Natural Killer Cell-mediated Cytotoxicity Research Articles

Genomic Basis for Individual Differences in Susceptibility to the Neurotoxic Effects of Diesel Exhaust.

Air pollution is a known environmental health hazard. A major source of air pollution includes diesel exhaust (DE). Initially, research on DE focused on respiratory morbidities; however, more recently, exposures to DE have been associated with neurological developmental disorders and neurodegeneration. In this study, we investigated the effects of sub-chronic inhalation exposure to DE on neuroinflammatory markers in two inbred mouse strains and both sexes, including whole transcriptome examination of the medial prefrontal cortex. We exposed aged male and female C57BL/6J (B6) and DBA/2J (D2) mice to DE, which was cooled and diluted with HEPA-filtered compressed air for 2 h per day, 5 days a week, for 4 weeks. Control animals were exposed to HEPA-filtered air on the same schedule as DE-exposed animals. The prefrontal cortex was harvested and analyzed for proinflammatory cytokine gene expression (Il1β, Il6, Tnfα) and transcriptome-wide response by RNA-seq. We observed differential cytokine gene expression between strains and sexes in the DE-exposed vs. control-exposed groups for Il1β, Tnfα, and Il6. For RNA-seq, we identified 150 differentially expressed genes between air and DE treatment related to natural killer cell-mediated cytotoxicity per Kyoto Encyclopedia of Genes and Genomes pathways. Overall, our data show differential strain-related effects of DE on neuroinflammation and neurotoxicity and demonstrate that B6 are more susceptible than D2 to gene expression changes due to DE exposures than D2. These results are important because B6 mice are often used as the default mouse model for DE studies and strain-related effects of DE neurotoxicity warrant expanded studies.

Co-expression of HLA-E and HLA-G in Porcine Endothelial Cells Attenuates Human Natural Killer Cell-Mediated Cytotoxicity

INTRODUCTION: Pig-to-human xenotransplantation (XTx) is a promising solution to the organ shortage. Genetically-modified (GM) pigs lacking major xenoantigens have reduced hyperacute rejection and prolonged xenograft survival. Despite these advancements, acute rejection remains a barrier to clinical XTx. Natural killer cells (NK) play a crucial role in transplantation. In the context of XTx, suppressing human NK activity by expressing HLA class I molecules in pig organs may protect xenografts from immune attack. Therefore, we aimed to co-express HLA class I molecules in GM pig endothelial cell line null for 5 key xenoantigens (5GKO) and to evaluate human NK response to 5GKO+HLA class I+ cells. METHODS: DNA sequences encoding HLA-E and HLA-G were designed, synthesized, and cloned into expression vectors. Three GM porcine cell lines (5GKO.HLA-E, 5GKO.HLA-G, 5GKO.HLA-E+G) were generated by transfection. IL-2–activated human peripheral blood mononuclear cells (PBMCs) from 5 donors were co-cultured with GM porcine cell lines to initiate NK cell–mediated immune responses. NK activation (CD107a expression) was evaluated by flow cytometry. Three experimental replicates were averaged and analyzed by one-way ANOVA and a post hoc Tukey test. RESULTS: We successfully co-expressed HLA-G, HLA-E, and HLA-E+G in 5GKO cells. Co-expression of HLA-E+G in 5GKO cells significantly reduced human NK activation compared with 5GKO, 5GKO.HLA-E, and 5GKO.HLA-G. CONCLUSION: Co-expression of HLA-E and HLA-G in porcine cells significantly reduces NK cell–mediated cytotoxicity and shows promise for incorporation into GM pigs for pig-to-human xenotransplantation.

The expression of cuproptosis-related genes in hepatocellular carcinoma and their relationships with prognosis

BackgroundThe mechanism of cuproptosis has recently been reported in lipoylated proteins of the tricarboxylic acid (TCA) cycle. Besides, the role of copper was previously recognized in cancer progression. We evaluated the prognostic value of cuproptosis-related gene expression in hepatocellular carcinoma (HCC).MethodsRemarkable genes were selected both in differential expression analysis and Kaplan-Meier survival analysis from ninety-six cuproptosis-related genes using The Cancer Genome Atlas (TCGA) database. The relationships between clinical characteristics and gene expression were performed with Wilcoxon signed-rank test, Kruskal-Wallis test, and logistic regression. Clinicopathologic factors correlated with overall survival in HCCs conducting univariate and multivariate Cox regression analysis. Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and Human Protein Atlas (HPA) databases were utilized to verify the results. Furthermore, Gene Set Enrichment Analysis (GSEA) identified the potential key pathways that dominate cuproptosis in HCC.ResultsElevated ATP7A, SLC25A3, SCO2, COA6, TMEM199, ATP6AP1, LIPT1, DLAT, PDHA1, MTF1, ACP1, FDX2, NUBP2, CIAPIN1, ISCA2 and NDOR1 expression, as well as declined AOC1, FDX1, MT-CO1, and ACO1 expression were significantly emerged in HCC tumor tissues and were significantly associated with HCCs poor survival. The expressions of screened cuproptosis-related genes were prominently related to clinical features. GSEA analysis reported many key signaling pathways (such as natural killer cell mediated cytotoxicity, TCA cycle, glutathione metabolism, ATP-binding cassette (ABC) transporters, Notch signaling pathway, ErbB signaling pathway, and metabolism of xenobiotics by cytochrome p450) were differentially enriched in HCCs with varying degrees of cuproptosis-related genes expression.ConclusionsThe twenty cuproptosis-related genes might be utilized as new candidate prognostic biomarkers for HCC.

Analysis of differentially expressed genes in individuals with noninfectious uveitis based on data in the gene expression omnibus database.

Noninfectious uveitis (NIU), an intraocular inflammation caused by immune-mediated reactions to eye antigens, is associated with systemic rheumatism and several autoimmune diseases. However, the mechanisms underlying the pathogenesis of uveitis are poorly understood. Therefore, we aimed to identify differentially expressed genes (DEGs) in individuals with NIU and to explore its etiologies using bioinformatics tools.GSE66936 and GSE18781 datasets from the gene expression omnibus (GEO) database were merged and analyzed. Functional enrichment analysis was performed, and protein-protein interaction (PPI) networks were constructed.A total of 89 DEGs were identified. Gene ontology (GO) enrichment analysis identified 21 enriched gene sets. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis identified four core enriched pathways: antigen processing and expression signaling, natural killer (NK) cell-mediated cytotoxicity signaling, glutathione metabolic signal transduction, and arachidonic acid metabolism pathways. PPI network analysis revealed an active component-target network with 40 nodes and 132 edges, as well as several hub genes, including CD27, LTF, NCR3, SLC4A1, CD69, KLRB1, KIR2DL3, KIR3DL1, and GZMK.The eight potential hub genes may be associated with the risk of developing NIU. NK cell-mediated cytotoxicity signaling might be the key molecular mechanism in the occurrence and development of NIU. Our study provided new insights on NIU, its genetics, molecular pathogenesis and new therapeutic targets.

HLA-B*27 Heavy Chain Homo-Oligomers Promote the Cytotoxicity of NK Cells via Activation of PI3K/AKT Signaling.

Background and Objectives: Ankylosing spondylitis (AS) is a chronic inflammatory disease and is highly linked with the expression of the human leukocytic antigen-B*27 (HLA-B*27) genotype. HLA-B*27 heavy chain (B*27-HC) has an innate characteristic to slowly fold, resulting in the accumulation of the misfolded B*27-HC and the formation of homo-oligomeric B*27-HC molecules. The homo-oligomeric B*27-HC can act as a ligand of KIR3DL2. Interaction of the homo-oligomeric B*27-HC molecules with KIR3DL2 will trigger the survival and activation of KIR3DL2-positive NK cells. However, the effects of homo-oligomeric B*27-HC molecules associated with KIR3DL2 on the cytotoxic activity of NK cells and their cytokine expressions remain unknown. Materials and Methods: HLA-B*-2704-HC was overexpressed in the HMy2.C1R (C1R) cell line. Western blotting and quantitative RT-PCR were used to analyze the protein expression and cytokine expression, respectively, when C1R-B*-2704 cells that overexpress B*2704-HC were co-cultured with NK-92MI cells. Flow cytometry was used to analyze the cytotoxicity mediated by NK-92MI cells. Results: Our results revealed that NK-92MI cells up-regulated the expression of perforin and enhanced the cytotoxic activity via augmentation of PI3K/AKT signaling after co-culturing with C1R-B*2704 cells. Suppression of the dimerized B*27-HC formation or treatment with an inhibitor of PI3K, LY294002, or with an anti-B*27-HC monoclonal antibody can reduce the perforin expression of NK-92MI after co-culturing with C1R-B*-2704. Co-culturing with C1R-B*-2704 cells suppressed the TNF-α and IL6 expressions of NK-92MI cells. Conclusion: Stimulation of NK cell-mediated cytotoxicity by homo-oligomeric B*27-HC molecules may contribute to the pathogenesis of AS.

AML-327 Novel Anti–B7-H3 Blocking Antibody Enhances NK Cell–Mediated Cytotoxicity and Improves Outcomes in AML-Bearing Mice

B7-H3 is an immune checkpoint molecule that is overexpressed in various human malignancies. Several monoclonal antibodies (mAbs) targeting B7-H3 have shown promising results against solid tumors. However, B7-H3's role in AML remains unexplored. i) To analyze the prognostic significance of B7-H3 in AML. ii) To investigate the immunomodulatory role of B7-H3 in AML in vitro and in vivo. We analyzed B7-H3 expression in 100 AML patients and 20 healthy donors by flow cytometry and tested for associations with clinical features. To investigate the role of B7-H3 in immunomodulation, B7-H3 was knocked down in AML cell lines using shRNA or using anti-B7-H3 blocking mAbs (T1-A5, HEK1B3A3, and 58B1A2). A human-mouse chimeric (chT-1A5) antibody was generated, and its binding site on B7-H3 protein was characterized by epitope mapping. Finally, we treated mice bearing AML cells with anti-B7-H3 antibodies and measured human AML engraftment with flow cytometry and bioluminescence imaging. Expression of B7-H3 was significantly higher in AML patients than in healthy donors (p<0.01) and in CD34+ AML cells than in CD34- cells (p<0.01). Clinically, we observed that high B7-H3 expression was associated with a poor prognosis. Furthermore, we observed that inhibition of B7-H3 expression or blocking of its activity in AML cells using a novel monoclonal antibody (T-1A5) significantly enhanced natural killer (NK) cell-mediated cytotoxicity in vitro and in vivo. Moreover, we observed that the human-mouse chimera of this antibody (ChT-1A5) induced dose-dependent cell-mediated cytotoxicity (ADCC) in B7-H3+ primary AML cells but not in normal hematopoietic cells, suggesting the specify of this antibody for AML cells. Epitope mapping using peptides derived from the B7-H3 protein identified the FG loop region as the binding site for the T1-A5 antibody. Finally, treatment with T-1A5 or chT-1A5 in combination with human NK cells significantly reduced leukemia burden and extended survival in AML xenograft and patient-derived xenograft models. Our data indicate that B7-H3 is overexpressed in AML and that an anti-B7-H3 antibody (T1-A5) not only blocks B7-H3's immunomodulatory function but also induces ADCC in AML cells in vitro and in vivo.

IL-2 Combined with IL-15 Enhanced the Expression of NKG2D Receptor on Patient Autologous NK Cells to Inhibit Wilms' Tumor via MAPK Signaling Pathway.

Objective The dysfunction of immune surveillance, a hot spot in cancer research, could lead to the occurrence and development in multicancers. However, the potential mechanisms of immunity in Wilms' tumor (WT) remain unclear on Wilms' tumor (WT). In this study, we aim to investigate the immune cell in WT and explore the underlying treatment strategy. Method We quantified stromal and immune scores by using ESTIMATE algorithm based on gene expression matrix of WT patients in TCGA and GEO databases. Different expression genes (DEGs) and functional enrichments were analyzed by R studio and DAVID tools. Flow cytometry, immunofluorescence staining, ELISA assay, and qRT-PCR were used for detecting the NK cells, cytotoxic cytokines (INF-γ, PRF, and GZMB), and NK cell receptor expression, respectively. WT patient autologous NK cells were stimulated by IL-2 and IL-15, and the cytotoxicity of NK cells against WT cell lines was detected by LDH assay. Western blot experiment was used for measuring the MAPK signaling pathway protein maker in NK cells. Results ESTIMATE indicated that WT tissue had a lower immune score than adjacent kidney tissue. Meanwhile, the low immune score group was associated with poorly outcomes. DEG functional enrichment analysis showed that NK cell-mediated cytotoxicity was significantly different in low and high immune score groups. Although few of proportion of NK cells in WT patients were increased, most of that were significantly lower than normal children. Moreover, the proportion of NK cells and the expression level of INF-γ, PRF, and GZMB in WT tissue were lower than adjacent kidney tissue. Importantly, the NKG2D expression level of NK cells was significantly lower in WT tissue. Furthermore, in vitro, compared with uncultured NK cells, IL-2 and IL-15 could effectively enhance the cytotoxicity of NK cells on killing the WT cell lines. The FACS and WB results showed that the NKG2D and p-PI3K ratio PI3K, MEK1/2, and p-ERK1/2 ratio ERK1/2 were significantly increased in IL-2 and IL15 group compared with uncultured groups. Conclusion The abnormal NK cell-mediated cytotoxicity may cause the occurrence of WT. Costimulation of WT patients autologous NK cells could effectively enhance the antitumor reaction which involved in activation of NKG2D-mediated MAPK signaling pathway.

Immunoglobulin-like transcript 2 blockade restores antitumor immune responses in glioblastoma.

Glioblastoma stands as the most frequent primary brain tumor. Despite the multimodal therapy for glioblastoma patients, the survival rate is very low, highlighting the need for novel therapies that improve patient outcomes. Immune checkpoint blockade strategies are achieving promising results in a myriad of tumors and several studies have reported its efficacy in glioblastoma at a preclinical level. ILT2 is a novel immune checkpoint that exerts an inhibitory effect via the interaction with classical and non-classical HLA class-I molecules. Herein, we report that ILT2 blockade promotes antitumor responses against glioblastoma. In silico and immunohistochemical analyses revealed that the expression of ILT2 and its ligands HLA-A, -B, -C, and -E are highly expressed in patients with glioblastoma. Disruption of ILT2 with blocking monoclonal antibodies increased natural killer cell-mediated IFN-γ production and cytotoxicity against glioblastoma, partially reverting the immunosuppression linked to this malignancy. In addition, co-treatment with temozolomide strengthened the antitumor capacity of anti-ILT2-treated immune cells. Collectively, our results establish the basis for future studies regarding the clinical potential of ILT2 blockade alone or in combination regimens in glioblastoma.

PSVIII-B-15 Potential Involvement of DNA Methylation of First Intron in Transcriptional Regulation During Bovine Subclinical Mastitis Caused by Staphylococcus Aureus

Abstract DNA methylation involvement in the regulation of mammary gland inflammatory defense against pathogens, including Staphylococcus aureus(SA) has been documented. However, the mechanisms are not clear. The possible influence of DNA methylation of first introns (DNA-MeFI) on transcriptional activities during subclinical mastitis was studied by profiling the methylome (whole genome methylation sequencing) and transcriptome (RNA-sequencing) of milk somatic cells from cows(n=15) with SA subclinical mastitis (SACs) and healthy cows (HCs)(n=13), followed by bioinformatics processing with standard tools. The DNA-MeFI was calculated, for each of 19411 genes found as expressed in the samples, by averaging the methylation levels of all CpG sites in the first intron. At genome-wide, the DNA-MeFIs was inversely correlated with gene expression (GE) (Pearson’s r=-0.173, p=1.60×10-122)(Figure 1-A). Similarly, the difference in the DNA-MeFI and GE between SACs and HCs was also inversely correlated (Pearson’s r=-0.149, p=4.25×10-97)(Figure 1-B). Application of machine learning by Gaussian Mixed model (GMM) using scikit-learn in python to the changes of DNA-MeFI and GE between SACs and HCs revealed 2866 outliers (GMM’s p&amp;lt; 0.005) that showed significant changes in the DNA-MeFI and/or GE. 644 genes with &amp;gt;10% difference in DNA-MeFI and |log2FC| &amp;gt;1 in GE were selected and referred to as differentially methylated and expressed genes (DME-genes). 644 DME-genes were significantly enriched in 5 GO terms and 11 KEGG pathways related to diseases and immune functions, including Staphylococcus aureus infection and Natural killer cell mediated cytotoxicity (Table 1). Furthermore, 74.84% DME-genes showed inverse changes in DNA-MeFI and GE. Besides, the DNA-MeFI of 264 DME-genes were found to correlate significantly with their GE (|rho| &amp;gt;0.3, FDR&amp;lt;0.05), including 225 DME-genes(85.23%) with inverse correlations(rho&amp;lt; -0.3, FDR&amp;lt; 0.05). 39 DME-genes with positive correlations were significantly enriched in 21 GO terms mainly related to metabolic processes. In conclusion, the DNA-MeFI possibly participate in the regulation of gene expression during bovine subclinical mastitis caused by SA.

Molecular classification of human papillomavirus-positive cervical cancers based on immune signature enrichment.

BackgroundHuman papillomavirus-positive (HPV+) cervical cancers are highly heterogeneous in clinical and molecular characteristics. Thus, an investigation into their heterogeneous immunological profiles is meaningful in providing both biological and clinical insights into this disease.MethodsBased on the enrichment of 29 immune signatures, we discovered immune subtypes of HPV+ cervical cancers by hierarchical clustering. To explore whether this subtyping method is reproducible, we analyzed three bulk and one single cell transcriptomic datasets. We also compared clinical and molecular characteristics between the immune subtypes.ResultsClustering analysis identified two immune subtypes of HPV+ cervical cancers: Immunity-H and Immunity-L, consistent in the four datasets. In comparisons with Immunity-L, Immunity-H displayed stronger immunity, more stromal contents, lower tumor purity, proliferation potential, intratumor heterogeneity and stemness, higher tumor mutation burden, more neoantigens, lower levels of copy number alterations, lower DNA repair activity, as well as better overall survival prognosis. Certain genes, such as MUC17, PCLO, and GOLGB1, showed significantly higher mutation rates in Immunity-L than in Immunity-H. 16 proteins were significantly upregulated in Immunity-H vs. Immunity-L, including Caspase-7, PREX1, Lck, C-Raf, PI3K-p85, Syk, 14-3-3_epsilon, STAT5-α, GATA3, Src_pY416, NDRG1_pT346, Notch1, PDK1_pS241, Bim, NF-kB-p65_pS536, and p53. Pathway analysis identified numerous immune-related pathways more highly enriched in Immunity-H vs. Immunity-L, including cytokine-cytokine receptor interaction, natural killer cell-mediated cytotoxicity, antigen processing and presentation, T/B cell receptor signaling, chemokine signaling, supporting the stronger antitumor immunity in Immunity-H vs. Immunity-L.ConclusionHPV+ cervical cancers are divided into two subgroups based on their immune signatures' enrichment. Both subgroups have markedly different tumor immunity, progression phenotypes, genomic features, and clinical outcomes. Our data offer novel perception in the tumor biology as well as clinical implications for HPV+ cervical cancer.

Abstract PR002: Antigen presentation and processing pathway is associated with early relapse after neoadjuvant immune checkpoint blockade (ICB) in dedifferentiated liposarcomas (DDLPS)

Abstract Background: We evaluated the activity of neoadjuvant ICB in localized resectable DDLPS (n=17) and undifferentiated pleomorphic sarcomas (UPS; n=10). DDLPS and UPS patients were randomized to neoadjuvant nivolumab or ipilimumab+nivolumab, with UPS patients receiving concurrent radiotherapy. We assessed genomic markers of early relapse (progression before surgery or relapse within 52 weeks following surgery) using longitudinally acquired tumor samples. Methods: RNA sequencing (RNAseq) and whole genome sequencing (WGS) were performed on longitudinally acquired samples (baseline biopsies and surgical specimens). Differential gene expression between any two groups of patients (i.e., non-early relapse [non-relapsers] vs early relapse [relapsers]) were selected (fold change&amp;gt;1.5 and p value&amp;lt;0.05). Gene set enrichment analyses (GSEA) of KEGG pathways were performed and a network-based approach used to identify genes/pathways associated with MHC-I. Results: At a median follow-up of 23 months, 12 patients (9 DDLPS, 3 UPS) relapsed, including 7 early relapses (relapsers: 5 DDLPS, 2 UPS). The median relapse-free survival was 22 months in DDLPS patients (6 months in relapsers; not reached [NR] in non-relapsers) and NR in UPS patients. At baseline, the most differentially upregulated pathways in non-relapsers compared to relapsers were “graft versus host disease” (GSEA Normalized Enrichment Score[NES]=2.25; False Discovery Rate[FDR] q= 0.009), “natural killer cell mediated cytotoxicity” (NES=2.17; FDR q=0.009), “antigen processing and presentation” (NES=2.16; FDR q=0.009), “allograft rejection” (NES=1.99; FDR q=0.019) and “B-cell receptor signaling pathway” (NES=1.87; FDR q=0.018). In DDLPS patients, the antigen presentation and processing pathway was the most upregulated pathway in non-relapsers compared to relapsers (NES=2.01; FDR q=0.025) while it was not significantly upregulated in UPS (NES=1.15; FDR q=0.62). When looking at pathways longitudinally, the antigen presentation and processing pathway was significantly upregulated at surgery compared to baseline in DDLPS. As antigen presentation and processing was significantly upregulated in DDLPS patients and associated with relapse, we looked for expressed neoantigens that may be processed and presented. Using WGS, we detected 5712 rearrangements at baseline in DDLPS, of which 230 were found in more than one tumor specimen. We also sought to identify genes associated with MHC-I. We selected genes upregulated during ICB comparing baseline to surgical specimens in DDLPS relapsers and looked at the top 10% of genes associated with MHC-I in order to identify potential therapeutic targets for combination. We identified 41 genes upregulated during ICB and associated with MHC-I in relapsers, for which up to 275 inhibitory compounds were found in drug databases. Conclusion: Antigen presentation and processing is a major driver of response to immunotherapy. Future efforts should focus on identifying which antigens are presented to find synergizing compounds in order to increase the clinical benefit of ICB. Citation Format: Elise F. Nassif, Chia-Chin Wu, Kadir Akdemir, Russell G. Witt, Raymond Traweek, Brandon Cope, Prapassorn Thirasastr, Taylor Tate, Grace Mathew, Shadarra Crosby, Randy Chu, Mohammad Mohammad, Kenna Shaw, Ingram Davis, Khalida Wani, Alexander J. Lazar, Wei-Lien Wang, Sheila Duncan, Ashleigh B. Guadagnolo, Andrew J. Bishop, Valerae Lewis, Justin E. Bird, Keila E. Torres, Kelly K. Hunt, Barry W. Feig, Christopher P. Scally, Ravin Ratan, Shreyaskumar Patel, Robert S. Benjamin, Robert Satcher, Kevin McBride, Wolf H. Fridman, Ignacio Wistuba, Andrew Futreal, Jennifer A. Wargo, Neeta Somaiah, Christina L. Roland, Emily Z. Keung. Antigen presentation and processing pathway is associated with early relapse after neoadjuvant immune checkpoint blockade (ICB) in dedifferentiated liposarcomas (DDLPS) [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr PR002.

Radiosensitization effect by HDAC inhibition improves NKG2D-dependent natural killer cytotoxicity in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Radiotherapy (RT) controls HCC unsatisfactorily and temporarily. Histone deacetylase inhibitor (HDACi) is a heterogeneous group of epigenetic therapeutics with promising anticancer effects and synergism in combination with RT. HDACi modulates natural killer (NK) cell ligand expression on tumor cells, and leads to immune evasion of cancer cells. Expressions of NK group 2D (NKG2D) ligands on cancer cells determine the cytotoxic effect by interacting with NKG2D receptor on NK cells. However, the role of NKG2D signaling in HCC upon combined RT and HDACi remains unclear.Method In vitro co-culture system with NK cells was tested for human and murine HCC cell lines. Pan-HDACi (panobinostat) and specific HDAC4 knockdown (HDAC4-KD) were used for HDAC inhibition. Clonogenic assay and flow cytometry examined HCC cell survival and NKG2D ligand expression, respectively. Syngeneic mouse model was used to validate the radiosensitizing effect in vivo.ResultsCombined RT and HDACi/HDAC4-KD significantly enhanced NK cell-related cytotoxicity and increased NKG2D ligands, MICA/MICB expressions in human and RAE-1/H60 expressions in murine HCC cells. Delayed tumor growth in vivo by the combinational treatment of RT and HDACi/HDAC4-KD was shown with the associated NKG2D ligand expressions. However, NKG2D receptor did not significantly change among tumors.ConclusionRadiosensitizing effect with combined RT and HDAC inhibition increased the expression of NKG2D ligands in HCC cells and enhanced their susceptibility to NK cell-mediated cytotoxicity. These findings imply the potential use of combined RT/HDACi and NK cell-directed immunotherapy.

Comprehensive Analysis of the Immune Implication of AKAP12 in Stomach Adenocarcinoma.

A kinase anchor protein 12 (AKAP12) as a tumor suppressor in various cancers has been extensively studied and confirmed. However, its immune implication in stomach adenocarcinoma (STAD) remains uncertain. Here, using The Cancer Genome Atlas (TCGA), Human Protein Atlas (HPA), Tumor Immune Estimation Resource (TIMER), Cancer Cell Line Encyclopedia (CCLE), integrated repository portal for tumor-immune system interactions (TISIDB), and Search Tool for the Retrieval of Interaction Gene/Proteins (STRING) database, we systematically analyzed the immune correlation of AKAP12 from three aspects including immune infiltration cells, immune-related pathways, and immunomodulators and developed a AKAP12-related 4-gene signature for prognosis prediction. Our results showed that AKAP12 mRNA and protein levels were downregulated in STAD patients, and its expression was positively related to CD4+ T cells and macrophages. In addition, the immune cell infiltration levels were associated with AKAP12 gene copy number deletion in STAD. Based on CCLE database, we found that AKAP12 coexpressed genes were enriched in several immune- and cancer-related pathways, which was further validated by Gene Set Enrichment Analysis (GSEA). Moreover, we identified 46 immunomodulators that were significantly related to AKAP12 expression using TISIDB database, and these immunomodulators were involved in immune-related pathways including Th17 cell differentiation and natural killer cell-mediated cytotoxicity. Additionally, based on the 46 AKAP12-related immunomodulators, a 4-gene risk prediction signature was developed using the Cox regression model. The risk signature was identified as an independent prognostic factor, which can accurately predict the prognosis of patients with STAD, showing good predictive performance. Furthermore, we constructed a prognostic nomogram and calibration to predict and assess patient survival probabilities by integrating the risk score and other clinical factors. In conclusion, our study provides strong evidence that AKAP12 is closely related to tumor immunity in STAD from three aspects: immune infiltration cells, immune pathways, and immunomodulators. More importantly, the AKAP12-related prognostic signature may have a good application prospect for clinical practice.

Defects in protective cytokine profiles in spontaneous miscarriage in the first trimester

To study differences in cytokine expression profiles between women with ongoing pregnancy and those experiencing spontaneous miscarriage, among women who presented with threatened miscarriage before week 16 of gestation. Prospective cohort study. Academic hospital. In this prospective cohort study, 155 pregnant women, comprising normal pregnant women recruited from antenatal clinics (n = 97) and women with threatened miscarriage recruited from an emergency walk-in clinic (n = 58). None. Sixty-five serum cytokines quantified using multiplex immunoassay correlated with miscarriage outcomes. Among women presenting with threatened miscarriage, those who eventually miscarried had significantly lower levels of interleukin (IL)-2, IL-12p70, IL-17A, B-cell-activating factor, B lymphocyte chemoattractant, basic nerve growth factor, interferon-γ, tumor necrosis factor-related apoptosis-inducing ligand, thymic stromal lymphopoietin, and tumor necrosis factor-α and higher levels of vascular endothelial growth factor A, IL-21, and stromal cell-derived factor 1α than those with ongoing pregnancy. Comparisons between normal pregnancies and women with threatened miscarriage who eventually miscarried revealed significant differences across 7 cytokines: B-cell-activating factor; B lymphocyte chemoattractant; basic nerve growth factor; IL-17A; fractalkine/CX3CL1; vascular endothelial growth factor A; and CCL22. Vascular endothelial growth factor A exhibited a negative correlation with the progesterone level (r = -0.270). The cluster of significant cytokines alludes to T cell proliferation, B-cell proliferation, natural killer cell-mediated cytotoxicity, and apoptosis as important pathways that determine pregnancy outcomes. Bioinformatic analysis further revealed alteration of the suppressor of cytokine signaling proteins family of Janus kinase-signal transducer and activator of transcription signaling axis by cytokines as a plausible key molecular mechanism in spontaneous miscarriage. This study demonstrates that the regulated balance between the proinflammatory and anti-inflammatory pathways is crucial to maintaining pregnancy. A better understanding of the cytokines associated with immunomodulatory effects may lead to novel targets for the prediction and treatment of spontaneous miscarriage.

The Role of N6-Methyladenosine-Associated lncRNAs in the Immune Microenvironment and Prognosis of Colorectal Cancer.

Background The role of N6-methyladenosine long noncoding RNAs (lncRNAs) in colorectal cancer (CRC) is elusive. Materials and Methods We identified m6A-associated lncRNAs by using the data gathered from The Cancer Genome Atlas (TCGA) and stratified CRC patients into different subgroups. Cox regression analysis was performed to construct an m6A-associated lncRNA signature. The role of this signature in the immune microenvironment and prognosis was dissected subsequently. Finally, a gene set enrichment analysis (GSEA) was conducted to predict the possible mechanisms based on the signature. Results Three m6A-associated clusters were constructed from 866 differentially expressed lncRNAs. Cluster 2 had poor prognosis and low immune cell infiltration. An m6A-associated lncRNA signature consisting of 14 lncRNAs was constructed and recognized as an independent prognostic indicator of CRC by using survival analysis and receiver operating characteristic (ROC) curves. The clinical features and immune cell infiltration status were significantly different in patients stratified by the risk score. Furthermore, GSEA showed that the P53 pathway and natural killer cell-mediated cytotoxicity were more enriched in the low-risk group. Conclusion Our data revealed that m6A-associated lncRNAs could be potential prognostic indicators of immunogenicity in CRC.

119P Characterisation of distinct subgroups of BRCA1-like triple-negative breast cancers with multiomics data analysis

Patients with triple-negative breast cancer (TNBC) that have a BRCA1-like phenotype - a specific pattern of DNA copy number aberrations similar to that of BRCA1-mutated tumours - benefit significantly from intensified platinum-based chemotherapy (IPB-chemo) compared to those without. To further molecularly characterise BRCA1-like TNBC tumours, we set out a multiomics analysis making use of published data sets. We made use of 3 clinical data sets (TCGA, RATHER, MATADOR). Sparse generalised canonical correlation analysis and logistic regression were performed on the multiomics data sets to identify features most strongly correlated with BRCA1-like status. Next, to characterise the subgroups, differential gene expression, active-subnetwork-oriented pathway enrichment analyses and cell type deconvolution of the bulk tissue mRNA-seq data were performed. We identified n = 84/1097, 62/126 and 46/495 BRCA1-like TNBCs in the TCGA, RATHER and MATADOR cohorts respectively. Two subgroups of BRCA1-like TNBCs were discerned that are characterised by a more or less profound copy-number loss on chromosome 5q. Deconvolution analyses revealed that BRCA1-like tumours with less 5q loss (n=65/130) are associated with higher tumour-infiltrating lymphocyte levels (p=0.001) and higher immune cell type percentages (p=0.006). This BRCA1-like subgroup was characterised by a gene expression profile that strongly associates with a number of cell-mediated immunity related pathways, such as the PD-L1/PD-1 pathway (p<0.001) and natural killer cell mediated cytotoxicity (p<0.001). By analysing multiomics data from n = 192 patients with BRCA1-like triple-negative breast cancer, we found indications for the presence of distinct subgroups of which one has immunomodulatory features. How these BRCA1-like, TNBC subgroups may direct immunotherapy strategies will be addressed in follow-up studies.

414.6: Porcine ULBP1 Disruption Does Not Significantly Reduce Human NK Cell Activation and Cytotoxicity in an in Vitro Model

Background: Pig-to-human xenotransplantation has the potential to address the critical organ shortage. Genetically-engineered pig organs have significantly prolonged survival in human and nonhuman primates by overcoming hyperacute rejection. Human natural killer (NK) cell-mediated acute xenograft rejection of porcine endothelial cells has been established. Porcine UL-Binding Protein (ULBP1) has been identified as a porcine ligand of human NK cell activating receptor NKG2D; interactions presumably lead to NK cell activation and ultimately porcine cell cytotoxicity. We sought to improve pig-to-human compatibility by eliminating ULBP1 ligand in an immortalized porcine liver-derived endothelial cell line (ipLDEC) with five-gene knockout (5GKO). The 5GKO cell line has mutations in GGTA1/CMAH/β4galNT2/SLA-I α-chain/β2M: three genes encoding enzymes responsible for xenoantigen production and two genes encoding swine leukocyte antigen class I. Methods: CRISPR/Cas9 technology was used to knockout the porcine ULBP1 gene in the 5GKO cell line to create a ULBP1-KO/5GKO cell line. Human peripheral blood mononuclear cells (PBMCs) from four donors were activated with human IL-2 for five days. Human PBMCs were co-cultured with 5GKO, ULBP1-KO/5GKO, and Wild-Type (WT) ipLDECs. Cells were stained with antibodies to CD45, CD3, CD56, and CD107a. Flow cytometry analysis was gated on the CD3-CD56+ NK cell population and monitored for expression of CD107a, a marker of NK cell degranulation. Calcein AM cytotoxicity assay was performed to determine human NK cell-mediated ipLDECs cell death. WT, 5GKO, ULBP1-KO/5GKO, HLA-G+/5GKO (positive control) were incubated in a calcein solution for 30 minutes at a concentration of 106 cells/mL. Stained porcine cells were co-cultured with activated human PBMCs at a 10:1 E:T ratio for 4 hours. Co-culture supernatant was harvested and calcein release was measured at an excitation wavelength of 485 nm and emission wavelength of 530 nm to calculate cytotoxicity percentages. Results: Porcine ULBP1 gene mutation was confirmed by Sanger sequencing (Figure 1a), establishing a porcine ULBP1-KO/5GKO cell line. There is no statistically significant difference in NK cell activation between co-cultures of human PBMCs (n=4) with pULBP1-KO/5GKO and 5GKO ipLDEC using CD107a. (Figure 1b). There is no statistically significant difference in NK-induced cytotoxicity between co-cultures of human PBMCs (n=6) with porcine ULBP1-KO/5GKO and 5GKO cell lines using Calcein AM assays (Figure 1c). As expected, HLA-G+/5GKO cytotoxicity was decreased (p = 0.0168) compared to WT (Figure 1c). Conclusion: We established a porcine ULBP1-KO/5GKO cell line. Our studies demonstrate no statistically significant difference in NK cell activation and NK cell-mediated cytotoxicity between 5GKO and ULBP1-KO/5GKO cell lines. Our studies show that ULBP1 is not a crucial ligand for human NKG2D. Future studies will target different human NK cell activating porcine ligands.

P2.10-04 Immunologic Subtype of Small Cell Lung Carcinoma Dictates Susceptibility to NK Cell-Mediated Cytotoxicity

Small cell lung carcinoma (SCLC) comprises approximately 15% of all lung cancer cases and SCLC patients initially respond to cytotoxic chemotherapy, but resistance inevitably emerges. Despite recent incorporation of immune checkpoint blockade (ICB) into first line treatment, predictive biomarkers of response remain lacking and patients with SCLC continue to have a poor prognosis with limited treatment options. Notably, recent transcriptomic profiling of SCLC cell lines and patient samples has indicated significant inter-tumoral and intra-tumoral heterogeneity.

Distinct patterns of the natural evolution of soft tissue sarcomas on pre-treatment MRIs captured with delta-radiomics correlate with gene expression profiles.

Radiomics of soft tissue sarcomas (STS) is assumed to correlate with histologic and molecular tumor features, but radiogenomics analyses are lacking. Our aim was to identify if distinct patterns of natural evolution of STS obtained from consecutive pre-treatment MRIs are associated with differential gene expression (DGE) profiling in a pathway analysis. All patients with newly diagnosed STS treated in a curative intent in our sarcoma reference center between 2008 and 2019 and with two available pre-treatment contrast-enhanced MRIs were included in this retrospective study. Radiomics features (RFs) were extracted from fat-sat contrast-enhanced T1-weighted imaging. Log ratio and relative change in RFs were calculated and used to determine grouping of samples based on a consensus hierarchical clustering. DGE and oncogenesis pathway analysis were performed in the delta-radiomics groups identified in order to detect associations between delta-radiomics patterns and transcriptomics features of STS. Secondarily, the prognostic value of the delta-radiomics groups was investigated. Sixty-three patients were included (median age: 63 years, interquartile range: 52.5-70). The consensus clustering identified 3 reliable delta-radiomics patient groups (A, B, and C). On imaging, group B patients were characterized by increase in tumor heterogeneity, necrotic signal, infiltrative margins, peritumoral edema, and peritumoral enhancement before the treatment start (p value range: 0.0019-0.0244), and, molecularly, by downregulation of natural killer cell-mediated cytotoxicity genes and upregulation of Hedgehog and Hippo signaling pathways. Group A patients were characterized by morphological stability of pre-treatment MRI traits and no local relapse (log-rank p = 0.0277). This study highlights radiomics and transcriptomics convergence in STS. Proliferation and immune response inhibition were hyper-activated in the STS that were the most evolving on consecutive imaging. • Three consensual and stable delta-radiomics clusters were identified and captured the natural patterns of morphological evolution of STS on pre-treatment MRIs. • These 3 patterns were explainable and correlated with different well-known semantic radiological features with an ascending gradient of pejorative characteristics from the A group to C group to B group. • Gene expression profiling stressed distinct patterns of up/downregulated oncogenetic pathways in STS from B group in keeping with its most aggressive radiological evolution.

Long noncoding RNA profiling reveals that LncRNA BTN3A2 inhibits the host inflammatory response to Eimeria tenella infection in chickens.

Coccidiosis is a widespread parasitic disease that causes serious economic losses to the poultry industry every year. Long noncoding RNAs (lncRNAs) play important roles in transcriptional regulation and are involved in a variety of diseases and immune responses. However, the lncRNAs associated with Eimeria tenella (E. tenella) resistance have not been identified in chickens. In addition, the expression profiles and functions of lncRNAs during E. tenella infection remain unclear. In the present study, high-throughput sequencing was applied to identify lncRNAs in chicken cecal tissues from control (JC), resistant (JR), and susceptible (JS) groups on day 4.5 post-infection (pi), and functional tests were performed. A total of 564 lncRNAs were differentially expressed, including 263 lncRNAs between the JS and JC groups, 192 between the JR and JS groups, and 109 between the JR and JC groups. Functional analyses indicated that these differentially expressed lncRNAs were involved in pathways related to E. tenella infection, including the NF-kappa B signaling, B cell receptor signaling and natural killer cell-mediated cytotoxicity pathways. Moreover, through cis regulation network analysis of the differentially expressed lncRNAs, we found that a novel lncRNA termed lncRNA BTN3A2 was significantly increased in both cecum tissue and DF-1 cells after coccidia infection or sporozoite stimulation. Functional test data showed that the overexpression of lncRNA BTN3A2 reduced the production of inflammatory cytokines, including IL-6, IL-1β, TNF-α and IL-8, while lncRNA BTN3A2 knockdown promoted the production of these inflammatory cytokines. Taken together, this study identify the differentially expressed lncRNAs during E. tenella infection in chickens for the first time and provide the direct evidence that lncRNA BTN3A2 regulates the host immune response to coccidia infection.