Expression Of Immunomodulatory Genes Research Articles

AIM high: Epigenetic modulation and immune stimulation in bladder cancer.

648 Background: Immune-checkpoint inhibitors (ICI) have emerged as valuable treatment options for patients with bladder cancer, particularly those who are ineligible for other therapies or have experienced disease progression on treatment. Response rates are only 20-30%, highlighting the need for biomarkers to predict treatment response and strategies to overcome resistance. Epigenetic modulators including the DNA methyltransferase (DNMT) inhibitor 5-azacitidine (AZA) and histone deacetylase inhibitors (HDACi) may represent co-targeting therapies in combination with ICI to enhance antitumor immunity. AZA has been demonstrated to increase the expression of a panel of immunomodulatory genes termed the “AZA Immune gene panel (AIM)” in breast, colorectal, and ovarian cancer cell lines. We hypothesized that bladder cancer with low AIM expression can be therapeutically converted by epigenetic modulation to “AIM-high” for future combination with ICI. Methods: Five human bladder cancer cell lines were selected: SCaBER, SW780, T24, RT4 and one developed at our institution (CoCaB1). The Cancer Cell Line Encyclopedia (CCLE) was used to screen the four included lines for intrinsic AIM expression, which were predicted to be AIM-high (n=1) and AIM-low (n=3). A cell viability assay was performed to screen for AZA and HDACi toxicity. Cells were treated with drug or vehicle for 72 hours and harvested at day 6, 14, and 21. RNA was extracted for RNA sequencing (RNA-seq) analyses. For analysis of AIM gene expression in clinical specimens, RNA-seq data was obtained from primary (n=6) and metastatic (n=83) bladder cancer specimens for 20 patients participating in the University of Washington Bladder Cancer Rapid Autopsy Program. Results: RNA-seq analyses of 5 cell lines demonstrated increased expression (>log 2-fold change, p<0.05) of a subset of AIM genes involved in inflammation, interferon, cytokine/chemokine signaling, and cancer testis antigens (CTA, a family of immunogenic proteins) with AZA treatment. Co-treatment with HDACi showed additional upregulation of AIM genes. Gene set enrichment analyses uncovered enrichment in T-cell pathway activation. In metastatic bladder cancer tissues, interferon, cytokine/chemokine, inflammation, and CTA AIM gene set categories were upregulated in a subset of patients. AIM gene enrichment displayed a patient-dependent pattern and was consistent across metastatic sites within a patient. Conclusions: Epigenetic priming therapy increases the expression of immunomodulatory and T-cell related genes in bladder cancer cell lines with low baseline AIM expression. Additional correlative studies in the rapid autopsy series will further determine clinical- and treatment-related factors contributing to intrinsic immune-related gene expression. Building on this paradigm, future in vivo and clinical studies could lead to novel combination therapies for patients with bladder cancer.

Leveraging diverse cell-death patterns to predict the clinical outcome of immune checkpoint therapy in lung adenocarcinoma: Based on muti-omics analysis and vitro assay.

Advanced LUAD shows limited response to treatment including immune therapy. With the development of sequencing omics, it is urgent to combine high-throughput multi-omics data to identify new immune checkpoint therapeutic response markers. Using GSE72094 (n = 386) and GSE31210 (n = 226) gene expression profile data in the GEO database, we identified genes associated with lung adenocarcinoma (LUAD) death using tools such as "edgeR" and "maftools" and visualized the characteristics of these genes using the "circlize" R package. We constructed a prognostic model based on death-related genes and optimized the model using LASSO-Cox regression methods. By calculating the cell death index (CDI) of each individual, we divided LUAD patients into high and low CDI groups and examined the relationship between CDI and overall survival time by principal component analysis (PCA) and Kaplan-Meier analysis. We also used the "ConsensusClusterPlus" tool for unsupervised clustering of LUAD subtypes based on model genes. In addition, we collected data on the expression of immunomodulatory genes and model genes for each cohort and performed tumor microenvironment analyses. We also used the TIDE algorithm to predict immunotherapy responses in the CDI cohort. Finally, we studied the effect of PRKCD on the proliferation and migration of LUAD cells through cell culture experiments. The study utilized the TCGA-LUAD cohort (n = 493) and identified 2,901 genes that are differentially expressed in patients with LUAD. Through KEGG and GO enrichment analysis, these genes were found to be involved in a wide range of biological pathways. The study also used univariate Cox regression models and LASSO regression analyses to identify 17 candidate genes that were best associated with mortality prognostic risk scores. By comparing the overall survival (OS) outcomes of patients with different CDI values, it was found that increased CDI levels were significantly associated with lower OS rates. In addition, the study used unsupervised cluster analysis to divide 115 LUAD patients into two distinct clusters with significant differences in OS timing. Finally, a prognostic indicator called CDI was established and its feasibility as an independent prognostic indicator was evaluated by Cox proportional risk regression analysis. The immunotherapy efficacy was more sensitive in the group with high expression of programmed cell death models. Relationship between programmed cell death (PCD) signature models and drug reactivity. After evaluating the median inhibitory concentration (IC50) of various drugs in LUAD samples, statistically significant differences in IC50 values were found in cohorts with high and low CDI status. Specifically, Gefitinib and Lapatinib had higher IC50 values in the high-CDI cohort, while Olaparib, Oxaliplatin, SB216763, and Axitinib had lower values. These results suggest that individuals with high CDI levels are sensitive to tyrosine kinase inhibitors and may be resistant to conventional chemotherapy. Therefore, this study constructed a gene model that can evaluate patient immunotherapy by using programmed cell death-related genes based on muti-omics. The CDI index composed of these programmed cell death-related genes reveals the heterogeneity of lung adenocarcinoma tumors and serves as a prognostic indicator for patients.

A novel system to culture human intestinal organoids under physiological oxygen content to study microbial-host interaction.

Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24-48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease.

M6A modification patterns are associated with copy number burden and tumor immune landscape in thyroid cancer

BackgroundThe association involving N6-methyladenosine (m6A) modification, molecular subtype and specific immune cell group in tumor microenvironment has been the focus of recent studies. The underlying function of m6A modification in thyroid cancer (TC) remains elusive.MethodsThe m6A modification regulations, molecular character and tumor immune profile of 461 TC patients were explored and then the correlation between them were comprehensively evaluated. The m6Ascore was established using principal component analysis (PCA) to quantify the m6A pattern of individual TC patients. The prognostic significance of the m6Ascore was evaluated by multivariate Cox regression analysis.ResultsFour m6Aclusters (mc1, 2, 3, 4)—characterized by differences in extent of aneuploidy, expression of immunomodulatory genes, mRNA or lncRNA expression pattern and prognosis were identified. T Preliminary validation of m6Ascore was a potential independent prognostic factor of TC involving in mc3. Finally, the prognostic value of the m6Ascore and its association with copy number variation (CNV) and tumor immune microenvironment (TIME) of TC in mc3 were verified.ConclusionsThe correlation between m6A modification, the copy number burden and tumor immune landscape in TC was demonstrated. A m6Acluster-mc3 with low m6Ascore and high CNV molecular subtype was identified with poor clinical prognosis, low infiltrating immunocyte and weak effector T cell. A three-gene clinical prognosis model for TC based on 4 m6a cluster expression was established. Understanding of TIME is enhanced by comprehensive assessment of m6A patterns in individual TC patients and gives a new insight toward improved immunotherapy strategies for TC cancer patients.

Role of HDAC6-STAT3 in immunomodulatory pathways in Colorectal cancer cells

Colorectal cancer (CRC) is one of the most common malignant neoplasms and the second leading cause of death from tumors worldwide. Therefore, there is a great need to study new therapeutical strategies, such as effective immunotherapies against these malignancies. Unfortunately, many CRC patients do not respond to current standard immunotherapies, making it necessary to search for adjuvant treatments. Histone deacetylase 6 (HDAC6) is involved in several processes, including immune response and tumor progression. Specifically, it has been observed that HDAC6 is required to activate the Signal Transducer and Activator of Transcription 3 (STAT3), a transcription factor involved in immunogenicity, by activating different genes in these pathways, such as PD-L1. Over-expression of immunosuppressive pathways in cancer cells deregulates T-cell activation. Therefore, we focused on the pharmacological inhibition of HDAC6 in CRC cells because of its potential as an adjuvant to avoid immunotolerance in immunotherapy. We investigated whether HDAC6 inhibitors (HDAC6is), such as Nexturastat A (NextA), affected STAT3 activation in CRC cells. First, we found that NextA is less cytotoxic than the non-selective HDACis panobinostat. Then, NextA modified STAT3 and decreased the mRNA and protein expression levels of PD-L1. Importantly, transcriptomic analysis showed that NextA treatment affected the expression of critical genes involved in immunomodulatory pathways in CRC malignancies. These results suggest that treatments with NextA reduce the functionality of STAT3 in CRC cells, impacting the expression of immunomodulatory genes involved in the inflammatory and immune responses. Therefore, targeting HDAC6 may represent an interesting adjuvant strategy in combination with immunotherapy.

Mechanosignaling YAP/TAZ-TEAD Axis Regulates the Immunomodulatory Properties of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have gained significant attention in cell therapies due to their multipotency and immunomodulatory capacities. The transcriptional co-activators YAP/TAZ, central to the mechanotransduction system in MSCs, dominantly direct MSCs lineage commitment. However, their role in immunomodulation remains elusive. Accordingly, this present study aimed to investigate the role of mechanotransducer YAP/TAZ and their binding target transcriptional factor, TEAD, in the immunomodulatory capacities of human bone marrow-derived MSCs. Reducing YAP/TAZ activity by altering the matrix stiffness, disrupting the F-actin integrity with chemical inhibitors, or using siRNAs increased the expression of immunomodulatory genes, such as TSG-6 and IDO, upon TNF-α stimulation. Similarly, transfection of TEAD siRNA also increased the immunomodulatory capacities in MSCs. RNA-seq analysis and inhibition assays demonstrated that the immunomodulatory capacities caused by YAP/TAZ-TEAD axis disruption were due to the NF-κB signaling pathway activation. Then, we also evaluated the in vivo anti-inflammatory efficacy of MSCs in a dextran sulfate sodium (DSS)-induced mice colitis model. The administration of human MSCs transfected with TEAD siRNA, which exhibited enhanced immunomodulatory properties in vitro, significantly ameliorated inflammatory bowel disease symptoms, such as body weight loss and acute colon inflammation, in the DSS-induced mice colitis model. Our findings underscore the mechanosignaling YAP/TAZ-TEAD axis as a regulator of MSCs immunomodulation. Targeting these signaling pathways could herald promising MSCs-based therapies for immune disorders.

Systematic analysis of DNA methylation-mediated TF dysregulation on lncRNAs reveals critical roles in tumor immunity

Emerging evidence suggests that DNA methylation affects transcriptional regulation and expression perturbations of long non-coding RNAs (lncRNAs) in cancer. However, a comprehensive investigation into the transcriptional control of DNA methylation-mediated dysregulation of transcription factors (TFs) on lncRNAs has been lacking. Here, we integrated the transcriptome, methylome, and regulatome across 21 human cancers and systematically identified the transcriptional regulation of DNA methylation-mediated TF dysregulations (DMTDs) on lncRNAs. Our findings reveal that TF regulation of lncRNAs is significantly impacted by DNA methylation. Comparative analysis of DMTDs on mRNAs revealed a conserved pattern of TFs involvement. Pan-cancer Methylation TFs (MethTFs) and Methylation LncRNAs (MethLncRNAs) were identified, and were found to be closely associated with cancer hallmarks and clinical features. In-depth analysis of co-expressed mRNAs with pan-cancer MethLncRNAs unveiled frequent disruptions in cancer immunity, particularly in the context of inflammatory response. Furthermore, we identified five immune-related network modules that contribute to immune cell infiltration in cancer. Immune-related subtypes were subsequently classified, characterized by high levels of immune cell infiltration, expression of immunomodulatory genes, and relevant immune cytolytic activity score, major histocompatibility complex score, response to chemotherapy, and prognosis. Our findings provide valuable insights into cancer immunity from the epigenetic and transcriptional regulation perspective.

Expansion of human bone marrow-derived mesenchymal stromal cells with enhanced immunomodulatory properties

BackgroundMesenchymal stromal cells (MSCs) have broad potential as a cell therapy including for the treatment of drug-resistant inflammatory conditions with abnormal T cell proliferation such as graft-versus-host disease (GVHD). Clinical success, however, has been complicated by the heterogeneity of culture-expanded MSCs as well as donor variability. Here, we devise culture conditions that promote expansion of MSCs with enhanced immunomodulatory functions both in vitro and in animal models of GVHD.MethodsHuman bone marrow-derived MSCs were expanded at high-confluency (MSCHC) and low-confluency state (MSCLC). Their immunomodulatory properties were evaluated with in vitro co-culture assays based on suppression of activated T cell proliferation and secretion of pro-inflammatory cytokines from activated T cells. Metabolic state of these cells was determined, while RNA sequencing was performed to explore transcriptome of these MSCs. Ex vivo expanded MSCHC or MSCLC was injected into human peripheral blood mononuclear cells (PBMC)-induced GVHD mouse model to determine their in vivo therapeutic efficacy based on clinical grade scoring, human CD45+ blood count and histopathological examination.ResultsAs compared to MSCLC, MSCHC significantly reduced both the proliferation of anti-CD3/CD28-activated T cells and secretion of pro-inflammatory cytokines upon MSCHC co-culture across several donors even in the absence of cytokine priming. Mechanistically, metabolic analysis of MSCHC prior to co-culture with activated T cells showed increased glycolytic metabolism and lactate secretion compared to MSCLC, consistent with their ability to inhibit T cell proliferation. Transcriptome analysis further revealed differential expression of immunomodulatory genes including TRIM29, BPIFB4, MMP3 and SPP1 in MSCHC as well as enriched pathways including cytokine–cytokine receptor interactions, cell adhesion and PI3K-AKT signalling. Lastly, we demonstrate in a human PBMC-induced GVHD mouse model that delivery of MSCHC showed greater suppression of inflammation and improved outcomes compared to MSCLC and saline controls.ConclusionOur study provides evidence that ex vivo expansion of MSCs at high confluency alters the metabolic and transcriptomic states of these cells. Importantly, this approach maximizes the production of MSCs with enhanced immunomodulatory functions without priming, thus providing a non-invasive and generalizable strategy for improving the use of MSCs for the treatment of inflammatory diseases.

Tumor cell-intrinsic c-Myb upregulation stimulates antitumor immunity in a murine colorectal cancer model.

The transcription factor c-Myb is overexpressed in many different types of solid tumors, including colorectal cancer. However, its exact role in tumorigenesis is unclear. In this study, we show that tumor-intrinsic c-Myb expression in mouse models of colon cancer and melanoma suppresses tumor growth. While no differences in proliferation, apoptosis, and angiogenesis of tumors were evident tumors with distinct levels of c-Myb expression, we observed changes in intratumoral immune cell infiltrates. MC38 tumors with upregulated c-Myb expression showed increased numbers of CD103+ dendritic cells and eosinophils, but decreased tumor-associated macrophages (TAMs). Concomitantly, an increase in the number of activated cytotoxic CD8+ T cells upon c-Myb upregulation was observed, which correlated with a pro-inflammatory tumor microenvironment and increased numbers of M1 polarized TAMs. Mechanistically, c-Myb upregulation in immunogenic MC38 colon cancer cells resulted in enhanced expression of immunomodulatory genes, including those encoding 2-microglobulin and IFNβ, and decreased expression of the gene encoding the chemokine receptor CCR2. The increased numbers of activated cytotoxic CD8+ T cells contributed to tumor growth attenuation. In poorly immunogenic CT26, LLC, and B16-BL6 tumor cells, c-Myb upregulation did not affect the immunomodulatory gene expression. Despite this, c-Myb upregulation led to reduced B16-BL6 tumor growth but it did not affect tumor growth of CT26 and LLC tumors. Altogether, we postulate that c-Myb functions as a tumor suppressor in a tumor cell-type specific manner and modulates antitumor immunity.

MMR gene patterns evaluation provides novel insights for personalized immunotherapy compared to neoadjuvant chemotherapy in lung adenocarcinama

BackgroundThe association involving mismatch repair (MMR) genes, molecular subtype and specific immune cell group in tumor microenvironment has been focused by more recent studies. Its prognosis value in lung adenocarcinoma (LUAD) neoadjuvant chemotherapy remains elusive.MethodsThe correlation between the MMR gene patterns and the immune landscape were comprehensively evaluated. The MMRScore was calculated using principal component analysis (PCA) after grouping using R/mclust package. The prognostic significance of the MMRScore was evaluated by Kaplan-merrier analysis. Then a cohort of 103 Chinese LUAD patients was collected for neoadjuvant chemotherapy prognosis evaluation and validation using MMRScore.ResultsFour MMRclusters (mc1, 2, 3, 4)-characterized by differences in extent of aneuploidy, expression of immunomodulatory (IM) genes, mRNA expression, lncRNA expression and prognosis were identified. We established MMRscore to quantify the MMR pattern of individual LUAD patients. As is shown in further analyses, the MMRscore was a potential independent prognostic factor of LUAD. Finally, the prognostic value of the MMRscore and its association with tumor immune microenvironment (TIME) of LUAD were verified in Chinese LUAD cohort.ConclusionsWe demonstrated the correlation between MMR gene pattern, the CNV and tumor immune landscape in LUAD. A MMRcluster mc2 with high MMRscore, high TMB and high CNV subtype was identified with poor prognosis and infiltrating immunocyte. The comprehensive evaluation of MMR patterns in individual LUAD patients enhances the understanding of TIME and gives a new insight toward improved immune treatment strategies for LUAD patients compared to neoadjuvant chemotherapy.

Characterizing the molecular landscape of early onset and average onset prostate cancer.

5059 Background: Prostate cancer (PC) has a median onset age of 66, however, recent evidence suggests an increase in incidence of PC diagnosis in males <55 years of age. Family history and increased mutational burden has been associated with early onset PC (EOPC), nonetheless, comprehensive molecular and immune signatures that cluster in EOPC and average onset PC (AOPC) is poorly understood. Here, we characterized EOPC and AOPC, and their association with molecular and immune signature. Methods: 5,305 PC samples (<55 yrs, n=575; ≥65 yrs, n=4730) were tested by NGS (592, NextSeq; WES, NovaSeq), WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). PC Patients with age <55 and ≥65 was classified as EOPC and AOPC, respectively. Microsatellite-instability (MSI) was tested by IHC and NGS. Tumor mutational burden (TMB) totaled somatic mutations per tumor (high≥10 mt/MB). Androgen receptor (AR) signature and Neuroendocrine Prostate Cancer (NEPC) score was calculated based on expression of a list of previously defined genes (Hieronymus et al. 2006, Beltran et al. 2016). Pathway enrichment determined by Gene Set Enrichment Analysis (Broad Inst). Immune cell fractions were calculated by deconvolution of WTS: Quantiseq. Statistical significance was determined using chi-square and Mann-Whitney U test (p <0.05) along with FDR-corrected p-values (q)<0.05. Results: EOPC had higher frequency of TMPRSS2 (35.8% vs 29.7%), ETV1 (5.2% vs 2.8%), ETV4 (3.4% vs 1.6%), and BRAF (1.9 vs 0.7%) fusions, but lower frequency of APC (4% vs 8%), CTNNB1 (2.4% vs 4.6%), RB1 (2.3% vs 4.7%) and AR (1% vs 4.9%) mutation, TMB high (2.1% vs 4.6%) and dMMR/MSI-H (2.1% vs 4%) compared to AOPC (all p<0.05). EOPC had pathway enrichment of myogenesis (NES: 1.5, q<0.01), but no difference in hedgehog signaling (NES:1.3, q=0.09) and epithelial mesenchymal transition (NES: 1.3, q=0.09) pathways compared to AOPC. EOPC had higher median NEPC score (0.359 vs 0.353, q=0.04), higher MAPK pathway activity score (MPAS) (3-fold, q=0.02) but no difference in median AR signature (q=0.39) compared to AOPC. In addition, EOPC had higher expression of PSA (1.2-fold, q<0.01) and immunomodulatory genes ( IL12A, CTLA4, FC:1.2, q<0.05) with reduced AR expression (1.3-fold, q<0.01), however there was no difference in IHC-AR (q=1) and PD-L1 (q=0.57). Analysis of inferred immune cell infiltrates showed that EOPC had increased infiltration of NK cells (4.5% vs 4.1%, q<0.01) and dendritic cells (1.9% vs 1.8% q<0.05) compared to AOPC. Conclusions: Our data suggest that EOPC is enriched in fusion events including TMPRSS2, ETV1, ETV4 and BRAF. Distinct transcriptomic features seen in EOPC included neuroendocrine differentiation, MAPK activations, immunomodulatory gene expression, and increased infiltration of NK cells and dendritic cells, suggesting inherent molecular differences and differential tumor immune microenvironment in EOPC and AOPC.

Transcriptional patterns of sexual dimorphism and in host developmental programs in the model parasitic nematode Heligmosomoides bakeri

BackgroundHeligmosomoides bakeri (often mistaken for Heligmosomoides polygyrus) is a promising model for parasitic nematodes with the key advantage of being amenable to study and manipulation within a controlled laboratory environment. While draft genome sequences are available for this worm, which allow for comparative genomic analyses between nematodes, there is a notable lack of information on its gene expression.MethodsWe generated biologically replicated RNA-seq datasets from samples taken throughout the parasitic life of H. bakeri. RNA from tissue-dwelling and lumen-dwelling worms, collected under a dissection microscope, was sequenced on an Illumina platform.ResultsWe find extensive transcriptional sexual dimorphism throughout the fourth larval and adult stages of this parasite and identify alternative splicing, glycosylation, and ubiquitination as particularly important processes for establishing and/or maintaining sex-specific gene expression in this species. We find sex-linked differences in transcription related to aging and oxidative and osmotic stress responses. We observe a starvation-like signature among transcripts whose expression is consistently upregulated in males, which may reflect a higher energy expenditure by male worms. We detect evidence of increased importance for anaerobic respiration among the adult worms, which coincides with the parasite’s migration into the physiologically hypoxic environment of the intestinal lumen. Furthermore, we hypothesize that oxygen concentration may be an important driver of the worms encysting in the intestinal mucosa as larvae, which not only fully exposes the worms to their host’s immune system but also shapes many of the interactions between the host and parasite. We find stage- and sex-specific variation in the expression of immunomodulatory genes and in anthelmintic targets.ConclusionsWe examine how different the male and female worms are at the molecular level and describe major developmental events that occur in the worm, which extend our understanding of the interactions between this parasite and its host. In addition to generating new hypotheses for follow-up experiments into the worm’s behavior, physiology, and metabolism, our datasets enable future more in-depth comparisons between nematodes to better define the utility of H. bakeri as a model for parasitic nematodes in general.Graphical

Abstract 4503: Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense desmoplasia which contributes to poor prognosis by hindering tumor entry of immune cells and therapeutics. This leads to PDAC being difficult to treat with gemcitabine, a current standard-of-care, with only a 22% 12-month survival rate. Therapeutic modalities are needed to tackle the PDAC tumor microenvironment (TME). An alternative target is polyamine metabolism which is required for tumor growth, survival, metastasis, and also for modulating generation, survival, and activity of immune cells. Myeloid-derived suppressor cells (MDSCs) are immune cells that have a key role in PDAC metastasis, invasion, progression, and in impeding anti-tumor immune response to therapeutics in PDAC. MDSCs include monocyte-like (M-MDSC) and neutrophil-like (PMN-MDSC) cells. Previous studies have shown that polyamine blockade therapy (PBT), consisting of difluoromethylornithine (DFMO) to block polyamine biosynthesis and a polyamine transport inhibitor (Trimer44NMe), elicits anti-tumor immune response, improved survival, and a decrease in MDSCs in other tumor types. This is of interest given our previous findings that PBT improved survival of PDAC-bearing mice. Here, we tested PBT in altering PDAC-stimulated MDSC survival and whether it shifts their immunosuppressive phenotype. Studies were conducted using CD11b+ bone marrow cells that were differentiated into MDSCs via GM-CSF and tumor-conditioned media from PDAC cells derived from a genetic mouse model for pancreatic cancer (KPC-mice). Bulk RNA-sequencing was conducted on MDSCs treated with PBT, gemcitabine or combination. MDSCs exhibited widespread downregulation of expression in the gemcitabine and combination treatment, but PBT alone exhibited more targeted downregulation. Specifically, Gene Ontology (GO) analysis revealed a decrease in genes associated with immune cell migration and chemotaxis, and KEGG analysis showed downregulation of inflammatory pathways. These findings indicate that PBT may modify MDSCs from their tumor-associated immunosuppressive and pro-inflammatory behavior. MDSC subtype-specific markers were investigated by flow cytometry. Results show subsets of immune cells, such as PMN-MDSCs, are reduced upon PBT treatment alone or in combination with gemcitabine, thereby highlighting the dependence of this subtype on polyamine metabolism. In conclusion, PBT alters the expression of MDSC immunomodulatory genes, decreases MDSC survival and supports the use of PBT for treatment of PDAC in combination with other immunomodulatory therapeutics. Citation Format: Joseph A. Goode, Sai Preethi Nakkina, Manav Gandhi, Otto Phanstiel, Deborah A. Altomare. Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4503.

Profiling of Immunomodulatory Genes and Quantification of CD25+ Cells in Different Types of Early Pregnancy Loss.

Maternal regulatory T (Treg) cells play a pivotal role in establishing general immune homeostasis in the decidua for maintenance of pregnancy. We aimed in this study to investigate the relationship between mRNA expression of immunomodulatory genes and CD25+ Treg cells with early pregnancy losses. Our study included 3 groups of early pregnancy losses including sporadic spontaneous abortions, recurrent spontaneous abortions, sporadic spontaneous abortions post IVF treatment and the control group. We performed RT-PCR for analyzing mRNA expression levels of 6 immunomodulatory genes and CD25 immunohistochemistry for quantification of Treg cells. Only FOXP3, CD274 (PDL1), and TGFβ1 mRNA expression levels were significantly decreased in the miscarriage groups in comparison to the control group, whereas there was no significant mRNA expression change of CD4, IL2RA, and IL10. We also found significantly lower number of CD25+ cells in the miscarriages. We conclude that decreased expression of FOXP3 and PD-L1 may play a significant role in the pathogenesis of spontaneous abortion cases whereas decreased expression of TGFβ1 gene may be associated with the occurrence of early loss in IVF-treated pregnancies. Additional immunoprofiling of Treg cell population is needed to quantify Treg cells in early pregnancy losses.

The Effect of the Histone Chaperones HSPA8 and DEK on Tumor Immunity in Hepatocellular Carcinoma.

Complex immune contexture leads to resistance to immunotherapy in hepatocellular carcinoma (HCC), and the need for new potential biomarkers of immunotherapy in HCC is urgent. Histone chaperones are vital determinants of gene expression and genome stability that regulate tumor development. This study aimed to investigate the effect of histone chaperones on tumor immunity in HCC. Bioinformatics analyses were initially performed using The Cancer Genome Atlas (TCGA) database, and were validated using the Gene Expression Omnibus (GEO) database and the International Cancer Genome Consortium (ICGC) database. Immune-related histone chaperones were screened with the Spearman rank coefficient. Consensus clustering was utilized to divide the HCC samples into two clusters. ESTIMATE, CIBERSORT and ssGSEA analyses were performed to assess immune infiltration. The expression of immunomodulatory genes, chemokines and chemokine receptors was analyzed to evaluate sensitivity to immunotherapy. The differentially expressed genes (DEGs) were included in weighted gene coexpression network analysis (WGCNA) to identify the hub genes. Enrichment analyses were used to investigate the functions of the hub genes. The Kaplan-Meier method and log-rank test were conducted to draw survival curves. A Cox regression analysis was utilized to identify independent risk factors affecting prognosis. HSPA8 and DEK were screened out from 36 known histone chaperones based on their strongest correlation with the ESTIMATE score. Cluster 2, with high HSPA8 expression and low DEK expression, tended to have stronger immune infiltration and better sensitivity to immunotherapy than Cluster 1, with low HSPA8 expression and high DEK expression. Furthermore, WGCNA identified 12 hub genes closely correlated with immune infiltration from the DEGs of the two clusters, of which FBLN2 was proven to be an independent protective factor of HCC patients. HSPA8 and DEK are expected to be biomarkers for precisely predicting the effect of immunotherapy, and FBLN2 is expected to be a therapeutic target of HCC.

Genetic Engineering of Immune Evasive Stem Cell-Derived Islets.

Genome editing has the potential to revolutionize many investigative and therapeutic strategies in biology and medicine. In the field of regenerative medicine, one of the leading applications of genome engineering technology is the generation of immune evasive pluripotent stem cell-derived somatic cells for transplantation. In particular, as more functional and therapeutically relevant human pluripotent stem cell-derived islets (SCDI) are produced in many labs and studied in clinical trials, there is keen interest in studying the immunogenicity of these cells and modulating allogeneic and autoimmune immune responses for therapeutic benefit. Significant experimental work has already suggested that elimination of Human Leukocytes Antigen (HLA) expression and overexpression of immunomodulatory genes can impact survival of a variety of pluripotent stem cell-derived somatic cell types. Limited work published to date focuses on stem cell-derived islets and work in a number of labs is ongoing. Rapid progress is occurring in the genome editing of human pluripotent stem cells and their progeny focused on evading destruction by the immune system in transplantation models, and while much research is still needed, there is no doubt the combined technologies of genome editing and stem cell therapy will profoundly impact transplantation medicine in the future.

Cuprotosis Patterns Are Associated with Tumor Mutation Burden and Immune Landscape in Lung Adenocarcinoma.

The association involving cuprotosis, molecular subtype, and specific immune cell groups in the tumor microenvironment has been focused on by more recent studies. In lung adenocarcinoma (LUAD), the potential functions of cuprotosis remain elusive. The cuprotosis regulations and tumor immune profile of 567 LUAD patients and the correlation between the cuprotosis patterns and the immune landscape were comprehensively evaluated. The cuprotosisScore was calculated using principal component analysis (PCA). The prognostic significance of the cuprotosisScore was evaluated by Cox regression statistics analysis. Five cuprotosisClusters (named mc1, 2, 3, 4, 5)-characterized by differences in expression of immunomodulatory genes, mRNA, or lncRNA expression, and prognosis were identified. We established cuprotosisScore to quantify the cuprotosis pattern of individual LUAD patients. As is shown in further analyses, the cuprotosisScore was a relatively potential independent prognostic factor of LUAD involved in mc1. Finally, the prognostic value of the cuprotosisScore and its association with tumor immune microenvironment (iTME) of LUAD in five cuprotosisClusters were verified. We demonstrated the correlation between cuprotosis modification, the molecular subtype, and tumor immune landscape in LUAD. The cuprotosisCluster with high cuprotosisScore and high tumor mutation burden (TMB) was identified with a good prognosis and immune functions. The comprehensive evaluation of cuprotosis patterns in individual LUAD patients enhances the understanding of iTME and gives a new insight toward improved immune treatment strategies for LUAD patients.

Effects of Saccharomyces cerevisiae hydrolysate on growth performance, immunity function, and intestinal health in broilers

The current study was performed to explore the effects of dietary supplementation of Saccharomyces cerevisiae hydrolysate (SCH) on growth performance, immune function, and intestinal health in broiler chicken. A total of 300 Ross 308 male broilers (1-day-old) were randomly assigned to 2 dietary treatments including a basal diet (control group), and a basal diet supplemented with SCH feed additive (500 mg/kg in starter and grower phase, and 250 mg/kg in finisher phase). Each treatment had 6 replicates with 25 birds each. The results showed that the addition of SCH promoted growth during d 15 to 28 (P < 0.05). Although the addition of SCH had no significant effect on the intestinal relative indexes, it significantly increased the jejunum villus height (VH) and the ratio of villus height to crypt depth (VCR) of jejunum, and decreased the crypt depth (CD) of ileum (P < 0.05). Furthermore, SCH addition significantly downregulated the mRNA expression of immunomodulatory genes (TNF-α, IL-1β, and IL-6), and upregulated the tight junction genes (ZO-1 and Claudin-1) (P < 0.05). High throughput sequencing analysis of bacterial 16S rRNA revealed that dietary SCH supplementation altered cecum microbiota. Alpha diversity analysis showed that a higher bacterial richness in cecum of broilers fed with SCH. The composition of cecum microbiota regulated by SCH addition was characterized by an increased abundance of Firmicutes and a reduced abundance of Bacteroidetes. At the genus level, dietary SCH resulted in a decrease of Bacteroides and an increase of short-chain fatty acids (SCFA) -producing bacteria including Lactobacillus and Faecalibacterium. Taken together, dietary SCH supplementation can stimulate the growth of broilers by regulating the intestinal immunity and barrier function, and improving the intestinal morphology, which may be related to the enhancement of bacterial diversity and the changes of intestinal microbial composition.

Expression of immunomodulatory and tissue regenerative biomarkers in human dental pulp derived-mesenchymal stem cells treated with curcumin: an in vitro study.

Human Dental pulp derived-mesenchymal stem cells (hDP-MSCs) have the capability of selfrenewal, multipotency, as well as immunosuppressive properties. They are ideal candidates for regenerating damaged dental tissue and treating inflammation-related diseases. However, methods (such as genetic variation) to improve the immunomodulatory and regenerative efficiency of MSCs in different diseases still need to be developed. Curcumin (CUR) is known for its broad applications in regenerative medicine and the treatment of inflammatory disorders via its anti-inflammatory and anti-oxidant effects. This study was conducted to investigate the effect and underlying mechanisms of CUR on the immunomodulatory and regenerative function of hDP-MSCs and whether treating these cells with CUR can improve therapeutic efficacy. hDP-MSCs were isolated from dental pulp and then treated with CUR. Cell viability rate was observed in hDP-MSCs after treatment of CUR by MTT assay. Real-time quantitative (RT-PCR) was applied to estimate the expression of immunomodulatory and regenerative genes after treatment of CUR. The RT-PCR results showed that VEGF-A and STAT3 markers were up-regulated while HLA-G5 and VCAM-1 markers were down-regulated by CUR (20 µM) treatment in hDP-MSCs (P < 0.001). Besides, this research indicated that there were no significant changes in the expressions of RelA and DSPP after 48h (P = 0.33, P = 1). Our findings demonstrate that CUR can enhance the immunomodulatory and regenerative effects of hDP-MSCs and improve their therapeutic efficacy. These findings can give an understanding of the mechanism for improving restorative and immunomodulatory activity in hDP-MSCs by curcumin.

Identification and validation of cellular senescence patterns to predict clinical outcomes and immunotherapeutic responses in lung adenocarcinoma

BackgroundAging and senescence can alter immune cell fitness and influence the efficacy of lung cancer treatments, especially immunotherapy. However, the correlations between cellular senescence and tumor microenvironment are still not clearly clarified and the value of cellular senescence-related genes in evaluating the immune infiltration and clinical outcomes of lung adenocarcinoma (LUAD) need further investigated.MethodsWe identified three cellular senescence clusters by NMF algorithm and correlated the cellular senescence clusters with the immune landscape in LUAD patients. A prognostic scoring system was established using random survival forest algorithm and validated in 4 external cohorts. Multivariate Cox regression analysis was performed to evaluate the prognostic value of the scoring system. Expression of LYPD3 was evaluated by immunohistochemistry in LUAD samples.ResultsBased on the mRNA expression profiles of 278 cellular senescence-related genes, three cellular senescence clusters with distinct prognosis were identified. We characterized three cellular senescence clusters by differences in biological processes, EMT score, expression of immunomodulatory genes, extent of intratumor heterogeneity and response to immunotherapy. Meanwhile, a cellular senescence-related scoring system (CSS) was established and validated as an independent prognostic factor and immunotherapy predictor of LUAD. Patients with low CSS was characterized by prolonged survival time. In response to anti-cancer drugs, patients with low CSS exhibited higher sensitivities to molecular drugs, such as Roscovitine (CDKs inhibitor), Lenaidornide (TNF-α inhibitor), MK2206 (Akt 1/2/3 inhibitor), and especially increased response to anti-PD-1/L1 immunotherapy.ConclusionsThis study demonstrated the correlations between cellular senescence patterns and tumor immune landscape in LUAD, which enhanced our understanding of the tumor immune microenvironment and provided new insights for improving the outcome of immunotherapy for LUAD patients.