Expression Analysis Of Immune-related Genes Research Articles

Deletion of speA and aroC genes impacts the pathogenicity of Vibrio anguillarum in spotted sea bass

Vibrio anguillarum is one of the major pathogens responsible for bacterial infections in marine environments, causing significant impacts on the aquaculture industry. The misuse of antibiotics leads to bacteria developing multiple drug resistances, which is detrimental to the development of the fisheries industry. In contrast, live attenuated vaccines are gradually gaining acceptance and widespread recognition. In this study, we constructed a double-knockout attenuated strain, V. anguillarum ΔspeA-aroC, to assess its potential for preparing a live attenuated vaccine. The research results indicate a significant downregulation of virulence-related genes, including Type VI secretion system, Type II secretion system, biofilm synthesis, iron uptake system, and other related genes, in the mutant strain. Furthermore, the strain lacking the genes exhibited a 67.47% reduction in biofilm formation ability and increased sensitivity to antibiotics. The mutant strain exhibited significantly reduced capability in evading host immune system defenses and causing in vivo infections in spotted sea bass (Lateolabrax maculatus), with an LD50 that was 13.93 times higher than that of the wild-type V. anguillarum. Additionally, RT-qPCR analysis of immune-related gene expression in spotted sea bass head kidney and spleen showed a weakened immune response triggered by the knockout strain. Compared to the wild-type V. anguillarum, the mutant strain caused reduced levels of tissue damage. The results demonstrate that the deletion of speA and aroC significantly reduces the biosynthesis of biofilms in V. anguillarum, leading to a decrease in its pathogenicity. This suggests a crucial role of biofilms in the survival and invasive capabilities of V. anguillarum.

The regulation effect of Atractylodis macrocephalae polysaccharides on the growth performance, antioxidant capacity and immune function in Litopenaeus vannamei

This study aims to investigate the effects of Atractylodis macrocephalae polysaccharides (AMP) on the growth performance, antioxidant capacity and non-specific immune function in Litopenaeus vannamei. Accordingly, three experimental diets including 0 (control group), 0.05% and 0.5% AMP were formulated and fed for 3 weeks. The result showed that compared with the control group, dietary 0.5% AMP supplementation significantly increased the weight gain (P < 0.05), serum biochemical indices (albumin, glutamate-pyruvate transaminase, glutamate-oxalacetate transaminase, glucose, triglyceride and total cholesterol) while 0.05% AMP supplementation had no significant influence on the above index (P > 0.05). Dietary 0.05% and 0.5% AMP supplementation significantly increased total antioxidant capacity (P < 0.05), superoxide dismutase, and glutathione peroxidase in tissues (hemolymph, hepatopancreas and intestine) while there was a decreasing trend on the malondialdehyde content with the increased AMP supplementation (P < 0.05). Tissue lysozyme, alkaline phosphatase, acid phosphatase and total nitric oxide synthase content were significantly increased with increased AMP levels in the diets and reached the highest values in the 0.5% AMP supplementation group (P < 0.05). Similar trends were observed in the further antioxidant and immune-related gene expression analysis. Gene expression of inflammatory signaling pathways (toll-like receptor 4/myeloid differentiation factor 88) and inflammatory cytokines (Tumour necrosis factor-α, apoptosis-inducing factor, member RAS oncogene family) was reduced with AMP supplementation in the diets. In conclusion, dietary AMP supplementation could improve growth performance, antioxidant ability and immune function in shrimp, and recommended dosage for safety was 0.05%.

Transcriptional differences between coronavirus disease 2019 and bacterial sepsis

BackgroundCoronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has led to major public health crises worldwide. Several studies have reported the comprehensive mRNA expression analysis of immune-related genes in patients with COVID-19, using blood samples, to understand its pathogenesis; however, the characteristics of RNA expression in COVID-19 and bacterial sepsis have not been compared. The current study aimed to address this gap.MethodsRNA-sequencing and bioinformatics analyses were used to compare the transcriptome expression of whole blood samples from patients with COVID-19 and patients with sepsis who were admitted to the intensive care unit of Osaka University Graduate School of Medicine.ResultsThe COVID-19 and sepsis cohorts showed upregulation of mitochondrial- and neutrophil-related transcripts, respectively. Compared with that in the control cohort, neutrophil-related transcripts were upregulated in both the COVID-19 and sepsis cohorts. In contrast, mitochondrial-related transcripts were upregulated in the COVID-19 cohort and downregulated in the sepsis cohort, compared to those in the control cohort. Moreover, transcript levels of the pro-apoptotic genes BAK1, CYCS, BBC3, CASP7, and CASP8 were upregulated in the COVID-19 cohort, whereas those of anti-apoptotic genes, such as BCL2L11 and BCL2L1, were upregulated in the sepsis cohort.ConclusionsThis study clarified the differential expression of transcripts related to neutrophils and mitochondria in sepsis and COVID-19 conditions. Mitochondrial-related transcripts were downregulated in sepsis than in COVID-19 conditions, and our results indicated suboptimal intrinsic apoptotic features in sepsis samples compared with that in COVID-19 samples. This study is expected to contribute to the development of specific treatments for COVID-19.

Abstract 781: Pretreatment immune-related gene expression analysis predicts survival of patients with metastatic lung cancer treated with anti PD-1 antibody

Abstract Introduction: Antibodies targeting PD-1 play a relevant role in the management of metastatic non-small cell lung cancer (NSCLC), although the majority of patients experience disease progression to treatment. We performed a pretreatment immune-related gene expression analysis to predict outcomes of patients with advanced stage NSCLC receiving pembrolizumab as first-line therapy. Methods: Pretreatment tissue samples from 48 metastatic NSCLC patients with PD-L1 high expression (&amp;gt; 50% 22C3 clone testing antibody), who undergone first line pembrolizumab therapy, were evaluated. Total RNA was isolated (RNeasy FFPE Kit Qiagen) and quantified (NanoDrop Fluorometer, Thermo Fisher Sci.). We performed a mRNA expression analysis of 770 immune-related genes (belonging to 21 pathways) by using the nCounter PanCancer Immune Profiling Panel (NanoString Tech.). Data normalization was performed in-house by using R functions. Patients were clustered by the K-means algorithm according to results of the gene expression analysis for each pathway. Advanced analyses were performed by nCounter Advanced Analysis plug in for nSolver Software (v. 2.0.134) and R package. Estimates of Overall Survival (OS) were done by Kaplan-Meier curves and tests of differences by log-rank test. Regression models were built including covariables. All tests were two-sided, with a statistical significance at p-value &amp;lt; 0.05. Results: The median age of patients was 72.5 years (IQR 64.3-77.0). Thirty-seven (77.1%) were male, 36 (75%) with non-squamous histology, 37 (77.1%) had an ECOG PS of 0 or 1, and 46 (95.8%) were former or current smokers. In 16 out of 48 cases (33.3%) the immune profile was performed on tumor-infiltrated lymph nodes. Disease Control Rate per RECIST 1.1 was 52.1% (16 partial and 2 complete responses, 7 disease stabilizations as best response). Median Progression-Free Survival was 2.96 months (95% CI 2.35-7.25), median OS was 13.43 months (95% CI 6.24-NR). The principal component analysis excluded 1 outlier subject by the gene expression analysis. Five gene expression signatures were significantly correlated with OS (Adhesion, Cytokines, Leukocyte Functions, Macrophage Functions, TNF Superfamily), all identifying 2 clusters of patients among each pathway. The 8-genes signature of the Leucocyte Functions pathway (CX3CL1, FUT7, HCK, IFNG, LCP1, SH2D1B, THBD, VEGFA) was the solely significantly correlated to OS regardless of age, sex, tumor histology, ECOG PS, smoke and site of diagnostic sampling (lymph node or non-nodal tissue), with a Hazard Ratio between the identified clusters of 0.29 (95% CI 0.24-0.60, p = 0.01). Conclusions: Our results support the hypothesis that pretreatment immune-related mRNA expression patterns, identified through the nCounter platform, may predict OS of patients with metastatic NSCLC treated with first line anti PD-1 therapy. Citation Format: Marco Tagliamento, Sara Sommariva, Cristina Campi, Giuseppa De Luca, Maria Giovanna Dal Bello, Marco Mora, Simona Coco, Mariella Dono, Carlo Genova. Pretreatment immune-related gene expression analysis predicts survival of patients with metastatic lung cancer treated with anti PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 781.

A multi-tissue study of immune gene expression profiling highlights the key role of the nasal epithelium in COVID-19 severity

Coronavirus Disease-19 (COVID-19) symptoms range from mild to severe illness; the cause for this differential response to infection remains unknown. Unravelling the immune mechanisms acting at different levels of the colonization process might be key to understand these differences. We carried out a multi-tissue (nasal, buccal and blood; n = 156) gene expression analysis of immune-related genes from patients affected by different COVID-19 severities, and healthy controls through the nCounter technology. Mild and asymptomatic cases showed a powerful innate antiviral response in nasal epithelium, characterized by activation of interferon (IFN) pathway and downstream cascades, successfully controlling the infection at local level. In contrast, weak macrophage/monocyte driven innate antiviral response and lack of IFN signalling activity were present in severe cases. Consequently, oral mucosa from severe patients showed signals of viral activity, cell arresting and viral dissemination to the lower respiratory tract, which ultimately could explain the exacerbated innate immune response and impaired adaptative immune responses observed at systemic level. Results from saliva transcriptome suggest that the buccal cavity might play a key role in SARS-CoV-2 infection and dissemination in patients with worse prognosis. Co-expression network analysis adds further support to these findings, by detecting modules specifically correlated with severity involved in the abovementioned biological routes; this analysis also provides new candidate genes that might be tested as biomarkers in future studies. We also found tissue specific severity-related signatures mainly represented by genes involved in the innate immune system and cytokine/chemokine signalling. Local immune response could be key to determine the course of the systemic response and thus COVID-19 severity. Our findings provide a framework to investigate severity host gene biomarkers and pathways that might be relevant to diagnosis, prognosis, and therapy.

Transcriptome analysis of immune-related gene expression in Yellow River carp (Cyprinus carpio var.) after challenge with Flavobacterium columnare

Yellow River carp (Cyprinus carpio) is an economically-important freshwater fish. It is the common host of the epizootic bacterium Flavobacterium columnare, a common fish pathogen that causes columnaris disease resulting in aquacultural losses. However, information on the functions and mechanisms of the immune system of Yellow River carp infected with F. columnare is limited. Therefore, the aim of this study is to evaluate the genetic and histopathological effects of an experimentally-induced F. columnare infection in Yellow River carp. Sixty fish were divided into control (CT group) and challenged groups. The gills were collected for histological and transcriptome analysis to understand the host immune response after challenge with F. columnare. The infected fish of the IF group presented typical columnaris disease symptoms and higher mortality, as well as histological changes. However, some challenged fish showed asymptomatic infection (IC group). Additionally, there were 1776 significant differentially-expressed genes (DEGs) between the IC and CT groups, 1853 DEGs between the IF and CT groups, and 1836 DEGs between the IF and IC groups, All the DEGs were classified into three gene ontology categories, which were allocated to 158 KEGG pathways. Moreover, immune-related genes were confirmed by qRT-PCR. we quantified the level of IL-1, IL-6, TNF-α and IL-8 by ELISA. The results showed the highest expression levels of inflammatory cytokines as well as stress proteins and the adhesion molecules in the lF group, which may contribute to severe infection, and a higher case fatality rate, while the high expression of chemokines, costimulatory molecules and the up regulation of antigen presentation function could help the carp resist F. columnare infection.

Immune-Related Gene Expression Analysis Revealed Three lncRNAs as Prognostic Factors for Colon Cancer.

Colorectal cancer (CRC) is one of the most common cancers. Almost 80% of CRC cases are colon adenocarcinomas (COADs). Several studies have indicated the role of immunotherapy in the treatment of various cancers. Our study aimed to identify immune-related long non-coding RNAs (lncRNAs) and to use them to construct a risk assessment model for evaluating COAD prognosis. Using differential expression, correlation, and Cox regression analyses, we identified three immune-related differentially expressed lncRNAs (IR-DELs) and used them to construct a risk assessment model. The area under the curve (AUC) for each receiver operating characteristic (ROC) curve at 3-, 5-, and 10-years were greater than 0.6. In addition, the risk assessment model was correlated with several immune cells and factors. The three IR-DELs (AC124067.4, LINC02604, and MIR4435-2HG) identified in this study can be used to predict outcomes for patients with COAD and might help in identifying those who can benefit from anti-tumor immunotherapy.

Immune related gene expression analysis of Macrobrachium nipponense in different hours post-infection by non-O1 Vibrio cholerae

Non-O1 Vibrio cholerae is a highly virulent pathogen which has seriously threatened the development of Macrobrachium nipponense in various farms. However, little information is available in relation to underlying mechanisms of host-bacteria interaction in M. nipponense. In this study, we performed transcriptome analysis of M. nipponense at 6 and 12 h post-infection (hpi), and differentially expressed genes (DEGs) (P<0.05) were revealed by comparison of gene expression levels between 6 and 12 hpi. The results showed that expression of different immune-related genes was found significantly regulated at 6 hpi than at 12 hpi by transcriptome analysis, which was verified by quantitative real-time PCR (qRT-PCR). A total of 189 DEGs including 104 up-regulated genes and 85 down-regulated genes from hepatopancreas at 6 hpi, 56 DEGs at 12 hpi were identified, which was much lower than that in the comparison of at 6 hpi and the up-regulated and down-regulated genes were 39 and 17, respectively. Many genes associated with Rap1 signaling pathway, MAPK signaling pathway, ECM-receptor interaction, Endocytosis, Hippo signaling pathway, Ubiquitin mediated proteolysis and Phagosome were positively modified after non-O1 V. cholerae challenge, and most the immune-related pathways were activated at 6 hpi. We selected 6 DEGs and validated their expression level by RT-qPCR. The data generated in this study may provide a valuable resource for further immune response research and offer improved strategies against non-O1 V. cholerae infection in shrimp.

A Potential Mechanism of Anticancer Immune Response Coincident With Immune-related Adverse Events in Patients With Renal Cell Carcinoma.

Some reports showed encouraging efficacy of immune checkpoint inhibitors among patients who experienced immune-related adverse events (irAEs). Thus, characterization of T-cell repertoire and immune signatures in peripheral blood mononuclear cells (PBMCs) and tumors before and after immune checkpoint inhibitors treatment should contribute to better understanding of irAE-provoked anticancer immune responses. We applied expression analysis of immune-related genes and T-cell receptor sequencing in tumor and PBMCs from five patients with renal cell carcinoma before combined immunotherapy and at the onset of severe irAEs. We found that the cluster of differentiation 8 (CD8)/forkhead box P3(FOXP3), granzyme B(GZMB)/CD3, perforin 1(PRF1)/CD3 and programmed cell death 1(PD1)/CD8 expression ratios were significantly elevated in PBMCs at the onset of irAEs. In addition, we found expansion of certain T-cell clones in metastatic tissue after irAEs, which were already increased in peripheral blood at the onset of irAEs. irAE-provoked T-cells may also circulate and attack distant tumors, leading to durable response in patients with irAEs.

Abstract A13: Implication of immune cell composition in biopsy specimens of triple-negative breast cancer for responsiveness to neoadjuvant chemotherapy

Abstract Triple-negative breast cancer (TNBC) is a subtype of breast carcinoma that has little or no expression of hormone receptors and human epidermal growth factor receptor 2 (HER2). Most patients with TNBC receive chemotherapy, since TNBC is an aggressive type of cancer and lacks targeted therapy. Recently, possible effectiveness of immunotherapy for TNBC has been identified. Here, we aim to elucidate predictive power of immune cell composition and immune-related gene expression in neoadjuvant chemotherapy (NAC) setting of TNBC. Expression profiles of 579 immune-related genes were examined in biopsy tissues obtained from 55 patients with primary TNBC treated with NAC (anthracycline, cyclophosphamide, and docetaxel) using NanoString nCounter GX Human Immunology Panel. Then, the composition of 22 subtypes of immune cells was estimated from the gene expression profiles using CIBERSORT. The optimal combination of immune cell subsets as well as immune-related genes to predict pathologic complete response (pCR) and clinical outcome (death) was inferred by univariate receiver operating characteristic (ROC) curve and subsequent multivariate ROC curve analyses. Among the 55 patients, 17 cases showed pCR whereas the others showed non-pCR. P values were provided by CIBERSORT for all the specimens but two were less than 0.05. Immune cell composition varied by patients, and only the proportion of resting dendritic cells was significantly different between the two groups, pCR vs. non-pCR (p=0.038). In our univariate ROC curve analysis, follicular helper T cell (area under the curve, AUC 0.646) and monocyte (AUC 0.625) were the most relevant immune cell subtypes to patients’ clinical outcome and pCR, respectively. Multivariate ROC curve-based exploratory analysis identified the highest AUC (0.647) for the prediction of clinical outcome from the combination of follicular helper T cell and naive B cell proportions. The combination of 18 out of 22 immune cell subsets showed highest AUC (0.745) for pCR. In terms of immune-related genes, univariate ROC curve analysis identified TNFSF13B (AUC 0.756) and MAPK1 (AUC 0.718) as the most relevant genes to patients’ survival and pCR, respectively. From our multivariate ROC curve analysis, the combination of 113 genes including RUNX1 had highest AUC (0.915) for survival outcome prediction. For the prediction of pCR, the combination of 107 genes including MAPK1, NFKB1, and TRAF1 showed highest AUC (0.946). Forty-one genes were overlapped between the two sets of genes. Our study showed not only immune-related gene expression but also composition of immune cell subtypes could predict responsiveness to neoadjuvant chemotherapy in biopsy specimens of TNBC, though its predictive power seemed to be lower than that of combination of gene expression. Further analysis of immune-related gene expression in each immune cell type might improve our understanding of immune microenvironment and provide higher predictive values in clinical response to chemotherapy in TNBC patients. Citation Format: In Ah Park, Jinho Jang, Hyoung-oh Jeong, Gyungyub Gong, Semin Lee, Hee Jin Lee. Implication of immune cell composition in biopsy specimens of triple-negative breast cancer for responsiveness to neoadjuvant chemotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A13.

Ginsenoside Rg3 inhibits grass carp reovirus replication in grass carp ovarian epithelial cells

Ginseng exhibits multiple medicinal properties, including the improvement of immune function and enhancing disease resistance. In this study, we investigated the inhibitory effects of ginsenoside Rg3 on grass carp reovirus (GCRV) infection of grass carp ovarian (CO) epithelial cells, in order to provide a baseline framework for future high-efficacy antiviral drug screening investigations. Ginsenoside Rg3 was added to GCRV-infected CO cells, and cells were cultured at 27 °C before cell proliferation was measured by MTT assays. Label-free real-time cellular analysis (RTCA) after 72 h of experimentation demonstrated that 100 μg/mL ginsenoside Rg3 treatment had the highest inhibitory effect on GCRV (among 1,10,100 μg/mL treatments). We then measured the capacity for cellular antioxidant ability. Cells treated with 1,10,100 μg/mL ginsenoside Rg3 exhibited increases in Total Antioxidant Capacity activity relative to controls, respectively. Furthermore, Antioxidant assay and reverse transcript quantitative polymerase chain reaction (RT-qPCR) showed that ginsenoside Rg3 were efficient to restrain the replication of GCRV in CO cells. Expression analysis of immune-related genes via RT-qPCR showed that treatment with ginsenoside Rg3 promoted expression of IRF-3 and IRF-7 increases, respectively. Moreover, expression of IFN-1 was induced, which then inhibition the expression of tumor necrosis factor-alpha (TNF-α). In conclusion, we demonstrated that ginsenoside Rg3 promotes CO cell proliferation, inhibits GCRV activity, promotes CO cell immune activities, and thereby enhances the resistance of CO to GCRV infection.

Intratumoral delivery of tavokinogene telseplasmid yields systemic immune responses in metastatic melanoma patients

Interleukin 12 (IL-12) is a pivotal regulator of innate and adaptive immunity. We conducted a prospective open-label, phase II clinical trial of electroporated plasmid IL-12 in advanced melanoma patients (NCT01502293). Patients with stage III/IV melanoma were treated intratumorally with plasmid encoding IL-12 (tavokinogene telseplasmid; tavo), 0.5 mg/ml followed by electroporation (six pulses, 1500 V/cm) on days 1, 5, and 8 every 90 days in the main study and additional patients were treated in two alternative schedule exploration cohorts. Correlative analyses for programmed death-ligand 1 (PD-L1), flow cytometry to assess changes in immune cell subsets, and analysis of immune-related gene expression were carried out on pre- and post-treatment samples from study patients, as well as from additional patients treated during exploration of additional dosing schedules beyond the pre-specified protocol dosing schedule. Response was measured by study-specific criteria to maximize detection of latent and potentially transient immune responses in patients with multiple skin lesions and toxicities were graded by the Common Terminology Criteria for Adverse Events version 4.0 (CTCAE v4.0). The objective overall response rate was 35.7% in the main study (29.8% in all cohorts), with a complete response rate of 17.9% (10.6% in all cohorts). The median progression-free survival in the main study was 3.7 months while the median overall survival was not reached at a median follow up of 29.7 months. A total of 46% of patients in all cohorts with uninjected lesions experienced regression of at least one of these lesions and 25% had a net regression of all untreated lesions. Transcriptomic and immunohistochemistry analysis showed that immune activation and co-stimulatory transcripts were up-regulated but there was also increased adaptive immune resistance. Intratumoral Tavo was well tolerated and led to systemic immune responses in advanced melanoma patients. While tumor regression and increased immune infiltration were observed in treated as well as untreated/distal lesions, adaptive immune resistance limited the response.

Characterization and expression analysis of immune-related genes in the red swamp crayfish, Procambarus clarkii in response to lipopolysaccharide challenge

To learn more about red swamp crayfish related genes in response to bacterial infections, we investigated immune-related genes induced by lipopolysaccharide (LPS) in the hepatopancreas using high-throughput sequencing method. In present the study, a total of 55,107 unigenes were identified, with an average length of 678 bp. A total of 2215 differentially expressed genes (DEGs) were found, including 669 up-regulated genes and 1546 down-regulated genes. The result of Gene ontology (GO) analysis revealed that 3017 DEGs were enriched in 19 biological process subcategories, 17 cellular component subcategories and 15 molecular function subcategories. The top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways showed that “ribosome” was the most abundant group, which had 34 DEGs. KEGG enrichment analysis identified several immune response pathways. Real-time quantitative reverse transcription-PCR (qRT-PCR) results exhibited that several immune responsive genes were greatly up-regulated following LPS stimulation as observed in the results of high-throughput sequencing. Overall, this study provides new insight into the immune defense mechanisms of P. clarkii against LPS infection.

Abstract 5006: Comprehensive gene expression analysis of the tumor microenvironment in patients with advanced cancer treated with a personalized neoantigen vaccine, NEO-PV-01, in combination with anti-PD1

Abstract Background: Neoantigens arise from DNA mutations in cancer cells and are important targets for T cell mediated anti-tumor immunity. NEO-PV-01 is a personal neoantigen vaccine of up to 20 peptides designed based on a patient’s neoantigen and HLA profile that is directed at inducing tumor-specific T cell responses to neoantigens. Here we report comprehensive immune-related gene expression analysis of longitudinal tumor biopsies from patients with metastatic melanoma, bladder, and non-small cell lung cancer treated on our NT-001 trial with NEO-PV-01 + adjuvant in combination with nivolumab (NCT02897765) to correlate with clinical outcomes. Methods: Tumor biopsies from all three tumor types were collected i) prior to treatment, ii) after 12 weeks of nivolumab monotherapy and iii) after completion of NEO-PV-01 vaccination. Targeted gene expression analysis on RNA extracted from FFPE blocks was performed using the NanoString™ nCounter platform. A custom set of 800 genes included markers for immune cell populations, cytolytic markers, immune activation and suppression, and the tumor microenvironment. Gene signatures of key immune features were calculated after normalization with housekeeping genes and used for subsequent analysis. Results: Changes in the immune cell populations and the tumor microenvironment were detected after treatment with nivolumab and NEO-PV-01. Increases in various immune cell subsets, including T cells and B cells, as well as an increase in cytolytic phenotype were observed in tumor biopsies following treatment. Moreover, changes in the tumor microenvironment, consistent with the absence of tumor by histologic evaluation, were detected in many of the post-vaccination biopsies. In addition, these observations were consistent with data from peripheral blood that demonstrated durable de novo neoantigen-specific immune responses after vaccination. Additional exploratory analyses of the data demonstrate differential gene expression in patients’ tumors that align with tumor responses to therapy. Conclusion: Treatment with nivolumab and NEO-PV-01 leads to changes in the tumor microenvironment that are consistent with cell types and phenotypes that could contribute to an anti-tumor response. Citation Format: Meghan E. Bushway, Ying Sonia Ting, Rana H. Besada, Tracey E. Sciuto, Jasmina Prabhakara, Julian Scherer, Kristen N. Balogh, April Lamb, Jennifer A. Kaplan, Lisa D. Cleary, Melissa A. Moles, Sarah E. Church, Yuqi Ren, Xing Ren, Richard B. Gaynor, Matthew J. Goldstein, Les H. Brail, Joel Greshock, Lakshmi Srinivasan. Comprehensive gene expression analysis of the tumor microenvironment in patients with advanced cancer treated with a personalized neoantigen vaccine, NEO-PV-01, in combination with anti-PD1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5006.

Transcriptome analysis of immune-related gene expression in hybrid snakehead (Channa maculata ♀ X Channa argus ♂) after challenge with Nocardia seriolae

Transcriptome analysis of immune-related gene expression in hybrid snakehead (Channa maculata ♀ X Channa argus ♂) after challenge with Nocardia seriolae

Preferential Infiltration of Unique Vγ9Jγ2-Vδ2 T Cells Into Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is clinically highly aggressive as a result of evolutionary dynamics induced by cross-talk between cancer cells and a heterogeneous group of immune cells in tumor microenvironment. The brain harbors limited numbers of immune cells with few lymphocytes and macrophages; thus, innate-like lymphocytes, such as γδ T cells, have important roles in antitumor immunity. Here, we characterized GBM-infiltrating γδ T cells, which may have roles in regulating the GBM tumor microenvironment and cancer cell gene expression. V(D)J repertoires of tumor-infiltrating and blood-circulating γδ T cells from four patients were analyzed by next-generation sequencing-based T-cell receptor (TCR) sequencing in addition to mutation and immune profiles in four GBM cases. In all tumor tissues, abundant innate and effector/memory lymphocytes were detected, accompanied by large numbers of tumor-associated macrophages and closely located tumor-infiltrating γδ T cells, which appear to have anti-tumor activity. The immune-related gene expression analysis using the TCGA database showed that the signature gene expression extent of γδ T cells were more associated with those of cytotoxic T and Th1 cells and M1 macrophages than those of Th2 cells and M2 macrophages. Although the most abundant γδ T cells were Vγ9Vδ2 T cells in both tumor tissues and blood, the repertoire of intratumoral Vγ9Vδ2 T cells was distinct from that of peripheral blood Vγ9Vδ2 T cells and was dominated by Vγ9Jγ2 sequences, not by canonical Vγ9JγP sequences that are mostly commonly found in blood γδ T cells. Collectively, unique GBM-specific TCR clonotypes were identified by comparing TCR repertoires of peripheral blood and intra-tumoral γδ T cells. These findings will be helpful for the elucidation of tumor-specific antigens and development of anticancer immunotherapies using tumor-infiltrating γδ T cells.

Kidney Transplant Outcome Is Associated with Regulatory T Cell Population and Gene Expression Early after Transplantation.

Successful long-term kidney allograft survival with parallel reduction of complications resulting from prolonged immunosuppressive treatment is a goal in kidney transplantation. We studied the immune changes in cell phenotypes and gene expression induced by kidney transplantation. Our goal was to find a phenotypic and/or transcriptional pattern that might be considered prognostic for the kidney transplant outcome. The analysis was performed prospectively on 36 KTx recipients sampled during the first year and followed for five years after transplantation and on 40 long-term KTx recipients (7.9 ± 2.2 y. post-KTx). The research involved flow cytometry assessment of lymphocyte subpopulations (including Tregs and CD3+CD8+CD28− lymphocytes) and gene expression analysis of immune-related genes (CD4, CD8, CTLA4, GZMB, FOXP3, IL10, IL4, ILR2A, NOTCH, PDCD1, PRF1, TGFB, and TNFA). The analysis of patterns observed over the first post-KTx year was confronted with control, pretransplant, and long-term transplant results. Treg counts at months one and three post-KTx correlated positively with the current and future allograft function. FOXP3 gene expression at month one post-KTx was also associated with long-term allograft function. The KTx-induced CD3+CD8+CD28− population correlated with GZMB and PRF1 expression and suggested their cytotoxic properties. The size of the Treg population and regulatory FOXP3 gene expression in the early period after transplantation are associated with kidney transplant outcome. The outlined predictive power of the Treg population needs to be investigated further to be confirmed as one of the immune monitoring strategies that may help achieve the best long-term kidney allograft outcomes.

Anti-inflammatory and immune-modulating effects of rice callus suspension culture (RCSC) and bioactive fractions in an in vitro inflammatory bowel disease model

BackgroundRice callus suspension culture (RCSC) has been shown to exhibit potent antiproliferative activity in multiple cancer cell lines. RCSC and its bioactive compounds can fill the need for drugs with no side effects. Hypothesis/PurposeThe anti-inflammatory potential of RCSC and its bioactive fractions on normal colon epithelial cell lines, was investigated. Study designThree cell lines, InEpC, NCM356 and CCD841-CoN were treated with proinflammatory cytokines followed by RCSC. Cytoplasmic and nuclear ROS were assayed with fluorescent microscopy and flow cytometer. Expression analysis of immune-related genes was performed in RCSC-treated cell lines. RCSC was fractionated using column chromatography and HPLC. Pooled fractions 10–18 was used to test for antiproliferative activity using colon adenocarcinoma cell line, SW620 and anti-inflammatory activity using CCD841-CoN. Mass spectrometric analysis was performed to identify candidate compounds in four fractions. ResultsRCSC treatment showed differential effects with higher cytoplasmic ROS levels in NCM356 and CCD841-CoN and lower ROS levels in InEpC. Nuclear generated ROS levels increased in all three treated cell lines. Flow cytometry analysis of propidium iodide stained cells indicated mitigation of cell death caused by inflammation in RCSC treated groups in both NCM356 and CCD841-CoN. Genes encoding transcription factors and cytokines were differentially regulated in NCM356 and CCD841-CoN cell lines treated with RCSC which provided insights into possible pathways. Analysis of pooled fractions 10–18 by HPLC identified 8 peaks. Cell viability assay with fractions 10–18 using SW620 showed that the number of viable cells were greatly reduced which was similar to 6X and 33X RCSC with very little effect on normal cells which similar to 1X RCSC. RCSC fractions increased nuclear and cytoplasmic ROS vs. both untreated and inflammatory control. Analysis of four fractions by mass spectrometry identified 4-deoxyphloridzin, 5′-methoxycurcumin, piceid and lupeol as candidate compounds which are likely to be responsible for the antiproliferative, anti-inflammatory and immune-regulating properties of RCSC. ConclusionRCSC and its fractions showed anti-inflammatory activity on inflamed colon epithelial cells. Downstream target candidate genes which are likely to mediate RCSC effects were identified. Candidate compounds responsible for the antiproliferative and anti-inflammatory activity of RCSC and its fractions provide possible drug targets.

Comparative Transcriptomic Analysis of Immune-Related Gene Expression in Duck Embryo Fibroblasts Following Duck Tembusu Virus Infection.

Duck is a major waterfowl species in China, providing high-economic benefit with a population of up to 20–30 billion per year. Ducks are commonly affected by severe diseases, including egg-drop syndrome caused by duck Tembusu virus (DTMUV). The immune mechanisms against DTMUV invasion and infection remain poorly understood. In this study, duck embryo fibroblasts (DEFs) were infected with DTMUV and harvested at 12 and 24 h post-infection (hpi), and their genomes were sequenced. In total, 911 (764 upregulated and 147 downregulated genes) and 3008 (1791 upregulated and 1217 downregulated) differentially expressed genes (DEGs) were identified at 12 and 24 hpi, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that DEGs were considerably enriched in immune-relevant pathways, including Toll-like receptor signaling pathway, Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, Chemokine signaling pathway, NOD-like receptor signaling pathway, and Hematopoietic cell lineage at both time points. The key DEGs in immune system included those of the cytokines (IFN α2, IL-6, IL-8L, IL-12B, CCR7, CCL19, and CCL20), transcription factors or signaling molecules (IRF7, NF-κB, STAT1, TMEM173, and TNFAIP3), pattern recognition receptors (RIG-I and MDA5), and antigen-presenting proteins (CD44 and CD70). This suggests DTMUV infection induces strong proinflammatory/antiviral effects with enormous production of cytokines. However, these cytokines could not protect DEFs against viral attack. Our data revealed valuable transcriptional information regarding DTMUV-infected DEFs, thereby broadening our understanding of the immune response against DTMUV infection; this information might contribute in developing strategies for controlling the prevalence of DTMUV infection.

Transcriptome analysis of immune-related gene expression in hybrid snakehead (Channa maculata ♀ × Channa argus ♂) after challenge with Nocardia seriolae

Hybrid snakehead fish (Channa maculata ♀ × Channa argus ♂), a new species used in freshwater aquaculture in China, is the common host of an epizootic bacterial infection by Nocardia seriolae. However, the information on the functions and mechanisms of hybrid snakehead immune pathways with the N. seriolae infection is limited. Thus, the peripheral blood lymphocytes from hybrid snakehead were used for transcriptome analysis to understand the host immune response after challenge with N. seriolae. A total of 49,839,332 and 50,059,283 raw reads were obtained from the N. seriolae-challenged group (Ns group) and phosphate-buffered saline control group (Ctr group), respectively. The 75.50% and 74.25% reads from the Ns and Ctr groups were matched to reference genomic sequence after cleaning the raw reads, respectively. Additionally, there were 2892 significant differentially expressed genes (DEGs) among the 17,196 expressed genes between the Ns and Ctr groups, including 1387 upregulated and 1505 downregulated genes. All the DEGs were classified into three gene ontology categories, and 2502 DEGs had significant matches, which were allocated to 246 Kyoto Encyclopedia of Genes and Genomes pathways. Immune-related genes were detected from immune system pathways among the top 20 enriched pathways. Moreover, the regulation of several observed effective genes was confirmed by real-time quantitative polymerase chain reaction. Altogether, this study offers deep-sequence data of hybrid snakehead peripheral blood lymphocyte via transcriptome analysis and lays the foundation for further study on the immunogenetics of hybrid snakehead. Moreover, it provides insights into the pathogenic mechanism of N. seriolae, facilitating the prevention and treatment of fish nocardiosis.