfGBP-130; Plasmodium falciparum; host-parasite interaction; infected RBC surface; infectious disease; microbiology

DOI:  https://doi.org/10.7554/eLife.110942
Methods Cell Biol. 2026 ;pii: S0091-679X(26)00090-7. [Epub ahead of print]207 237-253

Characterization of anti-tumor activity in different NK cell subsets by multiparametric flow cytometry.

Carola Perrone, Paolo Canevali, Veronica Colamassaro, Elena Giusto, Silvia Tamburini, Gianluca Ubezio, Guido Ferlazzo, Massimo Vitale, Raffaella Meazza.

  The ever more insightful dissection of the NK cell population in humans has produced a plethora of (more or less) defined subpopulations with quite differentiated functional properties. Considering the potential clinical perspectives offered by peripheral blood NK cells, it is crucial to design strategies to functionally assess a large number of cell subsets directly from the peripheral mononuclear cells (PBMCs). In this chapter, we describe a simple method to quantify cytotoxic degranulation and IFN-γ production in classical NK cell subsets, focusing, particularly on the NKG2C+ "adaptive" NK cells.

Keywords:  Adaptive NK cells; NK cell functions; NK cell subsets; Nonlinear dimensionality reduction algorithms

DOI:  https://doi.org/10.1016/bs.mcb.2026.02.022
Med Sci (Paris). 2026 May;42(5): 451-458

[A new nomenclature for natural killer cells].

Lucas Rebuffet, Éric Vivier.

Recent advances in single-cell transcriptomics have profoundly renewed our understanding of immune cells. They have enabled us to better characterize the heterogeneity of Natural Killer (NK) cells and establish a classification that integrates all their characteristics. These approaches have also shed light on the behavior of NK cells infiltrating the tumor microenvironment, the mechanisms of their dysfunction, and the limits of their antitumor activity. Finally, they are essential for understanding the mechanisms of action of new therapies targeting NK cells.

DOI: https://doi.org/10.1051/medsci/2026080
Immunol Cell Biol. 2026 May 27.

CD49a expression defines a metabolically robust, cytokine-biased liver NK cell subset in rhesus macaques during lentivirus infection.

Andrew Hudson, Melissa Xin Yu Wong, Michael Verdolin, R Keith Reeves, Cordelia Manickam.

  Liver-resident natural killer (LrNK) cells are critical regulators of hepatic immune homeostasis and demonstrate antiviral activity, yet their role in lentivirus infections remains poorly defined. Using a rhesus macaque (RM) model of Simian immunodeficiency virus (SIV) and Simian-human immunodeficiency virus (SHIV) infections, we investigated the phenotypes, metabolic profiles and functional responses of hepatic NK cells across naïve, acutely SIV-infected and chronically SHIV-infected RM. We identified liver-resident (Lr) NK cells as CD3-CD159a/c+CD49a+, whereas CD3-CD159a/c+CD49a- NK cells include circulating NK cell subsets in the liver. Across infection states, CD49a+ LrNK cells exhibited elevated expression of activation markers, including NKG2D, NKp30, NKp46 and CD69, as well as increased expression of metabolic markers, including CD98, Glut1 and MitoTracker. In contrast, Ki-67 expression was reduced in CD49a+ LrNK cells compared with their CD49a- counterparts. Unsupervised clustering revealed that CD49a+ LrNK cells were also marked by high CD69 expression. While total NK cell cytokine secretion was elevated in acute SIV-infected animals, CD49a+ LrNK cells exhibited robust polyfunctional cytokine responses and reduced expression of the cytotoxic marker CD107a upon mitogen stimulation in comparison with CD49a- LrNK cells regardless of infection status. These findings reveal distinct metabolic and functional specialization of hepatic NK cell subsets, underscoring the unique phenotype and functions of CD49a+ LrNK cells in RM, which may offer novel insights and therapeutic opportunities for treating liver inflammation in HIV infection and other chronic liver diseases.

Keywords:  SHIV infection; SIV; liver; resident NK cells; rhesus macaques

DOI:  https://doi.org/10.1111/imcb.70135
Discov Oncol. 2026 May 25. pii: 784. [Epub ahead of print]17(1):

Characterization of natural killer (NK) cells in lung adenocarcinoma and construction of an NK risk signature based on single-cell and macromolecular RNA-seg data.

Yikun Guo, Chuanxi Tian, Daowen Yang.

   BACKGROUND/AIMS: Natural killer (NK) cells play a crucial role in tumor cell apoptosis, immune milieu regulation, and angiogenesis inhibition. This study aims to analyze the NK signature in lung adenocarcinoma (LUAD) and establish an NK cell-based risk signature for predicting the prognosis of LUAD patients.
METHODS: Single-cell RNA sequencing (scRNA-seq) data were obtained from the GEO database, while RNA-seq and microarray data from LUAD were simultaneously obtained from the TCGA and GEO databases. The scRNA-seq data were processed using the Seurat R package to identify NK clusters based on NK markers. Differentially expressed genes (DEGs) between normal and tumor samples were identified through differential expression analysis of LUAD-related data. Pearson correlation analysis was used to identify DEGs associated with NK clusters, followed by one-way Cox regression analysis to identify NK cell-related prognostic genes. Subsequently, Lasso regression analysis was employed to construct a risk signature based on NK cell-related prognostic genes. Finally, a column-line diagram model was constructed based on the risk signature and clinicopathological features.
RESULTS: Based on the scRNA-seq data, we identified five Natural killer (NK)cells clusters in lung adenocarcinoma (LUAD), with four of them showing associations with prognosis in LUAD. Out of 19,495 differentially expressed genes (DEGs), a total of 725 genes significantly associated with NK clusters were pinpointed and further narrowed down to form a risk profile comprising 13 genes. These 13 genes were primarily linked to 21 signaling pathways, including vascular smooth muscle contraction, RNA polymerase, and pyrimidine metabolism. Additionally, the risk profile exhibited significant associations with stromal and immune scores, as well as various immune cells. Multifactorial analysis indicated that the risk profile served as an independent prognostic factor for LUAD, and its efficacy in predicting the outcome of immunotherapy was validated. Furthermore, a novel column-line diagram integrating staging and NK-based risk profiles was developed, demonstrating strong predictability and reliability in prognostic forecasting for LUAD.
CONCLUSION: The NK cell-based risk signature proves to be a valuable tool for predicting the prognosis of patients with LUAD. Furthermore, a comprehensive understanding of NK cell characterization in LUAD could potentially unveil insights into the response of LUAD to immunotherapies and offer novel strategies for cancer treatment.

Keywords:  Columnar plots; Differentially expressed genes; Lung adenocarcinoma; Natural killer cells; Risk signature

DOI:  https://doi.org/10.1007/s12672-026-04552-w
bioRxiv. 2026 May 12. pii: 2026.05.07.722994. [Epub ahead of print]

Antibody Blockade of Ly49/MHC-I interactions enhances Innate and Adaptive Immunity Against Cancer Metastasis.

Abir K Panda, Surajit Sinha, Kannan Natarajan, Jiansheng Jiang, Sruthi Chempati, Soha Kazmi, Yong-Hee Kim, Suveena Sharma, Paul Schaughency, Lisa F Boyd, Jonathan M Hernandez, David H Margulies, Ethan M Shevach.

Background: Antibody-mediated blockade of innate receptor-MHC-I interactions represents a promising strategy to enhance anti-tumor immunity, particularly against metastatic cancers resistant to conventional checkpoint inhibitors. In this study, we investigated the effects of the pan anti-MHC-I monoclonal antibody M1/42, which targets MHC-I interactions with Ly49, selectively expressed on murine NK cell subsets.
Methods: We administered M1/42 to mice and assayed the proliferation and activation immune cells. Anti-tumor activity of growth and metastasis of checkpoint inhibitor-resistant pancreatic ductal adenocarcoma (PDAC) and B16F10 melanoma were assessed, complemented by extensive cellular phenotypic and RNA expression analysis. Binding and cryo-electron microscopic (cryo-EM) and X-ray crystallographic structural studies of M1/42 complexed with the mouse MHC-I molecule, H2-D d , examined the Ab interaction site in comparison with those of Ly49 inhibitory receptors.
Results: M1/42 administration in mice robustly unleashed the proliferation and activation of natural killer (NK) cells, memory CD4 + and CD8 + T cells, dendritic cells, and macrophages in both lymphoid and non-lymphoid tissues, independent of Fcγ receptors. M1/42 significantly restricted the growth and metastasis of checkpoint inhibitor-resistant pancreatic ductal adenocarcinoma (PDAC) and B16F10 melanoma in the liver and lungs, accompanied by increased tumor infiltration of effector CD8 + T cells, reduction of T regulatory cells, and a pro-inflammatory cytokine milieu. The anti-tumor effects of M1/42 depend on NK cells and are associated with upregulation of genes involved in antigen processing, interferon gamma responsiveness, and Th1 cytokine production, while downregulating inhibitory PD1/11 signaling. Structural analysis indicated that the effect of M1/42 on Ly49/MHC-I interactions was not due to direct steric competition.
Conclusions: Collectively, these findings demonstrate that M1/42 unleashes coordinated innate and adaptive immune responses, overcoming tumor-induced immunosuppression and resistance to checkpoint blockade. This approach represents a paradigm shift in cancer immunotherapy, offering potential for more effective treatment of metastatic cancers that evade immune surveillance through MHC-I modulation.
KEY MESSAGES: What is already known on this topic: A pan anti-mouse MHC-I mAb (M1/42) blocks interaction with several NK inhibitory receptors (Ly49A or Ly49C) resulting in NK cell activation and anti-viral and anti-tumor responses in vitro and in vivo . Other pan anti-human MHC-I mAbs (DX17 and W6/32) function similarly, blocking LILRB inhibitory receptor interaction of myeloid cells and NK cells. These stimulate human immune cells in humanized mouse models. What this study adds: This study analyzes the effects of the pan anti-mouse MHC-I mAb on NK and myeloid cell activation in detail, in the absence of T or B cells, and independent of FcR interaction. Additionally we analyze several mouse models of metastatic tumor progression, indicative of the progressive activation not only of the innate immune response, but also adaptive responses. The molecular mechanism of the mAb blocking of inhibitory receptors is revealed by cryo-EM and X-ray structures of M1/42 Fab/MHC-I (H2-D d ) complexes. How this study might affect research, practice, or policy: Elucidation of the details of the inhibitory effects of the mouse pan anti-mouse MHC-I mAb provides not only a more advanced understanding of the murine model system, but suggests additional functional avenues to be explored using the parallel an anti-human MHC-I mAbs.

DOI: https://doi.org/10.64898/2026.05.07.722994
Front Immunol. 2026 ;17 1797290

Cytokine preactivation and ADCC: a potent strategy for enhancing human NK cell effector functions against 3D tumor models.

Ainara Lopez-Pardo, Ainhoa Amarilla-Irusta, Ainhoa Iturbe-Larrondo, Itxaso San Juan, Víctor Sandá, Olatz Zenarruzabeitia, Francesca D Ciccarelli, Francisco Borrego, Laura Amo.

  The adoptive transfer of NK cells has shown clinical promise in hematologic malignancies, but its efficacy in solid tumors remains limited. Three-dimensional (3D) models reproduce tumor architecture and immunosuppressive microenvironments more accurately than conventional two-dimensional (2D) cultures. Here, we employed colorectal cancer (CRC) and lung cancer 3D tumor models to evaluate the anti-tumor activity of cytokine-induced memory-like (CIML) NK cells and to test whether cetuximab augments these responses through antibody-dependent cellular cytotoxicity (ADCC). Human NK cells were preactivated overnight with interleukin (IL)-12/15/18 (pre-CIML), then co-cultured with tumor spheroids and patient-derived organoids in the presence or absence of cetuximab. Pre-CIML NK cells showed significantly enhanced effector functions compared to control NK cells, including increased degranulation, higher IFN-γ and TNF-α production, and superior cytotoxicity against both spheroids and organoids. Additionally, pre-CIML NK cells that infiltrated tumor spheroids displayed a more uniform distribution within the tumor mass than control NK cells, which may contribute to their improved killing capacity. Cetuximab-mediated ADCC further enhanced NK cell activity against spheroid models, while in organoids, the enhancement was tumor- and context-dependent. Overall, these findings demonstrate that pre-CIML NK cells exhibit robust anti-tumor activity in clinically relevant 3D tumor models, and identify ADCC as an additional, but context-restricted, mechanism by which targeted antibodies such as cetuximab can further enhance NK cell functionality. These findings underscore the translational potential of NK cell-based immunotherapies that combine cytokine preactivation and ADCC induction to overcome current challenges in the treatment of solid tumors.

Keywords:  ADCC; NK cells; cetuximab; colorectal cancer; cytokines; immunotherapy; lung cancer; memory-like

DOI:  https://doi.org/10.3389/fimmu.2026.1797290
Hum Gene Ther. 2026 May 27. 10430342261453858

Ablation of Cbl-b in ROBO1 CAR-NK92 Cells Enhances Their Antitumor Efficacy.

Jingjing Hu, Jun Zhang, Yu Shao, Huashun Li, Yi Yang, Jinping Zhang.

  Emerging evidence suggests CAR-NK cell therapy shows great promise in cancer treatment. ROBO1 is highly expressed in various cancer types, including glioblastoma, hepatocellular carcinoma, lung cancer, breast cancer, and uterine cancer. Our and other laboratories' studies have shown that ROBO1 CAR-NK cells exhibit promising tumor therapeutic effects. However, the results still have some limitations. Cbl-b, an E3 ubiquitin ligase, has been reported to negatively regulate NK cell activation, homeostasis, and antitumor immunity.1 Therefore, we attempted to further enhance the antitumor activity of ROBO1 CAR-NK92 cells by knocking out Cbl-b using CRISPR/Cas9 gene-editing technology. In this study, we conjugated Cbl-b sgRNA with Cas9 protein to form ribonucleoprotein complexes, which were then delivered into ROBO1 CAR-NK92 and NK-92 cells (control cells) via electroporation. Through fluorescence-activated cell sorting, limiting dilution, and sequencing, we obtained monoclonal Cbl-b-knock-out (KO) cell lines. Both in vitro cytotoxicity assays and in vivo tumor xenograft experiments were conducted to examine whether Cbl-b knockout enhances the target cell killing and tumor suppression capacities of ROBO1 CAR-NK92 cells. In this study, monoclonal cell lines of ROBO1 CAR-NK92-Cbl-b-KO and NK92-Cbl-b-KO were successfully established. In vitro, at an effector-to-target (E:T) ratio of 0.1:1, ROBO1 CAR-NK92-Cbl-b-KO (50.55%) cells exhibited significantly higher cytolytic activity against ROBO1-positive T47D target cells after 3 h of coculture than ROBO1 CAR-NK92 (34.10%), NK92-Cbl-b-KO (22.22%), and parental NK-92 cells (3.28%). In vivo, tumor volume and weight measurements demonstrated that mice treated with ROBO1 CAR-NK92-Cbl-b-KO cells developed significantly smaller tumors than all control groups, achieving a tumor growth inhibition (TGI) rate of 32.45%, indicating enhanced antitumor efficacy conferred by Cbl-b knockout. In vitro and in vivo data confirmed that Cbl-b knockout potentiates the antitumor efficacy of ROBO1 CAR-NK92 cells. The overall cytotoxic capability ranked as follows: ROBO1 CAR-NK92-Cbl-b-KO > ROBO1 CAR-NK92 > NK92-Cbl-b-KO > NK-92.

Keywords:  CAR-NK; Cbl-b; ROBO1

DOI:  https://doi.org/10.1177/10430342261453858
Oncoimmunology. 2026 Dec 31. 15(1): 2679315

Umbilical cord blood natural killer cells improve anti-GD2 antibody efficacy in neuroblastoma: from mouse to human.

Mengjia Song, Yingxia Lan, Xiya Tan, Linnan Wu, Jia Zhu, Suying Lu, Feifei Sun, Min Gao, Junting Huang, Yi Que, Yu Zhang, Xiaona Fang, Mengzhen Li, Yumiao Mai, Juan Wang, Tong Xiang, Yizhuo Zhang.

  Despite advances in anti-disialoganglioside (GD2) immunotherapy, high-risk neuroblastoma (NB) remains a clinical challenge. Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity is a potent mechanism of action of anti-GD2 antibody. However, dose-intensive chemotherapy causes NK cell depletion. Here, a positive association between intratumoral NK cell infiltration and anti-GD2 efficacy was first observed in clinical samples. We then established the unique advantages of ex vivo expanded and activated umbilical cord blood (UCB)-derived NK cells in terms of cytotoxicity, persistence, exhaustion resistance, and safety from preclinical models to clinical cases compared with patient-derived PB NK cells. Anti-GD2 antibody further enhanced the superiority of UCB NK cells in terms of their functional status, persistence, and capacity to remodel an immune-activated tumor microenvironment. Combined treatment with UCB NK cells and an anti-GD2 led to synergistic effects in vitro and in mice and achieved complete and partial responses in two patients with relapsed/refractory NB, with no additive toxicity. Mechanistically, the anti-GD2 antibody induced a "high activating-low inhibitory" phenotype in UCB NK cells, and donor UCB NK cells showed dynamically lower levels of immune checkpoints but higher levels of memory-like markers than those of recipient NK cells when combined with anti-GD2 therapy. Collectively, this study is the first translational investigation of UCB NK cells combined with anti-GD2 therapy in NB, bridging preclinical mechanistic insights into an early clinical proof-of-concept. A phase I study of UCB NK cell infusion combined with anti-GD2 therapy in children with high-risk, relapsed/refractory NB is ongoing at our center (NCT06631391).

Keywords:  Neuroblastoma; anti-GD2 therapy; combined immunotherapy; tumor microenvironment; umbilical cord blood natural killer cells

DOI:  https://doi.org/10.1080/2162402X.2026.2679315
Apoptosis. 2026 May 24. pii: 155. [Epub ahead of print]31(6):

Osteopontin-driven TGF-β1 signaling via integrin αvβ3-NF-κB axis impairs NK cell function in head and neck squamous cell carcinoma.

Zhe Shen, Xin Li, Shi Luo, Yuzhang Gao, Imadoudini Hassimi Safia, Jie Jiang, Xin Qi, Wei Huang, Yalan Liu, Xing Fang, Yaoyun Tang, Ping Wu.

  In head and neck squamous cell carcinoma (HNSCC), an immunosuppressive tumor microenvironment (TME) frequently leads to natural killer (NK) cell dysfunction, but the molecular mechanisms underlying this impairment remain unclear. Here, we combined flow cytometry and multiplex immunohistochemistry to evaluate NK cells infiltration and functional status in HNSCC tumors. Bulk RNA-seq datasets from patient cohorts and single-cell RNA-seq data were analyzed to identify transcriptional correlates of NK cells dysfunction. Mechanistic studies in tumor cell lines assessed the role of osteopontin (OPN)-integrin αvβ3/NF-κB signaling in modulating TGF-β1 production and its effects on NK cell activity. NK cells in HNSCC tumors exhibited reduced infiltration and diminished activation marker expression, coinciding with high OPN levels in the TME. Transcriptomic analyses demonstrated a strong inverse correlation between tumor SPP1 (encoding OPN) expression and NK cells abundance. Mechanistically, tumor-derived OPN engaged integrin αvβ3 to activate NF-κB signaling, inducing robust TGF-β1 secretion that suppressed NK cell activation and cytotoxic function. Single-cell RNA-seq analysis further confirmed the exhausted functional states of NK cells within the HNSCC TME. Tumor-derived OPN promotes immune evasion in HNSCC by driving TGF-β1-mediated suppression of NK cell activity. These findings highlight the OPN-TGF-β1 axis as a potential therapeutic target for restoring NK cell-mediated immunity in HNSCC.

Keywords:  Head and neck squamous cell carcinoma; NK cells; Osteopontin; TGF-β1; Tumor microenvironment

DOI:  https://doi.org/10.1007/s10495-026-02333-y
BMC Immunol. 2026 May 23.

Deficient TRPM3-linked mitochondrial Ca2+ influx in natural killer cells associated with myalgic encephalomyelitis/chronic fatigue syndrome.

Chandi Tabeth Magawa, Natalie Eaton-Fitch, Katsuhiko Muraki, Sonya Marshall-Gradisnik.

   INTRODUCTION: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystemic illness, commonly associated with dysregulation of the immune system including reduced cytotoxicity of natural killer (NK) cells and post-exertional neuroimmune exhaustion. Previously, transient receptor potential melastatin 3 (TRPM3) ion channel impairment associated with reduced Ca2+ mobilisation in NK cells from ME/CFS patients was reported. To further explore the pathomechanisms involved in ME/CFS, we investigated the downstream impact of TRPM3 ion channel dysfunction on mitochondrial Ca2+ mobilisation in NK cells.
METHOD: Fluorescence live-cell imaging was used to investigate Ca2+ mobilisation in NK cells of (N = 10) ME/CFS, classified using Canadian Consensus Criteria, and (N = 10) healthy control (HC) participants. Cytoplasmic and mitochondrial Ca2+ entry was determined using Fluo-8 AM and Rhod-2 AM Ca2+ indicators, respectively. The effect of TRPM3 modulation on Ca2+ mobilisation ex vivo, was examined using pregnenolone sulfate and ononetin to activate and inhibit the channel, respectively.
RESULTS: Cytosolic Ca2+ influx amplitude and slope were significantly reduced (p < 0.001), with a significantly shorter T1/2 response (p = 0.001) in ME/CFS compared to HC. Ca2+ influx amplitude (p < 0.001) and slope (p < 0.041) into the mitochondria were significantly higher in ME/CFS compared to HC. TRPM3 activation triggered pronounced cytosolic response (P < 0.001) accompanied by mitochondrial Ca2+ increase in HC. TRPM3-dependent cytosolic and mitochondrial Ca2+ mobilisation (P < 0.015) were significantly reduced with a shorter T1/2 response (p < 0.02) in ME/CFS compared to HC.
CONCLUSION: The results demonstrate that altered TRPM3-mediated cytosolic Ca2+ influx may significantly impact Ca2+ mobilisation into the mitochondria of people with ME/CFS. Alterations that interfere with the optimal function of Ca2+ permeable channels may cumulatively impact downstream signalling, leading to detrimental cellular consequences. Collectively these findings provide an avenue for further studies on the physiological functions of TRPM3 ion channel and its role in ME/CFS.

Keywords:  Calcium signalling; Chronic fatigue syndrome; ME/CFS; Mitochondria; Myalgic encephalomyelitis; Natural killer cells; TRP channels; TRPM3

DOI:  https://doi.org/10.1186/s12865-026-00849-1
Adv Sci (Weinh). 2026 May 25. e75655

Triple-Negative Breast Cancer Cells Utilize IL8 and CXCL1 to Suppress NK Cells' Function and Facilitate Cancer Metastasis.

Mingheng Yuan, Hongmei Yang, Renfei Wu, Meng Hao, Xiangpeng Chu, Chuxia Deng, Kathy Qian Luo.

  Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with high metastatic potential and limited treatment options. Natural killer (NK) cells represent a promising immunotherapy strategy due to their innate tumor-killing capacity, but their efficacy against TNBC remains limited. We found that TNBC cells, particularly the mesenchymal-like subtype, exhibited greater resistance to NK cells compared to non-TNBC cells. Mechanistic studies indicate that TNBC cells' survival in response to NK cells occurs in three phases. First, within 1 h of NK cell co-culture, TNBC cells accumulate reactive oxygen species (ROS), which upregulate C-X-C motif chemokine ligand 1 (CXCL1) and interleukin 8 (IL8) expression in an NF-κB- and ERK/JNK-AP-1-dependent manner. Second, secreted CXCL1/IL8 binds to C-X-C motif chemokine receptor 1/2 (CXCR1/2), activating the AKT-BCL-2 pathway to enhance cancer cell survival and suppress NK cell function by downregulating NKG2D, TRAIL, and IFN-γ expression. Third, CXCL1/IL8-CXCR1/2 autocrine loop further amplifies their own synthesis and induces programmed cell death 1 ligand 1 (PD-L1) expression via NF-κB and ERK/JNK-AP-1 pathways. High CXCL1/IL8 expression correlates with reduced NK cell infiltration and shorter distant-metastasis-free survival in breast cancer patients. Combinatorial application of CXCR1/2 inhibitor with anti-PD-L1 antibody can overcome NK cell dysfunction and reduce TNBC metastasis.

Keywords:  C‐X‐C motif chemokine ligand 1 (CXCL1); co‐culture; interleukin 8 (IL8); metastasis; natural killer (NK) cell; reactive oxygen species (ROS); triple‐negative breast cancer (TNBC)

DOI:  https://doi.org/10.1002/advs.75655
Mol Ther Oncol. 2026 Jun 18. 34(2): 201223

Natural killer cell immunotherapy for pancreatic cancer: Release the brakes.

Stacey N Lee, Jeanette E Boudreau.

DOI: https://doi.org/10.1016/j.omton.2026.201223
Mol Ther Adv. 2026 Jun 11. 34(2): 201749

Efficient NK cell transduction with VSV-G-pseudotyped lentiviral vectors.

Emmi Järvelä, Jan Koski, Farhana Jahan, Antti Tuhkala, Mira Saari, Manar Elmadani, Kari Salokas, Laurens Veltman, Lotta Andersson, Erja Kerkelä, Hekim Can, Marie Nyman, Seppo Ylä-Herttuala, Markku Varjosalo, Diana Schenkwein, Henrik Paavilainen, Kim Vettenranta, Matti Korhonen, Helka Göös.

  The need for safe, allogeneic cell therapies for cancer is driving a growing interest in CAR-NK-based therapies, which, unlike CAR-T cell therapies, offer the potential for off-the-shelf administration. Lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) are commonly used for genetic modification of cell therapy products. Their use in NK cells, however, is limited by low transduction efficiency. This study explores the complexities of NK cell transduction using lentiviral vectors pseudotyped with VSV-G. We demonstrate that efficient transduction depends on multiple factors, such as NK cell activation, domain organization of CAR construct, lentivirus pseudotype selection, and the use of transduction enhancers. By optimizing these elements, we achieved effective transduction, facilitating the use of VSV-G-pseudotyped lentiviral vectors for therapeutic NK cell production. Our optimized workflow comprises NK cell activation with interleukins, followed by transduction with an NK-cell-specific CAR construct using VSV-G-pseudotyped LVs in the presence of BX795 and retronectin, resulting in excellent transduction efficiency (91% using GFP and 80% using CAR) without compromising NK cell phenotype, growth, or cytotoxicity. This allows for the use of a widely used gene transfer vector with an excellent safety record for producing therapeutic NK cell products.

Keywords:  BX795; CAR-NK; VSV-G; chimeric antigen receptor; natural killer cell; retronectin; transduction; vesicular stomatitis virus glycoprotein

DOI:  https://doi.org/10.1016/j.omta.2026.201749
Ann Med. 2026 Dec;58(1): 2677931

Overexpression of SLC44A4 suppresses ferroptosis and reduces lipid peroxidation via noncanonical NF-κB signaling in a NIK-dependent manner.

Tian Li, Yan Xu, Wenyu Song, Huafan Chang, Junqi He, Suli Liu, Zhicheng Liu, Yuance Xu, Qin Yao, Kejuan Song.

   BACKGROUND: Ovarian cancer (OC) is one of the most common gynecologic malignancies worldwide and is characterized by a high recurrence rate driven by drug resistance, contributing to poor prognosis and increased mortality. Consequently, there is an urgent need to develop novel therapeutic strategies to improve patient outcomes. Although SLC44A4 is known to be highly expressed in various tumor types, its precise functional role and underlying mechanisms in OC remain largely unexplored.
OBJECTIVE: This study aims to investigate the effects and underlying mechanisms of SLC44A4 overexpression on the biological behavior of OC cells.
RESULTS: SLC44A4 enhances the proliferation and migration of OC cells both in vitro and in vivo. Moreover, SLC44A4 overexpression reduces lipid peroxidation, suppresses ferroptosis, and is associated with activation of noncanonical NF-κB signaling, resulting in decreased sensitivity to the ferroptosis inducer erastin.
CONCLUSION: Our findings suggest that SLC44A4 overexpression reduces lipid peroxidation and suppresses ferroptosis, with these effects being linked to the activation of noncanonical NF-κB signaling. Thus, SLC44A4 may serve as a potential target for modulating ferroptosis in OC; however, further validation, including loss-of-function studies and patient-derived sample analyses, is required.

Keywords:  NF-κB signaling; Ovarian cancer; ferroptosis; lipid peroxidation

DOI:  https://doi.org/10.1080/07853890.2026.2677931
Front Immunol. 2026 ;17 1779381

Rapid assessment of immune effector cell-mediated cytotoxicity using mRNA lipid nanoparticles.

Philip Mollica, Jian Li, Sang-Hoon Kim, Beatriz Goncalves, Yingshi Chen, Aditya Bharti, Dillon O'Neill, Evan Weber, Mohamad Alameh, Leyuan Ma.

  Cellular immunotherapy has revolutionized cancer treatment by enabling more targeted and personalized disease management. As the field progresses, there is an increasing need for high-throughput in vitro assays to efficiently assess the cytotoxicity of therapeutic cells. Conventional cytotoxicity assays pose various limitations in the workflow and scalability. Here, we present an mRNA lipid nanoparticle (mRNA-LNP) approach to efficiently and robustly deliver reporter genes to target cells for assessing immune effector cell-mediated cytotoxicity. This approach enables the rapid, homogenous reporter expression without compromising the viability of target cells. The cytotoxicity results obtained using mRNA-LNP-transfected cells are highly consistent and comparable to those obtained using cell lines with stable reporter gene expression. Finally, we highlight the mRNA-LNP approach's compatibility across a diverse range of tumor models, including primary tumor-derived models, enabling rapid and high-throughput assessment of the potency of various cytotoxic therapeutic cells.

Keywords:  CAR T; NK cells; cytotoxicity; immune effector cells; mRNA-LNP; reporter assay

DOI:  https://doi.org/10.3389/fimmu.2026.1779381