Natural Killer Cell Line Research Articles

Optogenetically engineered Septin-7 enhances immune cell infiltration of tumor spheroids

Chimeric antigen receptor T cell therapies have achieved great success in eradicating some liquid tumors, whereas the preclinical results in treating solid tumors have proven less decisive. One of the principal challenges in solid tumor treatment is the physical barrier composed of a dense extracellular matrix, which prevents immune cells from penetrating the tissue to attack intratumoral cancer cells. Here, we improve immune cell infiltration into solid tumors by manipulating septin-7 functions in cells. Using protein allosteric design, we reprogram the three-dimensional structure of septin-7 and insert a blue light-responsive light-oxygen-voltage-sensing domain 2 (LOV2), creating a light-controllable septin-7-LOV2 hybrid protein. Blue light inhibits septin-7 function in live cells, inducing extended cell protrusions and cell polarization, enhancing cell transmigration efficiency through confining spaces. We genetically edited human natural killer cell line (NK92) and mouse primary CD8+ T-cells expressing the engineered protein, and we demonstrated improved penetration and cytotoxicity against various tumor spheroid models. Our proposed strategy to enhance immune cell infiltration is compatible with other methodologies and therefore, could be used in combination to further improve cell-based immunotherapies against solid tumors.

Three-Dimensional Model Analysis Revealed Differential Cytotoxic Effects of the NK-92 Cell Line and Primary NK Cells on Breast and Ovarian Carcinoma Cell Lines Mediated by Variations in Receptor-Ligand Interactions and Soluble Factor Profiles.

Background/objectives: The functional activity of a certain tumor determines the effectiveness of primary NK cells and NK-92 cell line-based cancer therapy; their therapeutic effectiveness against different tumors can vary. This work provides a direct simultaneous comparison of the cytotoxic effects of in vitro-activated peripheral NK (pNK) cells and NK-92 cells in spheroid models of BT-474, MCF7 and SKOV-3 carcinomas and uncovers the reasons for the differential effectiveness of NK cells against tumors. Methods: Tumor spheroids of similar size and shape, obtained from agarose molds, were incubated with NK-92 or pNK cells for 24 h. Tumor cell death was detected using flow cytometry or confocal microscopy. Cytokine production, granzyme B levels and NK cell degranulation analyses were performed, along with pNK and target-cell phenotypic characterization. Results: While NK-92 and pNK cells lysed BT-474 spheroids with comparably low efficiency, pNK cells were more capable of eliminating MCF7 and SKOV-3 spheroids than NK-92 cells were. The results of the functional and phenotypic analyses strongly support the participation of the NKG2D-NKG2DL pathway in pNK cell activation induced by the most sensitive cytotoxic attack on SKOV-3 spheroids, whereas the CX3CR1-CX3CL1 axis appears to be involved in the pNK reaction against MCF-7 spheroids. Conclusions: We provide a new approach for the preliminary identification of the most promising NK cell receptors that can alter the effectiveness of cancer therapy depending on the specific tumor type. Using this approach, NK-92 cells or pNK subsets can be selected for further accumulation and/or genetic modification to improve specificity and reactivity.

Cytokine-armed vaccinia virus promotes cytotoxicity toward pancreatic carcinoma cells via activation of human intermediary CD56dimCD16dim natural killer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains a particularly aggressive disease with few effective treatments. The PDAC tumor immune microenvironment (TIME) is known to be immune suppressive. Oncolytic viruses can increase tumor immunogenicity via immunogenic cell death (ICD). We focused on tumor-selective (vvDD) and cytokine-armed Western-reserve vaccinia viruses (vvDD-IL2 and vvDD-IL15) and infected carcinoma cell lines as well as patient-derived primary PDAC cells. In co-culture experiments, we investigated the cytotoxic response and the activation of human natural killer (NK). Infection and virus replication were assessed by measuring virus encoded YFP. We then analyzed intracellular signaling processes and oncolysis via in-depth proteomic analysis, immunoblotting and TUNEL assay. Following the co-culture of mock or virus infected carcinoma cell lines with allogenic PBMCs or NK cell lines, CD56+ NK cells were analyzed with respect to their activation, cytotoxicity and effector function. Both, dose- and time-dependent release of danger signals following infection weremeasured. Viruses effectively entered PDAC cells, emitted YFP signals and resulted in concomitant oncolysis. The proteome showed reprogramming of normally active core signaling pathways in PDAC (e.g., MAPK-ERK signaling). Danger-associated molecular patterns were released upon infection and stimulated co-cultured NK cells for enhanced effector cytotoxicity. NK cell subtyping revealed enhanced numbers and activation of a rare CD56dimCD16dim population. Tumor cell killing was primarily triggered via Fas ligands rather than granule release, resulting in marked apoptosis. Overall, the cytokine-armed vaccinia viruses induced NK cell activation and enhanced cytotoxicity toward human PDAC cells in vitro. We could show that cytokine-armed virus targets the carcinoma cells and thus hasgreat potential to modulate the TIME in PDAC.

Mesenchymal Stem Cells Derived from Human Urine-Derived iPSCs Exhibit Low Immunogenicity and Reduced Immunomodulatory Profile.

Human-induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) represent a promising and renewable cell source for therapeutic applications. A systematic evaluation of the immunological properties and engraftment potential of iMSCs generated from urine-derived iPSCs is lacking, which has impeded their broader application. In this study, we differentiated urine-derived iPSCs into iMSCs and assessed their fundamental MSC characteristics, immunogenicity, immunomodulatory capacity and in vivo engraftment. Compared to umbilical cord-derived MSCs (UCMSCs), iMSCs demonstrated an enhanced proliferative capacity, a higher level of regenerative gene expression, and lower immunogenicity, strengthening resistance to apoptosis induced by allogeneic peripheral blood mononuclear cells (PBMCs) and the NK-92 cell line. In addition, iMSCs exhibited a diminished ability to inhibit T cell proliferation and activation compared with UCMSCs. Transcriptomic analyses further revealed the decreased expression of immune regulatory factors in iMSCs. After transfusion into mouse models, iMSCs engrafted in the lungs, liver, and spleen and exhibited the ability to migrate to tumor tissues. Our results indicated that iMSCs generated from urine-derived iPSCs have a significant replicative capacity, low immunogenicity and unique immunomodulatory properties, and hence offer obvious advantages in immune privilege and allogenic therapeutic application prospects.

Fetal bone engraftment reconstitutes the immune system in pigs with severe combined immunodeficiency.

Genetic modification of genes such as recombination activating gene 2 (RAG2) or interleukin-2 receptor-γ (IL2RG) results in pigs exhibiting severe combined immunodeficiency (SCID). Pigs presenting a SCID phenotype are important animal models that can be used to establish xenografts and to study immune system development and various immune-related pathologies. However, due to their immunocompromised nature, SCID pigs have shortened lifespans and are notoriously difficult to maintain. The failure-to-thrive phenotype makes the establishment of a breeding population of RAG2/IL2RG double-knockout pigs virtually impossible. Here, to overcome this limitation, we investigated whether reconstituting the immune system of SCID piglets with a fetal bone allograft would extend their lifespan. Following intramuscular transplantation, allografts gave rise to lymphocytes expressing T cell (CD3, CD4 and CD8), B cell (CD79α) and natural killer cell (CD335) lineage markers, which were detected in circulation as well in the spleen, liver, bone marrow and thymic tissues. The presence of lymphocytes indicates broad engraftment of donor cells in the recipient SCID pigs. Unlike unreconstituted SCID pigs, the engrafted animals thrived and reached puberty under standard housing conditions. This study demonstrates a novel method to extend the survival of SCID pigs, which may improve the availability and use of SCID pigs as a biomedical animal model.

Oncolytic virotherapy augments self-maintaining natural killer cell line cytotoxicity against neuroblastoma

BackgroundNeuroblastoma is the most common extracranial solid tumor in children and accounts for 15% of pediatric cancer related deaths. Targeting neuroblastoma with immunotherapies has proven challenging due to a paucity of immune cells in the tumor microenvironment and the release of immunosuppressive cytokines by neuroblastoma tumor cells. We hypothesized that combining an oncolytic Herpes Simplex Virus (oHSV) with natural killer (NK) cells might overcome these barriers and incite tumor cell death.MethodsWe utilized MYCN amplified and non-amplified neuroblastoma cell lines, the IL-12 expressing oHSV, M002, and the human NK cell line, NK-92 MI. We assessed the cytotoxicity of NK cells against neuroblastoma with and without M002 infection, the effects of M002 on NK cell priming, and the impact of M002 and priming on the migratory capacity and CD107a expression of NK cells. To test clinical applicability, we then investigated the effects of M002 and NK cells on neuroblastoma in vivo.ResultsNK cells were more attracted to neuroblastoma cells that were infected with M002. There was an increase in neuroblastoma cell death with the combination treatment of M002 and NK cells both in vitro and in vivo. Priming the NK cells enhanced their cytotoxicity, migratory capacity and CD107a expression.ConclusionsTo the best of our knowledge, these investigations are the first to demonstrate the effects of an oncolytic virus combined with self-maintaining NK cells in neuroblastoma and the priming effect of neuroblastoma on NK cells. The current studies provide a deeper understanding of the relation between NK cells and neuroblastoma and these data suggest that oHSV increases NK cell cytotoxicity towards neuroblastoma.

Efficient generation of human NOTCH ligand-expressing haemogenic endothelial cells as infrastructure for in vitro haematopoiesis and lymphopoiesis

Arterial endothelial cells (AECs) are the founder cells for intraembryonic haematopoiesis. Here, we report a method for the efficient generation of human haemogenic DLL4+ AECs from pluripotent stem cells (PSC). Time-series single-cell RNA-sequencing reveals the dynamic evolution of haematopoiesis and lymphopoiesis, generating cell types with counterparts present in early human embryos, including stages marked by the pre-haematopoietic stem cell genes MECOM/EVI1, MLLT3 and SPINK2. DLL4+ AECs robustly support lymphoid differentiation, without the requirement for exogenous NOTCH ligands. Using this system, we find IL7 acts as a morphogenic factor determining the fate choice between the T and innate lymphoid lineages and also plays a role in regulating the relative expression level of RAG1. Moreover, we document a developmental pathway by which human RAG1+ lymphoid precursors give rise to the natural killer cell lineage. Our study describes an efficient method for producing lymphoid progenitors, providing insights into their endothelial and haematopoietic ontogeny, and establishing a platform to investigate the development of the human blood system.

Enhanced non-viral gene delivery via calcium phosphate/DNA co-precipitates with low-voltage pulse electroporation in NK-92 cells for immunocellular therapy.

Achieving high cell transfection efficiency is essential for various cell types in numerous disease applications. However, the efficient introduction of genes into natural killer (NK) cells remains a challenge. In this study, we proposed a design strategy for delivering exogenous genes into the NK cell line, NK-92, using a modified non-viral gene transfection method. Calcium phosphate/DNA nanoparticles (pDNA-CaP NPs) were prepared using co-precipitation methods and combined with low-voltage pulse electroporation to facilitate NK-92 transfection. The results demonstrated that the developed pDNA-CaP NPs exhibited a uniform diameter of approximately 393.9 nm, a DNA entrapment efficiency of 65.8%, and a loading capacity of 15.9%. Furthermore, at three days post-transfection, both the transfection efficiency and cell viability of NK-92 were significantly improved compared to standalone plasmid DNA (pDNA) electroporation or solely relying on the endocytosis pathway of pDNA-CaP NPs. This study provides valuable insights into a novel approach that combines calcium phosphate nanoparticles with low-voltage electroporation for gene delivery into NK-92 cells, offering potential advancements in cell therapy.

A synthetic cytotoxic T cell platform for rapidly prototyping TCR function

Current tools for functionally profiling T cell receptors with respect to cytotoxic potency and cross-reactivity are hampered by difficulties in establishing model systems to test these proteins in the contexts of different HLA alleles and against broad arrays of potential antigens. We have implemented a granzyme-activatable sensor of T cell cytotoxicity in a universal prototyping platform which enables facile recombinant expression of any combination of TCR-, peptide-, and class I MHC-coding sequences and direct assessment of resultant responses. This system consists of an engineered cell platform based on the immortalized natural killer cell line, YT-Indy, and the MHC-null antigen-presenting cell line, K562. These cells were engineered to furnish the YT-Indy/K562 pair with appropriate protein domains required for recombinant TCR expression and function in a non-T cell chassis, integrate a fluorescence-based target-centric early detection reporter of cytotoxic function, and deploy a set of protective genetic interventions designed to preserve antigen-presenting cells for subsequent capture and downstream characterization. Our data show successful reconstitution of the surface TCR complex in the YT-Indy cell line at biologically relevant levels. We also demonstrate successful induction and highly sensitive detection of antigen-specific response in multiple distinct model TCRs. Additionally, we monitored destruction of targets in co-culture and found that our survival-optimized system allowed for complete preservation after 24 h exposure to cytotoxic effectors. With this bioplatform, we anticipate investigators will be empowered to rapidly express and characterize T cell receptor responses, generate knowledge regarding the patterns of T cell receptor recognition, and optimize therapeutic T cell receptors.

Therapeutic effect of small extracellular vesicles from cytokine-induced memory-like natural killer cells on solid tumors

Small extracellular vesicles (sEV) derived from diverse natural killer (NK) cell lines have proven their exceptional antitumor activities. However, sEV from human primary NK cells, especially memory-like NK cells, are rarely utilized for cancer treatment. In this study, we obtained sEV from IL-12, IL-15 and IL-18 cultured human memory-like NK cells (mNK-sEV) that showed strong cytokine-secretory ability. It was uncovered that mNK-sEV entered cancer cells via macropinocytosis and induced cell apoptosis via caspase-dependent pathway. Compared to sEV from conventionally cultured NK cells (conNK-sEV), mNK-sEV inhibited tumor growth to a greater extent. Concomitantly, pharmacokinetics and biodistribution results validated a higher accumulation of mNK-sEV than conNK-sEV in tumors of xenografted murine models. Notably, elevated containment of granulysin (GNLY) within mNK-sEV, at least in part, may contribute to the enhanced therapeutic effect. Herein our results present that mNK-sEV can be a novel class of therapeutic reagent for effective cancer treatment.Graphical

NK cell line modified to express a potent, DR5 specific variant of TRAIL, show enhanced cytotoxicity in ovarian cancer models

ObjectiveOvarian cancer is a lethal gynaecological malignancy with unsatisfactory 5 year survival rates of 30–50 %. Cell immunotherapy is a promising new cancer treatment where immune cells, such as Natural Killer (NK) cells, are administered to enable the patient to fight cancer through direct cytotoxicity. NK cells orchestrate an adaptive immune response by enabling the release of tumour antigens. NK cell cytotoxicity and effector responses are largely driven by TRAIL engagement. In this study we investigated the cytotoxic potential of a human NK cell line that were modified to express a potent DR5 specific TRAIL variant. We hypothesised that this modification would enhance NK cell cytotoxicity against TRAIL sensitive and resistant ovarian cancer cell lines in vitro. MethodsKHYG-1 human NK cells were modified with a TRAIL variant targeting DR5 (TRAILv-KHYG-1). Human ovarian cancer cell lines, OVCAR-3 and SKOV-3, were cultured with modified or non-modified NK cells at different effector:target (E:T) ratios for 4 or 16 h. Apoptosis was assessed by Annexin-APC and 7-AAD and measured using flow cytometry. Apoptotic cells were defined as annexin V 7-AAD double positive. Cytokine expression was measured by multiplex ELISA, and analysed by flow cytometry. ResultsModified and non-modified NK cells significantly reduced OVCAR-3 cell viability as compared to OVCAR-3 cells that were cultured alone after 4 and 16 h treatment. OVCAR-3 cell viability was reduced after treatment with 1:1 E:T ratio with TRAILv-KHYG-1 cells after 16 h. On the contrary, neither NK cell line had any effect of SKOV-3 cell viability despite SKOV-3 cells having more DR5 surface expression compared to OVCAR-3 cells. ConclusionsTRAILv-KHYG-1 cells significantly reduced OVCAR-3 cell viability as compared to non-modified NK cells. However, no significant reduction in viability was observed when SKOV-3 cell were cultured with either NK cells, despite having more DR5 surface expression compared to OVCAR-3 cells. These data indicate that mechanisms other than DR5 expression drive TRAIL resistance in ovarian cancer.

Spleen Tyrosine Kinase (SYK) negatively regulates ITAM-mediated human NK cell signaling and CD19-CAR NK cell efficacy.

NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca 2+ influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylate SYK genes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination of SYK resulted in more robust Ca ++ influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereas ZAP70 deletion diminished these responses. By contrast, ZAP70 depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca 2+ flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion of SYK in NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablate SYK or ZAP70 . Deletion of SYK resulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.

NK Cytotoxicity Mediated by NK-92 Cell Lines Expressing Combinations of Two Allelic Variants for FCGR3.

Natural killer (NK) cells play an important role in the surveillance of viral infections and cancer. NK cell antibody-dependent cellular cytotoxicity (ADCC) and direct cytotoxicity are mediated by the recognition of antibody-coated target cells through the Fc gamma receptor IIIA (FcγRIIIa/CD16) and by ligands of activating/inhibitory NK receptors, respectively. Allelic variants of the FCGR3A gene include the high-affinity single-nucleotide polymorphism (SNP) rs396991 (V176F), which is associated with the efficacy of monoclonal antibody (mAb) therapies, and the SNP rs10127939 (L66H/R). The contribution of FCGR3A SNPs to NK cell effector functions remains controversial; therefore, we generated a panel of eight NK-92 cell lines expressing specific combinations of these SNPs and tested their cytotoxicities. NK-92 cells were stably transfected with plasmids containing different combinations of FCGR3A SNPs. Messenger RNA and FcγRIIIa/CD16 cell surface expressions were detected using new generation sequencing (NGS) and flow cytometry, respectively. All FcγRIIIa/CD16-transfected NK-92 cell lines exhibited robust ADCC against three different target cell lines with minor differences. In addition, enhanced direct NK cytotoxicity against K562 target cells was observed, suggesting a mechanistic role of FcγRIIIa/CD16 in direct NK cytotoxicity. In conclusion, we generated eight FcγRIIIa/CD16-transfected NK-92 cell lines carrying different combinations of two of the most studied FCGR3A SNPs, representing the major genotypes described in the European population. The functional characterization of these cell lines revealed differences in ADCC and direct NK cytotoxicity that may have implications for the design of adoptive cancer immunotherapies using NK cells and tumor antigen-directed mAbs.

Blockade of the TIGIT-CD155/CD112 axis enhances functionality of NK-92 but not cytokine-induced memory-like NK cells toward CD155-expressing acute myeloid leukemia

TIGIT is an alternative checkpoint receptor (CR) whose inhibition promotes Graft-versus-Leukemia effects of NK cells. Given the significant immune-permissiveness of NK cells circulating in acute myeloid leukemia (AML) patients, we asked whether adoptive transfer of activated NK cells would benefit from additional TIGIT-blockade. Hence, we characterized cytokine-induced memory-like (CIML)-NK cells and NK cell lines for the expression of inhibitory CRs. In addition, we analyzed the transcription of CR ligands in AML patients (CCLE and Beat AML 2.0 cohort) in silico and evaluated the efficacy of CR blockade using in vitro cytotoxicity assays, CD69, CD107a and IFN-γ expression. Alternative but not classical CRs were abundantly expressed on healthy donor NK cells and even further upregulated on CIML-NK cells. In line with our finding that CD155, one important TIGIT-ligand, is reliably expressed on AMLs, we show improved killing of CD155+-AML blasts by NK-92 but interestingly not CIML-NK cells in the presence of TIGIT-blockade. Additionally, our in silico data (n = 671) show that poor prognosis AML patients rather displayed a CD86low CD112/CD155high phenotype, whereas patients with a better outcome rather exhibited a CD86high CD112/CD155low phenotype. Collectively, our data evidence that the complex CR ligand expression profile on AML blasts may be one explanation for the intrinsic NK cell exhaustion observed in AML patients which might be overcome with adoptive NK-92 transfer in combination with TIGIT-blockade.

Microbiota-Derived Short-Chain Fatty Acids Boost Antitumoral Natural Killer Cell Activity.

Background: The intestinal microbiota can regulate numerous host functions, including the immune response. Through fermentation, the microbiota produces and releases microbial metabolites such as short-chain fatty acids (SCFAs), which can affect host homeostasis. There is growing evidence that the gut microbiome can have a major impact on cancer. Specific gut microbial composition and metabolites are associated with tumor status in the host. However, their effects on the antitumor response have scarcely been investigated. Natural killer (NK) cells play an important role in antitumor immunity due to their ability to directly identify and eliminate tumor cells. Methods: The aim of this study was to investigate the effects of SCFAs on antitumoral NK cell activity, using NK-92 cell line. Results: Here, we describe how SCFAs can boost antitumoral NK cell activity. The SCFAs induced the release of NK extracellular vesicles and reduced the secretion of the anti-inflammatory cytokine IL-10. The SCFAs also increased the cytotoxicity of the NK cells against multiple myeloma cells. Conclusions: Our results indicate, for the first time, the enormous potential of SCFAs in regulating antitumoral NK cell defense, where modulation of the SCFAs' production could play a fundamental role in cancer immunotherapy.

Enhancing natural killer cells proliferation and cytotoxicity using imidazole-based lipid nanoparticles encapsulating interleukin-2 mRNA

mRNA applications have undergone unprecedented applications-from vaccination to cell therapy. Natural killer (NK) cells are recognized to have a significant potential in immunotherapy. NK-based cell therapy has drawn attention as allogenic graft with a minimal graft-versus-host risk leading to easier off-the-shelf production. NK cells can be engineered with either viral vectors or electroporation, involving high costs, risks, and toxicity, emphasizing the need for alternative way as mRNA technology. We successfully developed, screened, and optimized novel lipid-based platforms based on imidazole lipids. Formulations are produced by microfluidic mixing and exhibit a size of approximately 100nm with a polydispersity index of less than 0.2. They are able to transfect NK-92 cells, KHYG-1 cells, and primary NK cells with high efficiency without cytotoxicity, while Lipofectamine Messenger Max and D-Lin-MC3 lipid nanoparticle-based formulations do not. Moreover, the translation of non-modified mRNA was higher and more stable in time compared with a modified one. Remarkably, the delivery of therapeutically relevant interleukin 2 mRNA resulted in extended viability together with preserved activation markers and cytotoxic ability of both NK cell lines and primary NK cells. Altogether, our platforms feature all prerequisites needed for the successful deployment of NK-based therapeutic strategies.

Preclinical efficacy of a HER2 synNotch/CEA-CAR combinatorial immunotherapy against colorectal cancer with HER2 amplification

HER2 amplification occurs in about 5% of colorectal cancer (CRC) cases and is associated only partially with clinical response to combined HER2/EGFR targeted treatment. An alternative approach based on adoptive cell therapy (ACT) using T-cells engineered with anti-HER2 chimeric antigen receptor (CAR) proved to be toxic due to "on-target off-tumor" activity. Here we describe a combinatorial strategy to safely target HER2 amplification and CEA expression in CRC using a synNotch-CAR based artificial regulatory network. The natural killer cell line NK-92 was engineered with an anti-HER2 synNotch receptor driving the expression of a CAR against CEA only when engaged. After being transduced and sorted for HER2-driven CAR expression, cells were cloned. The clone with optimal performances in terms of specificity and amplitude of CAR induction demonstrated significant activity in vitro and in vivo specifically against HER2amp/CEA+ CRC models, with no effects on cells with physiological HER2 levels. The HER2-synNotch/CEA-CAR-NK system provides an innovative, scalable and safe off-the shelf cell therapy approach with potential against HER2amp CRC resistant or partially responsive to HER2/EGFR blockade.

CD56bright NK cell expansion correlated with EBV reactivation control post allogeneic hematopoietic stem cell transplantation.

Natural killer (NK) cells are equipped with anti-Epstein-Barr virus (EBV) function, however, whether EBV infection will affect NK cells reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains unclear. To identify the characteristics of NK cells, we prospectively enrolled 11 patients who occurred EBV reactivation post allo-HSCT and 11 patients without EBV infection as control. We found that that EBV infection induced the expansion of CD56bright and NKG2A+KIR- NK subsets,and decreased the cytotoxicity function of NK cells. The frequency of NKG2A+KIR- NK cells were higher in patients who progressed into post-transplant lymphoproliferative disorder (PTLD) than EBV viremia patients, which also correlated with decreased proliferation and cytotoxic function. By screening the activation receptors of NK cells, we found the DNAM-1+CD56bright NK cells is significantly increased after EBV stimulation, further we demonstrated that DNAM-1 is essential for EBV induced NK cells activation as the cytokine release against EBV-transformed lymphoblastoid cell lines(EBV-LCLs) of CD56bright NK cells were significantly decreased after DNAM-1 blockade. NK cells infusion suppressed the progression of EBV-related tumor mice model. A prospective cohort indicated that old donor age was an independent risk factor for EBV infection. Rapid CD56bri expansion and high expression of DNAM-1 on CD56bri NK cells in response to EBV reactivation correlated with rapid EBV clearance post allo-HSCT in patients with younger donors. In summary, our data showed that high expression of DNAM-1 receptors on NK cell may participate protective CD56bri NK cells response to EBV infection after allo-HSCT.

Dynamic Transcriptional Programs During Single NK Cell Killing: Connecting Form to Function in Cellular Immunotherapy.

Natural killer (NK) cell-based therapies are a promising new method for treating indolent cancer, however engineering new therapies is complex and progress towards therapy for solid tumors is slow. New methods for determining the underlying intracellular signaling driving the killing phenotype would significantly improve this progress. We combined single-cell RNA sequencing with live cell imaging of a model system of NK cell killing to correlate transcriptomic data with functional output. A model of NK cell activity, the NK-92 cell line killing of HeLa cervical cancer cells, was used for these studies. NK cell killing activity was observed by microscopy during co-culture with target HeLa cells and killing activity subsequently manually mapped based on NK cell location and Annexin V expression. NK cells from this culture system were profiled by single-cell RNA sequencing using the 10× Genomics platform, and transcription factor activity inferred using the Viper and DoRothEA R packages. Luminescent microscopy of reporter constructs in the NK cells was then used to correlate activity of inferred transcriptional activity with killing activity. NK cells had heterogeneous killing activity during 10h of culture with target HeLa cells. Analysis of the single cell sequencing data identified Nuclear Factor Kappa B (NF-κB), Signal Transducer and Activator of Transcription 1 (STAT1) and MYC activity as potential drivers of NK cell functional phenotype in our model system. Live cell imaging of the transcription factor activity found NF-κB activity was significantly correlated with past killing activity. No correlation was observed between STAT1 or MYC activity and NK cell killing. Combining luminescent microscopy of transcription factor activity with single-cell RNA sequencing is an effective means of assigning functional phenotypes to inferred transcriptomics data. The online version contains supplementary material available at 10.1007/s12195-024-00812-3.

Bortezomib promotes the TRAIL-mediated killing of resistant rhabdomyosarcoma by ErbB2/Her2-targeted CAR-NK-92 cells via DR5 upregulation

Treatment resistance and immune escape are hallmarks of metastatic rhabdomyosarcoma (RMS), underscoring the urgent medical need for therapeutic agents against this disease entity as a key challenge in pediatric oncology. Chimeric antigen receptor (CAR)-based immunotherapies, such as the ErbB2 (Her2)-CAR-engineered natural killer (NK) cell line NK-92/5.28.z, provide antitumor cytotoxicity primarily through CAR-mediated cytotoxic granule release and thereafter - even in cases with low surface antigen expression or tumor escape - by triggering intrinsic NK cell-mediated apoptosis induction via additional ligand/receptors. In this study, we showed that bortezomib increased susceptibility towards apoptosis in clinically relevant RMS cell lines RH30 and RH41, and patient-derived RMS tumor organoid RMS335, by upregulation of the TRAIL receptor DR5 in these metastatic, relapsed/refractory (r/r) RMS tumors. Subsequent administration of NK-92/5.28.z cells significantly enhanced antitumor activity in vitro. Applying recombinant TRAIL instead of NK-92/5.28.z cells confirmed that the synergistic antitumor effects of the combination treatment were mediated via TRAIL. Western blot analyses indicated, that the combination treatment with bortezomib and NK-92/5.28.z cells increased apoptosis by interacting with the NF-κB, JNK and caspase pathways. Overall, bortezomib pretreatment can sensitize r/r RMS tumors to CAR- and by upregulating DR5, to TRAIL-mediated cytotoxicity of NK-92/5.28.z cells.