TLR agonists polarize interferon responses in conjunction with dendritic cell vaccination in malignant glioma: a randomized phase II Trial.

In this randomized phase II clinical trial, we evaluated the effectiveness of adding the TLR agonists, poly-ICLC or resiquimod, to autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination in patients with newly-diagnosed or recurrent WHO Grade III-IV malignant gliomas. The primary endpoints were to assess the most effective combination of vaccine and adjuvant in order to enhance the immune potency, along with safety. The combination of ATL-DC vaccination and TLR agonist was safe and found to enhance systemic immune responses, as indicated by increased interferon gene expression and changes in immune cell activation. Specifically, PD-1 expression increases on CD4+ T-cells, while CD38 and CD39 expression are reduced on CD8+ T cells, alongside an increase in monocytes. Poly-ICLC treatment amplifies the induction of interferon-induced genes in monocytes and T lymphocytes. Patients that exhibit higher interferon response gene expression demonstrate prolonged survival and delayed disease progression. These findings suggest that combining ATL-DC with poly-ICLC can induce a polarized interferon response in circulating monocytes and CD8+ T cells, which may represent an important blood biomarker for immunotherapy in this patient population.Trial Registration: ClinicalTrials.gov Identifier: NCT01204684.

Dendritic Cell Vaccination in Conjunction with a TLR Agonist Polarizes Interferon Immune Responses in Malignant Glioma Patients.

Autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination is a promising immunotherapy for patients with high grade gliomas, but responses have not been demonstrated in all patients. To determine the most effective combination of autologous tumor lysate-pulsed DC vaccination, with or without the adjuvant toll-like receptor (TLR) agonists poly-ICLC or resiquimod, we conducted a Phase 2 clinical trial in 23 patients with newly diagnosed or recurrent WHO Grade III-IV malignant gliomas. We then performed deep, high-dimensional immune profiling of these patients to better understand how TLR agonists may influence the systemic immune responses induced by ATL-DC vaccination. Bulk RNAseq data demonstrated highly significant upregulation of type 1 and type 2 interferon gene expression selectively in patients who received adjuvant a TLR agonist together with ATL-DC. CyTOF analysis of patient peripheral blood mononuclear cells (PBMCs) showed increased expression of PD-1 on CD4+ T-cells, decreases in CD38 and CD39 on CD8+ T cells and elevated proportion of monocytes after ATL-DC + TLR agonist administration. In addition, scRNA-seq demonstrated a higher expression fold change of IFN-induced genes with poly-ICLC treatment in both peripheral blood monocytes and T lymphocytes. Patients who had higher expression of interferon response genes lived significantly longer and had longer time to progression compared to those with lower expression. The results suggest that ATL-DC in conjunction with adjuvant poly-ICLC induces a polarized interferon response in circulating monocytes and specific activation of a CD8+ T cell population, which may represent an important blood biomarker for immunotherapy in this patient population. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01204684.

Immune checkpoint blockade induces distinct alterations in the microenvironments of primary and metastatic brain tumors.

In comparison with responses in recurrent glioblastoma (rGBM), the intracranial response of brain metastases (BrM) to immune checkpoint blockade (ICB) is less well studied. Here, we present an integrated single-cell RNA-Seq (scRNA-Seq) study of 19 ICB-naive and 9 ICB-treated BrM samples from our own and published data sets. We compared them with our previously published scRNA-Seq data from rGBM and found that ICB led to more prominent T cell infiltration into BrM than rGBM. These BrM-infiltrating T cells exhibited a tumor-specific phenotype and displayed greater activated/exhausted features. We also used multiplex immunofluorescence and spatial transcriptomics to reveal that ICB reduced a distinct CD206+ macrophage population in the perivascular space, which may modulate T cell entry into BrM. Furthermore, we identified a subset of progenitor exhausted T cells that correlated with longer overall survival in BrM patients. Our study provides a comprehensive immune cellular landscape of ICB's effect on metastatic brain tumors and offers insights into potential strategies for improving ICB efficacy for brain tumor patients.

Antigen presentation by clonally diverse CXCR5+ B cells to CD4 and CD8 T cells is associated with durable response to immune checkpoint inhibitors.

Introduction: Increased T cell infiltration and interferon gamma (IFNγ) pathway activation are seen in tumors of melanoma patients who respond to ICI (immune checkpoint inhibitor) or MAPK pathway inhibitor (MAPKi) therapies. Yet, the rate of durable tumor control after ICI is almost twice that of MAPKi, suggesting that additional mechanisms may be present in patients responding to ICI therapy that are beneficial for anti-tumor immunity. Methods: We used transcriptional analysis and clinical outcomes from patients treated with ICI or MAPKi therapies to delineate immune mechanisms driving tumor response. Results: We discovered response to ICI is associated with CXCL13-driven recruitment of CXCR5+ B cells with significantly higher clonal diversity than MAPKi. Our in vitro data indicate that CXCL13 production was increased in human peripheral blood mononuclear cells by anti-PD1, but not MAPKi, treatment. Higher B cell infiltration and B cell receptor (BCR) diversity allows presentation of diverse tumor antigens by B cells, resulting in activation of follicular helper CD4 T cells (Tfh) and tumor reactive CD8 T cells after ICI therapy. Higher BCR diversity and IFNγ pathway score post-ICI are associated with significantly longer patient survival compared to those with either one or none. Conclusions: Response to ICI, but not to MAPKi, depends on the recruitment of CXCR5+ B cells into the tumor microenvironment and their productive tumor antigen presentation to follicular helper and cytotoxic, tumor reactive T cells. Our study highlights the potential of CXCL13 and B cell based strategies to enhance the rate of durable response in melanoma patients treated with ICI.

Luteolin Binds Streptolysin O Toxin and Inhibits Its Hemolytic Effects and Cytotoxicity.

Group A streptococcus (GAS, Streptococcus pyogenes) is a common pathogen that can cause a variety of human diseases. Streptolysin O (SLO) is an exotoxin produced by GAS. It is a pore-forming toxin (PFT) that exhibits high in vivo toxicity. SLO enables GAS to evade phagocytosis and clearance by neutrophils, induces eukaryotic cell lysis, and activates inflammatory bodies. Luteolin is a natural compound that is produced by a wide range of plant species, and recent studies have shown that luteolin can inhibit the growth and alter the morphological of GAS. Here, we reported that luteolin can weaken the cytotoxicity and hemolytic activity of SLO in vitro. Briefly, luteolin bound SLO with high affinity, inhibited its dissolution of erythrocytes, affected its conformational stability and inhibited the formation of oligomers. To further verify the protective effect of luteolin, we used an in vitro SLO-induced human laryngeal carcinoma epithelial type-2 cells (HEp-2) model. Notably, our results showed luteolin protected HEp-2 cells from SLO induced cytotoxicity and changed in cell membrane permeability. In addition, we explored the role of luteolin in protecting mice from GAS-mediated injury using an aerosolized lung delivery model, and our results indicate that luteolin increases murine survival rate following inoculation with a lethal dose of GAS, and that survival was also associated with decreased pathological damage to lung tissue. Our results suggest that luteolin may be a novel drug candidate for the treatment of GAS infection.

The roles of TGF-ß and VEGF pathways in the suppression of antitumor immunity in melanoma and other solid tumors.

Immune checkpoint blockade (ICB) has become well-known in cancer therapy, strengthening the body's antitumor immune response rather than directly targeting cancer cells. Therapies targeting immune inhibitory checkpoints, such as PD-1, PD-L1, and CTLA-4, have resulted in impressive clinical responses across different types of solid tumors. However, as with other types of cancer treatments, ICB-based immunotherapy is hampered by both innate and acquired drug resistance. We previously reported the enrichment of gene signatures associated with wound healing, epithelial-to-mesenchymal, and angiogenesis processes in the tumors of patients with innate resistance to PD-1 checkpoint antibody therapy; we termed these the Innate Anti-PD-1 Resistance Signatures (IPRES). The TGF-ß and VEGFA pathways emerge as the dominant drivers of IPRES-associated processes. Here, we review these pathways' functions, their roles in immunosuppression, and the currently available therapies that target them. We also discuss recent developments in the targeting of TGF-ß using a specific antibody class termed trap antibody. The application of trap antibodies opens the promise of localized targeting of the TGF-ß and VEGFA pathways within the tumor microenvironment. Such specificity may offer an enhanced therapeutic window that enables suppression of the IPRES processes in the tumor microenvironment while sparing the normal homeostatic functions of TGF-ß and VEGFA in healthy tissues.

MMiRNA-Viewer2, a bioinformatics tool for visualizing functional annotation for MiRNA and MRNA pairs in a network.

BACKGROUND: Although there are many studies on the characteristics of miRNA-mRNA interactions using miRNA and mRNA sequencing data, the complexity of the change of the correlation coefficients and expression values of the miRNA-mRNA pairs between tumor and normal samples is still not resolved, and this hinders the potential clinical applications. There is an urgent need to develop innovative methodologies and tools that can characterize and visualize functional consequences of cancer risk gene and miRNA pairs while analyzing the tumor and normal samples simultaneously. RESULTS: We developed an innovative bioinformatics tool for visualizing functional annotation of miRNA-mRNA pairs in a network, known as MMiRNA-Viewer2. The tool takes mRNA and miRNA interaction pairs and visualizes mRNA and miRNA regulation network. Moreover, our MMiRNA-Viewer2 web server integrates and displays the mRNA and miRNA gene annotation information, signaling cascade pathways and direct cancer association between miRNAs and mRNAs. Functional annotation and gene regulatory information can be directly retrieved from our web server, which can help users quickly identify significant interaction sub-network and report possible disease or cancer association. The tool can identify pivotal miRNAs or mRNAs that contribute to the complexity of cancer, while engaging modern next-generation sequencing technology to analyze the tumor and normal samples concurrently. We compared our tools with other visualization tools. CONCLUSION: Our MMiRNA-Viewer2 serves as a multitasking platform in which users can identify significant interaction clusters and retrieve functional and cancer-associated information for miRNA-mRNA pairs between tumor and normal samples. Our tool is applicable across a range of diseases and cancers and has advantages over existing tools.

Inhibitory Effect of the Traditional Chinese Medicine Ephedra sinica granules on Streptococcus pneumoniae Pneumolysin.

Streptococcus pneumoniae (S. pneumoniae) is an opportunistic pathogen that causes pneumonia, meningitis and bacteremia in humans and animals. Pneumolysin (PLY), a major pore-forming toxin that is important for S. pneumoniae pathogenicity, is a promising target for the development of anti-infective agents. Ephedra sinica granules (ESG) is one of the oldest medical preparation with multiple biological activities (such as a divergent wind and cold effect); however, the detailed mechanism remains unknown. In this study, we found that ESG treatment significantly inhibited the oligomerization of PLY and then reduced the activity of PLY without affecting S. pneumoniae growth and PLY production. In a PLY and A549 cell co-incubation system, the addition of ESG resulted in significant protection against PLY-mediated cell injury. Furthermore, S. pneumoniae-infected mice showed decreased mortality, and alleviated tissue damage and inflammatory reactions following treatment with ESG. Our results indicate that ESG is a potential candidate treatment for S. pneumoniae infection that targets PLY. This finding partially elucidates the mechanism of the Chinese herbal formula ESG in the treatment of pneumococcal disease.

Identifying Interaction Clusters for MiRNA and MRNA Pairs in TCGA Network.

Existing methods often fail to recognize the conversions for the biological roles of the pairs of genes and microRNAs (miRNAs) between the tumor and normal samples. We have developed a novel cluster scoring method to identify messenger RNA (mRNA) and miRNA interaction pairs and clusters while considering tumor and normal samples jointly. Our method has identified 54 significant clusters for 15 cancer types selected from The Cancer Genome Atlas project. We also determined the shared clusters across tumor types and/or subtypes. In addition, we compared gene and miRNA overlap between lists identified in our liver hepatocellular carcinoma (LIHC) study and regulatory relationships reported from human and rat nonalcoholic fatty liver disease studies (NAFLD). Finally, we analyzed biological functions for the single significant cluster in LIHC and uncovered a significantly enriched pathway (phospholipase D signaling pathway) with six genes represented in the cluster, symbols: DGKQ, LPAR2, PDGFRB, PIK3R3, PTGFR and RAPGEF3.

Clustering analysis of microRNA and mRNA expression data from TCGA using maximum edge-weighted matching algorithms.

BACKGROUND: microRNA (miRNA) is a short RNA (~ 22 nt) that regulates gene expression at the posttranscriptional level. Aberration of miRNA expressions could affect their targeting mRNAs involved in cancer-related signaling pathways. We conduct clustering analysis of miRNA and mRNA using expression data from the Cancer Genome Atlas (TCGA). We combine the Hungarian algorithm and blossom algorithm in graph theory. Data analysis is done using programming language R and Python. METHODS: We first quantify edge-weights of the miRNA-mRNA pairs by combining their expression correlation coefficient in tumor (T_CC) and correlation coefficient in normal (N_CC). We thereby introduce a bipartite graph partition procedure to identify cluster candidates. Specifically, we propose six weight formulas to quantify the change of miRNA-mRNA expression T_CC relative to N_CC, and apply the traditional hierarchical clustering to subjectively evaluate the different weight formulas of miRNA-mRNA pairs. Among these six different weight formulas, we choose the optimal one, which we define as the integrated mean value weights, to represent the connections between miRNA and mRNAs. Then the Hungarian algorithm and the blossom algorithm are employed on the miRNA-mRNA bipartite graph to passively determine the clusters. The combination of Hungarian and the blossom algorithms is dubbed maximum weighted merger method (MWMM). RESULTS: MWMM identifies clusters of different sizes that meet the mathematical criterion that internal connections inside a cluster are relatively denser than external connections outside the cluster and biological criterion that the intra-cluster Gene Ontology (GO) term similarities are larger than the inter-cluster GO term similarities. MWMM is developed using breast invasive carcinoma (BRCA) as training data set, but can also applies to other cancer type data sets. MWMM shows advantage in GO term similarity in most cancer types, when compared to other algorithms. CONCLUSIONS: miRNAs and mRNAs that are likely to be affected by common underlying causal factors in cancer can be clustered by MWMM approach and potentially be used as candidate biomarkers for different cancer types and provide clues for targets of precision medicine in cancer treatment.

Identification of genome-wide non-canonical spliced regions and analysis of biological functions for spliced sequences using Read-Split-Fly.

BACKGROUND: It is generally thought that most canonical or non-canonical splicing events involving U2- and U12 spliceosomes occur within nuclear pre-mRNAs. However, the question of whether at least some U12-type splicing occurs in the cytoplasm is still unclear. In recent years next-generation sequencing technologies have revolutionized the field. The "Read-Split-Walk" (RSW) and "Read-Split-Run" (RSR) methods were developed to identify genome-wide non-canonical spliced regions including special events occurring in cytoplasm. As the significant amount of genome/transcriptome data such as, Encyclopedia of DNA Elements (ENCODE) project, have been generated, we have advanced a newer more memory-efficient version of the algorithm, "Read-Split-Fly" (RSF), which can detect non-canonical spliced regions with higher sensitivity and improved speed. The RSF algorithm also outputs the spliced sequences for further downstream biological function analysis. RESULTS: We used open access ENCODE project RNA-Seq data to search spliced intron sequences against the U12-type spliced intron sequence database to examine whether some events could occur as potential signatures of U12-type splicing. The check was performed by searching spliced sequences against 5'ss and 3'ss sequences from the well-known orthologous U12-type spliceosomal intron database U12DB. Preliminary results of searching 70 ENCODE samples indicated that the presence of 5'ss with U12-type signature is more frequent than U2-type and prevalent in non-canonical junctions reported by RSF. The selected spliced sequences have also been further studied using miRBase to elucidate their functionality. Preliminary results from 70 samples of ENCODE datasets show that several miRNAs are prevalent in studied ENCODE samples. Two of these are associated with many diseases as suggested in the literature. Specifically, hsa-miR-1273 and hsa-miR-548 are associated with many diseases and cancers. CONCLUSIONS: Our RSF pipeline is able to detect many possible junctions (especially those with a high RPKM) with very high overall accuracy and relative high accuracy for novel junctions. We have incorporated useful parameter features into the pipeline such as, handling variable-length read data, and searching spliced sequences for splicing signatures and miRNA events. We suggest RSF, a tool for identifying novel splicing events, is applicable to study a range of diseases across biological systems under different experimental conditions.

Dissecting the biological relationship between TCGA miRNA and mRNA sequencing data using MMiRNA-Viewer.

BACKGROUND: MicroRNAs (miRNA) are short nucleotides that interact with their target genes through 3' untranslated regions (UTRs). The Cancer Genome Atlas (TCGA) harbors an increasing amount of cancer genome data for both tumor and normal samples. However, there are few visualization tools focusing on concurrently displaying important relationships and attributes between miRNAs and mRNAs of both cancer tumor and normal samples. Moreover, a deep investigation of miRNA-mRNA target and biological relationships across multiple cancer types by integrating web-based analysis has not been thoroughly conducted. RESULTS: We developed an interactive visualization tool called MMiRNA-Viewer that can concurrently present the co-relationships of expression between miRNA-mRNA pairs of both tumor and normal samples into a single graph. The input file of MMiRNA-Viewer contains the expression information including fold changes between normal and tumor samples for mRNAs and miRNAs, the correlation between mRNA and miRNA, and the predicted target relationship by a number of databases. Users can also load their own input data into MMiRNA-Viewer and visualize and compare detailed information about cancer-related gene expression changes, and also changes in the expression of transcription-regulating miRNAs. To validate the MMiRNA-Viewer, eight types of TCGA cancer datasets with both normal and control samples were selected in this study and three filter steps were applied subsequently. We performed Gene Ontology (GO) analysis for genes available in final selected 238 pairs and also for genes in the top 5 % (95 percentile) for each of eight cancer types to report a significant number of genes involved in various biological functions and pathways. We also calculated various centrality measurement matrices for the largest connected component(s) in each of eight cancers and reported top genes and miRNAs with high centrality measurements. CONCLUSIONS: With its user-friendly interface, dynamic visualization and advanced queries, we also believe MMiRNA-Viewer offers an intuitive approach for visualizing and elucidating co-relationships between miRNAs and mRNAs of both tumor and normal samples. We suggest that miRNA and mRNA pairs with opposite fold changes of their expression and with inverted correlation values between tumor and normal samples might be most relevant for explaining the decoupling of mRNAs and their targeting miRNAs in tumor samples for certain cancer types.