Expression Profiles Of Immune-related Genes Research Articles

A pilot study of intratumoral SD-101 (toll-like receptor 9 agonist), nivolumab, and radiotherapy for treatment of chemotherapy-refractory metastatic pancreatic adenocarcinoma.

TPS782 Background: Pancreatic adenocarcinoma is an aggressive disease projected to be the second leading cause of cancer-related death. A majority of patients have advanced disease on diagnosis. Combination chemotherapy is first-line for advanced disease but limited by toxicity and median survival under 1 year. SD-101 is a toll-like receptor 9 agonist that is injected intratumorally to increase immunogenicity in the tumor microenvironment. Localized radiation can further enhance this via antigen release and potential abscopal effects. Immunotherapy has revolutionized care for various solid organ malignancies but not yet for pancreatic cancer. Therefore, the combination of SD-101, localized radiation, and checkpoint inhibitor is a promising therapeutic strategy for metastatic pancreatic adenocarcinoma. Methods: Six patients with chemotherapy-refractory, liver-metastatic pancreatic adenocarcinoma will be evaluated for combination SD-101, radiation, and nivolumab. SD-101 is injected intratumorally into a liver metastasis on days 1, 8, 15, 29 with optional dosing days 43 and 57. Localized radiation (6-10 Gy per fraction) to the injected lesion will be given on days 1, 3, 5, 8, and 10. Nivolumab will be given at 240 mg every 2 weeks starting day 2 until progression or unacceptable toxicity. Blood samples will be collected at baseline and at regular intervals while on treatment. Biopsies will be obtained at baseline and on day 29. Primary objectives are to evaluate safety and tolerability, defined so if ≥5 patients reach day 29 without experiencing grade ≥3 treatment-related toxicity. Secondary objectives include preliminary efficacy as defined by disease control rate, duration of response, progression-free survival, and overall survival. Exploratory objectives include objective response rate and biomarker correlatives (T-cell clonality, tumor mutational burden, tumor infiltrative immune cell subsets, and immune-related gene expression profile). Blood and biopsy specimens will be analyzed using flow cytometry, qRT-PCR, RNA sequencing, and whole exome sequencing including immunohistochemistry on biopsy specimens. Clinical trial information: NCT04050085.

Phloroglucinol Treatment Induces Transgenerational Epigenetic Inherited Resistance Against Vibrio Infections and Thermal Stress in a Brine Shrimp (Artemia franciscana) Model.

Emerging, infectious diseases in shrimp like acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus and mortality caused by other Vibrio species such as Vibrio harveyi are worldwide related to huge economic losses in industrial shrimp production. As a strategy to prevent disease outbreaks, a plant-based phenolic compound could be used as a biocontrol agent. Here, using the brine shrimp (Artemia franciscana) as a model system, we showed that phloroglucinol treatment of the parental animals at early life stages resulted in transgenerational inherited increased resistance in their progeny against biotic stress, i.e., bacteria (V. parahaemolyticus AHPND strain and V. harveyi) and abiotic stress, i.e., lethal heat shock. Increased resistance was recorded in three subsequent generations. Innate immune-related gene expression profiles and potential epigenetic mechanisms were studied to discover the underlying protective mechanisms. Our results showed that phloroglucinol treatment of the brine shrimp parents significantly (P < 0.05) enhanced the expression of a core set of innate immune genes (DSCAM, proPO, PXN, HSP90, HSP70, and LGBP) in subsequent generations. We also demonstrated that epigenetic mechanisms such as DNA methylation, m6A RNA methylation, and histone acetylation and methylation (active chromatin marker i.e., H3K4Me3, H3K4me1, H3K27me1, H3 hyperacetylation, H3K14ac and repression marker, i.e., H3K27me3, H4 hypoacetylation) might play a role in regulation of gene expression leading toward the observed transgenerational inheritance of the resistant brine shrimp progenies. To our knowledge, this is the first report on transgenerational inheritance of a compound-induced robust protected phenotype in brine shrimp, particularly protected against AHPND caused by V. parahaemolyticus and vibriosis caused by V. harveyi. Results showed that epigenetic reprogramming is likely to play a role in the underlying mechanism.

Duckling short beak and dwarfism syndrome virus infection activates host innate immune response involving both DNA and RNA sensors

Duckling short beak and dwarfism syndrome virus (SBDSV), a newly identified goose parvovirus, causes devastating disease in domestic waterfowl and considerable economic losses to Chinese waterfowl industry. The molecular pathogenesis of SBDSV infection, nature and dynamics of host immune responses against SBDSV infection remained elusive. In this study, we systematically explored the relative mRNA expression profiles of major innate immune-related genes in SBDSV infected duck embryo fibroblasts. We found that SBDSV infection effectively activated host innate immune responses and resulted in significant up-regulation of IFN-β and several vital IFN-stimulated genes (ISGs). These up-regulation responses were mainly attributed to viral genomic DNA and dsRNA replication intermediates. Importantly, the expression of cGAS was significantly induced, whereas the expression of other DNA receptors including DDX41, STING, ZBP1, LSM14A and LRRFIP1 have no significant change. Furthermore, SBDSV infection also activates the up-regulation of TLR3 and inhibited the expression of TLR2 and TLR4; however, no effect was observed on the expression of TLR1, TLR5, TLR7, TLR15 and TLR21. Intriguingly, SBDSV infection significantly up-regulated the expression of RNA sensors such as MDA5 and LGP2, and resulted in a delayed but significant up-regulation of RIG-I gene. Taken together, these data indicate that host multiple sensors including DNA sensor (cGAS) and RNA sensors (TLR3, MDA5 and LGP2) are involved in recognizing a variety of different pathogen associated molecular patterns (PAMPs) including viral genomic ssDNA and dsRNA replication intermediates, which trigger an effective antiviral innate immune response.

1017P - Immune-related gene expression profiling after neoadjuvant chemotherapy (NACT) of ovarian high-grade serous carcinoma

BackgroundIn patients with stage III or IV HGSOC who are not suitable for primary surgery (PDS), 3 cycles of platinum based NACT followed by interval surgery (IDS) and adjuvant chemotherapy is an accepted treatment approach. NACT enhances host immune response by increasing levels of PD1, CTLA4 and PDL1 (Böhm S et al. Clin Cancer Res 2016). Gene expression profiling of tumors has identified prognostic signatures for patient selection with immunotherapy (Ribas A et al. J Clin Oncol 2015). MethodsThe purpose of this study is to assess the effect of neoadjuvant chemotherapy (NACT) on immune activation in stage IIIC/IV tubo-ovarian high-grade serous carcinoma (HGSOC). We obtained pre- and post-treatment omental biopsies from a total of 45 patients undergoing platinum-based NACT followed by IDS. We measured T-cell density and phenotype, immune activation, and markers of cancer-related inflammation using IHC. Messenger RNA expression was analyzed on the nCounter system using the PanCancer IO360 Panel. ResultsThere was evidence of T-cell activation in omental biopsies after NACT. CD4+ T cells showed enhanced IFNgproduction and antitumor Th1 gene signatures were increased. T-cell activation correlated with therapeutic response to NACT. The CD8 T-cell and CD45RO memory cell density in the tumor microenvironment was unchanged after NACT, but biopsies after a good therapeutic response had significantly fewer FoxP3+(Treg) cells. Biopsies of good therapeutic responders also showed areduction in a Treg gene signature in post versus pre-NACT samples.Preliminary results according to gene expression of the immune compartment reveal that tumor infiltrating lymphocytes were more abundant in samples from patients showing a good response over those with no or incomplete response to NACT, as well as an increase in gene signatures associated with antigen presentation and Cytokine and Chemokine Signaling. When comparing the expression profiles between samples obtained before or after NACT in patients showing incomplete response,we obtained an expected decreased in cellular proliferation signature. ConclusionsNACT may promote an immune modulatory effect that could improve or favour the further use of specific immunotherapy in HGSOC patients. Legal entity responsible for the studyFundación para la Investigación 12 de Octubre. FundingTesaro SL. DisclosureL.M. Manso: Research grant / Funding (self): Tesaro; Advisory / Consultancy: Roche, Novartis, Tesaro, AstraZeneca, Pfizer, Clovis; Speaker Bureau / Expert testimony: Roche, Novartis, Tesaro, AstraZeneca, Pfizer, Clovis; Travel / Accommodation / Expenses: Roche, Novartis, Tesaro, Pfizer. S. Wang: Full / Part-time employment: Tesaro. All other authors have declared no conflicts of interest.

Clinical and immune profiling for cancer of unknown primary site

BackgroundImmune checkpoint inhibitors (ICIs) confer a survival benefit in many cancer types. Given that the survival outcome for cancer of unknown primary site (CUP) remains poor, we investigated the potential of CUP for immunotherapy.MethodsA total of 164 patients with CUP (favorable subset, 34 patients; unfavorable subset, 130 patients) who were treated between January 2009 and March 2017 was identified from a review of medical records at Kindai University Hospital. They included 92 patients for whom pretreatment tumor tissue was available both for determination of programmed cell death–ligand 1 expression and tumor-infiltrating lymphocyte (TIL) density by immunohistochemistry (IHC) and for immune-related gene expression profiling (irGEP). The results of irGEP for CUP were compared with published data for ICI-treated solid cancers classified into progressive disease (PD) and non-PD subsets according to their best response to ICIs.ResultsThe median overall survival of all CUP patients was 29.3 months (95% confidence interval [CI], 15.7–not reached) and 7.1 months (95% CI, 5.0–9.4) for favorable and unfavorable subsets, respectively. IHC and irGEP revealed that pretreatment immune activity—including expression of immune checkpoint molecules—for CUP was similar to that for ICI-responsive malignancies (antitumor immune cell signatures: CUP versus PD, P = 0.002–0.067; CUP versus non-PD, P = 0.591–0.999), although VEGFA expression was associated with suppression of antitumor immunity in CUP (P = 0.008, false discovery rate = 0.010). In addition, one case of CUP in the unfavorable subset that was associated with prominent PD-L1 expression on TILs and showed a durable response to nivolumab is presented.ConclusionsThe survival outcome of CUP remains unsatisfactory. However, our clinical and immune profiling of CUP has revealed a potential to benefit from immunotherapy, with ICIs thus being a potential option for CUP treatment.

Citrobacter freundii infection in red swamp crayfish (Procambarus clarkii) and host immune-related gene expression profiles

Citrobacter freundii is widely distributed in nature as a typical conditional pathogen. In this study, the pathogenicity and histopathology of crayfish (Procambarus clarkii) infected with C. freundii were investigated, and immune-related gene expression profiling was performed. Challenge experiments confirmed that the isolated LJ1 strain of C. freundii had high virulence, leading to significant deaths of otherwise healthy shrimp. Histopathological analysis revealed that the hepatopancreas, intestines, and gills of diseased crayfish exhibited obvious inflammatory responses to C. freundii infection. Quantitative real-time PCR (qRT-PCR) was also performed to investigate the expression pattern of twelve immune-related genes in gill, intestine, hepatopancreas, and hemocyte samples. The results showed various expression profiles and clear transcriptional activation of these immune related genes in the tested tissues. These results will contribute to further study of C. freundii infection and help to understand P. clarkii immune function.

Abstract 2242: Roles of FGFR inhibition on murine RCC cells in combination antitumor activity of lenvatinib with anti-PD1 antibody via regulatingIFN gamma signaling pathway

Abstract Lenvatinib mesilate (LEN) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of VEGFR1-3, in addition to other proangiogenic and oncogenic pathway-related RTKs including FGFR1-4; PDGFRα; KIT; and RET. LEN plus everolimus for advanced renal cell carcinoma (RCC) after one prior anti-VEGF therapy was approved in the US and EU in 2016. Currently, Phase1b/2 clinical trial of LEN in combination with pembrolizumab with selected solid tumors including RCC and Phase3 study of LEN in combination with everolimus or pembrolizumab for metastatic RCC patients are in progress. In this study, we investigated the antitumor and immunomodulatory activities of LEN alone and in combination with anti-PD1 Ab and explored the mechanism of action underlying the activities of LEN and PD1 blockade in preclinical syngeneic RCC models. We examined antitumor activity of combination treatment of LEN at 10 mg/kg (po, qd) and anti-PD1 Ab at 10 mg/kg (ip, twice weekly) in the murine subcutaneous RCC model using RAG cell line. Immune cell population analyses in tumor tissues were performed by flow cytometer. Immune-related gene expression profile in tumor tissues or cultured cells was analyzed by qPCR and RNA seq. Effects on FGFR and IFN gamma (IFNγ) signaling pathways and their downstream molecules in cultured cells were analyzed by western blot. In the murine syngeneic RAG tumor model, the combination treatment of LEN and anti-PD1 Ab showed tumor shrinkage, and some of tumors were regressed to nonpalpable sizes with long duration of response. LEN alone and in combination with anti-PD1 Ab significantly decreased the population of tumor associated macrophage in tumor tissues by FCM analysis. Gene expression analysis for immune cell responses in tumor tissues revealed the upregulation of IFNγ and its downstream genes including PD-L1 with combination of LEN and anti-PD1 Ab. Western blot analysis elucidated that FGFR signaling pathway inhibited the activation of IFNγ signaling pathway and the induction of PD-L1 expression in murine RCC cells, and LEN-treatment reactivated the IFNγ signaling pathway by inhibition of FGFR signaling. Combination of LEN and anti-PD1 Ab showed greater antitumor activity including tumor shrinkage with long duration of response in the murine RCC model. Modulation of immune microenvironment by LEN and combination of LEN with PD1 Ab may underlie the strong antitumor activity in preclinical RCC model. Inhibitory activity of LEN against FGFR reactivated the IFNγ pathway and PD-L1 expression suppressed by activation of FGFR signaling pathway, and this inhibitory activity of LEN may potentiate the antitumor activity of combination treatments with anti-PD1 Ab. These preclinical results provide one of the unique mechanisms of combinational effect of LEN with PD1 blockade in the preclinical RCC model. Citation Format: Yusuke Adachi, Kenji Ichikawa, Yu Kato, Yasuhiro Funahashi. Roles of FGFR inhibition on murine RCC cells in combination antitumor activity of lenvatinib with anti-PD1 antibody via regulatingIFN gamma signaling pathway [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 2242.

Immune-related gene expression profiles of hypothermia adipocytes: Implications for Bell's palsy.

To identify immune-related gene expression profiles of adipocytes under low temperatures with RNA sequencing as a model for Bell's palsy implications. Adipocytes were harvested from the white adipose tissue of male Sprague-Dawley rats and cultured under different acute-grade cold exposure conditions of 30, 20, and 10°C, and their genomes were sequenced for RNA sequencing analysis. The differentially expressed genes (DEGs) were validated with reverse transcription polymerase chain reaction. In total, 55 (35 upregulated and 20 downregulated), 121 (76 upregulated and 45 downregulated), and 92 (64 upregulated and 28 downregulated) DEGs were identified under 30, 20, and 10°C compared with the control, respectively. KEGG and GO analysis revealed that the DEGs were considerably enriched in immune-related pathways (leukocyte transendothelial migration and platelet activation) and infection (bacterial invasion of epithelial cells and Salmonella infection). The levels of key inflammatory chemokines (CSF1, CXCL1, CCL2, and CCL7) were enhanced after cold exposure. These findings broaden our understanding of the immune responses to cold exposure in adipocytes. The molecular profiles of adipocyte immune function will help clarify the potential mechanism impacting myelin, which might contribute to the development of strategies to control Bell's palsy.

Interference of tumour mutational burden with outcome of patients with head and neck cancer treated with definitive chemoradiation: a multicentre retrospective study of the German Cancer Consortium Radiation Oncology Group

BackgroundTumour mutational burden (TMB) estimated from whole exome sequencing or comprehensive gene panels has previously been established as predictive factor of response to immune checkpoint inhibitors (ICIs). Its predictive value for the efficacy of concurrent chemoradiation (cCRTX), a potential combination partner of ICI, remains unknown. MethodsThe accuracy of TMB estimation by an in-house 327-gene panel was established in the Cancer Genome Atlas (TCGA) head and neck squamous cell carcinoma (HNSCC) data set. Interference of TMB with outcome after cCRTX was determined in a multicentre cohort of patients with locally advanced HNSCC uniformly treated with cCRTX. Targeted next-generation sequencing was successfully applied in 101 formalin-fixed, paraffin-embedded pretreatment tumour samples. In a subset of cases (n = 40), tumour RNA was used for immune-related gene expression profiling by the nanoString platform. TMB was correlated with TP53 genotype, human papilloma virus (HPV) status, immune expression signatures and survival parameters. Results were validated in the TCGA HNSCC cohort. ResultsA high accuracy of TMB estimation by the 327-gene panel was established. High TMB was significantly associated with an increased prevalence of TP53 mutations and immune gene expression patterns unrelated to T cell–inflamed gene expression profiles. Kaplan-Meier analysis revealed significantly reduced overall survival in the patient group with high TMB (hazard ratio for death: 1.79, 95% confidence interval: 1.02–3.14; P = 0.042) which remained significant after correcting for confounding factors in the multivariate model. The prognostic value of TMB was confirmed in the TCGA HNSCC cohort. ConclusionHigh TMB identifies HNSCC patients with poor outcome after cCRTX who might preferentially benefit from CRTX-ICI combinations.

Comparative susceptibilities and immune-related gene expressions of brown trout strains and their hybrids infected with Lactococcus garvieae and Yersinia ruckeri

Brown trout are polymorphic salmonid species, and it is of importance to investigate whether hybridization affects disease resistance. In this study, susceptibility of brown trout (Salmo trutta Abant, Anatolian, Black Sea, and Caspius) strains and their hybrids to Lactococcus garvieae and Yersinia ruckeri as well as their immune-related gene expression profiles were studied. Results indicated that reciprocal hybridization did not affect disease resistance in brown trout strains. Purebred Black Sea strain of brown trout was the most resistant group against Y. ruckeri, followed by other Black Sea strain hybrids. On the other hand, purebred Anatolian strain was the most resistant group to L. garvieae, followed by other Anatolian strain hybrids. Expression pattern of target genes differed in families, but the overall gene expression was comparatively high in Y. ruckeri infected families. Upregulations were mainly significant at 7 and 28 d post infection while marginal regulations were observed 8 h after infection. Disease resistance status of strains was supported by high expression of immune-related genes such as major histocompatibility complex class I (MHC-I), immunoglobulin light chain (IgL), and antioxidant- and hemoglobin-related gene expression. Therefore, our findings suggest that Black Sea and Anatolian strains could be used to develop fish stock that are resistant for yersiniosis and lactocaccosis, respectively.

Effects of microcystin-LR on the immune dysfunction and ultrastructure of hepatopancreas in giant freshwater prawn Macrobrachium rosenbergii

Microcystins (MCs), produced by cyanobacteria, can strongly inhibit the activity of protein phosphatase, and exhibit strong hepatotoxicity. Macrobrachium rosenbergii is an important aquaculture economic species. Cyanobacterial blooms occur frequently during the culture of M. rosenbergii. However, the effects of MCs on the M. rosenbergii immune function have not been studied. In the present study, M. rosenbergii were exposed to environment-related concentrations of MC-LR type (0.5 and 5 μg/L) for 3 weeks. Hepatopancreatic histology was investigated, and antioxidant enzymes activity, acid phosphatase, alkaline phosphatase and lysozyme activity in hepatopancreas were also analyzed. Results showed that MC-LR could damage M. rosenbergii hepatopancreas, induce hepatopancreatic apoptosis and antioxidant dysfunctions. The expression profiles of major immune-related genes after MC-LR exposure were also detected. Some genes with antibacterial functions were suppressed, and the expression of apoptosis-related genes were up-regulated. After MC-LR exposure, the cumulative mortality of M. rosenbergii infected with Vibrio vulnificus and Aeromonas hydrophila were much higher than the control in a time-dose dependent manner. These results indicated the potential negative influence of MC-LR on the immune function of M. rosenbergii.

Development of a prognostic index based on an immunogenomic landscape analysis of papillary thyroid cancer

Background: Papillary thyroid cancer (PTC) is the most common subtype of thyroid cancer, and inflammation relates significantly to its initiation and prognosis. Systematic exploration of the immunogenomic landscape therein to assist in PTC prognosis is therefore urgent. The Cancer Genome Atlas (TCGA) project provides a large number of genetic PTC samples that enable a comprehensive and reliable immunogenomic study.Methods: We integrated the expression profiles of immune-related genes (IRGs) and progression-free intervals (PFIs) in survival in 493 PTC patients based on the TCGA dataset. Differentially-expressed and survival-associated IRGs in PTC patients were estimated a computational difference algorithm and COX regression analysis. The potential molecular mechanisms and properties of these PTC-specific IRGs were also explored with the help of computational biology. A new prognostic index based on immune-related genes was developed by using multivariable COX analysis.Results: A total of 46 differentially expressed immune-related genes were significantly correlated with clinical outcome of PTC patients. Functional enrichment analysis revealed that these genes were actively involved in a cytokine-cytokine receptor interaction KEGG pathway. A prognostic signature based on RGs (AGTR1, CTGF, FAM3B, IL11, IL17C, PTH2R and SPAG11A) performed moderately in prognostic predictions and correlated with age, tumor stage, metastasis, number of lesions, and tumor burden. Intriguingly, the prognostic index based on IRGs reflected infiltration by several types of immune cells.Conclusions: Together, our results screened several IRGs of clinical significance, revealed drivers of the immune repertoire, and demonstrated the importance of a personalized, IRG-based immune signature in the recognition, surveillance, and prognosis of PTC.

Transcriptome profile in bursa of Fabricius reveals potential mode for stress-influenced immune function in chicken stress model

BackgroundThe molecular mechanisms underlying stress-influenced immune function of chicken (Gallus Gallus) are not clear. The stress models can be established effectively by feeding chickens corticosterone (CORT) hormone. The bursa of Fabricius is a unique central immune organ of birds. RNA-Seq technology was used to investigate differences in the expression profiles of immune-related genes and associated pathways in the bursa of Fabricius to clarify molecular mechanisms. The aim of this study was to broaden the understanding of the stress-influenced immune function in chickens.ResultsDifferentially expressed genes (DEGs) in the bursa of Fabricius between experimental group (basal diet with added CORT 30 mg/kg; C_B group) and control group (basal diet; B_B group) were identified by using RNA-seq technology. In total, we found 1434 significant DEGs (SDEGs), which included 199 upregulated and 1235 downregulated genes in the C_B group compared with the B_B group. The immune system process GO term was the top significantly GO term, including MYD88, TLR4, IL15, VEGFA gene and so on. The cytokine-cytokine receptor interaction pathway and the Toll-like receptor signaling pathway were the key pathways affected by stress. The protein-protein interaction (PPI) analysis of the SDEGs showed that VEGFA, MyD88 and IL15 were hub genes and module analysis showed that MYD88, TLR4 and VEGFA play important roles in response to stress.ConclusionThis study showed that the VEGFA and ILs (such as IL15) via the cytokine-cytokine receptor interaction pathway, MYD88 and TLR4 via the Toll-like receptor signaling pathway may play important roles in the regulation of immune function under stress condition with CORT administration. The results of this study provide a reference for further studies of the molecular mechanisms of stress-influenced immune function.

Digital PCR-Based T-cell Quantification–Assisted Deconvolution of the Microenvironment Reveals that Activated Macrophages Drive Tumor Inflammation in Uveal Melanoma

Uveal melanoma progression can be predicted by gene expression profiles enabling a clear subdivision between tumors with a good (class I) and a poor (class II) prognosis. Poor prognosis uveal melanoma can be subdivided by expression of immune-related genes; however, it is unclear whether this subclassification is justified; therefore, T cells in uveal melanoma specimens were quantified using a digital PCR approach. Absolute T-cell quantification revealed that T-cell influx is present in all uveal melanomas associated with a poor prognosis. However, this infiltrate is only accompanied by differential immune-related gene expression profiles in uveal melanoma with the highest T-cell infiltrate. Molecular deconvolution of the immune profile revealed that a large proportion of the T-cell-related gene expression signature does not originate from lymphocytes but is derived from other immune cells, especially macrophages. Expression of the lymphocyte-homing chemokine CXCL10 by activated macrophages correlated with T-cell infiltration and thereby explains the correlation of T-cell numbers and macrophages. This was validated by in situ analysis of CXCL10 in uveal melanoma tissue with high T-cell counts. Surprisingly, CXCL10 or any of the other genes in the activated macrophage-cluster was correlated with reduced survival due to uveal melanoma metastasis. This effect was independent of the T-cell infiltrate, which reveals a role for activated macrophages in metastasis formation independent of their role in tumor inflammation. IMPLICATIONS: The current report uses an innovative digital PCR method to study the immune environment and demonstrates that absolute T-cell quantification and expression profiles can dissect disparate immune components.

Effects of dietary Bacillus subtilis and Shewanella algae in expression profile of immune-related genes from hemolymph of Litopenaeus vannamei challenged with Vibrio parahaemolyticus

B. subtilis and S. algae effects in growth, survival and innate immunity were assessed on L. vannamei juveniles. During 60 days, shrimp were reared in three treatments: Bs, fed with 106 CFU of B. subtilis per gram of commercial feed, Sa, fed with 106 CFU of S. algae per gram of commercial feed and Control (without bacterial addition). Then, the animals were subjected to a V. parahaemolyticus challenge. For this purpose, four treatments were established: Control (shrimp not submitted to probiotic treatments), Vibrio (Vibrio challenged shrimp), Vibrio + Bs (Bs challenged shrimp) and Vibrio + Sa (Sa challenged shrimp). Shrimp hemolymph was sampled 45-days after rearing and 24 h post-challenge for quantification of prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) and hemocyanin (HEM) transcripts by qPCR. Moreover, shrimp final weight and survival were also verified. B. subtilis administration enhanced shrimp growth and improved proPO, LGBP and HEM expression levels before and after challenge. After 60-days of feeding, Sa final weight was higher than the Control, whereas Vibrio + Sa cumulative mortality after 48 h of Vibrio challenge was lower than Vibrio group. These results could be correlated with the proPO and LGBP up regulation in Vibrio + Sa compared to Vibrio group, protecting L. vannamei from the bacterial infection. Together, these results suggest the probiotic potential of B. subtilis e S. algae in the modulation of immune-related genes as a tool to control V. parahaemolyticus infection inside shrimp.

Histopathological analysis and the immune related gene expression profiles of mandarin fish (Siniperca chuatsi) infected with Aeromonas hydrophila

Hemorrhagic septicemia of mandarin fish (Siniperca chuatsi) was mainly caused by Aeromonas hydrophila which was an opportunistic pathogen. In recent years, the disease has caused tremendous economic loss with high morbidity and mass mortality in the mandarin fish breeding industry. Histopathological analysis and the immune related gene expression profiles of mandarin fish (S. chuatsi) infected with A. hydrophila were investigated in this study. Transmission electron microscopy (TEM) images showed that the cells of A. hydrophila densely covered with a mass of fimbriae. Histopathological analysis revealed that inflammation, vacuolization and extensive necrosis existed in the gill, liver, spleen and head kidney of the diseased fish. Quantitative real-time PCR was performed to measure mRNA expression levels for six immune related genes in mandarin fish after A. hydrophila infection. The transcriptional analysis of these immune related genes demonstrated that the expression levels of major histocompatibility complex class II (MHC II), T cell receptor α (TCRα), tumor necrosis factor α (TNFα), CC chemokine 3, interleukin 8 (IL-8) and Hepcidin were strongly up-regulated in spleen and head kidney of mandarin fish post-infection. These results will contribute to further study on the pathogenesis and host defensive system in A. hydrophila infection.

Development of a Prognostic Index Based on an Immunogenomic Landscape Analysis of Papillary Thyroid Cancer

Papillary thyroid cancer (PTC) is the most common subtype of thyroid cancer, and inflammation relates significantly to its initiation and prognosis. Systematic exploration of the immunogenomic landscape therein to assist in PTC prognosis is therefore urgent. The Cancer Genome Atlas (TCGA) project provides a large number of genetic PTC samples that enable a comprehensive and reliable immunogenomic study. Hence, we integrated the expression profiles of immune-related genes (IRGs) and progression-free intervals (PFIs) in survival in 493 PTC patients based on the TCGA dataset. We first defined differentially-expressed and survival-associated IRGs in PTC patients with a computational difference algorithm and COX regression analysis. The potential molecular mechanisms and properties of these PTC-specific IRGs were also explored with the help of computational biology. A new prognostic index based on immune-related genes was developed, which proved adept at evaluating the PFIs of PTC patients. Relationships between the prognostic index and several clinical and demographic characteristics were also characterized. Intriguingly, the prognostic index based on IRGs reflected infiltration by several types of immune cells. Together, our results screened several IRGs of clinical significance, revealed drivers of the immune repertoire, and demonstrated the importance of a personalized, IRG-based immune signature in the recognition, surveillance, and prognosis of PTC. Funding: This study was supported by grants from Innovation Project of Guangxi Graduate Education (YCSW2018104), Guangxi Medical University Training Program for Distinguished Young Scholars, Medical Excellence Award Funded by the Creative Research Development Grant from the First Affiliated Hospital of Guangxi Medical University and Fund of Guangxi Provincial Health Bureau Scientific Research Project (Z2018). Declaration of Interest: The authors have no conflicts of interest.

Nile tilapia fry fed on antimicrobial peptide Epinecidin-1-expressing Artemia cyst exhibit enhanced immunity against acute bacterial infection

Artemia are often used as a live feed for fry in aquaculture. We have previously demonstrated that supplementing adult zebrafish feed with Artemia, which express an Epinephelus coioides-derived antimicrobial peptide, Epinecidin-1 (Epi-1), protects against bacterial infection. Thus, Artemia may serve as a bioreactor for producing biofunctional molecules. However, the application of Epi-1 transgenic Artemia in larval aquaculture of commercial fish species has not been investigated. Here we used a Tol2-transposon system to generate stable Epi-1 expressing Artemia. Nile tilapia (Oreochromis niloticus) fry were then fed with decapsulated transgenic cysts and acutely challenged with Gram-positive Streptococcus iniae or Gram-negative Vibrio vulnificus (204). Survival analysis revealed that tilapia fry fed with Epi-1 transgenic cysts were resistant to acute bacterial infection. Immune-related gene expression profiling showed that S. iniae and V. vulnificus inoculations produced distinct immunomodulatory effects in the tilapia fry. Upon S. iniae infection, tilapia fry fed on control diet exhibited an immune response dominated by Tlr-7/MyD88, wherein Tnf-α, Il-8 and Cxcl-10 expression were all induced; conversely, the tilapia fry fed with Epi-1 transgenic cysts showed a Tlr-2/Tlr-5-dominant immune response, marked by the induction of Il-1β, Il-8 and Il-12 expression. However, after V. vulnificus (204) infection control fry exhibited a Tlr-2/MyD88/Traf-6-dominant response with activation of Tnf-α and Il-8 expression; meanwhile tilapia fry fed on Epi-1 transgenic cyst showed a dominant Tlr-2/Tlr-5-mediated immune response, including induction of Il-1β, Il-8, Il-12, and Cxcl-10 expression. These findings suggest that feeding larval fish fry with Epi-1 transgenic Artemia cysts confers enhanced immunity toward bacterial challenge. Epi-1 transgenic cysts should therefore be considered as a potential functional feed for larval aquaculture.

Equine mesenchymal stromal cells from different tissue sources display comparable immune-related gene expression profiles in response to interferon gamma (IFN)-γ

Mesenchymal stromal cells (MSC) have the therapeutic potential to decrease inflammation due to their immunomodulatory properties. They can be isolated from various tissue sources such as bone marrow, adipose tissue, and blood, but it is unknown how the tissue source of origin affects the responses of MSC to inflammatory stimuli. Here, we conceptually addressed this question by evaluating the immune-related gene expression profiles of equine MSC from different tissue sources in response to interferon gamma (IFN-γ) stimulation, with the goal to determine if there is a preferable MSC source for clinical application in an inflammatory environment. The salient findings from this initial study were that the baseline expression of all immune related genes analyzed, with the exception of prostaglandin-endoperoxide synthase 2 (PTGS2), was variable in MSC depending on tissue source. Following IFN-γ stimulation, however, gene expression profiles became more similar across all tissue sources, suggesting that MSC from different sources will likely respond similarly in an inflammatory environment when used clinically.

Targeting the TGF\u03b2 pathway with galunisertib, a TGF\u03b2RI small molecule inhibitor, promotes anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint blockade

BackgroundTGFβ signaling plays a pleotropic role in tumor biology, promoting tumor proliferation, invasion and metastasis, and escape from immune surveillance. Inhibiting TGFβ’s immune suppressive effects has become of particular interest as a way to increase the benefit of cancer immunotherapy. Here we utilized preclinical models to explore the impact of the clinical stage TGFβ pathway inhibitor, galunisertib, on anti-tumor immunity at clinically relevant doses.ResultsIn vitro treatment with galunisertib reversed TGFβ and regulatory T cell mediated suppression of human T cell proliferation. In vivo treatment of mice with established 4T1-LP tumors resulted in strong dose-dependent anti-tumor activity with close to 100% inhibition of tumor growth and complete regressions upon cessation of treatment in 50% of animals. This effect was CD8+ T cell dependent, and led to increased T cell numbers in treated tumors. Mice with durable regressions rejected tumor rechallenge, demonstrating the establishment of immunological memory. Consequently, mice that rejected immunogenic 4T1-LP tumors were able to resist rechallenge with poorly immunogenic 4 T1 parental cells, suggesting the development of a secondary immune response via antigen spreading as a consequence of effective tumor targeting. Combination of galunisertib with PD-L1 blockade resulted in improved tumor growth inhibition and complete regressions in colon carcinoma models, demonstrating the potential synergy when cotargeting TGFβ and PD-1/PD-L1 pathways. Combination therapy was associated with enhanced anti-tumor immune related gene expression profile that was accelerated compared to anti-PD-L1 monotherapy.ConclusionsTogether these data highlight the ability of galunisertib to modulate T cell immunity and the therapeutic potential of combining galunisertib with current PD-1/L1 immunotherapy.