NK-92MI Cells Research Articles

Photoporation of NK-92MI cells with biodegradable polydopamine nanosensitizers as a promising strategy for the generation of engineered NK cell therapies

Chimeric antigen receptor (CAR)-T cells have made significant advancements in the field of adoptive immune cell therapies and the treatment of hematological malignancies. However, there are several drawbacks associated with the production and administration of these therapies. As a result, there has been interest in using natural killer (NK) cells to develop allogeneic CAR-NK cell therapies instead. While viral transduction is powerful for engineering T cells, NK cells have shown limited efficacy and high toxicity with this method. Therefore, efforts are being made to optimize non-viral transfection technologies for engineering NK cells. One such emerging technology is photoporation, which has demonstrated high efficiency and versatility for transfecting different immune cells. In this study, we evaluated the potential of nanoparticle-sensitized photoporation for genetic engineering of NK cells. Our findings show that both FD500 and eGFP mRNA can be successfully delivered into NK-92MI cells with high efficiency and low toxicity. When compared to state-of-the-art electroporation, photoporation proved to be more efficient, gentle, and capable of preserving the phenotype of NK-92MI cells. Overall, our work highlights the promising prospects of photoporation for NK cell engineering.

Association of HLA-E single nucleotide polymorphisms with human myeloid leukemia.

Single nucleotide polymorphisms (SNPs) of HLA-E are related to the occurrence of many diseases, but their functions remain unclear. In this study, the function of SNPs at HLA-E rs76971248 and rs1264457 on the myeloid leukemia cells was analyzed by a progressive procedure, included genotyping, mRNA transcription, regulatory element, protein expression, and anti-tumor effect. The frequencies of rs76971248 G and rs1264457 G were found higher in myeloid leukemia patients than those in healthy blood donors (p < 0.05). For myeloid leukemia, rs76971248 T was protective, while rs1264457 G was susceptible. We also found that rs76971248 affected HLA-E mRNA transcription and membrane HLA-E (mHLA-E) expression in K562 cells through differently binding to transcription factor HOXA5 (p < 0.0001), while rs1264457 affected mHLA-E expression by changing mRNA transcription and an encoding amino acid (p < 0.01). In contrast, the expression of soluble HLA-E (sHLA-E) was not influenced by both rs1264457 and rs76971248. The higher HLA-E expression was detected among myeloid leukemia patients, and the K562 cells with higher HLA-E molecules played a significant inhibitory effect on the killing activity of NK-92MI cells (p < 0.05). In conclusion, the higher HLA-E expression of myeloid leukemia cells is promoted by rs76971248 G and rs1264457 G, which helps escape from NK-92MI cells' killing.

Abstract 3602: TRAIL-NK-92 MI as a novel adoptive therapy for castration-resistant prostate cancer (CRPC): Preliminary in vitro results

Abstract Introduction: Natural killer (NK) cell tumor infiltration and higher expression of activating receptors NKp30 and NKp46 are associated with improved clinical outcomes in prostate cancer (PC). Activation of NK cells by androgen receptor inhibitors enhanced the killing of PC cell lines (Schwermann 2023). These findings support the therapeutic potential of strategies inducing NK cell activation to treat CRPC. NK cells induce apoptosis of cancer cells via TNF-related apoptosis-inducing ligand) (TRAIL) binding of death receptor 5 (DR5), which is overexpressed in PC cells. This is the first report of adoptive therapy utilizing high expression of TRAIL as a novel therapeutic strategy. Methods: We transduced NK-92-MI and CD4+ (Jurkat) cell lines with human TRAIL (pLVX-EF1alpha-TRAIL-IRES-eGFP).Transduced cells were pre-treated with simvastatin or atorvastatin (4uM) for 36h, followed by stimulation (IL-2 100 IU/ml and IL-12 100 ng/ml) for 2h. The eGFP-expressing cells were then sorted by FACS. PC (PC3, DU145, LNCaP, and 22Rv1), fibroblast (IMR-90), and brain endothelial (hCMEC/D3) cell lines were used in co-culture experiments with non-transduced and transduced NK cells and CD4+ cells. These experiments were quantified using the ImageXpress Confocal HT instrument. Viability assays were performed using Annexin-PI and flow cytometry staining for caspases 3/7. Results: Transduced NK-92 MI cells with TRAIL displayed increased surface expression of TRAIL (control[C]: 4.38%±1; TRAIL-overexpression[NK-TRAIL]: 78.54%±3.76; p&amp;lt;0.001). Co-culture of PC cell lines with TRAIL-NK-92 MI cells resulted in significant tumor-induced apoptosis compared to co-culture with non-transduced NK cells after 48 hours (LNCaP + NK [C]: 16.52%±5, LNCaP + NK-TRAIL: 73.19%±6; PC [C]: 14.4%±3, PC3 + NK-TRAIL: 79%±8; 22Rv1 [C]: 16.78%±1.7, 22Rv1 + NK-TRAIL: 76%±8; DU145 [C]: 13%±4, DU145 + NK-TRAIL: 59.1%±10; p&amp;lt;0.001). This enhanced cytotoxic function was also observed in CD4+ cells expressing high levels of TRAIL. Annexin-PI staining as well as Caspase 3 and 7 showed increased apoptosis in PC co-cultured with NK-TRAIL. Co-culture of NK-TRAIL cells did not affect the viability of the non-malignant cells (IMR-90 and hCMEC/D3) over 48h (IMR-90 + NK: 4.6%±1, IMR-90 + NK-TRAIL: 4.47±2, p=0.9; hCMEC/D3 + NK: 2.7%±0.8, hCMEC/D3 + NK-TRAIL: 2.2±0.5, p&amp;gt;0.99). These results align with the lower expression of DR5 on non-malignant cells compared to PC cell lines (IMR-90: 1.46%±0.5, hCMEC/D3: 1.9%±0.8, LNCaP: 92.3%±4.5). Conclusion: Transduced NK-92 MI cells with human TRAIL-induced higher levels of apoptosis of PC cell lines compared with non-transduced NK cells in co-culture experiments. The results highlight the potential of TRAIL-expressing adoptive cell therapy for CRPC. Engaging adoptive therapies with specific targets (anti-PSMA CAR) might increase the therapeutic potential of this strategy. Citation Format: Maximilian Schwermann, Praveen Srinivasan, William MacDonald, Vida Tajiknia, Andrea Schmidt, Shengliang Zhang, Lindsey Carlsen, Andrew George, Connor Purcell, Shaolei Lu, Matthew Hadfield, Anthony Mega, Benedito Carneiro, Wafik El-Deiry. TRAIL-NK-92 MI as a novel adoptive therapy for castration-resistant prostate cancer (CRPC): Preliminary in vitro results [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3602.

Abstract 721: Novel and distinctive anti-HLA-G antibody, IMB-201, inhibits tumor progression by breaking immune escape mechanism and enhancing cytotoxicity

Abstract Human leukocyte antigen-G (HLA-G), a type of MHC class I, is known to be expressed only in the placenta and acts as an immune suppressor to protect the fetus from maternal immunity. Recent studies have revealed that HLA-G suppresses various tumor-infiltrated immune cells, such as T cells, NK cells, and macrophages, through interaction with inhibitory receptors Ig-like transcript 2 (ILT2). HLA-G has recently emerged as an attractive anticancer target because HLA-G is a highly specific tumor antigen and has a role in immune evasion of cancer cells. Additionally, the expression pattern of ILT2 differs from that of PD-1 in tumor-infiltrated CD8+ T cells. Therefore, an HLA-G targeting strategy is expected to be an alternative to address unmet medical needs for those not responding to anti-PD(L)1 therapies. In this study, we screened the novel anti-HLA-G antibody, IMB-201, using phage display technique and confirmed that IMB-201 selectively bound to the membrane HLA-G of cancer cells. It exhibited superior activities in ILT2 blockade, NK cell activation, and antibody-dependent cellular cytotoxicity (ADCC) compared to the reference antibody. Furthermore, IMB-201 demonstrated more efficient suppression of tumor growth compared to the reference antibody in subcutaneous gastric cancer xenograft model. Notably, the group administered IMB-201 with NK92MI cells (CD16-negative NK cells) in the ovarian cancer xenograft model showed stronger tumor growth inhibition compared to the group treated only IMB-201, indicating that NK92MI cells were activated by IMB-201. This result indicates that one of the mechanisms of action (MoA) of IMB-201 worked effectively in vivo. In addition, to maximize the efficacy of IMB-201 and differentiate it from competitors, various efficacy enhancing modality could be applicable. We aim to develop biopharmaceuticals by selecting the optimized modality. Based on these results, IMB-201 could overcome the limitations of existing cancer immunotherapy, potentially becoming highly influential in cancer treatment as a novel immune checkpoint inhibitor. It is also expected to exhibit higher anticancer efficacy in combination therapy with immune checkpoint inhibitors. Citation Format: Hyeonju Kang, Seungil Baek, Inyoung Lee, Manseok Ju, Seonggu Han, Eunjung Han, Sungmuk Kang, Jihye Koo, Yoojin Kim, Suho Park, Eunyoung Cho, Chungmin Lee, Gyongsik Ha. Novel and distinctive anti-HLA-G antibody, IMB-201, inhibits tumor progression by breaking immune escape mechanism and enhancing cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 721.

Abstract 2705: Hyaluronic acid impairs NK killing towards breast tumor spheroids despite elevated cytotoxicity

Abstract The tumor microenvironment (TME) is often regarded as a critical component in tumor progression and metastasis. Among the main structural components of the TME, such as collagen and glycosaminoglycans (GAGs), hyaluronic acid (HA) is highly elevated in the breast TME, and is associated with poor prognosis. In recent years, immunotherapy has gained tremendous success in the battle against hematological cancers. Yet, solid tumors have remained a significant clinical challenge for immunotherapy due to the physical constraints they impose on immune cells. Mechanically, our previous work has shown that the abundance of high molecular weight (HMW) HA significantly reduces matrix pore sizes and softens the collagen and HA cogel, and impairs breast tumor invasion. However, it is still unclear how modulating tumor invasion through the mechanics of the TME may correlate with the efficacy of immunotherapy. In this study, we aim to investigate the effect of HMW HA on natural killer (NK) cell-based immunotherapy using a tumor spheroid model (GFP-labeled MCF-7). We introduced NK-92 MI cells into tumor spheroid embedded collagen gels with or without HA, and followed spheroid-NK cell interactions through live cell imaging. Preliminary data shows that while tumor spheroids were still able to invade in the collagen only gel, they underwent apoptosis in the presence of NK cells. More importantly, the presence of HA within the ECM reduced NK cells’ ability to kill tumor spheroids. Specifically, when surrounded by NK cells, tumor spheroids remained intact and viable in the collagen-HA cogel, while they underwent apoptosis in collagen only gels. In a parallel experiment, multicolor flow cytometry data showed that HA increased NK activation and certain cytotoxic markers (interferon-gamma, perforin, and granzyme-B), despite the fact that spheroids appeared to be more resistant towards NK cells in the presence of HA. Taken together, we speculate that the presence of HMW HA in the TME may protect solid tumors from immune attacks by modulating the mechanical environment. Citation Format: Brian C. Cheung, Alan T. Li, Jeffrey E. Segall, Mingming Wu. Hyaluronic acid impairs NK killing towards breast tumor spheroids despite elevated cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2705.

Abstract 4780: TRAIL-inducing imipridone ONC201/TIC10 demonstrates anti-neoplastic effects in colonic adenoma-derived organoids

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death inthe United States and screening is the most effective method to reduce mortality. Chemoprevention, which aims to prevent CRC through eradication or prevention of precursor colonic adenomas, represents an alternative strategy to address this problem. The imipridone dordaviprone (ONC201) has been shown to induce apoptosis in cancer cells through both upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the integrated stress response. More recently, we have shown that ONC201 induces TRAIL and apoptosis in a dose-dependent manner and reduces adenoma formation in the Apcmin/+ CRC mouse model. We hypothesized that ONC201 would similarly enhance human colonic adenoma cell death through both TRAIL-dependent and TRAIL-independent mechanisms. We therefore evaluated the effects of ONC201 in two adenoma-derived organoid lines: one developed from an adenoma obtained from an individual with familial adenomatous polyposis (FAP) and the second from a sessile serrated adenoma (SSA). Methods: Sensitivity of the FAP and SSA organoid lines to ONC201 was assessed using CellTiter-Glo® luminescent cell viability assay. Organoids were plated in triplicates and treated with ONC201 (concentration ranging from 0 uM to 20 uM). Results were analyzed after 72 hours of incubation. Western blotting was performed on lysates generated from organoids treated with either 1.69 uM DMSO, 1.69 uM ONC201, or 3.38 uM ONC201 for FAP and 1.23 uM DMSO, 1.23 uM ONC201, or 2.46 uM ONC201 for SSA for 0, 24, or 48 hours. Co-culture experiments were performed with the adenoma-derived organoids (dyed using blue CMAC dye) and human NK-92MI cells (dyed with green CMFDA dye) treated with either 845 nM DMSO, 845 nM ONC201, or 1.69 uM ONC201 for FAP and 615 nM DMSO, 615 nM ONC201, or 1.23 uM ONC201 for SSA. Fluorescent images following administration of red live/dead dye were performed using DAPI, FITC, and cherry red channels on ImageXpress® at 0, 24, 48, and 72hours. Results: ONC201 demonstrated antineoplastic effects in adenoma-derived FAP and SSA organoids. Half maximal inhibitory concentrations (IC50) of ONC201 in FAP and SSA organoids were 1.69 uM and 1.23 uM, respectively. As was noted in rodent studies, the mechanism of action of ONC201 appears to be mediated through modulation of multiple pathways including the TRAIL pathway (TRAIL and DR5 were both upregulated) and the integrated stress response (ATF4 was upregulated). Co-culture with NK cells revealed an increase in NK-mediated organoid cell death. The ability of ONC201 to enhance NK-mediated killing is still being evaluated. Further analysis of the effects of ONC201 on stemness markers and on T-cell-mediated organoid cell killing are currently underway and may reveal other potential biomarkers and chemopreventive strategies for ONC201. Citation Format: Alexis J. Lannigan, Jasper Chan, Maximilian Schwermann, Lanlan Zhou, Varun V. Prabhu, Michael Dame, Dean Brenner, Wafik S. El-Deiry, Alexander G. Raufi. TRAIL-inducing imipridone ONC201/TIC10 demonstrates anti-neoplastic effects in colonic adenoma-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4780.

Small-molecule inhibitor HI-TOPK-032 improves NK-92MI cell infiltration into ovarian tumours.

The effectiveness of natural killer (NK) cells transferred adoptively in combating solid tumours is limited by challenges such as their difficulty in penetrating tumours from the bloodstream and maintaining viability without the support of interleukin-2 (IL-2). Genetically modified NK-92MIcells, which can release IL-2 to sustain their viability, have been identified as a promising alternative. This adaptation addresses the negative consequences of systemic IL-2 administration. The role of PSD-95/discs large/ZO-1 (PDZ)-binding kinase (PBK) in cancer development is recognized, but its effects on immunity are not fully understood. This study explores how PBK expression influences the ability of NK-92MI cells to infiltrate ovarian tumours. Elevated levels of PBK expression have been found in various cancers, including ovarian cancer (OV), with analyses showing higher PBK mRNA levels in tumour tissues compared to normal ones. Immunohistochemistry has confirmed increased PBK expression in OV tissues. Investigations into PBK's role in immune regulation reveal its association with immune cell infiltration, indicating a potentially compromised immune environment in OV with high PBK expression. The small-molecule inhibitor HI-TOPK-032, which inhibits PBK, enhances the cytotoxicity of NK-92MI cells toward OV cells. It increases the production of interferon-γ and tumour necrosis factor-α, reduces apoptosis and encourages cell proliferation. Mechanistic studies showed that contact with OV cells treated with HI-TOPK-032 upregulates CD107a on NK-92 cells. In vivo studies demonstrated that HI-TOPK-032 improves the antitumour effects of NK-92MI cells in OVCAR3Luc xenografts, extending survival without significant side effects. Safety assessments in mice confirm HI-TOPK-032's favourable safety profile, highlighting its potential as a viable antitumour therapy. These results suggest that combining NK-92MI cells with HI-TOPK-032 enhances antitumour effectiveness against OV, indicating a promising, safe and effective treatment strategy that warrants further clinical investigation.

The Three-dimensional Environment of Type Ⅰ Collagen Gels With Varying Stiffness Modulates the Immunological Functions of NK Cells

To construct type Ⅰ collagen gels with different stiffness and to investigate the effects of three-dimensional (3D) culture environments of the gels on the morphology, free migration ability, and cell killing function of natural killer (NK) cells. Type Ⅰ collagen was isolated from the tails of Sprague Dawley (SD) rats and collagen gels with different levels of stiffnesses were prepared accordingly. The microstructure of the collagen gels was observed by laser confocal microscopy. The stiffness of the collagen gels was assessed by measuring the plateau modulus with a rheometer. NK-92MI cells were cultured in collagen gels with different levels of stiffness. The morphology of NK-92MI cells was observed by inverted microscope. High content imaging system was used to record the free migration process of NK-92MI cells and analyze the migration speed and distance. NK-92MI cells were cultured with type Ⅰ collagen gels with different levels of stiffness for 24 h and 48 h and, then, co-cultured with human colorectal DLD-1, a human adenocarcinoma epithelial cell line. CCK8 assay was performed to determine the proliferation rate of DLD-1 cells and analyze the cell killing ability of NK-92MI cells. Low-stiffness type Ⅰ collagen gel and high-stiffness type Ⅰ collagen gel with the respective stiffness of (10.970±2.10) Pa and (114.50±3.40) Pa were successfully prepared. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, the NK-92MI cells in the high-stiffness type Ⅰ collagen gel showed a more elongated shape (P<0.05), the mean area of the cells was reduced ([69.88±26.97] μm2 vs. [46.59±21.62] μm2, P<0.05), the roundness of the cells decreased (0.82±0.12 vs. 0.78±0.18, P<0.05), cell migration speed decreased ([2.50±0.91] μm/min vs. [1.70±0.72] μm/min, P<0.001) and the migration distance was shortened ([147.10±53.74] μm vs. [98.03± 40.95] μm, P<0.0001), with all the differences being statistically significant. Compared with those cultured with the low-stiffness type Ⅰ collagen gel, NK-92MI cells cultured with high-stiffness type Ⅰ collagen gel for 24 h could promote DLD-1 cell proliferation, with the proliferation rate being (46.39±12.79)% vs. (65.87±4.45)% (P<0.05) and reduce the cell killing ability. Comparison of the cells cultured for 48 h led to similar results, with the proliferation rates being (31.36±2.88)% vs. (74.57±2.16)% (P<0.05), and the differences were all statistically significant. The 3D culture environment of type Ⅰ collagen gels with different levels of stiffness alters the morphology, migration ability, and killing function of NK-92MI cells. This study provides the research basis for exploring and understanding the mechanisms by which the biomechanical microenvironment affects the immune response of NK cells, as well as laying the theoretical foundation for optimizing immunotherapy protocols.

LINC00707 impairs the Natural Killer cell antitumour activity in hepatocellular carcinoma through decreasing YTHDF2 stability.

Hepatocellular carcinoma (HCC) is the fifth most frequently diagnosed cancer and ranks third in cancer-related fatalities. The recognized involvement of long noncoding RNAs (lncRNAs) in several cancer types, including HCC, inspired this study to explore a novel lncRNA's functional importance in the progression of HCC. To achieve this, lncRNA microarray analysis was conducted on three distinct sets of HCC tissues, revealing LINC00707 as the most significantly upregulated lncRNA. Further research into its biological functions has revealed that LINC00707 acts as an oncogene, driving HCC progression by enhancing the proliferation, migration and invasion of HCC cells. Mechanistic insights were provided, demonstrating that LINC00707 interacts with YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2), thus facilitating the ubiquitination-dependent degradation of the YTHDF2 protein. Furthermore, LINC00707 was found to influence the cytotoxicity of NK-92MI cells against HCC cells through its interactions with YTHDF2. These findings significantly contribute to a deeper understanding of the role played by LINC00707 in the progression of HCC.

NK-92MI Cells Engineered with Anti-claudin-6 Chimeric Antigen Receptors in Immunotherapy for Ovarian Cancer.

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR-NK cell efficacy. Claudin-6 (CLDN6) has been reported to be overexpressed in ovarian cancer and may be an attractive target for CAR-NK cells immunotherapy. However, the feasibility of using anti-CLDN6 CAR-NK cells to treat ovarian cancer remains to be explored. Methods: CLDN6 expression in primary human ovarian cancer, normal tissues and cell lines were detected by immunohistochemistry and western blot. Two types of third-generation CAR NK-92MI cells targeting CLDN6, CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) and CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB) were constructed by lentivirus transfection, sorted by flow cytometry and verified by western blot and qPCR. OVCAR-3, SK-OV-3, A2780, Hey and PC-3 cells expressing the GFP and luciferase genes were transduced. Subcutaneous and intraperitoneal tumor models were established via NSG mice. The ability of CLDN6-CAR NK cells to kill CLDN6-positive ovarian cancer cells were evaluated in vitro and in vivo by live cell imaging and bioluminescence imaging. Results: Both CLDN6-CAR1 and CLDN6-CAR2 NK-92MI cells could specifically killed CLDN6-positive ovarian cancer cells (OVCAR-3, SK-OV-3, A2780 and Hey), rather than CLDN6 negative cell (PC-3), in vitro. CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) exhibited stronger cytotoxicity than CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB). Furthermore, CLDN6-CAR1 NK cells could effectively eliminate ovarian cancer cells in subcutaneous and intraperitoneal tumor models. More importantly, CAR-NK cells combined with immune checkpoint inhibitors, anti-PD-L1, could synergistically enhance the antitumor efficacy of CLDN6-targeted CAR-NK cells. Conclusions: These results indicate that CLDN6-CAR NK cells possess strong antitumor activity and represent a promising immunotherapeutic modality for ovarian cancer.

Interleukin-2 is required for NKp30-dependent NK cell cytotoxicity by preferentially regulating NKp30 expression.

Natural killer (NK) cells are key effectors in cancer immunosurveillance, eliminating a broad spectrum of cancer cells without major histocompatibility complex (MHC) specificity and graft-versus-host diseases (GvHD) risk. The use of allogeneic NK cell therapies from healthy donors has demonstrated favorable clinical efficacies in treating diverse cancers, particularly hematologic malignancies, but it requires cytokines such as IL-2 to primarily support NK cell persistence and expansion. However, the role of IL-2 in the regulation of activating receptors and the function of NK cells expanded for clinical trials is poorly understood and needs clarification for the full engagement of NK cells in cancer immunotherapy. Here, we demonstrated that IL-2 deprivation significantly impaired the cytotoxicity of primary expanded NK cells by preferentially downregulating NKp30 but not NKp46 despite their common adaptor requirement for expression and function. Using NK92 and IL-2-producing NK92MI cells, we observed that NKp30-mediated cytotoxicity against myeloid leukemia cells such as K562 and THP-1 cells expressing B7-H6, a ligand for NKp30, was severely impaired by IL-2 deprivation. Furthermore, IL-2 deficiency-mediated NK cell dysfunction was overcome by the ectopic overexpression of an immunostimulatory NKp30 isoform such as NKp30a or NKp30b. In particular, NKp30a overexpression in NK92 cells improved the clearance of THP-1 cells in vivo without IL-2 supplementation. Collectively, our results highlight the distinct role of IL-2 in the regulation of NKp30 compared to that of NKp46 and suggest NKp30 upregulation, as shown here by ectopic overexpression, as a viable modality to harness NK cells in cancer immunotherapy, possibly in combination with IL-2 immunocytokines.

Screening and Characteristics Analysis of Polysaccharides from Orah Mandarin (Citrus reticulata cv. Orah).

This study aimed to screen out polysaccharides with the ability to activate NK cells. Ten polysaccharides (OP) were isolated from orah mandarin (Citrus reticulata cv. Orah) peel using hot-water extraction combined with the alcohol precipitation method and the ultrafiltration-membrane separation method. After measuring the effects of 10 OPs on NK-92MI cell proliferation and cytotoxicity, it was found that the polysaccharide OP5 had the highest activity in vitro. OP5 can significantly promote the proliferation of and increase the gene expression of perforin, granzyme B and IFN-γ in NK-92MI cells. Its molecular weight was between 50 and 70 kDa. The identification results of monosaccharide composition indicated that OP5 was composed of arabinose (31.52%), galacturonic acid (22.35%), galactose (16.72%), glucose (15.95%), mannose (7.67%), rhamnose (2.39%), fucose (1.41%), xylose (1.30%), glucuronic acid (0.42%) and ribose (0.27%). The sugar ring of the β-configuration was the main, and that of the α-configuration was the auxiliary. These results would provide a foundation for the functional product development of OPs.

Ginsenoside Rh2 enhances immune surveillance of natural killer (NK) cells via inhibition of ERp5 in breast cancer

BackgroundOne critical component of the immune system that prevents breast cancer cells from forming distant metastasis is natural killer (NK) cells participating in immune responses to tumors. Ginsenoside Rh2 (GRh2) as one of the major active ingredients of ginseng has been employed in treatment of cancers, but the function of GRh2 in modulating the development of breast cancer remains elusive. PurposeThis study was to dissect the effect of GRh2 against breast cancer and its potential mechanisms associated with NK cells, both in vitro and in vivo. MethodsMDA-MB-231 and 4T1 cells were used to establish in situ and hematogenous mouse models. MDA-MB-231 and MCF-7 were respectively co-cultured with NK92MI cells or primary NK cells in vitro. Anti-tumor efficacy of GRh2 was verified by immunohistochemistry (IHC), Cell Counting Kit-8 (CCK8), high resolution micro-computed tomography (micro-CT) scanning of lungs and hematoxylin and eosin (H&E) staining. Lactate dehydrogenase (LDH) cytotoxicity assay, flow cytometry, in vivo depletion of NK cells, enzyme-linked immunosorbent assay (ELISA), western blot, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunofluorescence and cell transfection were performed for investigating the anti-tumor mechanisms of GRh2. Molecular docking, microscale thermophoresis (MST) and cellular thermal shift assay (CETSA) were employed to determine the binding between endoplasmic reticulum protein 5 (ERp5) and GRh2. ResultsWe demonstrated that GRh2 exerted prominent impacts on retarding the growth and metastasis of breast cancer through boosting the cytotoxic function of NK cells, as validated by the elevated release of perforin, granzyme B and interferon-γ (IFN-γ). Mechanistical studies revealed that GRh2 was capable of diminishing the expression of ERp5 and GRh2 directly bound to ERp5 in MDA-MB-231 cells as well as on a recombinant protein level. GRh2 prevented the formation of soluble MICA (sMICA) and upregulated the expression level of MICA in vivo and in vitro. Importantly, the reduced lung metastasis of breast cancer by GRh2 was almost abolished upon the depletion of NK cells. Moreover, GRh2 was able to insert into the binding pocket of ERp5 directly. ConclusionWe firstly demonstrated that GRh2 played a pivotal role in augmenting NK cell activity by virtue of modulating the NKG2D-MICA signaling axis via directly binding to ERp5, and may be further optimized to a therapeutic agent for the treatment of breast cancer.

IMMU-55. THERAPEUTIC WINDOW ENABLING ERADICATION OF RESIDUAL GLIOMA STEM CELLS BY INTRACRANIAL NK CELL AND EXTRACELLULAR VESICLE-MEDIATED THERAPY FOLLOWING TEMOZOLOMIDE-INDUCED TUMOUR DEPOPULATION

Abstract Glioblastoma (GBM) is an incurable brain cancer, where dismal outcomes result from disease recurrence driven by tumour-initiating glioma stem cells (GSCs). GSCs survive and expand in the brain after surgery, radiation and temozolomide (TMZ) amidst weak immune and natural killer (NK) cell surveillance. NK cell-mediated killing of GBM cells occurs at effector to target rations precluding effective eradication as enforced by clinical trials with autologous NK cells in an adjuvant setting. Here we explore in a human GSC xenograft model whether tumor depopulation using high dose Temozolomide (TMZ) could create a window of curative opportunity for endogenous or exogenous NK cells. We observed that while subcutaneous (sc) xenografts of patient derived GSCs are infiltrated by endogenous functional (NCR1+) NK cells in SCID mice, the corresponding intracranial (ic) tumours remained NK cell depleted. In NK-deficient NSG mice, neither site contained functional NK cells. Notably, while TMZ caused near complete regression of GSC xenografts in both strains of mice and both locations (sc and ic), this was followed by recurrence of drug resistant lesions in all cases except NK-infiltrated sc SCID xenografts. This effect was obliterated by anti-sialo GM1 treatment. To explore whether direct delivery of NK cells into depopulated intracranial tumours would change the lethal course of the disease, mice were injected with GSCs ic, and upon tumour formation were treated with a sequence of TMZ (systemically), and two days later with irradiated NK92MI. Remarkably, this combined therapy completely obliterated recurrent disease. To assess whether NK92MI cells could be replaced by their derived extracellular vesicles (NK-EVs), the latter were injected i.c. post TMZ in GSC xenograft bearing mice. A single injection of NK-EVs resulted in tumour eradication in some but not all mice. Thus, chemotherapy-dependent tumour depopulation may create a unique window of opportunity for curative NK-mediated immunotherapy in GBM.

Single-cell profiling reveals immune disturbances landscape and HLA-F-mediated immune tolerance at the maternal-fetal interface in preeclampsia.

Preeclampsia is a pregnancy-specific disorder that always causes maternal and fetal serious adverse outcome. Disturbances in maternal immune tolerance to embryo at the maternal-fetal interface (MFI) may be associated with preeclampsia onset. Recent studies have revealed the reduced expression pattern of HLA-F at the MFI in preeclampsia, while the mechanism of it mediating maternal fetal immune tolerance has not been revealed. Single-cell RNA sequencing on placental decidua was performed to reveal the immune disturbances landscape at the MFI in preeclampsia. Human Jar cells and NK-92MI cells were employed to study the role of HLA-F in trophoblasts and lymphocyte. A total of 101,250 cells were classified into 22 cell clusters. Disease-related IGFBP1+SPP1+ extracellular villus trophoblast (EVT) was identified in the preeclamptic placental decidua, accompanied by newly discovered immune cellular dysfunction such as reduced ribosomal functions of NK populations and abnormal expression of antigen-presenting molecules in most cell clusters. Certain genes that are characteristic of the intermediate stage of myeloid or EVT cell differentiation were found to have unexplored but important functions in the pathogenesis of preeclampsia; specifically, we detected enhanced cell cross-talk between IGFBP1+SPP1+ EVT2 or SPP1+M1 cells and their receptor cell populations at the MFI of PE patients compared to controls. With respect to HLA-F, mIF staining confirmed its reduced expression in PE samples compared to controls. Over-expression of HLA-F in Jar cells promoted cell proliferation, invasion, and migration while under-expression had the opposite effect. In NK-92MI cells, over-expression of HLA-F increased the secretion of immunoregulation cytokines such as CSF1 and CCL22, and promoted adaptive NKG2C+NK cell transformation. We revealed the immune disturbance landscape at the MFI in preeclampsia. Our findings regarding cellular heterogeneity and immune cellular dysfunction, as revealed by scRNA-seq, and the function of HLA-F in cells provide new perspectives for further investigation of their roles in the pathogenesis of preeclampsia, and then provide potential new therapeutic target.

Cordycepin Enhances the Cytotoxicity of Human Natural Killer Cells against Cancerous Cells.

Cancer treatment with natural killer (NK) cell immunotherapy is promising. NK cells can recognize and kill cancer cells without sensitization, making them a potential cancer treatment alternative. To improve clinical efficacy and safety, more research is needed. Enhancing NK cell function improves therapeutic efficacy. Due to its potent apoptosis induction, Cordycepin, a bioactive compound from Cordyceps spp., inhibits cancer cell growth. Cordycepin has immunoregulatory properties, making it a promising candidate for combination therapy with NK cell-based immunotherapy. Cordycepin may enhance NK cell function and have clinical applications, but more research is needed. In this study, cordycepin treatment of NK-92 MI cells increased THP-1 and U-251 cell cytotoxicity. Cordycepin also significantly increased the mRNA expression of cytokine-encoding genes, including tumour necrosis factor (TNF), interferon gamma (IFNG), and interleukin 2 (IL2). NK-92 MI cells notably secreted more IFNG and granzyme B. Cordycepin also decreased CD27 and increased CD11b, CD16, and NKG2D in NK-92 MI cells, which improved its anti-cancer ability. In conclusion, cordycepin could enhance NK cell cytotoxicity against cancerous cells for the first time, supporting its use as an alternative immunoactivity agent against cancer cells. Further studies are needed to investigate its efficacy and safety in clinical settings.

Oridonin enhances cytotoxic activity of natural killer cells against lung cancer

BackgroundOridonin is a Chinese herbal medicine exhibiting anti-tumor properties; however, its immune modulation capacity has yet to be elucidated. Our objective in this study was to determine whether oridonin enhances the anti-tumor activity of natural killer (NK) cells against lung cancer cells. MethodsLDH-releasing assays were used to investigate the effects of oridonin on NK-92MI cell activity against lung cancer cells. Flow cytometry and real-time PCR were used to examine the effects of oridonin on degranulation markers, cytotoxic factors, activating receptors on NK-92MI cells, and ligands in lung cancer cells. Western blot analysis provided insight into the mechanisms underlying the observed effects. ResultsOridonin enhanced the cytotoxic effects of NK-92MI cells against A549 lung cancer cells. This effect involved upregulating the expression of the degranulation marker CD107a and IFN-γ as well as activating receptors on NK cells and their ligand MICA/B. Oridonin also inhibited STAT3 phosphorylation in A549 cells and NK-92MI cells. A lung cancer mouse model confirmed the anti-tumor effects of oridonin and NK-92MI cells, wherein both treatments alone suppressed tumor growth. Oridonin was also shown to have a synergistic effect on the anti-tumor activity of NK-92MI cells. ConclusionsThe ability of oridonin to enhance the cytotoxic effects of NK cells indicates its potential as a novel therapeutic agent for the treatment of lung cancer.

Wedelolactone induces natural killer cell activity and the improvement to bioavailability using polysaccharides from Ligustri Lucidi Fructus

Wedelolactone (WDL) is the major bioactive component in Ecliptae Herba. This present study investigated the effects of WDL on natural killer cell functions and possible underlying mechanisms. It was proved that wedelolactone enhanced the killing ability of NK92-MI by upregulating the expression of perforin and granzyme B through the JAK/STAT signaling pathway. Additionally, wedelolactone could induce the migration of NK-92MI cells by promoting CCR7 and CXCR4 expressions. However, the application of WDL is limited due to poor solubility and bioavailability. Accordingly, this study investigated the impact of polysaccharides from Ligustri Lucidi Fructus (LLFPs) on WDL. The biopharmaceutical properties and pharmacokinetic characteristics were determined to compare WDL individually and in combination with LLFPs. The results showed that LLFPs could benefit the biopharmaceutical properties of WDL. Specifically, stability, solubility, and permeability were increased by 1.19–1.82-fold, 3.22-fold, and 1.08-fold higher than those of WDL alone, respectively. Furthermore, the pharmacokinetic study revealed that LLFPs could remarkably improve AUC(0-t) (150.34 vs. 50.47 ng/mL ∗ h), t1/2 (40.78 vs. 2.81 h), and MRT(0-∞) (46.64 vs. 5.05 h) for WDL. In conclusion, WDL would be considered a potential immunopotentiator, and LLFPs could overcome the instability and insolubility, ultimately improving the bioavailability of this plant-derived phenolic coumestan.

25KDa branched polyethylenimine increases interferon-γ production in natural killer cells via improving translation efficiency

BackgroundEx vivo cultivation is a promising strategy for increasing the number of NK cells and enhancing their antitumor activity prior to clinical application. Recent studies show that stimulation with 25KDa branched polyethylenimine (25KbPEI) generates NK cells with enhanced antitumor activity. To better understand how 25KbPEI primes NK cells, we explored the mechanism underlying increase in production of IFN-γ.MethodsChemical priming was performed on NK-92MI cells by incubating them with 5 μg/ml of 25KbPEI. The production of IFN-γ was evaluated by RT-qPCR, ELISA, and Flow cytometry. By evaluating the effect of pharmacological inhibition of ERK/mTOR-eIF4E signaling pathways on IFN-γ translation, the function of these signaling pathways in IFN-γ translation was examined. To comprehend the level of 25KbPEI activity on immune-related components in NK cells, RNA sequencing and proteomics analyses were conducted.Results25KbPEI enhances the production of IFN-γ by NK cells without transcriptional activation. Activation of ERK and mTOR signaling pathways was found to be associated with 25KbPEI-mediated calcium influx in NK cells. The activation of ERK/mTOR signaling was linked to the phosphorylation of 4E-BP1, which resulted in the activation of translation initiation complex and subsequent IFN-γ translation. Analysis of RNA sequencing and proteomics data revealed that the activity of 25KbPEI to improve translation efficiency in NK cells could be extended to additional immune-related molecules.ConclusionsThis study provides substantial insight into the process by which 25KbPEI primes NK cells. Our data demonstrated that the 25KbPEI mediated activation of ERK/mTOR signaling and subsequent stimulation of eIF4E is the primary mechanism by which the chemical stimulates translation of IFN-γ in NK cells.BTHQ_5BYqzK7H1pGe9W3hFVideo abstract

MAP kinase activating death domain deficiency is a novel cause ofimpaired lymphocyte cytotoxicity.

Most hereditary forms of hemophagocytic lymphohistiocytosis (HLH) are caused by defects of cytotoxicity, including the vesicle trafficking disorder Griscelli syndrome type 2 (GS2, RAB27A deficiency). Deficiency of the mitogen-activated protein kinase activating death domain protein (MADD) results in a protean syndrome with neurological and endocrinological involvement. MADD acts as a guanine nucleotide exchange factor for small guanosine triphosphatases, including RAB27A. A homozygous splice site mutation in MADD was identified in a female infant with syndromic features, secretory diarrhea, and features of HLH. Aberrant splicing caused by this mutation leads to an in-frame deletion of 30 base pairs and favors other aberrant variants. Patient natural killer (NK) cells and cytotoxic T cells showed a severe degranulation defect leading to absent perforin-mediated cytotoxicity. Platelets displayed defective adenosine triphosphate secretion, similar to that in GS2. To prove causality, we introduced a CRISPR/Cas9-based MADD knockout in the NK cell line NK-92mi. MADD-deficient NK-92mi cells showed a degranulation defect and impaired cytotoxicity similar to that of the patient. The defect of cytotoxicity was confirmed in another patient with MADD deficiency. In conclusion, RAB27A-interacting MADD is involved in vesicle release by cytotoxic cells and platelets. MADD deficiency causes a degranulation defect and represents a novel disease predisposing to an HLH phenotype.