Natural Killer Cell-mediated Cytotoxicity Research Articles

Differences in Tumor Growth and Differentiation in NSG and Humanized-BLT Mice; Analysis of Human vs. Humanized-BLT-Derived NK Expansion and Functions.

There is significant interest and debate regarding the best mouse model of human disease, since studies in wild-type mice may not always recapitulate human diseases. The NSG mouse model has been one of the most commonly used mouse models to study cancer; however, this mouse model, even though it has several advantages in regard to the ease of tumor implantation and financial feasibility, does not represent human disease due to the immunodeficient nature of this model. In this study, we performed oral and pancreatic tumor studies in NSG and hu-BLT mice and found several distinguishing features that make hu-BLT model more suitable for studying human cancer. In addition, we compared the immune function of humans to hu-BLT mice to understand the differences and similarities of the models. Oral and pancreatic cancer stem cells were implanted in NSG and hu-BLT mice. Both tumors grew robustly in NSG mice and killed them within a short period of time. On the contrary, unlike NSG mice, tumor-bearing hu-BLT mice survived longer, grew smaller tumors, and the grown tumors exhibited lower rates of expansion, with a higher surface expression of MHC-class I and lower NK cell-mediated cytotoxicity that was previously shown to have more of a differentiated phenotype. Although the peripheral blood of hu-BLT mice in comparison to that of humans had lower percentages of NK cells and cytotoxic function, it mediated a higher secretion of IFN-γ, likely contributing to the differentiation of the tumor cells and subsequent decrease in the tumor size in the hu-BLT mice in comparison to the NSG mice. Spleen-derived hu-BLT mouse NK cells were able to expand in the presence of autologous osteoclasts and substantially increase both cytotoxicity and secretion of IFN-γ, similar to those seen in peripheral blood-derived human NK cells, indicating that NK cells from hu-BLT mice are capable of expansion and functional activation when activating signals are given. Thus, the many similarities between human and hu-BLT mouse immune systems make this mouse model more appropriate to study human cancer. In particular, it is well-suited for studies of allogeneic NK cell-based immunotherapy in cancer treatment. The advantages and challenges of hu-BLT mice in cancer studies are also discussed in this report.

Red Ginseng Extract and γ-Aminobutyric Acid Synergistically Enhance Immunity Against Cancer Cells and Antitumor Metastasis Activity in Mice.

The effects of combined administration of red ginseng (RG) extracts and gamma-aminobutyric acid (GABA) on immunostimulatory activity and tumor metastasis inhibition were investigated in mice. For the immunostimulatory activity, splenocyte proliferation, natural killer (NK) cell activity, including the production of granzyme B (GrB) and interferon gamma (IFN-γ), and serum level of cytokine such as IFN-γ, interleukin (IL)-17, and IL-21 were assessed. Peyer's patch cells obtained from mice administered with RG+GABA were cultured, and the cytokine level in the culture supernatant and bone marrow (BM) cell proliferation activity were examined. The proliferative activity of splenocytes was significantly higher in the RG-GABA treatment group than in RG or GABA alone (P < .05). In the experimental tumor metastasis model, oral administration of RG+GABA showed a higher antitumor metastatic effect compared to that of RG or GABA alone. Oral administration of RG+GABA significantly augmented NK cell-mediated cytotoxicity against YAC-1 tumor cells. In addition, the production of GrB and IFN-γ was stimulated in the culture supernatant of NK cells and YAC-1 cells. Serum concentrations of IFN-γ, IL-17, and IL-21 in mice with RG+GABA were significantly higher compared to the corresponding blood levels in mice administered with RG or GABA alone. The RG+GABA group showed significant BM cell proliferation and increased production of IL-6 and granulocyte-macrophage colony-stimulating factor compared to that in the monotherapy groups. Therefore, RG may have a synergistic effect with GABA for enhancing the host defense system such as BM proliferation and NK cell activity in a tumor metastasis model.

Triptolide leads to hepatic intolerance to exogenous lipopolysaccharide and natural-killer-cell mediated hepatocellular damage by inhibiting MHC class I molecules

BackgroundTripterygium wilfordii Hook. F (TWHF) is used as a traditional Chinese medicine, called thunder god vine, based on its efficacy for treating inflammatory diseases. However, its hepatotoxicity has limited its clinical application. Triptolide (TP) is the major active and toxic component of TWHF. Previous studies reported that a toxic pretreatment dose of TP leads to hepatic intolerance to exogenous lipopolysaccharide (LPS) stimulation, and to acute liver failure, in mice, but the immune mechanisms of TP-sensitised hepatocytes and the TP-induced excessive immune response to LPS stimulation are unknown. PurposeTo identify both the key immune cell population and mechanism involved in TP-induced hepatic intolerance of exogenous LPS. Study DesignIn vitro and in vivo experiments were conducted to investigate the inhibitory signal of natural killer (NK) cells maintained in hepatocytes, and the ability of TP to impair that signal. MethodsFlow cytometry was performed to determine NK cell activity and hepatocyte histocompatibility complex (MHC) class I molecules expression; the severity of liver injury was determined based on blood chemistry values, and drug- or cell-mediated hepatocellular damage, by measuring lactate dehydrogenase (LDH) release. In vivo H-2Kb transduction was carried out using an adeno-associated viral vector. ResultsInterferon (IFN)-γ-mediated necroptosis occurred in C57BL/6N mice treated with 500 μg TP/kg and 0.1 mg LPS/kg to induce fulminant hepatitis. Primary hepatocytes pretreated with TP were more prone to necroptosis when exposed to recombinant murine IFN-γ. In mice administered TP and LPS, the intracellular IFN-γ levels of NK cells increased significantly. Subsequent study confirmed that NK cells were activated and resulted in potent hepatocellular toxicity. In vivo and in vitro TP administration significantly inhibited MHC class I molecules in murine hepatocytes. An in vitro analysis demonstrated the susceptibility of TP-pretreated hepatocytes to NK-cell-mediated cytotoxicity, an effect that was significantly attenuated by the induction of hepatocyte MHC-I molecules by IFN-α. In vivo induction or overexpression of hepatocyte MHC-I also protected mouse liver against TP and LPS-induced injury. ConclusionThe TP-induced inhibition of hepatocyte MHC-I molecules expression leads to hepatic intolerance to exogenous LPS and NK-cell mediated cytotoxicity against self-hepatocytes. These findings shed light on the toxicity of traditional Chinese medicines administered for their immunomodulatory effects

Integrated bioinformatics analysis of IFITM1 as a prognostic biomarker and investigation of its immunological role in prostate adenocarcinoma.

Prostate adenocarcinoma (PRAD) is a highly aggressive malignancy with high mortality and poor prognosis, and its potential mechanism remains unclear. Our study aimed to identify novel markers for the prognosis of PRAD using bioinformatics technology. The GSE32571 dataset was downloaded from the GEO database, and analyzed via the limma R package to identify differentially expressed genes (DEGs) and differentially expressed immune score-related genes (DEISRGs). The immune-related genes (IRGs) were further obtained by overlapping DEISRGs and DEGs, and the core gene was identified via survival analysis. Furthermore, the expression level, prognostic value, and potential functions of the core gene were evaluated via multiple bioinformatics databases. A total of 301 IRGs were identified from the GSE32571 dataset, and IFITM1 was a down-regulated gene in several types of cancer, including PRAD. Besides, low expression of IFITM1 was associated with a poor prognosis in PRAD. GSEA indicated that the vital pathways of IFITM1-associated genes were mainly enriched in primary immunodeficiency, Th17 cell differentiation, Th1, and Th2 cell differentiation, natural killer cell-mediated cytotoxicity, myeloid dendritic cell activation, regulation of leukocyte activation, etc. Furthermore, IFITM1 was closely correlated with 22 types of tumor-infiltrating immune cells. IFITM1 was a prognostic biomarker for PRAD patients, and it can be acted as a potential immune therapy target in PRAD.

Upregulated antimicrobial immune response mediated by neutrophils in the development from allergic rhinitis to asthma.

Allergic rhinitis (AR) and asthma are closely related, and AR is regarded as an important risk factor for the onset of asthma. However, the pathogenesis of the development of asthma from AR is still undefined. The aim of this study was to investigate the mechanisms underlying the development of asthma from AR by comparing the transcriptome features of patients with AR with and without asthma. Patients with AR with or without asthma caused by weed pollen who presented to the Allergy Clinic of Peking Union Medical College Hospital were recruited for this study. Peripheral blood samples of all the patients were collected during the weed pollen season (September) when the patients had allergic symptoms and outside the pollen season (November) when the patients had no symptoms. Transcriptomic analysis was conducted, and the differentially expressed genes (DEGs) and enriched immune pathways between the patients with AR with asthma (AR-asthma group) and those without asthma (AR group) were identified. In addition, the expression levels of some pivotal differentially expressed RNAs were quantified using quantitative polymerase chain reaction (PCR). During the weed pollen season, the immune-related Gene Ontology (GO) terms with P value < 0.05, enriched by the upregulated genes in the AR-asthma group compared to the AR group included antifungal humoral response, neutrophil-mediated killing of bacterium, antibacterial humoral response, antimicrobial humoral immune response mediated by antimicrobial peptides, and regulation of the T cell receptor signaling pathway. The immune-related GO terms with P values <0.05 enriched by downregulated genes were positive regulation of natural killer cell-mediated cytotoxicity, microglial cell activation, natural killer cell activation, and leukocyte-mediated cytotoxicity. The GO term of antimicrobial humoral immune response mediated by antimicrobial peptides was upregulated both during and outside the pollen season, and the upregulated expression of three DEGs (LTF, PF4, and ELANE) included in this term was verified through quantitative PCR. The activation of the antimicrobial immune response mediated by neutrophils and the depression of cytotoxicity mediated by natural killer cells may play roles in the progression from AR to asthma.

Mebendazole Increases Anticancer Activity of Radiotherapy in Radiotherapy-Resistant Triple-Negative Breast Cancer Cells by Enhancing Natural Killer Cell-Mediated Cytotoxicity

Breast cancer is the most commonly diagnosed cancer worldwide and ranks first in terms of both prevalence and cancer-related mortality in women. In this study, we aimed to evaluate the anticancer effect of mebendazole (MBZ) and radiotherapy (RT) concomitant use in triple-negative breast cancer (TNBC) cells and elucidate the underlying mechanisms of action. Breast cancer mouse models and several types of breast cancer cells, including TNBC-derived RT-resistant (RT-R) MDA-MB-231 cells, were treated with MBZ and/or RT. In mice, changes in body weight, renal and liver toxicity, tumor volume, and number of lung metastases were determined. In cells, cell viability, colony formation, scratch wound healing, Matrigel invasion, and protein expression using western blotting were determined. Our findings showed that MBZ and RT combined treatment increased the anticancer effect of RT without additional toxicity. In addition, we noted that cyclin B1, PH2AX, and natural killer (NK) cell-mediated cytotoxicity increased following MBZ + RT treatment compared to unaided RT. Our results suggest that MBZ + RT have an enhanced anticancer effect in TNBC which acquires radiation resistance through blocking cell cycle progression, initiating DNA double-strand breaks, and promoting NK cell-mediated cytotoxicity.

PP4 inhibition sensitizes ovarian cancer to NK cell-mediated cytotoxicity via STAT1 activation and inflammatory signaling

BackgroundIncreased infiltration of T cells into ovarian tumors has been repeatedly shown to be predictive of enhanced patient survival. However, despite the evidence of an active immune response in ovarian...

PP 4.7 – 00097 Signatures of HIV-Infected CD4+ T Cell Resistance to NK Cell-Mediated Cytotoxicity

PP 4.7 – 00097 Signatures of HIV-Infected CD4+ T Cell Resistance to NK Cell-Mediated Cytotoxicity

CircRNF10 Regulates Tumorigenic Properties and Natural Killer Cell-Mediated Cytotoxicity against Breast Cancer through the miR-934/PTEN/PI3k-Akt Axis

Circular RNA (circRNA), a type of non-coding RNA, has received a great deal of attention with regard to the initiation and progression of tumors. However, the molecular mechanism and function of circRNAs in breast cancer (BC) remain unclear. In the current study, we discovered that hsa_circ_0028899 (also called circRNF10) was significantly reduced in BC tissues, and a higher level of circRNF10 was markedly related to a favorable prognosis. The results of CCK8, colony formation, Transwell, ELISA, and NK cell-mediated cytotoxicity assays indicated that increased circRNF10 expression could significantly repress the proliferation, invasion, and migration of BC cells and enhance the killing efficiency of NK cells against BC cells. According to these biological functions, the possible role and molecular mechanism of circRNF10 in BC cells were further investigated. We used bioinformatics prediction tools to predict circRNF10-bound miRNAs, which were verified by many experimental studies, including FISH, luciferase reporter assays, RIP, and Western blots. These data suggest that circRNF10 serves as a molecular sponge for miR-934 to further regulate PTEN expression and PI3k/Akt/MICA signaling in vitro and tumor growth in vivo. Altogether, these findings reveal that circRNF10 functions as a novel anti-oncogene in BC via sponging miR-934 and suppressing the PI3K/Akt/MICA pathway.

Identification of two robust subclasses of sepsis with both prognostic and therapeutic values based on machine learning analysis.

Sepsis is a heterogeneous syndrome with high morbidity and mortality. Optimal and effective classifications are in urgent need and to be developed. A total of 1,936 patients (sepsis samples, n=1,692; normal samples, n=244) in 7 discovery datasets were included to conduct weighted gene co-expression network analysis (WGCNA) to filter out candidate genes related to sepsis. Then, two subtypes of sepsis were classified in the training sepsis set (n=1,692), the Adaptive and Inflammatory, using K-means clustering analysis on 90 sepsis-related features. We validated these subtypes using 617 samples in 5 independent datasets and the merged 5 sets. Cibersort method revealed the Adaptive subtype was related to high infiltration levels of T cells and natural killer (NK) cells and a better clinical outcome. Immune features were validated by single-cell RNA sequencing (scRNA-seq) analysis. The Inflammatory subtype was associated with high infiltration of macrophages and a disadvantageous prognosis. Based on functional analysis, upregulation of the Toll-like receptor signaling pathway was obtained in Inflammatory subtype and NK cell-mediated cytotoxicity and T cell receptor signaling pathway were upregulated in Adaptive group. To quantify the cluster findings, a scoring system, called, risk score, was established using four datasets (n=980) in the discovery cohorts based on least absolute shrinkage and selection operator (LASSO) and logistic regression and validated in external sets (n=760). Multivariate logistic regression analysis revealed the risk score was an independent predictor of outcomes of sepsis patients (OR [odds ratio], 2.752, 95% confidence interval [CI], 2.234-3.389, P<0.001), when adjusted by age and gender. In addition, the validation sets confirmed the performance (OR, 1.638, 95% CI, 1.309-2.048, P<0.001). Finally, nomograms demonstrated great discriminatory potential than that of risk score, age and gender (training set: AUC=0.682, 95% CI, 0.643-0.719; validation set: AUC=0.624, 95% CI, 0.576-0.664). Decision curve analysis (DCA) demonstrated that the nomograms were clinically useful and had better discriminative performance to recognize patients at high risk than the age, gender and risk score, respectively. In-depth analysis of a comprehensive landscape of the transcriptome characteristics of sepsis might contribute to personalized treatments and prediction of clinical outcomes.

Sleep disturbance is associated with perturbations in immune-inflammatory pathways in oncology outpatients undergoing chemotherapy

Objective/backgroundSleep disturbance is a common problem in patients receiving chemotherapy. Purpose was to evaluate for perturbations in immune-inflammatory pathways between oncology patients with low versus very high levels of sleep disturbance. Patients/methodsSleep disturbance was evaluated using the General Sleep Disturbance Scale six times over two cycles of chemotherapy. Latent profile analysis was used to identify subgroups of patients with distinct sleep disturbance profiles. Pathway impact analyses were performed in two independent samples using gene expression data obtained from RNA sequencing (n = 198) and microarray (n = 162) technologies. Fisher's combined probability test was used to identify significantly perturbed pathways between Low versus Very High sleep disturbance classes. ResultsIn the RNA sequencing and microarray samples, 59.1% and 51.9% of patients were in the Very High sleep disturbance class, respectively. Thirteen perturbed pathways were related to immune-inflammatory mechanisms (i.e., endocytosis, phagosome, antigen processing and presentation, natural killer cell mediated cytotoxicity, cytokine-cytokine receptor interaction, apoptosis, neutrophil extracellular trap formation, nucleotide-binding and oligomerization domain-like receptor signaling, Th17 cell differentiation, intestinal immune network for immunoglobulin A production, T-cell receptor signaling, complement and coagulation cascades, and tumor necrosis factor signaling). ConclusionsFirst study to identify perturbations in immune-inflammatory pathways associated with very high levels of sleep disturbance in oncology outpatients. Findings suggest that complex immune-inflammatory interactions underlie sleep disturbance.

Cryo-EM reveals the conformational epitope of human monoclonal antibody PAM1.4 broadly reacting with polymorphic malarial protein VAR2CSA.

Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4's broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria.

Single-Cell Transcriptomic Analysis Reveals Reduction of Cytotoxic NK Cells in a Subset of Newly Diagnosed Multiple Myeloma Patients Impacting Outcome after Daratumumab Therapy

Introduction Anti-CD38 antibody-based therapies in multiple myeloma (MM) rely to a large degree on natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Classically, NK cells are divided into cytotoxic CD56dim NK cells, important for ADCC, and cytokine-producing CD56bright NK cells. Accumulating evidence in other types of cancer suggests that alterations in the balance between CD56dim and CD56bright NK cells is associated with inferior clinical outcome. We hypothesized that changes in NK cell subset composition in the MM microenvironment would impact therapy-responses during NK cell-driven immunotherapies. Here, we used single-cell RNA sequencing and flow cytometry to investigate the heterogeneity of the NK cell compartment in the bone marrow (BM) of newly diagnosed MM (NDMM) patients, and the effects of altered NK cell subset composition on therapy response in patients undergoing first-line daratumumab-containing therapy. Results We performed single-cell RNA sequencing of the BM CD38+ immune cell compartment from 19 NDMM patients. NK cells were identified in silico resulting in a single-cell transcriptomic dataset of 24,664 NK cells. UMAP dimensionality reduction identified 5 transcriptomic distinct NK cell clusters, present in all NDMM patients. Conventional CD56bright and CD56dim NK cells were identified by increased transcription of GZMK or GZMB, respectively. To study the balance of cytotoxic vs cytokine-producing NK cells in MM we compared the relative abundance of cytokine-producing GZMK+CD56bright NK cells vs cytotoxic GZMB+CD56dim NK cells in individual patients in our cohort. The MM BM was characterized by heterogeneity in the ratio of cytotoxic vs cytokine-producing NK cell cluster composition, in which most patients had a predominance of cytotoxic NK cells, while a subset of patients presented with a relative decrease in cytotoxic NK cell clusters (Figure 1A). The reduction in cytotoxic NK cell subsets in this patient subgroup was reflected in an altered overall transcriptome of the total BM NK cell population, with enrichment for inhibitory receptors such as KLRB1 and KLRC1, and a relative loss of activating receptors such as FCGR3A (CD16), NCR3 (NKp30) and CD226 (DNAM-1), and cytotoxic effector genes including NKG7 and GNLY. To define the relative abundance of a reduced cytotoxic NK cell potential in NDMM patients, we performed flow cytometric analyses of 246 NDMM patients from 2 independent clinical cohorts. This revealed a reduced frequency of cytotoxic CD56dim NK cells, defined as < 90% CD56dim NK cells, in 17.9 % of 162 transplant-eligible treatment-naïve NDMM and in 28.6% of 84 treatment-naïve frail or unfit transplant ineligible MM patients. To test whether such relative decrease in cytotoxic NK cells would impact responses to daratumumab, we tested the association of BM NK cell composition with clinical outcome in the context of first-line daratumumab therapy in a cohort of frail NDMM patients ineligible for transplant from the HO143 trial (n=43). NDMM patients with reduced frequencies of BM cytotoxic NK cells presented with significantly shorter progression-free survival (PFS) (Figure 1B, PFS, p = 0.031, log-rank test), while OS was not impacted (p = 0.18, log-rank test). Univariate Cox regression analyses confirmed the correlation between reduced frequencies of CD56dim NK cells in the BM and significantly shorter PFS (hazard ratio (HR) 1.38; 95% confidence interval: 1.057 - 1.803; P = 0.018). Whether reduced frequencies of cytotoxic CD56dim NK cells affect outcome in transplant-eligible patients warrants additional study. Conclusion Here we show that about 20% of NDMM patients have an altered BM NK cell composition with a relative reduction in cytotoxic NK cells. Such altered NK cell composition correlates with significantly shorter PFS following daratumumab-containing therapy in a cohort of frail NDMM patients. Our data suggest that defining BM NK cell composition could stratify patients and identify a subgroup less likely to benefit from therapies driven by NK cell-mediated ADCC. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibition of MICA and MICB Shedding Enhances Cytokine-Induced Memory-like NK Cell-Mediated Activity Against Multiple Myeloma

Introduction Natural Killer (NK) cells are important mediators of cytotoxic immune responses against MM, and the efficacy of existing MM therapies relies substantially on NK cell function. However, MM cells develop mechanisms to escape NK cell recognition by loss of ligands for activating receptors and by inducing functional defects. To overcome reduced NK cell functionality, adoptive transfer of cytokine-induced memory-like (CIML) NK cells are of particular interest, as these cells acquire memory-like features resulting in enhanced activity upon restimulation, greater IFN-γ response, and greater anti-tumor cytotoxicity. The success of adoptive transfer of CIML NK cells in MM is dependent on the ability of NK cells to recognize malignant plasma cells (PCs). NKG2D, one of the major activating receptors on NK cells, is upregulated on CIML NK cells. NKG2D plays an important role in the immune recognition of malignant PCs through interaction with its ligands MICA and MICB. However, MM cells evade this recognition by proteolytic shedding of MICA/B from the cell surface, leading to NKG2D internalization. Rationally designed antibodies like the 7C6 monoclonal antibody (mAb) targeting the proteolytic MICA α3 domain stabilize cell surface MICA/B expression and can enhance NK cell recognition and susceptibility to NK cell-mediated cytotoxicity. Furthermore, it has been shown that Bortezomib, an FDA-approved proteasome inhibitor with potent anti-MM activity, augments MICA expression in MM. We hypothesized that use of the 7C6 mAb would allow simultaneous blockade of MICA/B shedding and ADCC by CIML NK cells resulting in enhanced targeting of MM cells, which would further synergize with Bortezomib treatment. Results Flowcytometric analysis of surface MICA/B levels on 10 human myeloma cell lines (HMCLs) demonstrated heterogeneous protein expression. MICA/B transcript levels for corresponding HMCLs were queried from the CCLE database. There was no correlation between transcript and protein expression, suggesting substantial post-translational modifications, such as proteolytic cleavage, determining surface expression levels of MICA/B in MM. To determine if proteolytic cleavage occurs in MM, we treated HMCL with the 7C6 mAb, targeting the proteolytic cleavage site. Flowcytometric analysis of MM.1S revealed that 7C6 was able to upregulate basal expression of MICA/B in a dose-dependent manner as well as inhibit MICA/B shedding. These results suggest proteolytic cleavage of MICA/B occurs in MM and that this process can be inhibited by a MICA/B stabilizing antibody. Previously, it has been shown that MICA/B levels can be upregulated in MM by Bortezomib. As heterogeneity in MICA/B expression levels was seen among HMCLs, we hypothesized that the use of 7C6 would synergize with Bortezomib. Indeed, the addition of Bortezomib to 7C6 substantially increased MICA/B surface expression. To assess the functional consequences of MICA/B stabilization, we examined the effects on NK cell activation in co-culture assays of NK cells with MM.1S pretreated with 7C6 or IC mAb. We observed enhanced NK cell function as assessed by increased CD107a degranulation, IFN-γ and TNF-α production in co-culture assays of conventional NK cells with MM.1S pre-treated with 7C6 vs IC. Next, we measured the activity of CIML NK cells to myeloma targets. CIML NK cells exhibited enhanced CD107a in response to MM targets when compared to control NK cells from the same donors (Figure 1A). Furthermore, CIML NK cells demonstrated significantly higher cytotoxicity against 7C6 pretreated MM.1S, which was dependent on increased MICA/B and NKG2D levels. CIML NK cells in NSG mice treated with 7C6-hIgG1 mAb were significantly more effective in controlling early MM tumor cell outgrowth compared to CIML NK cells in mice treated with isotype control antibodies (Figure 1B). Conclusion Here we demonstrate that inhibition of MICA/B shedding with a novel antibody 7C6 enhances CIML NK cell-mediated function against MM in vitro and in a xenograft mouse model. Preliminary data demonstrate that this treatment strategy could synergize with current treatment regimens including Bortezomib. Our data support the hypothesis that combination strategies to enhance NK cell activation can be effective to enhance NK cell-mediated immune surveillance in MM. This combined approach highlights their potential use as novel immunotherapy in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abatacept As Salvage Therapy for Graft-Versus-Host Disease in Allogenic Hematopoietic Stem Cell Transplantation : A Systematic Review

Introduction Graft vs host disease (GVHD) is one of the major complications after allogenic - Hematopoietic stem cell transplantation (allo-HSCT). It has been reported in around 70 % of patients after allo-HSCT and leads to increased non-relapse mortality (NRM)/transplant-related mortality (TRM) and also contributes to impaired quality of life. Cytotoxic T-lymphocyte-associated protein 4 (CTLA4), expressed on the surface of regulatory T cells and activated conventional T cells play a role in inhibiting T cell activation by modulating the costimulatory CD28 signal. The CTLA4-Fc fusion proteins abatacept and belatacept lead to immunosuppression induced by blocking CD28-costimulation. CTLA4Ig (Abatacept) has recently been approved by US Food and Drug Administration for prophylaxis of acute GVHD following mismatched unrelated donor (URD) HSCT. Methods We performed a comprehensive literature search on PubMed and ClinicalTrials.gov. We used the MeSH terms for Abatacept and Stem Cell Transplantation and their associated keywords. The search yielded 153 articles. Screening and data extraction were done according to PRISMA guidelines. After excluding case reports, review articles, 4 clinical studies (n=236) were included. Results: The total number of patients tested in the Abatacept group was 193. (Table 1.) In a retrospective trial by Wertheimer T et al., patients with severe cGVHD receiving abataceptdid not need new immunosuppressive treatments (80%) within 3 months of starting abatacept therapy. Patients with lung involvement (n = 9) showed significant benefit with an overall response rate of 89% based on lung severity score (n = 6), and lung function as measured by FEV1. In the phase II trial by Watkins et al addition of T-cell co-stimulation blockade with abatacept to calcineurin inhibitor (CNI)/methotrexate (MTX)-based GVHD prophylaxis, lead to reduced aGVHD, and improved day +180 severe-aGVHD-free-survival. Incidence of grade 3-4 aGVHD in 8/8-HLA-matched URD, was 6.8% (abatacept) versus 14.8% (placebo) (P = .13, hazard ratio = 0.45). Severe-aGVHD-free-survival (SGFS) was 93.2% (CNI/MTX + abatacept) versus 82% (CNI/MTX + placebo, P = .05). In the smaller 7/8-HLA-mismatched URD cohort, grade 3-4 aGVHD was 2.3% (CNI/MTX + abatacept), which compared favorably with a nonrandomized matched cohort of CNI/MTX (30.2%, P < .001), with improved SGFS (97.7% v 58.7%, P < .001). In retrospective study by Khandelwal, P, et al, addition of Abatacept to post-transplantation cyclophosphamide (PTCy) based haploidentical transplantation for patients with thalassemia major (TM, n=5) and sickle cell disease (SCD, n=5), showed that none of the patients in the abatacept group, had grades III-IV aGVHD by day +100 compared with 50% in the group with standard aGVHD prophylaxis (P = 0.001). Thalassemia-free survival after HSCT was 100% in the patients who received abatacept compared to 62.5% in the cohort with standard aGVHD prophylaxis at follow-up (P = 0.007) .In another study by Jaiswal, et al, patients with refractory aggressive B-cell lymphoma (R-ABCL) following autologous HSCT received CTLA4Ig-based reduced-intensity conditioning and sequential CTLA4Ig-primed donor lymphocyte infusions (DLIs) on days +7, +21, and +35. aGVHD was not seen in any patient and cGVHD was seen in 2 patients. 3 patients had disease-progression at 100 days, progression-free and GVHD-free survival at 2 years was 75%. Conclusion: This systematic review shows that abatacept could be a promising treatment option for GvHD after HSCT. Abatacept is a novel treatment modality to markedly reduce GVHD and infections and yet augment NK cell-mediated cytotoxicity following HSCT. The addition of abatacept to routine GVHD prophylaxis reduced the incidence of GVHD without impacting engraftment or survival. Additional randomized clinical trials are needed to establish the efficacy and safety of to prevent GVHD after HSCT with a larger number of patients to confirm these results.

The Novel Trifunctional Anti-BCMA NK Cell Engager SAR'514 Has Potent in-Vitro and in-Vivo Anti-Myeloma Effect through Dual NK Cell Engagement

Multiple Myeloma (MM) is the second most prevalent hematopoietic malignancy, representing 10% of total blood cancers. Despite the emergence of new therapies in the recent years, it is still an incurable disease, with a 5-year overall survival of 56% in patients (https://cancerstatisticscenter.cancer.org/). Thus, innovative approaches that would be safe, potent and with long lasting beneficial effects are required. BCMA (B Cell Maturation Antigen) is a cell surface receptor highly and selectively expressed on normal and malignant plasma cells and which function is to promote cell proliferation and survival upon binding of its ligands APRIL (A PRoliferation Inducing Ligand) and BAFF (B cell Activating Factor). BCMA has a very high prevalence among MM patients and its expression on tumor cells is maintained after treatments with standard of care such as anti-CD38 therapies, or even BCMA-targeting agents (Cohen AD et al. J Clin Invest 2019). The ability of NK cells to intrinsically kill tumor cells, leaving healthy cells unharmed, with minimal pro-inflammatory cytokine release induction as compared to T cell-based therapies makes NK cells ideal immune cells for a safe and efficacious therapeutic approach. We developed a NK Cell Engager (NKCE) SAR'514 that activates NK cells through a dual engagement of NKp46 and CD16a, two major NK cell activating receptors highly expressed on NK cells in MM patients, and which redirects the activated NK cells to engage and kill BCMA+ tumor cells. The trifunctional NK cell engager molecule SAR'514 was designed to specifically and efficiently activate NK cells to induce potent cytotoxic activity against BCMA+ MM cells. SAR'514 exhibits high affinity for the 3 targets BCMA, CD16 and NKp46. The ability of SAR'514 to induce NK cell-mediated cytotoxicity in vitro was tested against a panel of MM cell lines with low, medium and high BCMA expression levels and compared to an afucosylated reference compound. SAR'514 exhibited a strong and similar potency against all tested cell lines and a better potency compared to this comparator (EC50 0.4 pM vs 7.8 pM) demonstrating the superiority of a dual targeting NK cell engager molecule as compared to an antibody approach. An optimized NK cell activation was obtained with a dual engagement of both NKp46 and CD16a compared to single CD16a or NKp46 engagement or the combination of two molecules engaging CD16a or NKp46. Binding of SAR'514 on NK cells promoted a strong NK cell activation and induced cytotoxic activity only in the presence of MM target cells with no fratricide effect on NK cells. This antitumor activity is associated with very low IL-1b, IL-6, TNFa and IFNg cytokine release (in the low pg/mL range) in human PBMC and whole blood settings in the presence of BCMA+ tumor target cells, and a good safety profile. In addition, the in vivo anti-tumor activity of an anti-murine NKp46 surrogate NK cell engager molecule at 0.05, 0.5 and 5 mg/kg was investigated versus a control isotype in huFcgR transgenic mice engrafted with the EL4-huBCMA murine thymoma model. As compared to the control group in which only 20% of mice survived with a median survival day of 44.5 days, SAR'514 induced a significant mouse survival from 0.5 mg/kg to 5 mg/kg with an overall survival of 90% and a median survival day over 90 days. In summary, these results demonstrate the efficacy of SAR'514 for controlling MM tumors in vivo and provide consistent support for its clinical development.

Perturbations in common and distinct inflammatory pathways associated with morning and evening fatigue in outpatients receiving chemotherapy.

Moderate to severe fatigue occurs in up to 94% of patients with cancer. Recent evidence suggests that morning and evening fatigue are distinct dimensions of physical fatigue. The purposes of this study were to evaluate the transcriptome for common and distinct perturbed inflammatory pathways in patients receiving chemotherapy who reported low versus high levels of morning or low versus high levels of evening cancer-related fatigue. Patients completed questionnaires during the week prior to their chemotherapy treatment. Severity of morning and evening fatigue was evaluated using the Lee Fatigue Scale. Gene expression and pathway impact analyses (PIA) were performed in two independent samples using RNA-sequencing (n=357) and microarray (n=360). Patterns of interactions between and among these perturbed pathways were evaluated using a knowledge network (KN). Across the PIA, nine perturbed pathways (FDR < 0.025) were common to both morning and evening fatigue, six were distinct for morning fatigue, and four were distinct for evening fatigue. KN (19 nodes, 39 edges) identified the phosphatidylinositol 3-kinase (PI3K)-Akt pathway node (perturbed in evening fatigue) with the highest betweenness (0.255) and closeness (0.255) centrality indices. The next highest betweenness centrality indices were seen in pathways perturbed in evening fatigue (i.e., nuclear factor kappa B: 0.200, natural killer cell-mediated cytotoxicity: 0.178, mitogen-activated protein kinase: 0.175). This study describes perturbations in common and distinct inflammatory pathways associated with morning and/or evening fatigue. PI3K-Akt was identified as a bottleneck pathway. The analysis identified potential targets for therapeutic interventions for this common and devastating clinical problem.

Cardiovascular adverse events in chronic myeloid leukemia patients treated with nilotinib or imatinib: A systematic review, meta-analysis and integrative bioinformatics analysis

The aim of this article is to assess the risk and potential mechanisms of cardiovascular adverse events in patients treated with nilotinib or imatinib by conducting a systematic review, meta-analysis and integrative bioinformatics analysis. Three databases were systematically searched for studies published from inception to May 29, 2022. Differential expression analysis and weighted gene coexpression network analysis (WGCNA) were performed to search for modules of genes most associated with cardiotoxicity. Protein-protein interaction (PPI) network analysis was then performed to identify hub genes for the cardiotoxicity of nilotinib. Molecular docking was used to analyze the effects of rosuvastatin and aspirin on these targets. Patients treated with nilotinib as first-line treatment were associated with a higher risk of CAE (OR = 3.43 [95% CI 2.77-4.25]), CAD (OR = 5.30 [95% CI 3.85-7.29]), ACS (OR 2.7 [95% CI 1.60-4.54]), CVA (OR 5.76 [95% CI 2.84-11.28]), PAOD (OR 5.57 [95% CI 3.26-9.50]) and arrhythmia (OR 2.34 [1.17,4.67]) than those treated with imatinib, while no significant difference was found in the risk of HF (OR 1.40 [95% CI 0.42-4.69]) between the two groups. Patients who were treated with more than 600 mg daily dosage of nilotinib or followed up for more than 5 years had a higher risk of ACS and CVA. IL6, CXCL8, CCL2, SOD2, NFKBIA, and BIRC3 were identified as the top 6 hub genes in the magenta module (human cardiomyocyte samples) and were mainly enriched in the NOD-like receptor signaling pathway, IL-17 signaling pathway, TNF signaling pathway, lipid and atherosclerosis signaling pathway. TYROBP and CSF1R were identified as hub genes in the turquoise module (liver samples from Mus musculus). GSEA results showed that type II diabetes mellitus, B-cell receptor, apoptosis, insulin, natural killer cell mediated cytotoxicity,mTOR, chemokine, and T-cell receptor signaling pathways were related to the higher risk of atherosclerosis caused by nilotinib. Rosuvastatin can effectively bind to most of the hub targets and proteins enriched in the inflammatory pathways above. CML patients who start with nilotinib have a higher risk of CAE than those with imatinib. Atherosclerosis caused by the inflammatory response and glycolipid metabolism disorder is the key mechanism of nilotinib cardiotoxicity. Rosuvastatin may be an effective treatment for the cardiotoxicity of nilotinib.

Genome-wide association study of red skin color in leopard coral grouper (Plectropomus leopardus) based on genome resequencing

Leopard coral grouper (Plectropomus leopardus) is an economically important grouper species which is popular in markets because of its red-skin color. However, currently, a large proportion of individuals in the artificial breeding of P. leopardus exhibit non-red skin colors, which increases breeding costs by reducing efficiency and restricts the development of the industry. To identify candidate genes and SNPs associated with the red-skin trait, this study collected 139 P. leopardus individuals from three farms (LED, QHH, and QHL), and performed a genome-wide association study (GWAS) based on whole-genome resequencing. The results identified 10 SNPs that are potentially associated with the red-skin color traits in P. leopardus, and a total of 35 potential genes were annotated within 50 kb of these SNPs. KEGG pathway analysis using these 35 candidate genes showed that natural killer cell mediated cytotoxicity, Ras signaling, MAPK signaling, and other pathways were significantly enriched (p < 0.05). Subsequently, GWAS was used for populations of LED, QHH, and QHL respectively, which showed that 7 genes (MIC13, SPIN1, PRS57, FLOT1, TARS, DHI1L, and FGF10) of the above 35 genes were shared in QHH populations. These results will greatly improve the P. leopardus industry by helping understand the molecular mechanisms underlying red-skin color traits. Furthermore, the identified SNPs can be used as markers to aid targeted breeding for skin color in P. leopardus.

Metformin increases natural killer cell functions in head and neck squamous cell carcinoma through CXCL1 inhibition

BackgroundMetformin slows tumor growth and progression in vitro, and in combination with chemoradiotherapy, resulted in high overall survival in patients with head and neck cancer squamous cell carcinoma (HNSCC) in...