Immune-associated Genes Research Articles

Comprehensive bioinformatics analysis identifies hub genes associated with immune cell infiltration in early-onset schizophrenia

BackgroundEarly-onset schizophrenia (EOS) occurs between the ages of 13 and 17 years, and neurobiological factors leading to cognitive deficits and psychotic symptoms with varying degrees of positive and negative symptoms. Numerous studies have demonstrated a broad link between immune dysregulation and the central nervous system in EOS, and its pathogenesis involves immune dysfunction, but the exact biological mechanisms have not been elucidated. This study employs immune infiltration analysis and bioinformatics to unveil the pathogenic mechanisms of EOS and identify potential diagnostic biomarkers, aiming for more precise clinical interventions.MethodsIn this study, we recruited 26 EOS patients and 27 healthy controls (HCs), and microarray data were collected. Crossover genes were identified using weighted gene co-expression network analysis (WGCNA) and differential expression genes (DEGs) analysis. These genes were subjected to genome enrichment analysis (GSEA) and gene ontology (GO) analysis. Hub genes were identified through protein-protein interactions (PPIs) and the GeneMANIA database. The diagnostic potential of immune-associated hub genes was evaluated using ROC analysis. Immune infiltration in EOS was analyzed with CIBERSORT. Regulatory miRNAs for the hub genes were predicted using miRNet, and the correlation between mRNAs and miRNAs was analyzed and validated in clinical samples.ResultsBy WGCNA and DEGs analysis, 330 relevant genes were screened in EOS patients compared to HCs. Functional enrichment analysis using Metascape showed significant enrichment in immune system pathways. Subsequently, a PPI network was constructed to select the top 10 potential hub genes, and functional analysis was performed by GeneMANIA, resulting in the identification of four immune-related genes. In addition, significant differences were observed among the four immune cell types in the two groups of samples. ROC analysis showed clinical relevance of the immune-related hub genes, and the AUC of all genes was greater than 0.7. A miRNA-mRNA regulatory network was constructed from miRNA data, and three miRNAs were found to be significantly associated with the immune-related hub genes.ConclusionOur findings demonstrated that CCL3, IL1B, CXCL8, CXCL10 and miR-34a-5p may be biomarkers that play crucial roles in the underlying mechanisms of EOS immune-related pathways. These findings contribute to the understanding of EOS pathophysiology and may help identify new diagnostic and therapeutic targets.

Potential Diagnostic Biomarkers in Heart Failure: Suppressed Immune-Associated Genes Identified by Bioinformatic Analysis and Machine Learning

Heart failure (HF) threatens tens of millions of people’s health worldwide, which is the terminal stage in the development of majority cardiovascular diseases. Recently, an increasing number of studies have demonstrated that bioinformatics and machine learning (ML) algorithms can offer new insights into the diagnosing and treating HF. To further discover HF diagnostic genes, we utilized least absolute shrinkage and selection operator (LASSO) and Support Vector Machine (SVM) to identify novel immune-related genes. The HF dataset was obtained from the gene expression omnibus (GEO) database and three candidate genes (LCN6, MUC4, and TNFRSF13C) were finally screened. In addition, the myocardial infarction (MI) modeling experiments on mice were performed to validate the expression of LCN6, MUC4, and TNFRSF13C on experimental HF mice. Altogether, these three candidate genes are promising targets for the prediction of HF with immunological perspective.

Chronic mucocutaneous candidiasis, mycobacterial infections and rosacea in a Mexican adult with STAT1 gain of function

STAT1 is a cytoplasmic transcription factor associated with cell growth regulation, differentiation, proliferation, metabolism, and apoptosis. IFN-mediated JAK/STAT signaling pathway is involved in eliminating intracellular pathogens and viruses. However, pathogenic variants in STAT1 can result in impaired or increased function. Increased activity or function in STAT1 was described in 2011 and is characterized by excessive phosphorylation of the transcription factor. Carriers can develop autoimmune and inflammatory diseases and are susceptible to fungal, viral, and bacterial infections. The early and common manifestation is chronic mucocutaneous candidiasis. Here, we report a clinical case of a patient with increased STAT1 activity or gain of function, which started in the first year of his life. He is currently 27 years old and has presented bacillus Calmette-Guérin and Mycobacterium tuberculosis infection, chronic mucocutaneous candidiasis, tinea capitis, and facial and ocular rosacea. HIV infection was ruled out. Given the clinical manifestations, an inborn error of immunity was suspected, specifically STAT1 with gain of function. The diagnosis was corroborated by the sequencing of multiple genes associated with inborn errors of immunity. The pathogenic variant c.961A>G (p.Arg321Gly) in the STAT1 gene, previously reported as a gain of function mutation, was found in the patient. Finally, this case illustrates that mutations in immune-associated genes can contribute to producing severe and recurrent infections, even in adult patients. Chronic mucocutaneous candidiasis should raise suspicion of gain of function in STAT1.

Construction of a prognostic survival model with tumor immune-related genes for breast cancer.

Numerous studies have demonstrated that immune cell infiltration is a significant predictor in the prognosis of those with breast cancer. This study aimed to develop a prognostic model for undifferentiated breast cancer using immune-related markers. Differentially expressed genes (DEGs) and prognostic factors were identified from The Cancer Genome Atlas (TCGA) database. Cancer immune-associated genes were filtered using the GeneCards database. Least absolute shrinkage and selection operator (LASSO) and Cox proportional hazards regression were employed to select prognostic indicators. The single-sample gene set enrichment analysis (ssGSEA) algorithm and the CIBERSORT algorithm were used to analyze the correlation of prognostic indicators with immune cells in breast cancer. We identified six tumor immune-related genes, including zic family member 2 (ZIC2), solute carrier family 7 member 5 (SLC7A5), forkhead box J1 (FOXJ1), C-X-C motif chemokine ligand 9 (CXCL9), tumor necrosis factor receptor superfamily member 18 (TNFRSF18), and serine protease 2 (PRSS2), for the development of a prognostic model for patients with breast cancer. Notably, the results of the correlation analysis indicated that CXCL9 was associated with antitumor immune cells, including CD8+ T cells, cytotoxic cells, M1 macrophages, and activated memory CD4 T cells, and with the enrichment of natural killer (NK) CD56dim cells. Furthermore, CXCL9 exhibited a significant negative association with the tumor-promoting M2 macrophage phenotype. Our study established a six-gene model for predicting breast cancer prognosis. Furthermore, we unexpectedly discovered that CXCL9 is integral to immune infiltration in breast cancer and may serve as a critical biomarker for evaluating immune response and therapeutic efficacy in breast cancer treatment.

Identification of immune-associated genes for the diagnosis of ulcerative colitis-associated carcinogenesis via integrated bioinformatics analysis.

UC patients suffer more from colorectal cancer (CRC) than the general population, which increases with disease duration. Early colonoscopy is difficult because ulcerative colitis-associated colorectal cancer (UCAC) lesions are flat and multifocal. Our study aimed to identify promising UCAC biomarkers that are complementary endoscopy strategies in the early stages. The datasets may be accessed from the Gene Expression Omnibus and The Cancer Genome Atlas databases. The co-expressed modules of UC and CRC were determined via weighted co-expression network analysis (WGCNA). The biological mechanisms of the shared genes were exported for analysis using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. To identify protein interactions and hub genes, a protein-protein interaction network and CytoHubba analysis were conducted. To evaluate gene expression, external datasets and experimental validation of human colon tissues were utilized. The diagnostic value of core genes was examined through receiver operating characteristic (ROC) curves. Immune infiltration analysis was employed to investigate the associations between immune cell populations and hub genes. Three crucial modules were identified from the WGCNA of UC and CRC tissues, and 33 coexpressed genes that were predominantly enriched in the NF-κB pathway were identified. Two biomarkers (CXCL1 and BCL6) were identified via Cytoscape and validated in external datasets and human colon tissues. CRC patients expressed CXCL1 at the highest level, whereas UC and CRC patients showed higher levels than the controls. The UC cohort expressed BCL6 at the highest level, whereas the UC and CRC cohorts expressed it more highly than the controls. The hub genes exhibited significant diagnostic potential (ROC curve > 0.7). The immune infiltration results revealed a correlation among the hub genes and macrophages, neutrophils and B cells. The findings of our research suggest that BCL6 and CXCL1 could serve as effective biomarkers for UCAC surveillance. Additionally, they demonstrated a robust correlation with immune cell populations within the CRC tumour microenvironment (TME). Our findings provide a valuable insight about diagnosis and therapy of UCAC.

Diversity of the immune microenvironment and response to checkpoint inhibitor immunotherapy in mucosal melanoma.

Mucosal melanoma (MucM) is a rare cancer with a poor prognosis and low response rate to immune checkpoint inhibition (ICI) compared with cutaneous melanoma (CM). To explore the immune microenvironment and potential drivers of MucM's relative resistance to ICI drugs, we characterized 101 MucM tumors (43 head and neck [H&N], 31 female urogenital, 13 male urogenital, 11 anorectal, and 3 other gastrointestinal) using bulk RNA-Seq and immunofluorescence. RNA-Seq data show that MucM has a significantly lower IFN-γ signature levels than CM. MucM tumors of the H&N region show a significantly greater abundance of CD8+ T cells, cytotoxic cells, and higher IFN-γ signature levels than MucM from lower body sites. In the subcohort of 35 patients with MucM treated with ICI, hierarchical clustering reveals clusters with a high and low degree of immune infiltration, with a differential ICI response rate. Immune-associated gene sets were enriched in responders. Signatures associated with cancer-associated fibroblasts, macrophages, and TGF-β signaling may be higher in immune-infiltrated, but ICI-unresponsive tumors, suggesting a role for these resistance mechanisms in MucM. Our data show organ region-specific differences in immune infiltration and IFN-γ signature levels in MucM, with H&N MucM displaying the most favorable immune profile. Our study might offer a starting point for developing more personalized treatment strategies for this disease.

Single-cell RNA sequencing highlights the role of proinflammatory fibroblasts, vascular endothelial cells, and immune cells in the keloid immune microenvironment.

Keloids, characterized by an aberrant wound-healing process and a highly complex immune microenvironment, pose significant challenges for clinical management. Fibroblasts and vascular endothelial cells (VEC) were identified as the leading cells of keloid development. However, their roles in the keloid immune landscape have yet to be thoroughly elucidated. To explore the functional state of cells in the immune landscape of keloids, we conducted a single-cell RNA sequencing analysis on the tissue from three keloid lesions and two specimens of healthy skin. We simultaneously utilized available keloid data from the public database for external validation. Specific subsets, such as proinflammatory fibroblasts (piF) and VEC, were markedly elevated in lesional skincompared to normal skin. Subsequent differential gene expression and Gene Ontology analyses indicated that these subsets may be involved in shaping the microenvironment. In keloids, there is an increased expression of immune-associated genes (P < 0.05), including TNFRSF6B, HGF, and TGFB3, alongside a decreased expression of inflammatory chemokines in the piF. Moreover, the significant upregulation of immune suppressive genes (P < 0.05), including CD39, CD73, and HIF1A, suggested the potential involvement of VEC as a conditional immune subpopulation in the keloid microenvironment. Immune cell communication analysis revealed preferential enrichment of macrophages and Tregs, highlighting intensified macrophage-centered interactions within the keloid microenvironment. Our study highlighted the role of piF and VEC in the immune microenvironment of keloids for the first time, providing potential targets for therapeutic development.

Potential tumor-specific antigens and immune landscapes identification for mRNA vaccine in thyroid cancer.

Tumor mRNA vaccines have been identified as a promising technology for cancer therapy in multiple cancer types, while their efficacy in thyroid cancer (THCA) is unclear. Immunotyping is strongly associated with the immune microenvironment and immune status in cancer, thus it is important in vaccination and therapeutic response. This study is to identify potential valuable antigens and novel immune subtypes of THCA for immune landscape construction, thus screening patients suitable for mRNA vaccination. The clinical information and gene expression files of 568 THCA cases were obtained from the TCGA dataset. The DNA copy number variation and the somatic mutation of THCA were visualized by the cBioPortal database. TIMER was used to investigate the immune infiltrating correlation with candidate antigens. Consensus clustering analysis was conducted to cluster data using the ConsensusClusterPlus package. The immune landscapes of THCA patients were visualized using the Monocle package. The critical hub genes for THCA mRNA vaccines were identified by WGCNA package. To validate, the immunohistochemistry and real-time quantitative PCR (RT-qPCR) were performed to detect the expression level of potential antigen for mRNA vaccine in tissue and cell lines in THCA. Thymidine kinase 1 (TK1) was identified as a potential biomarker of mRNA vaccine against THCA. It was confirmed to be significantly upregulated in THCA tissues and cells lines. Moreover, three novel immune subtypes of THCA were obtained based on the expression consistency of immune-associated genes. The S2 subtype was characterized as an immunological "cold" phenotype with a high expression of immunogenic cell death modulators. S1 and S3 subtypes were immunological "hot" phenotypes with immune checkpoints upregulation. Further, the immune landscape of THCA patients was visualized and ten hub genes for mRNA vaccines were identified. TK1 was a tumor-specific antigen of mRNA vaccines. The patients belonging to the S2 subtype ("cold" tumor) were suitable for mRNA vaccine therapy in THCA. Notably, ten hub genes were conducted as potential biomarkers for identifying suitable patients for mRNA vaccination. These findings provided novel insights into mRNA vaccine development against THCA.

Abstract B048: Lipopolysaccharide (LPS) mediated Toll like receptor-4 (TLR4) agonism immuno-modulates pancreatic cancer niche via CD4+ T cells-Myeloid cells axis

Abstract Introduction: Pancreatic cancer is a major cause of cancer-related mortality with a dismally low overall survival. Despite advancement in the cancer immunotherapy, pancreatic cancer remains unresponsive due to its immunosuppressives. We have established that lipopolysaccharide (LPS) reduces tumor burden in pancreatic cancer mouse-models by stimulating anti-tumor immunity and infiltrating cytotoxic CD4+ T-cells in the tumor-microenvironment. Here, we are investigating how LPS immuno-modulates pancreatic cancer niche and brings anti-cancerous effect. Methods: Pancreatic cancer-cells (KPCs) were implanted in C57BL/6, TRIF KO, MyD88 KO and TLR4 flox/flox LysmCre (TLR4f/f) mice subcutaneously and were treated with LPS (1mg/kg) weekly. The animals were sacrificed at endpoints and tumors were evaluated. Subcutaneous KPC-LPS model in BL/6 mice were also coupled with clodronate treatment to deplete cells of myeloid lineage. Tumor samples were analyzed using flow-cytometry and RT- PCR for immunocyte infiltration and expression of immune-associated genes. Dendritic cells (DCs) were isolated from BL/6 and TLR4f/f mice, pulsed with KPC cancer cell antigen, treated with LPS and taken to flow cytometry. Such DCs were also co-cultured with CD4+ T-cells, which were then subjected to cytotoxicity against cancer cells Results: LPS therapy in wild-type pancreatic cancer mouse-model significantly reduced the tumor burden by stimulating anti- tumor immunity in tumor-microenvironment and caused significant upregulation of activated DCs and macrophages. The tumor regressing effect of LPS disappeared in absence of TRIF as well as MyD88 receptors in TRIF KO and MyD88 KO mice respectively. Macrophage depletion by clodronate attenuated influence of LPS on tumor burden, which again proved futile in TLR4f/f model. On antigenic stimulation and LPS treatment, DCs from BL/6 mice expressed higher frequency of co-stimulatory molecules such as CD40, CD80 and CD86 whereas TLR4f/f mice derived DCs did not show such a trend. When such BL/6 DCs were co-cultured with CD4+ T- cells, they expressed higher cytotoxicity against cancer cells. Conclusions: LPS treatment reduces tumor burden in pancreatic cancer model by stimulating anti-tumor immunity in tumor- microenvironment wherein cells of the myeloid lineage are indispensable. This anti-cancer effect executed by CD4+ T-cells in an MHC II dependent manner, seems to be mediated through dendritic cells. Designer TLR4-agonists have the potential to emerges as a novel immunotherapeutic agent and could be combined with available immune-checkpoint blockade therapies. Citation Format: Utpreksha Vaish, Tejeshwar Jain, Dhanisha S Suresh, Maria F Salcedo-Noriega, Srikanth Iyer, Troy Randall, Vikas Dudeja. Lipopolysaccharide (LPS) mediated Toll like receptor-4 (TLR4) agonism immuno-modulates pancreatic cancer niche via CD4T cells-Myeloid cells axis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B048.

Distinct Gene Expression Patterns Identify Patients who Relapse After Neoadjuvant Pembrolizumab and Radical Cystectomy in the PURE-01 Study

PurposeThe PURE-01 clinical trial reported the use of neoadjuvant treatment with pembrolizumab prior to radical cystectomy (RC) in patients with muscle-invasive bladder. Specific molecular subtypes and immune signatures were reported to be associated with a favorable survival. However, reports on the detailed tumor biology of patients relapsing after neoadjuvant pembrolizumab are lacking. Materials and MethodsMicroarray data from transurethral resection of the bladder tumor (TURBT; n = 102) and matched RC (N = 25) tissue from patients in PURE-01 who experience a disease relapse were analyzed, with gene expression signatures and molecular subtypes. The Kaplan–Meier method was used to estimate differences in patient outcomes. Immune-signatures were split by median for survival analysis. All significance testing used a two-sided t-test at a threshold of P < .05. ResultsThe study cohort consisted of 102 patients, of whom N = 19 (19%) experienced relapse. Molecular subtyping revealed that neuroendocrine-like tumors had the worst outcomes, while tumors classified as Claudin-low did show only one recurrence event. Differential gene expression analysis identified genes associated with relapse, including KRT20, H19, and immune-associated genes such as CXCL9 and CXCL11. ConclusionThis study provides a detailed characterization of patients who relapsed after neoadjuvant pembrolizumab and RC and identifies distinct gene expression patterns associated with relapse. These findings may have implications for predicting patient response and guiding treatment decisions in the neoadjuvant setting.

Populations of Triple Negative and Hormone Receptor Positive HER2 Negative Breast Tumors Share Immune Gene Profiles

Background: Immune therapy has revolutionized the treatment of certain cancers including melanoma and lung cancer. Breast cancer has fewer tumors with high immune infiltrate and Immune Checkpoint Inhibitor (ICI) therapy has shown the most significant responses in the Triple Negative (TN) subtype although roles are emerging in the other subtypes. The presence of Tumor Infiltrating Lymphocytes (TIL) is predictive of response to ICI therapy in metastatic TN and associated with better survival in local TN disease. Hormone Receptor (HR) positive HER2 negative (HR+HER2-) breast tumors do not show improved prognosis with increasing TIL and only a small portion of HR+HER2- tumors respond to ICI in both metastatic and local disease. We therefore sought to identify genetic differences between TN and HR+HER2- tumors to identify how these may contribute to their differences in immune infiltrate. Methods: We contrasted immune-associated gene expression between 119 TN and 475 HR+HER2- breast tumors from The Cancer Genome Atlas (TCGA) and confirmed our findings from 299 TN and 1369 HR+HER2- breast tumors in METABRIC. Results: We found that TN and HR+HER2- tumors can be grouped into immune-high or -low tumors, with both subtypes represented in the immune-high group (40% HR+HER2- tumors of both Luminal A and Luminal B type and 71% TN tumors) in the TCGA. The genes upregulated in the immune-high cluster included GZMB, CXCL9, and IDO1 (Log2FC &gt; 2; Adj. p &lt; 10X10-40). The largest expression difference between the immune-high TN and HR+HER2- tumors was that TN tumors had more abundant Th1 and Th2 CD4+ T cells while HR+HER2- tumors had more abundant fibroblasts (Log2FC &gt; 0.3; Adj p &lt; 10X10-10). Conclusions: Our data suggest that an immune-high signature is not dictated by breast cancer subtype. However, fibroblasts, particularly fibroblast subsets associated with worse outcome, are more abundant in the immune-high HR+HER2- tumors.

Identification of immune-associated genes with altered expression in the spleen of mice enriched with probiotic Lactobacillus species using rna-seq profiling.

Probiotics are living microorganisms that can provide health benefits when consumed. Here, we investigated the effects of probiotics on gene expression in the spleen of mice using RNA-sequencing analysis between negative control and probiotic groups (including 4 Lactobacillus strains: Lactobacillus fermentum, L. casei, L. plantarum, and L. brevis). Mice exposed with probiotic in 4 weeks by intragastric administration. Then, spleen tissues of the control and probiotics groups were collected on days 14 and 28 for RNA sequencing. In total, 665, 186, and 81 DEGs were significantly expressed on day 14 vs. control, day 28 vs. control groups, and probiotics day 28 vs. day 14 groups, respectively. On the other hand, 12 Toll-like receptor (TLR) genes underwent additional validation through qRT-PCR, affirming the increased alignment between qRT-PCR and RNA-Seq findings. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses revealed that the DEGs were predominantly enriched in defense responses to pathogens, including inflammatory bowel diseases, malaria, leukaemia virus 1, and herpes virus, as well as immune processes related to immune response and signal transduction. This study represents the first investigation into mice's gene expression in the spleen exposed to probiotics using Lactobacillus spp. isolated from a field strain in Vietnam. Our results provide valuable insights into the impacts and functions of probiotics on mammalian development, offering crucial information for the potential therapeutic use of probiotics in defending against pathogens in Vietnam. The findings from this study highlight the potential of probiotics in modulating gene expression in the spleen, which may have implications for immune function and overall health in mice.

Populations of triple negative and hormone receptor positive HER2 negative breast tumors share immune gene profiles.

In breast cancer, triple negative (TN) breast cancer has most responses to immune checkpoint inhibitor (ICI) therapy. Lymphocyte infiltrate does not impact prognosis in Hormone receptor positive HER2 negative (HR + HER2-) breast tumors and few HR + HER2- tumors respond to ICI. We contrasted immune-associated gene expression between 119 TN and 475 HR + HER2- breast tumors from The Cancer Genome Atlas (TCGA) and confirmed our findings in 299 TN and 1369 HR + HER2- breast tumors in the METABRIC database. TN and HR+ HER2- tumors grouped into immune-high or -low tumors, both subtypes were represented in the immune-high group. The largest difference between the immune-high TN and HR + HER2- tumors was TN tumors had more abundant Th1 and Th2 CD4+ T cells while HR + HER2- tumors had more abundant fibroblasts (log2FC > 0.3; p < 10×10-10). This suggests an immune-high signature is not dictated by breast cancer subtype, but fibroblast subsets associated with worse outcome were higher in the immune-high HR + HER2- tumors.

Identification of immune-associated genes involved in latent Mycobacterium marinum infection

Tuberculosis (TB) is a high mortality infectious disease caused by Mycobacterium tuberculosis (Mtb), and often develops into latent infection. About 5～10% of latent infections turn into active tuberculosis when the host immune system becomes deficient. Therefore, exploring the latent infection mechanism of Mtb is pivotal for the prevention and treatment of tuberculosis. We first established the zebrafish latent infection model and the chronic infection model utilizing Mycobacterium marinum, which has the highly similar gene background to Mtb. Using the latent infection model, we characterized the gene expression profiles and found 462 genes expressed differentially in the latent period and chronic tuberculosis infection. These differentially expressed genes are involved in various biological processes including transcription, transcriptional regulation, organism development, and immune responses. Among them, nineteen immune-related genes were found to express differentially in the latent period. By analyzing immune related protein network, the genes in the center of the network, including Nos2b, TNFα, IL1, TNFβ, TLR1, TLR2, and TLR4b, displayed significant deferential expression in latent infection and chronic infection period of zebrafish, suggesting that these genes might play an important role in controlling latent infection of Mtb. Identifying immune biomarker related to the status of tuberculosis latent infection might lead to novel strategy for diagnosis and treatment.

Identification and experimental validation of KMO as a critical immune-associated mitochondrial gene in unstable atherosclerotic plaque

BackgroundThe heightened risk of cardiovascular and cerebrovascular events is associated with the increased instability of atherosclerotic plaques. However, the lack of effective diagnostic biomarkers has impeded the assessment of plaque instability currently. This study was aimed to investigate and identify hub genes associated with unstable plaques through the integration of various bioinformatics tools, providing novel insights into the detection and treatment of this condition.MethodsWeighted Gene Co-expression Network Analysis (WGCNA) combined with two machine learning methods were used to identify hub genes strongly associated with plaque instability. The cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) method was utilized to assess immune cell infiltration patterns in atherosclerosis patients. Additionally, Gene Set Variation Analysis (GSVA) was conducted to investigate the potential biological functions, pathways, and mechanisms of hub genes associated with unstable plaques. To further validate the diagnostic efficiency and expression of the hub genes, immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA) were performed on collected human carotid plaque and blood samples. Immunofluorescence co-staining was also utilized to confirm the association between hub genes and immune cells, as well as their colocalization with mitochondria.ResultsThe CIBERSORT analysis demonstrated a significant decrease in the infiltration of CD8 T cells and an obvious increase in the infiltration of M0 macrophages in patients with atherosclerosis. Subsequently, two highly relevant modules (blue and green) strongly associated with atherosclerotic plaque instability were identified. Through intersection with mitochondria-related genes, 50 crucial genes were identified. Further analysis employing least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine recursive feature elimination (SVM-RFE) algorithms revealed six hub genes significantly associated with plaque instability. Among them, NT5DC3, ACADL, SLC25A4, ALDH1B1, and MAOB exhibited positive correlations with CD8 T cells and negative correlations with M0 macrophages, while kynurenine 3-monooxygenas (KMO) demonstrated a positive correlation with M0 macrophages and a negative correlation with CD8 T cells. IHC and RT-qPCR analyses of human carotid plaque samples, as well as ELISA analyses of blood samples, revealed significant upregulation of KMO and MAOB expression, along with decreased ALDH1B1 expression, in both stable and unstable samples compared to the control samples. However, among the three key genes mentioned above, only KMO showed a significant increase in expression in unstable plaque samples compared to stable plaque samples. Furthermore, the expression patterns of KMO in human carotid unstable plaque tissues and cultured mouse macrophage cell lines were assessed using immunofluorescence co-staining techniques. Finally, lentivirus-mediated KMO silencing was successfully transduced into the aortas of high-fat-fed ApoE-/- mice, with results indicating that KMO silencing attenuated plaque formation and promoted plaque stability in ApoE-/- mice.ConclusionsThe results suggest that KMO, a mitochondria-targeted gene associated with macrophage cells, holds promise as a valuable diagnostic biomarker for assessing the instability of atherosclerotic plaques.

A PLA2 deletion mutant using CRISPR/Cas9 coupled to RNASeq reveals insect immune genes associated with eicosanoid signaling.

Eicosanoids mediate insect immune responses and synthesized by the catalytic activity of phospholipase A2 (PLA2). A uniquely encoded secretory PLA2 (sPLA2) is associated with immune responses of a lepidopteran insect, Spodoptera exigua. Its deletion mutant was generated using a CRISPR/Cas9 genome editing technology. Both wild and mutant lines were then immune-challenged, and the resulting transcripts were compared with their naïve transcripts by RNASeq using the Illumina-HiSeq platform. In total, 12,878 unigenes were further analyzed by differentially expressed gene tools. Over 69% of the expressed genes in S. exigua larvae are modulated in their expression levels by eicosanoids, recorded from CRISPR/Cas9 mutagenesis against an eicosanoid-synthetic gene, Se-sPLA2. Further, about 36% of the immune-associated genes are controlled by the eicosanoids in S. exigua. Indeed, the deletion mutant suffered significant immunosuppression in both cellular and humoral responses in response to bacterial challenge as well as severely reduced developmental and reproductive potentials.

Characteristics of the immune environment in prostate cancer as an adjunct to immunotherapy.

The tumor microenvironment (TME) exerts an important role in carcinogenesis and progression. Several investigations have suggested that immune cell infiltration (ICI) is of high prognostic importance for tumor progression and patient survival in many tumors, particularly prostate cancer. The pattern of immune infiltration of PCa, on the other hand, has not been thoroughly understood. The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) datasets on PCa were obtained, and several datasets were merged into one data set using the "ComBat" algorithm. The ICI profiles of PCa patients were then to be uncovered by two computer techniques. The unsupervised clustering method was utilized to identify three ICI patterns in tumor samples, and Principal Component Analysis (PCA) was conducted to estimate the ICI score. Three different clusters of three ICIs were identified in 1341 PCa samples, which also correlated with different clinical features/characteristics and biological pathways. Patients with PCa are classified into high and low subtypes based on the ICI scores extracted from immune-associated signature genes. High ICI score subtypes are associated with a worse prognosis, which may intrigue the activation of cancer-related and immune-related pathways such as pathways involving Toll-like receptors, T-cell receptors, JAK-STAT, and natural killer cells. The ICI score was linked to tumor mutation load and immune/cancer-relevant signaling pathways, which explain prostate cancer's poor prognosis. The findings of this study not only advanced our knowledge of the mechanism of immune response in the prostate tumor microenvironment but also provided a novel biomarker, that is, the ICI score, for disease prognosis and guiding precision immunotherapy.

Decoding the immune landscape: a comprehensive analysis of immune-associated biomarkers in cervical carcinoma and their implications for immunotherapy strategies.

Cervical cancer, a prevalent gynecological malignant tumor, poses a significant threat to women's health and lives. Immune checkpoint inhibitor (ICI) therapy has emerged as a promising avenue for treating cervical cancer. For patients with persistent or recurrent metastatic cervical cancer, If the sequence of dead receptor ligand-1 (PD-L1) is positive, ICI show significant clinical efficacy. PD-L1 expression serves as a valuable biomarker for assessing ICI therapeutic efficacy. However, the complex tumor immune microenvironment (TIME), encompassing immune cell composition and tumor-infiltrating lymphocyte (TIL) status, also exerts a profound influence on tumor immunity and prognosis. Given the remarkable strides made by ICI treatments in improving the survival rates of cervical cancer patients, it becomes essential to identify a comprehensive biomarker that integrates various TIME aspects to enhance the effectiveness of ICI treatment. Therefore, the quest for biomarkers linked to multiple facets of TIME in cervical cancer is a vital pursuit. In this study, we have developed an Immune-Associated Gene Prognostic Index (IRGPI) with remarkable prognostic value specifically for cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). The Cancer Genome Atlas CESC dataset (n = 305) was meticulously analyzed to pinpoint key immune-related genes via weighted gene co-expression network analysis and differential gene expression assays. Subsequently, we employed Cox regression analysis to construct the IRGPI. Furthermore, the composition of immune cells and TIL status were examined using CIBERSORT and TIDE. Tumor expression of Epigen, LCN10, and P73 were determined with immunohistochemistry. The resulting IRGPI, composed of EPGN, LCN10, and TP73 genes, displayed a strong negative correlation with patient survival. The discovery was validated with a patient cohort from our hospital. The IRGPI not only predicts the composition of immune cell subtypes such as Macrophages M1, NK cells, Mast cells, Plasma cells, Neutrophils, Dendritic cells, T cells CD8, and T cells CD4 within CESC, but also indicates TIL exclusion, dysfunction, and PD-1 and PD-L1 expression. Therefore, the IRGPI emerges as a promising biomarker not only for prognostic assessment but also for characterizing multiple immune features in CESC. Additionally, our results underscored the significant associations between the IRGPI and immune cell composition, TIL exclusion, and dysfunction, along with PD-1 and PD-L1 expression in the TIME. Consequently, the IRGPI stands out as a biomarker intimately connected to both the survival and TIME status of CESC patients, offering potential insights into immunotherapy strategies for CESC.

Interaction between VEGF-A and immune checkpoint targets in triple-negative breast cancer and mechanisms of immune evasion and tumor progression.

1096 Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment triple negative breast cancer (TNBC). However, not all patients with TNBC benefit from the addition of ICIs due to factors such as intrinsic and acquired resistance to ICIs, and complex interplay between tumor and immune cells in the tumor microenvironment (TME). Vascular endothelial growth factor (VEGF) promotes angiogenesis and potentially modulates tumor immune evasion. A phase I clinical trial has shown that a bispecific antibody to VEGF-A and PD-L1 produces encouraging tumor responses in advanced TNBC. In this study, we investigated the interaction between VEGF and various immune gene expression signatures including PD-L1 in a real-world breast cancer patient population. Methods: Comprehensive immune profiling, including the RNA-seq based gene expression assessment of 395 immune-associated genes, was performed on 120 formalin-fixed paraffin-embedded breast cancer samples. PD-L1 expression (Combined Positive Score, CPS ≥ 10%) was measured by immunohistochemistry (IHC; 22C3). Clinicopathologic variables were collected. Statistical associations between quantitative biomarkers, including 28 immune checkpoint genes, were calculated using Pearson correlations (r), and comparisons of quantitative biomarkers between groups were performed using the proportions test. For statistical significance, p&lt;0.05 was required. Results: The study cohort included 29 patients (24.2%) with TNBC and 91 (75.2%) with non-TNBC (including ER+/HER2- and HER2+). Most patients had metastatic tumors (n=92, 76.7%) and invasive ductal carcinoma (n=83, 69.2%). Among the TNBC group, 21 patients had PD-L1 positive tumors (72.4%), defined by CPS ≥ 10.In non-TNBC, VEGF has significant, though weak (r&lt;0.35) correlations with the expression of three checkpoint genes: AKT1 (r=0.35, p=0.00063), ICOS (r=0.34, p=0.00099), and TIM3 (r=0.29, p=0.0061). In TNBC, VEGF has significant, strong (r&gt;0.47) correlations with the expression of three other checkpoint genes: PD-1 (r=0.47, p=0.0094), PD-L1 (r=0.57, p=0.0012), and PIK3CA (r=0.6, p=0.00051) However, in TNBC, VEGF gene expression does not have a statistical association with PD-L1 IHC (CPS ≥/&lt; 10 (p=0.9)). Conclusions: Our findings suggest that there is an interplay between angiogenesis and an immunosuppressive tumor immune microenvironment in TNBC to a greater degree than in non-TNBCs. This supports the notion that angiogenesis mediators such as VEGF-A may enhance the expression of immunosuppressive checkpoint molecules, leading to immune evasion and tumor progression. Combination treatments of ICIs, especially anti-PD-1 therapy, with VEGF antibodies may be promising approaches to enhance the immune responses in TNBC, especially in tumors with CPS&lt;10, where the current frontline treatment is cytotoxic chemotherapy.

Immune infiltrate analysis to reveal distinct immune classes for leiomyosarcomas.

11546 Background: Leiomyosarcomas (LMS) are aggressive malignancies of smooth-muscle origin. Few targeted molecular options exist and immunotherapy (IO) agents against T cells have been disappointing. In this study, we examine the immune infiltrate of LMS tumors by immunohistochemistry (IHC) and gene expression. Our objective was to identify actionable targets for immunotherapy in patients with LMS. Methods: Tumor samples from a series of LMS patients were prospectively collected and banked for DNA and RNA sequencing as part of the MD Anderson Patient Mosaic. Associated clinical data for the patients were collected and maintained. IHC was performed on available separate tissue microarrays. Differential gene expression (DGE) analysis was done using the DESeq2 package for R and the Benjamini-Hochberg procedure used to control for false discovery rate. Immune cell fractions were estimated using the MCPcounter package. All gene expression analyses were repeated on The Cancer Genome Atlas (TCGA) dataset as an independent dataset. Results: A total of 104 TCGA samples and 72 samples from Mosaic from 53 patients with uterine or soft tissue LMS were included in the analysis. Tumors in the TCGA group were treatment naive. Of the samples in the Mosaic group, 61 (85%) had been previously treated with chemotherapy, 8 (11%) had been radiated, and 5 (7.5%) had been previously treated with immunotherapy. Thirty-one (43%) tumors were metastatic. Fifty-one (70%) were soft tissue LMS and 21 (30%) were uterine LMS. Four distinct immune classes were seen in both the TGCA and Mosaic: a predominantly B-cell group, a monocyte/macrophage group, a mixed infiltrate group and a group with a neutrophil signature. DGE performed between the subgroups revealed a unique gene expression pattern for tumors in the B cell and monocyte/macrophage subgroups. Interrogation of specific immune-associated genes revealed low levels of expression in the neutrophil group, suggesting this may be the most immunologically “cold” subset. Some of these results were reproduced by IHC using a TMA: the presence of a cluster of samples with B-cells and higher expression of immune checkpoints, a macrophage driven cluster, and an immunologically cold cluster. Conclusions: Analyses of LMS samples across two large datasets demonstrate the existence of 4 distinct immune classes of tumors that persist despite variation in clinical and treatment conditions: B cell, monocytic/macrophage, mixed-infiltrate and neutrophil rich. The B cell and monocytic groups have increased expression of immune markers which may be amenable to targeted therapies.