Increased Natural Killer Cell Research Articles

Isoxazole-based molecules restore NK cell immune surveillance in hepatocarcinogenesis by targeting TM4SF5 and SLAMF7 linkage

Dynamic communication between hepatocytes and the environment is critical in hepatocellular carcinoma (HCC) development. Clinical immunotherapy against HCC is currently unsatisfactory and needs more systemic considerations, including the identification of new biomarkers and immune checkpoints. Transmembrane 4 L six family member 5 (TM4SF5) is known to promote HCC, but it remains unclear how cancerous hepatocytes avoid immune surveillance and whether avoidance can be blocked. We investigated how TM4SF5-mediated hepatic tumorigenesis avoids surveillance by natural killer (NK) cells, which are prevalent in the liver, and whether the avoidance can be blocked by anti-TM4SF5 agents. We used comprehensive structure activity relationship analysis to identify TM4SF5-specific isoxazole (TSI)-based small molecules that inhibit TM4SF5-mediated effects. TM4SF5 expressed by hepatocytes reduced NK cell cytotoxicity by downregulating stimulatory ligands/receptors, including signaling lymphocytic activation molecule family member 7 (SLAMF7). TM4SF5 bound SLAMF7 depending on N-glycosylation and caused intracellular trafficking of SLAMF7 from the plasma membrane to lysosomes for degradation. TSI treatments in cell lines and animal models of HCC blocked this binding, intracellular trafficking, and downregulation, resulting in higher levels of stimulatory NK cell ligands. In mouse xenograft models, TSI treatment abrogated HCC development by increasing the abundance and dispersion of Slamf7-positive cells in liver tissues, recapitulating the phenotype of Tm4sf5-knockout mice and indicating TSI-mediated restoration of NK cell surveillance. These findings suggest that TSIs can inhibit TM4SF5-mediated liver carcinogenesis by increasing NK cell surveillance.

Cellular Senescence in Hepatocellular Carcinoma: Immune Microenvironment Insights via Machine Learning and In Vitro Experiments.

Hepatocellular carcinoma (HCC), a leading liver tumor globally, is influenced by diverse risk factors. Cellular senescence, marked by permanent cell cycle arrest, plays a crucial role in cancer biology, but its markers and roles in the HCC immune microenvironment remain unclear. Three machine learning methods, namely k nearest neighbor (KNN), support vector machine (SVM), and random forest (RF), are utilized to identify eight key HCC cell senescence markers (HCC-CSMs). Consensus clustering revealed molecular subtypes. The single-cell analysis explored the tumor microenvironment, immune checkpoints, and immunotherapy responses. In vitro, RNA interference mediated BIRC5 knockdown, and co-culture experiments assessed its impact. Cellular senescence-related genes predicted HCC survival information better than differential expression genes (DEGs). Eight key HCC-CSMs were identified, which revealed two distinct clusters with different clinical characteristics and mutation patterns. By single-cell RNA-seq data, we investigated the immunological microenvironment and observed that increasing immune cells allow hepatocytes to regain population dominance. This phenomenon may be associated with the HCC-CSMs identified in our study. By combining bulk RNA sequencing and single-cell RNA sequencing data, we identified the key gene BIRC5 and the natural killer (NK) cells that express BIRC5 at the highest levels. BIRC5 knockdown increased NK cell proliferation but reduced function, potentially aiding tumor survival. These findings provide insights into senescence-driven HCC progression and potential therapeutic targets.

Emerging mutation in SARS-CoV-2 facilitates escape from NK cell recognition and associates with enhanced viral fitness.

In addition to adaptive immunity, natural killer (NK) cells of the innate immune system contribute to the control of viral infections. The HLA-E-restricted SARS-CoV-2 Nsp13232-240 epitope VMPLSAPTL renders infected cells susceptible to NK cells by preventing binding to the inhibitory receptor NKG2A. Here, we report that a recently emerged methionine to isoleucine substitution at position 2 (pM2I) of Nsp13232-240 impairs binding of the mutated epitope to HLA-E and diminishes HLA-E/peptide complex stability. Structural analyses revealed altered occupancy of the HLA-E B-pocket as the underlying cause for reduced presentation and stability of the mutated epitope. Functionally, the reduced presentation of the mutated epitope correlated with elevated binding to NKG2A as well as with increased NK cell inhibition. Moreover, the pM2I mutation associated with enhanced estimated viral fitness and was transmitted to descendants of the SARS-CoV-2 BQ.1 variant. Interestingly, the mutated epitope resembles sequences of related peptides found in endemic common cold-causing human coronaviruses. Altogether, these findings indicate compromised peptide presentation as a viral adaptation to evade NK cell-mediated immunosurveillance by enabling enhanced presentation of self-peptide and restoring NKG2A-dependent inhibition of NK cells.

Protein phosphatase 2A inhibitor modulates natural killer cell homeostasis in peripheral tissues

Although natural killer (NK) cell responses to tumor and viral infection have been studied, the mechanisms underlying NK cell homeostasis in vivo remain unclear. In this study, we demonstrate the pharmacological action of cytostatin, a protein phosphatase 2A (PP2A) specific inhibitor (PP2Ai), on NK cells in regulating NK cell homeostasis in the peripheral tissues. We found that PP2Ai treatment decreased NK cell percentages in the bone marrow and secondary lymphoid tissues while increasing NK cell percentages in peripheral tissues such as the lung and liver. In the peripheral tissues of PP2Ai-treated mice, Ki-67 expression and BrdU uptake in NK cells were upregulated, and an initial increase in the pre-mature CD11bhiCD27hi NK subset was observed, followed by an increase in the terminally differentiated mature CD11bhiCD27lo NK subset. In addition, bone marrow Ki-67+ NK cells predominantly expressed CX3CR1 in the PP2Ai-treated mice and were further mobilized to the peripheral tissues. Among various target molecules of PP2A, we found that the upregulation of c-Myc pathway and its phosphorylation, along with its downstream cyclin E expression and G1/S cell cycle transition in PP2Ai-treated mice NK cells. Our results suggest that PP2Ai modulates NK cell proliferation through c-Myc and cyclin E, leading to their maturation and trafficking from the bone marrow to the peripheral tissues.

Abstract C038: Harnessing NK cells to improve targeted therapy therapeutic efficacy in BRAF- mutant melanoma

Abstract Malignant melanoma is the most lethal form of skin cancer, with BRAFV600E as the most common driver mutation. Despite the success of BRAF/MEK inhibitors (BRAF/MEKi) as a standard-of-care, drug resistance occurs at high frequency and remains a concern to be overcome. In addition to suppressing cancer cell proliferation and survival, BRAF/MEKi treatment alters the tumor microenvironment to favor anti-tumor immunity, with extensive previous studies focusing on the T cell compartment. Investigating other immune components, such as natural killer (NK) cells, may offer new insights into improving the overall effectiveness of BRAF/MEKi. NK cells are innate immune cells that provide activating signals to cytotoxic T cells, in addition to their critical roles in tumor surveillance and metastasis. However, the role of NK cells as a component of the BRAF/MEKi response in melanoma remains largely unknown. To investigate the role of NK cells in BRAF/MEKi-mediated tumor control, we depleted NK cells in melanoma-bearing mice. We found that the tumors developed full resistance faster in the NK cell-depleted group than in controls. Moreover, quantification of tumor-infiltrating NK cells in different melanoma phases, including off-therapy growth, therapy-induced regression, drug- tolerant residual disease, and on-therapy regrowth and resistance, revealed a positive correlation between NK cell infiltration and the tumor response to BRAF/MEKi. We further increased NK cell infiltration during drug-tolerant residual disease by inhibiting Ptpn22, a potential negative regulator of activity in NK and other immune cell populations, resulting in a delayed onset of therapy resistance. These results demonstrate the indispensable role of NK cells in the BRAF/MEKi tumor response and their potential to improve the therapeutic efficacy of BRAF/MEKi. Citation Format: Chia-Hsin Hsu, Andrew C. White. Harnessing NK cells to improve targeted therapy therapeutic efficacy in BRAF- mutant melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C038.

Altered serum metabolome is associated with disease activity and immune responses in rheumatoid arthritis

Rheumatoid arthritis (RA) is widespread globally, with the emergence of metabolites derived from both the host and microbes playing a pivotal role in its pathogenesis. This study aims to elucidate the relationships between serum metabolites and the immunological and clinical features of RA. Serum samples were collected from 35 RA patients and 37 healthy controls (HC). Metabolite profiling was performed using gas chromatography-mass spectrometry (GC/MS). Principal component analysis revealed a significant distinction between the RA and HC cohorts. Employing univariate statistical analysis, we identified 36 differential metabolites. Among these, 9 metabolites, including galactose and glucose, were found to be enriched, while the remaining metabolites, such as citric acid, fumaric acid, and inosine, were depleted in RA. These diverse metabolites encompassed various metabolic processes, including the biosynthesis of fatty acids, amino acids, and glucose. The enrichment of glucose and galactose in RA exhibited a substantial correlation with elevated IgG levels, as determined through correlation analysis. Conversely, the depletion of citric acid was correlated with elevated levels of C3 and CRP. Methionine, which also declined in RA patients, displayed a negative correlation with ESR. Furthermore, galactose and glucose exhibited significant positive correlations with naïve B cells, while the decreased eicosanoic acid level in RA was significantly associated with an increase in natural killer cells. Our findings suggest that the altered serum metabolite profile in RA is closely linked to disease severity and the dysregulated immune responses observed in RA patients. Key Points• Identified nine metabolites with upregulated expression and twenty-seven metabolites with downregulated expression.• Established a correlation between alterations in serum metabolite levels and inflammatory markers in RA patients.• Discovered a significant association between changes in serum metabolites and immune cell profiles in RA patients.

CD56 does not contribute to the anti-tumor, tissue homing, and glycolytic capacity of human NK cells.

Natural Killer (NK) cells are critical innate immune cells involved in the clearance of virally infected and malignant cells. Human NK cells are distinguished by their surface expression of CD56 and a lack of CD3. While CD56 expression and cell surface density has long been used as the prototypic marker to characterize primary human NK cell functional subsets, the exact functional role of CD56 in primary human NK cells is still not fully understood. Here we eliminated the expression of CD56 in human ex vivo expanded NK cells (CD56bright) using CRISPR/Cas9 in order to assess the function of CD56 in this highly activated and cytotoxic NK cell population. We show that the expression of CD56 has no effect on NK cell proliferative capacity or expression of various activation and inhibitory markers. Further, CD56 does not contribute to NK cell-mediated cytotoxicity, inflammatory cytokine production, or the ability of NK cells to control tumor engraftment in vivo. We also found that while deletion of CD56 did not impact NK cell glycolytic metabolism it did increase NK cell reliance on oxidative phosphorylation. Lastly, CD56 does not alter expanded NK cell in vivo tissue trafficking. Our results indicate that while CD56 expression could be used to indicate a hyper-functional state of NK cells, it does not directly influence the anti-tumor functions of expanded NK cells.

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa/CD16a structure.

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa/CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues, we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step, with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating that the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa/CD16a structure

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa/CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues, we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step, with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating that the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.

The Consumption of Lacticaseibacillus rhamnosus HDB1258 Changes Human Gut Microbiota and Induces Immune Enhancement Through NK Cell Activation.

The gut microbiota can play an important role in enhancing the host's complex immune system. In this regard, many studies indicate that probiotics consumption has a beneficial impact on alterations in the composition of the gut microbiota. Our previous study demonstrated that the oral administration of Lacticaseibacillus rhamnosus HDB1258 (HDB1258) enhances immune cell activity and alters the composition of gut microbiota in C57BL/6 mice, thereby showing its potential as a novel immunostimulatory ingredient. Therefore, this clinical trial assessed the effects of HDB1258 on human natural killer (NK) cell activity and changes in gut microbiota. It also investigated the correlation between gut microbiota and NK cell activity following HDB1258 supplementation. Participants (n = 71) were randomized into placebo and HDB1258 groups, and NK cell activity and gut microbiota were investigated at baseline (week 0) and endline (week 8). The present study showed that HDB1258 significantly increased NK cell activity and resulted in positive regulatory effects on the gut microbial balance in subjects compared to the placebo group. HDB1258 affected the gut microbial balance by inducing the growth of beneficial bacteria such as Lactococcus and Sutterella. Especially, the changes in Escherichia-Shigella composition were negatively correlated with the changes in NK cell activity after HDB1258 consumption. There was also a positive correlation between the NK cell activity in the HDB1258 group and the composition of Prevotella 9 and Adlercreutzia. These findings suggest that HDB1258 may improve the host's intestinal environment by regulating gut bacteria related to immune response and promote NK cell activation. This study was registered at clinical research information service (CRIS: KCT0008204).

Abstract A052: RNF43 p.G659fs mutation in MSI-high colorectal cancer leads to natural killer cell dysfunction

Abstract RNF43_p.G659fsV*41 mutation (RNF43659mut) is found in approximately 8% of colorectal cancers (CRCs) and is enriched in microsatellite-instability high (MSI-high) tumors. Previously, we reported that RNF43659mut promotes tumor growth independent of WNT signaling and activates PI3K/AKT signaling in CRC, conferring vulnerability to PI3K/MTOR inhibition. In addition, RNF43659mut was associated with decreased interferon signaling suggesting a link with anti-tumor immunity. Thus, in the current work we investigated the role of RNF43659mut in CRC immunomodulation. The immune-cell landscape was evaluated in RNF43659mut compared with RNF43-wild type (wt) MSI-high CRCs in two cohorts; single-cell (sc)-RNA-sequencing data from treatment-naïve primary tumors from 34 patients (cohort-1) and multiplex immunofluorescence / whole exome sequencing data from an independent cohort of 91 patients (cohort-2). Healthy donor blood-derived natural killer (NK) cell responses and cytotoxicity were investigated in co-cultures with RNF43659mut isogenic tumor lines and with patient-derived organoids harboring RNF43659mut or overexpressing RNF43wt. A third patient cohort with MSI-high CRCs treated with immune checkpoint blockade was analyzed to understand the impact of RNF43659mut on immunotherapy efficacy. Interrogation of sc-RNASeq data in cohort-1 revealed a significant enrichment of NK cells in the RNF43659mut patient samples compared to the RNF43wt ones. This finding was also confirmed by multiplex immunofluorescence in cohort-2. Single-cell transcriptomic analyses in cohort-1 showed increased inhibitory markers on NK cells despite their enrichment in RNF43659mut tumors. RNAseq analysis of sorted NK cells co-cultured with RNF43659mut isogenic tumor lines further supported their dysfunctional state. Flow cytometric analyses of NK cell maturation, activation, and inhibitory markers in NK cells co-cultured with RNF43659mut isogenic tumor lines and organoids highlighted decreased levels of NCAM1 (CD56)dim and NCAM1 (CD56)bright subsets, FCGR3A (CD16), and KLRK1 (NKG2D), along with increased levels of KLRC1 (NKG2A), KIR3DL1 (NKB1) and TIGIT inhibitory markers. There was reduced NK cell mediated tumor cell killing in RNF43659mut isogenic tumor lines and organoids. We postulated that aberrant signaling of the PI3K/AKT pathway with RNF43659mut could lead to NK cell dysfunction. Indeed, siRNA-mediated blocking of PI3K/AKT pathway restored the NK-cell dysfunctional phenotype and tumor cell killing in co-cultures with RNF43659mut isogenic tumor lines and organoids. Conversely, overexpression of WT RNF43 in RNF43659mut organoids downregulated PI3K/AKT signaling and increased NK cell cytotoxicity. Lastly, MSI-high CRC tumors harboring RNF43659mut in patients treated with immunotherapy showed less benefit from immune checkpoint blockade. PI3K/AKT activation due to RNF43659mut in MSI-high CRC leads to NK cell dysfunction and reduced NK cell cytotoxicity. This could be responsible for immunotherapy failure, warranting further investigation. Citation Format: Pushpamali De Silva, Samantha Fitzgerald, Jules Cazaubiel, Matan Hofree, Tomotaka Ugai, Juha P Väyrynen, Dane Ford-Roshon, Lishan Fang, Ana Garrido-Castro, Nir Hacohen, Kimmie Ng, Shuji Ogino, Jonathan A Nowak, Marios Giannakis. RNF43 p.G659fs mutation in MSI-high colorectal cancer leads to natural killer cell dysfunction [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A052.

Increased peritoneal TGF-β1 is associated with ascites-induced NK-cell dysfunction and reduced survival in high-grade epithelial ovarian cancer.

Natural killer (NK) cell therapy represents an attractive immunotherapy approach against recurrent epithelial ovarian cancer (EOC), as EOC is sensitive to NK cell-mediated cytotoxicity. However, NK cell antitumor activity is dampened by suppressive factors in EOC patient ascites. Here, we integrated functional assays, soluble factor analysis, high-dimensional flow cytometry cellular component data and clinical parameters of advanced EOC patients to study the mechanisms of ascites-induced inhibition of NK cells. Using a suppression assay, we found that ascites from EOC patients strongly inhibits peripheral blood-derived NK cells and CD34+ progenitor-derived NK cells, albeit the latter were more resistant. Interestingly, we found that higher ascites-induced NK cell inhibition correlated with reduced progression-free and overall survival in EOC patients. Furthermore, we identified transforming growth factor (TGF)-β1 to correlate with ascites-induced NK cell dysfunction and reduced patient survival. In functional assays, we showed that proliferation and anti-tumor reactivity of CD34+ progenitor-derived NK cells are significantly affected by TGF-β1 exposure. Moreover, inhibition of TGF-β1 signaling with galunisertib partly restored NK cell functionality in some donors. For the cellular components, we showed that the secretome is associated with a different composition of CD45+ cells between ascites of EOC and benign reference samples with higher proportions of macrophages in the EOC patient samples. Furthermore, we revealed that higher TGF-β1 levels are associated with the presence of M2-like macrophages, B cell populations and T-regulatory cells in EOC patient ascites. These findings reveal that targeting TGF-β1 signaling could increase NK cell immune responses in high-grade EOC patients.

MicroRNA-146a deficiency enhances host protection against murine cytomegalovirus.

Natural killer (NK) cells are innate lymphoid cells that protect a host from viral infections and malignancies. MicroRNA-146a (miR-146a) is an important regulator of immune function that is highly expressed in NK cells and is further upregulated during murine cytomegalovirus (MCMV) infection. Here we utilized mice with a global targeted deletion of miR-146a to understand its impact on the innate immune responses to MCMV infection. MiR-146a-/- mice were protected from lethal MCMV infection, which was intrinsic to the hematopoietic compartment based on bone marrow chimera experiments. NK cell depletion abrogated this protection, implicating NK cells as critical for the miR-146a-/- protection from MCMV. Surprisingly, NK cells from miR-146a-deficient mice were largely similar to control NK cells with respect to development, maturation, trafficking, and effector functions. However, miR-146a-/- mice had increased NK cell numbers and frequency of the most mature Stage IV (CD27-CD11b+) NK cells in the liver at baseline, enhanced STAT1 phosphorylation, and increased selective expansion of Ly49H+ NK cells and T cells during MCMV infection. This study demonstrates a critical role for miR-146a in the host response to MCMV, arising from mechanisms that include increased NK cell numbers and early T-cell expansion.

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.

One N-glycan regulates natural killer cell antibody-dependent cell-mediated cytotoxicity and modulates Fc γ receptor IIIa / CD16a structure.

Both endogenous antibodies and a subset of antibody therapeutics engage Fc gamma receptor (FcγR)IIIa / CD16a to stimulate a protective immune response. Increasing the FcγRIIIa/IgG1 interaction improves the immune response and thus represents a strategy to improve therapeutic efficacy. FcγRIIIa is a heavily glycosylated receptor and glycan composition affects antibody-binding affinity. Though our laboratory previously demonstrated that natural killer (NK) cell N-glycan composition affected the potency of one key protective mechanism, antibody-dependent cell-mediated cytotoxicity (ADCC), it was unclear if this effect was due to FcγRIIIa glycosylation. Furthermore, the structural mechanism linking glycan composition to affinity and cellular activation remained undescribed. To define the role of individual amino acid and N-glycan residues we measured affinity using multiple FcγRIIIa glycoforms. We observed stepwise affinity increases with each glycan truncation step with the most severely truncated glycoform displaying the highest affinity. Removing the N162 glycan demonstrated its predominant role in regulating antibody-binding affinity, in contrast to four other FcγRIIIa N-glycans. We next evaluated the impact of the N162 glycan on NK cell ADCC. NK cells expressing the FcγRIIIa V158 allotype exhibited increased ADCC following kifunensine treatment to limit N-glycan processing. Notably, an increase was not observed with cells expressing the FcγRIIIa V158 S164A variant that lacks N162 glycosylation, indicating the N162 glycan is required for increased NK cell ADCC. To gain structural insight into the mechanisms of N162 regulation, we applied a novel protein isotope labeling approach in combination with solution NMR spectroscopy. FG loop residues proximal to the N162 glycosylation site showed large chemical shift perturbations following glycan truncation. These data support a model for the regulation of FcγRIIIa affinity and NK cell ADCC whereby composition of the N162 glycan stabilizes the FG loop and thus the antibody-binding site.

Inhibitory Effects of Dioscoreae Rhizoma and Polygalae Radix Mixture Combined with Sorafenib in Hepatoma Xenografts Model

Background Hepatocellular carcinoma (HCC) is one of the most common malignancies and a leading cause of cancer-related mortality and morbidity worldwide. Purpose This study evaluated the enhanced efficacy of a combination of herbal medicine with sorafenib (MIX) in a transplantation tumor model induced by HepG2 cells in HCC. Materials and Methods The HepG2 cell transplantation tumor model was established by injecting HepG2 cells subcutaneously into BALB/c nude mice. The impact of MIX on tumor growth was observed. Granzyme B was used to measure natural killer (NK) cell activity. Prognostic markers of HCC, telomerase reverse transcriptase (TERT), and alpha-fetoprotein (AFP) were analyzed via real-time polymerase chain reaction. Additionally, inflammation and apoptosis-related markers were assessed through serum analysis and Western blotting. Results Among the MIX groups, MIX25 (sorafenib 10 mg/kg + Dioscoreae rhizoma and Polygalae radix mixture 25 mg/kg) demonstrated the most significant effect. MIX25 effectively suppressed tumor volume and weight, enhanced apoptosis-promoting proteins (Bax, Cytochrome c, and Caspase-3), and reduced apoptosis-inhibiting proteins like survivin. Furthermore, MIX25 inhibited the mitogen-activated protein kinase inflammatory response and inflammatory markers, including tumor necrosis factor-alpha and cyclooxygenase-2. The MIX25 group exhibited superior effects on NK cell activity and cancer marker genes (AFP and TERT) compared to other drug-treated groups. Conclusion MIX25 effectively suppressed the proliferation of HepG2 cell transplantation tumors in BALB/c nude mice. It not only inhibited inflammatory markers but also enhanced cell apoptosis. Additionally, MIX25 significantly increased NK cell activity and reduced mRNA expressions of AFP and TERT. Overall, the combination of MIX25 with sorafenib shows promise as a potential treatment for patients with HCC.

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

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

Decreased Peripheral Blood Natural Killer Cell Count in Untreated Juvenile Dermatomyositis Is Associated with Muscle Weakness.

Juvenile Dermatomyositis (JDM) is the most common inflammatory myopathy in pediatrics. This study evaluates the role of Natural Killer (NK) cells in Juvenile Dermatomyositis (JDM) pathophysiology. The study included 133 untreated JDM children with an NK cell count evaluation before treatment. NK cell subsets (CD56low/dim vs. CD 56bright) were examined in 9 untreated children. CD56 and perforin were evaluated in situ in six untreated JDM and three orthopedic, pediatric controls. 56% of treatment-naive JDM had reduced circulating NK cell counts, designated "low NK cell". This low NK group had more active muscle disease compared to the normal NK cell group. The percentage of circulating CD56low/dim NK cells was significantly lower in the NK low group than in controls (0.55% vs. 4.6% p < 0.001). Examination of the untreated JDM diagnostic muscle biopsy documented an increased infiltration of CD56 and perforin-positive cells (p = 0.023, p = 0.038, respectively). Treatment-naive JDM with reduced circulating NK cell counts exhibited more muscle weakness and higher levels of serum muscle enzymes. Muscle biopsies from treatment-naive JDM displayed increased NK cell infiltration, with increased CD56 and perforin-positive cells.

Effect of preoperative natural killer cell on postoperative pulmonary complications in patients of lung cancer − A single-center retrospective cohort study

BackgroundThe effect of preoperative natural killer (NK) cell abnormalities on postoperative pulmonary complications (PPCs) after thoracoscopic radical resection of lung cancer is still unclear. The main purpose of this study was to investigate the relationship between the preoperative NK cell ratio and PPCs. MethodsThe patients who underwent thoracoscopic radical resection for lung cancer were divided into a normal group and an abnormal group according to whether the proportion of preoperative NK cells was within the reference range. The main outcome was the incidence of PPCs during postoperative hospitalization. The demographic and perioperative data were collected. Propensity score matching was used to exclude systematic bias. Univariate logistic regression was used to test the relationship between the preoperative NK cell ratio and the incidence of PPCs. The restrictive cubic spline curve was used to analyze the dose–effect relationship between the preoperative NK cell ratio and the incidence of PPCs. ResultsA total of 4161 patients were included. After establishing a matching cohort, 910 patients were included in the statistical analysis. The incidence of PPCs in the abnormal group was greater than that in the normal group (55.2% vs. 31.6%). The incidence of PPCs first decreased and then increased with increasing NK cell ratio. The proportion of patients with Grade 3 or higher PPCs in the normal group was lower than that in the abnormal group [108 (23.7%) vs. 223 (49%)]. The indwelling time of the thoracic drainage tube in the abnormal group was longer than that in the normal group [3 (3, 4) vs. 3 (3, 5)]. A preoperative abnormal NK cell ratio constituted a risk factor for PPCs in each subgroup. ConclusionLung cancer patients with an abnormal proportion of peripheral blood NK cells before surgery were more likely to develop PPCs, their disease degree was more severe, and they had a prolonged duration of chest tube indwelling. Compared with those with abnormally high NK cell ratios, those with abnormally low NK cell ratios had more pronounced PPCs.

Combinatorial cellular therapy in pediatric solid tumors with natural killer (NK) and genetically engineered myeloid cells (GEMys).

2561 Background: Early human studies with Natural Killer (NK) cells have shown promise, harnessing their ability to infiltrate into tumors and be tumoricidal. However, these effects can be limited by adenosine, reactive oxygen species, and TGF-β in the highly immune suppressive tumor microenvironment (TME). TGF-β and downstream SMAD3 signaling in Natural Killer (NK) cells have been shown to cause decreased cytokine production, downregulation of activating cell surface receptors, and attenuated cytotoxic function against solid tumors. Exposure to TGF-β during cell culture generates expanded NK cells with increased resistance to TGF-β signaling, dubbed TGF-β imprinted NK cells. In vitrostudies have shown that these cells have increased cytokine production and cytotoxic function in the presence of TGF-β. However, their in vivo trafficking, modulation of the TME, and therapeutic efficacy remain understudied. Our lab has previously shown that when given to tumor-bearing mice, myeloid cells modified to produce IL-12 (IL-12-Genetically Engineered Myeloid cells, aka IL-12 GEMys) are effective at homing to tumor and metastatic sites and, via IL-12 secretion and TME modulation, increase NK cell presence and activation in these sites. They hold great promise as combination therapy, enhancing the effectiveness of other cellular therapies such as NK cell therapy. A current clinical trial exploring TGF-β Imprinted, Ex Vivo Expanded, Universal Donor NK Cell Infusions in relapsed sarcomas is underway (NCT05634369). Methods: In vitro experiments were conducted with the xCELLigence real-time cell analysis system. The human osteosarcoma cell line 143B and human rhabdomyosarcoma cell line RH-30 were evaluated for cell death after co-culture with healthy donor NK cells or TGF-β imprinted NK cells (E:T ratio of 5:1) in isolation and in combination with IL-12 GEMys (E:T ratio of 2:1). Results: TGF-β imprinted NK cells are significantly more effective at tumor cell killing of both 143B and RH30 cell lines in-vitro compared to regular expanded NK cells (WT NK cells), with &gt;90% decrease in cell index compared to 50-60%, respectively, after 48 hours of co-culture. IL-12 GEMys also significantly enhance the cytotoxic effects of WT NK cells, increasing cell killing to 70-80% compared to co-culture with untransduced GEMy controls. Conclusions: These initial in-vitro experiments provide insight into the promising effectiveness of NK-myeloid cell combination therapies in the treatment of pediatric sarcomas. Ongoing in vitroexperiments include cytokine quantification by ELISA and flow cytometric analysis of NK cell activation and proliferation markers. Our preliminary results provide rationale to continue studying the in vivo efficacy of TGF-β imprinted NK cell monotherapy and in combination with IL-12 GEMys in the treatment of tumor-bearing NSG mice.