Integrated Single-cell and Transcriptome Sequencing Analyses Identified PREX1 as an Immune-related Prognostic Biomarker for Liver Hepatocellular Carcinoma.

Background: PtdIns (3,4,5) P3-dependent Rac exchanger 1 (PREX1), also known as PREX1, a member of the Rac guanine nucleotide exchange factors (Rac-GEF) family. Studies have suggested that PREX1 plays a role in mediating oncogenic pathway activation and controlling various biological mechanisms in different types of cancer, including liver hepatocellular carcinoma (LIHC). However, the function of PREX1 in the pathogenesis of LIHC and its potential role on immunological regulation is not clearly elucidated. Methods: The expression level and the clinical role of PREX1 in LIHC was analyzed based on database from the Cancer Genome Atlas (TCGA), TNM plotter and University of Alabama Cancer Database (UALCAN). We investigated the relationship between PREX1 and immunity in LIHC by TISIDB, CIBERSORT and single cell analysis. Immunotherapy responses were assessed by the immunophenoscores (IPS). Moreover, biological functional assays were performed to further investigate the roles of PREX1 in liver cancer cell lines. Results: Higher expression of PREX1 in LIHC tissues than in normal liver tissues was found based on public datasets. Further analysis revealed that PREX1 was associated with worse clinical characteristics and dismal prognosis. Pathway enrichment analysis indicated that PREX1 participated in immune-related pathways. Through CIBERSORT and single cell analysis, we found a remarkable correlation between the expression of PREX1 and various immune cells, especially macrophages. In addition, high PREX1 expression was found to be associated with a stronger response to immunotherapy. Furthermore, in vitro assays indicated that depletion of PREX1 can suppress invasion and proliferation of LIHC cells. Conclusion: Elevated expression of PREX1 indicates poor prognosis, influences immune modulation and predicts sensitivity of immunosuppression therapy in LIHC. Our results suggested that PREX1 may be a prognostic biomarker and therapeutic target, offering new treatment options for LIHC.

Distinct Localization, Transcriptional Profiles, and Functionality in Early Life Tonsil Regulatory T Cells.

CD4+ regulatory T cells (Tregs) are key orchestrators of the immune system, fostering the establishment of protective immunity while preventing deleterious responses. Infancy and childhood are crucial periods of rapid immunologic development, but how Tregs mediate immune responses at these earliest timepoints of human life is poorly understood. In this study, we compare blood and tissue (tonsil) Tregs across pediatric and adult subjects to investigate age-related differences in Treg biology. We observed increased FOXP3 expression and proportions of Tregs in tonsil compared with paired blood samples in children. Within tonsil, early life Tregs accumulated in extrafollicular regions with cellular interactions biased toward CD8+ T cells. Tonsil Tregs in both children and adults expressed transcriptional profiles enriched for lineage defining signatures and canonical functionality compared with blood, suggesting tissue as the primary site of Treg activity. Early life tonsil Tregs transcriptional profiles were further defined by pathways associated with activation, proliferation, and polyfunctionality. Observed differences in pediatric tonsil Treg transcriptional signatures were associated with phenotypic differences, high proliferative capacity, and robust production of IL-10 compared with adult Tregs. These results identify tissue as a major driver of Treg identity, provide new insights into developmental differences in Treg biology across the human lifespan, and demonstrate unique functional properties of early life Tregs.

Histopathology and transcriptome profiling reveal features of immune responses in gills and intestine induced by Spring viremia of carp virus.

The immune system of bony fish closely resembles that of mammals, comprising both specific (adaptive) and non-specific (innate) components. Notably, the mucosa-associated lymphoid tissue (MALT) serves as the first line of defense within the non-specific immune system, playing a critical role in protecting these aquatic organisms against invading pathogens. MALT encompasses a network of immune cells strategically distributed throughout the gills and intestines, forming an integral part of the mucosal barrier that interfaces directly with the surrounding aquatic environment. Spring Viremia of Carp Virus（SVCV）, a highly pathogenic agent causing substantial harm to common carp populations, has been designated as a Class 2 animal disease by the Ministry of Agriculture and Rural Affairs of China. Utilizing a comprehensive array of research techniques, including Hematoxylin and Eosin (HE)、Alcian Blue Periodic Acid-Schiff (AB-PAS)、transcriptome analysis for global gene expression profiling and Reverse Transcription-Polymerase Chain Reaction (RT-qPCR), this study uncovered several key findings: SVCV is capable of compromising the mucosal architecture in the gill and intestinal tissues of carp, and stimulate the proliferation of mucous cells both in gill and intestinal tissues. Critically, the study revealed that SVCV's invasion elicits a robust response from the carp's mucosal immune system, demonstrating the organism's capacity to resist SVCV invasion despite the challenges posed by the pathogen.

Senescent fibroblasts and innate immune cell activation might play a role in the pathogenesis of elderly atopic dermatitis.

BACKGROUND: Elderly atopic dermatitis (AD) is a subtype of AD defined by age (≥ 60 years). The molecular characteristics of elderly AD remain to be clarified. OBJECTIVE: We sought to characterize the molecular features of skin lesions and peripheral blood mononuclear cells (PBMCs) in patients with AD across different age, focusing on elderly AD. METHODS: Skin and PBMCs samples were used for RNA sequencing. Analysis of differentially expressed genes and gene set variation analysis were performed. Immunofluorescence staining, quantitative real-time PCR (qRT-PCR), flow cytometry and transwell assay were used for validation. RESULTS: Compared with healthy controls, the skin transcriptome of AD patients showed common signatures of AD, like barrier dysfunction and enhanced Th1/Th2/Th17 immune pathways. In PBMCs, the expression of Th1/Th2 response genes was more remarkable in adult AD, while expression of Th17-related genes was significantly higher in childhood AD. The gene modules associated with natural killer (NK) cells were downregulated in elderly AD. In skin lesions, elderly AD exhibited enrichment of macrophages, fibroblasts and senescence-associated secretory phenotype (SASP) related genes. The correlation among fibroblasts, SASP and innate immune cells were revealed by the co-localization of fibroblasts, macrophages and NK cells in the lesions across different age groups. Fibroblasts under inflammation or senescence could induce stronger chemotaxis of macrophages and NK cells. CONCLUSION: We identified the molecular phenotypes of skin lesions and PBMCs in elderly AD individuals. Fibroblasts, innate immune cells, and SASP might play important roles in the pathogenesis of elderly AD.

Exploring the causal role of immune cells in cerebral aneurysm through single-cell transcriptomics and Mendelian randomization analysis.

Cerebral aneurysm (CA) represents a significant clinical challenge, characterized by pathological dilation of cerebral arteries. Recent evidence underscores the crucial involvement of immune cells in CA pathogenesis. This study aims to explore the complex interplay between immune cells and CA formation. We analyzed single-cell RNA sequencing data from the GSE193533 dataset, focusing on unruptured CA and their controls. Comprehensive cell-type identification and pseudo-time trajectory analyses were conducted to delineate the dynamic shifts in immune cell populations. Additionally, a two-sample Mendelian randomization (MR) approach was employed to investigate the causal influence of various immunophenotypes on CA susceptibility and the reciprocal effect of CA formation on immune phenotypes. Single-cell transcriptomic analysis revealed a progressive loss of vascular smooth muscle cells (VSMCs) and an increase in monocytes/macrophages (Mo/MΦ) and other immune cells, signifying a shift from a structural to an inflammatory milieu in CA evolution. MR analysis identified some vital immunophenotypes, such as CD64 on CD14+ CD16+ monocytes (OR: 1.236, 95% CI: 1.064-1.435, P = 0.006), as potential risk factors for CA development, while others, like CD28- CD8br %CD8br (OR: 0.883, 95% CI: 0.789-0.988, P = 0.030), appeared protective. Reverse MR analysis demonstrated that CA formation could modulate specific immunophenotypic expressions, highlighting a complex bidirectional interaction between CA pathology and immune response. This study underscores the pivotal role of immune cells in this process through the integration of single-cell transcriptomics with MR analysis, offering a comprehensive perspective on CA pathogenesis, and potentially guiding future therapeutic strategies targeting specific immune pathways.

Exploration of the immune response of grass carp (Ctenopharyngodon idellus) erythrocytes during bacterial infection.

In teleost blood, red blood cells (RBCs) are the most common type of cell, and they differ from mammalian RBCs in having a nucleus and other organelles. As nucleated cells, teleost RBCs contribute to the immune response against pathogens, but their antibacterial mechanism remains unclear. Here, we utilized RNA-Seq to analyze gene expression patterns of grass carp (Ctenopharyngodon idellus) RBCs (GcRBCs) stimulated by Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus. Our transcriptomic data showed that bacterial stimulation generated many differentially expressed genes (DEGs). Furthermore, several inflammatory pathways responded to bacterial activation, and the TLR, IL-17, and tumor necrosis factor (TNF) signaling pathways were significantly activated based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Furthermore, the findings of qRT-PCR showed markedly elevated expression of various cytokines, including IL-1ß, IL4, IL6, IL8, IL12, and TNFα, in GcRBCs after incubation with bacteria. Reactive oxygen species (ROS) production in GcRBCs was markedly increased after the cells were stimulated with the three bacteria, and the expression of superoxide dismutase, glutathione peroxidase, and antioxidant enzymes, including catalase, was altered. Flow cytometry analysis showed that the apoptosis rate of GcRBCs was enhanced after stimulation with the three bacteria for different times. In summary, our findings reveal that bacterial stimulation activates the immune response of GcRBCs by regulating ROS release, cytokine expression, and the antioxidant system, leading to apoptosis of GcRBCs.

Humoral immune transcriptome signature in myelomeningocele patients.

INTRODUCTION: Myelomeningocele (MMC) results from incomplete closure of the neural tube, and has a complex multifactorial etiology, including an inflammatory microenvironment. OBJECTIVE: We evaluated the contribution of humoral immune response for development of inflammatory milieu. METHODS: Using public repository Gene Expression Omnibus (GEO), we retrieve dataset transcriptome from the amniotic fluid of ten fetuses with myelomeningocele and ten healthy control fetuses to found differential gene expression associated with disturbances and inflammatory signatures in MMC. The identified DEGs were submitted to enrichment, network, and matrix correlation analyses. RESULTS: Our initial analysis revealed 90 DEGs in MMC, mainly associated with signaling pathways of inflammation, including the immune modules, humoral immune response and IFN-type I signatures. Protein-protein analysis (PPI) revealed an association with three protein networks; positive regulation of B cell proliferation constituted the largest network. Matrix correlation analyses showed that MMC alters the co-expression of genes related to inflammatory processes that promote microenvironment inflammation. CONCLUSION: These results revealed an altered humoral immune response in MMC patients, contributing to an inflammatory profile and providing opportunities for identifying potential biomarkers in myelomeningocele disease.

Zika Virus Infection Induces Interleukin-1ß-Mediated Inflammatory Responses by Macrophages in the Brain of an Adult Mouse Model.

During the 2015-2016 Zika virus (ZIKV) epidemic, ZIKV-associated neurological diseases were reported in adults, including microcephaly, Guillain-Barre syndrome, myelitis, meningoencephalitis, and fatal encephalitis. However, the mechanisms underlying the neuropathogenesis of ZIKV infection are not yet fully understood. In this study, we used an adult ZIKV infection mouse model (Ifnar1-/-) to investigate the mechanisms underlying neuroinflammation and neuropathogenesis. ZIKV infection induced the expression of proinflammatory cytokines, including interleukin-1ß (IL-1ß), IL-6, gamma interferon, and tumor necrosis factor alpha, in the brains of Ifnar1-/- mice. RNA-seq analysis of the infected mouse brain also revealed that genes involved in innate immune responses and cytokine-mediated signaling pathways were significantly upregulated at 6 days postinfection. Furthermore, ZIKV infection induced macrophage infiltration and activation and augmented IL-1ß expression, whereas microgliosis was not observed in the brain. Using human monocyte THP-1 cells, we confirmed that ZIKV infection promotes inflammatory cell death and increases IL-1ß secretion. In addition, expression of the complement component C3, which is associated with neurodegenerative diseases and known to be upregulated by proinflammatory cytokines, was induced by ZIKV infection through the IL-1ß-mediated pathway. An increase in C5a produced by complement activation in the brains of ZIKV-infected mice was also verified. Taken together, our results suggest that ZIKV infection in the brain of this animal model augments IL-1ß expression in infiltrating macrophages and elicits IL-1ß-mediated inflammation, which can lead to the destructive consequences of neuroinflammation. IMPORTANCE Zika virus (ZIKV) associated neurological impairments are an important global health problem. Our results suggest that ZIKV infection in the mouse brain can induce IL-1ß-mediated inflammation and complement activation, thereby contributing to the development of neurological disorders. Thus, our findings reveal a mechanism by which ZIKV induces neuroinflammation in the mouse brain. Although we used adult type I interferon receptor IFNAR knockout (Ifnar1-/-) mice owing to the limited mouse models of ZIKV pathogenesis, our conclusions contributed to the understanding ZIKV-associated neurological diseases to develop treatment strategies for patients with ZIKV infection based on these findings.

New insight of variable lymphocyte receptor-likes in anti-bacteria activity from Eriocheir sinensis.

The Chinese mitten crab, Eriocheir sinensis, is a vital freshwater aquaculture species in China, however, is also facing various crab disease threats. In the present study, we identify three novel variable lymphocyte receptor-like (VLR-like) genes-VLR-like1, VLR-like3 and VLR-like4-from E. sinensis, which play vital roles in adaptive immune system of agnathan vertebrates. The bacterial challenge, bacterial binding and antibacterial-activity experiments were applied to study immune functions of VLR-likes, and the transcriptomic data from previous E. sinensis bacterial challenge experiments were analyzed to speculate the possible signaling pathway. VLR-like1 and VLR-like4 can respond to Staphylococcus aureus challenge and inhibit S. aureus specifically. VLR-like1 and VLR-like4 possess broad-spectrum bacteria-binding ability whereas VLR-like3 do not. VLR-likes in E. sinensis could associate with the Toll-like receptor (TLR) signaling pathway. The above results suggest that VLR-likes defend against bacteria invasion though exerting anti-bacteria activity, and probably connect with the TLR signaling pathway. Furthermore, studying the immune functions of these VLR-likes will provide a new insight into the disease control strategy of crustacean culture.

Transcriptomic Signatures of Human Immunodeficiency Virus Post-Treatment Control.

Posttreatment controllers (PTCs) are rare HIV-infected individuals who can limit viral rebound after antiretroviral therapy interruption (ATI), but the mechanisms of this remain unclear. To investigate these mechanisms, we quantified various HIV RNA transcripts (via reverse transcription droplet digital PCR [RT-ddPCR]) and cellular transcriptomes (via RNA-seq) in blood cells from PTCs and noncontrollers (NCs) before and two time points after ATI. HIV transcription initiation did not significantly increase after ATI in PTCs or in NCs, whereas completed HIV transcripts increased at early ATI in both groups and multiply-spliced HIV transcripts increased only in NCs. Compared to NCs, PTCs showed lower levels of HIV DNA, more cell-associated HIV transcripts per total RNA at all times, no increase in multiply-spliced HIV RNA at early or late ATI, and a reduction in the ratio of completed/elongated HIV RNA after early ATI. NCs expressed higher levels of the IL-7 pathway before ATI and expressed higher levels of multiple cytokine, inflammation, HIV transcription, and cell death pathways after ATI. Compared to the baseline, the NCs upregulated interferon and cytokine (especially TNF) pathways during early and late ATI, whereas PTCs upregulated interferon and p53 pathways only at early ATI and downregulated gene translation during early and late ATI. In NCs, viral rebound after ATI is associated with increases in HIV transcriptional completion and splicing, rather than initiation. Differences in HIV and cellular transcription may contribute to posttreatment control, including an early limitation of spliced HIV RNA, a delayed reduction in completed HIV transcripts, and the differential expression of the IL-7, p53, and TNF pathways. IMPORTANCE The findings presented here provide new insights into how HIV and cellular gene expression change after stopping ART in both noncontrollers and posttreatment controllers. Posttreatment control is associated with an early ability to limit increases in multiply-spliced HIV RNA, a delayed (and presumably immune-mediated) ability to reverse an initial rise in processive/completed HIV transcripts, and multiple differences in cellular gene expression pathways. These differences may represent correlates or mechanisms of posttreatment control and may provide insight into the development and/or monitoring of therapeutic strategies that are aimed at a functional HIV cure.

Alterações sistêmicas na assinatura do ciclo celular de pacientes com COVID-19 / Systemic changes in the cell cycle signature of patients with COVID-19

Com base nas perturbações fosfoproteômicas de moléculas associadas ao ciclo celular em células infectadas pelo coronavírus causador da síndrome respiratória aguda grave (SARSCoV)-2, a hipótese de inibidores do ciclo celular como uma terapia potencial para a doença de coronavírus 2019 (COVID-19) foi proposta. No entanto, o cenário das alterações do ciclo celular em COVID-19 permanece inexplorado. Aqui, realizamos uma análise integrativa de sistemas imunológicos de proteoma publicamente disponível (espectrometria de massa) e dados de transcriptoma (sequenciamento de RNA em massa e de célula única [scRNAseq]), com o objetivo de caracterizar mudanças globais na assinatura do ciclo celular de pacientes com COVID-19. Além de módulos de co-expressão de genes significativos enriquecidos associados ao ciclo celular, encontramos uma rede interconectada de proteínas diferencialmente expressas associadas ao ciclo celular (DEPs) e genes (DEGs) integrando dados moleculares de 1.480 indivíduos (974 pacientes infectados por SARS-CoV-2 e 506 controles [controles saudáveis ou indivíduos com outras doenças respiratórias]). Entre esses DEPs e DEGs estão várias ciclinas (CCNs), ciclo de divisão celular (CDCs), quinases dependentes de ciclinas (CDKs) e proteínas de manutenção de minicromossomos (MCMs). Embora os pacientes com COVID-19 compartilhem parcialmente o padrão de expressão de algumas moléculas associadas ao ciclo celular com outras doenças respiratórias, eles exibiram uma expressão significativamente maior de moléculas associadas ao ciclo celular relacionadas à gravidade da doença. Notavelmente, a assinatura do ciclo celular predominou nos leucócitos do sangue dos pacientes, mas não nas vias aéreas superiores. Os dados de scRNAseq de 229 indivíduos (159 pacientes com COVID- 19 e 70 controles) revelaram que as alterações das assinaturas do ciclo celular predominam nas células B, T e NK. Esses resultados fornecem uma compreensão global única das alterações nas moléculas associadas ao ciclo celular em pacientes com COVID-19, sugerindo novas vias putativas para intervenção terapêutica

Based on phosphoproteomics perturbations of cell cycle-associated molecules in severe acute respiratory syndrome coronavirus (SARS-CoV)-2-infected cells, the hypothesis of cell cycle inhibitors as a potential therapy for Coronavirus disease 2019 (COVID-19) has been proposed. However, the landscape of cell cycle alterations in COVID-19 remains mostly unexplored. Here, we performed an integrative systems immunology analysis of publicly available proteome (mass spectrometry) and transcriptome data (bulk and single-cell RNA sequencing [scRNAseq]), aiming to characterize global changes in the cell cycle signature of COVID-19 patients. Beyond significant enriched cell cycle-associated gene co-expression modules, we found an interconnected network of cell cycle-associated differentially expressed proteins (DEPs) and genes (DEGs) by integrating molecular data of 1,480 individuals (974 SARS-CoV- 2 infected patients and 506 controls [either healthy controls or individuals with other respiratory illness]). Among these DEPs and DEGs are several cyclins (CCNs), cell division cycle (CDCs), cyclin-dependent kinases (CDKs), and mini-chromosome maintenance proteins (MCMs). Although COVID-19 patients partially shared the expression pattern of some cell cycleassociated molecules with other respiratory illnesses, they exhibited a significantly higher expression of cell cycle-associated molecules associated with disease severity. Notably, the cell cycle signature predominated in the patients blood leukocytes but not in the upper airways. The scRNAseq data from 229 individuals (159 COVID-19 patients and 70 controls) revealed that the alterations of cell cycle signatures predominate in B, T, and NK cells. These results provide a unique global comprehension of the alterations in cell cycle-associated molecules in COVID-19 patients, suggesting new putative pathways for therapeutic intervention

Prognostic value and immune relevancy of a combined autophagy-, apoptosis- and necrosis-related gene signature in glioblastoma.

BACKGROUND: Glioblastoma (GBM) is considered the most malignant and devastating intracranial tumor without effective treatment. Autophagy, apoptosis, and necrosis, three classically known cell death pathways, can provide novel clinical and immunological insights, which may assist in designing personalized therapeutics. In this study, we developed and validated an effective signature based on autophagy-, apoptosis- and necrosis-related genes for prognostic implications in GBM patients. METHODS: Variations in the expression of genes involved in autophagy, apoptosis and necrosis were explored in 518 GBM patients from The Cancer Genome Atlas (TCGA) database. Univariate Cox analysis, least absolute shrinkage and selection operator (LASSO) analysis, and multivariate Cox analysis were performed to construct a combined prognostic signature. Kaplan-Meier survival, receiver-operating characteristic (ROC) curves and Cox regression analyses based on overall survival (OS) and progression-free survival (PFS) were conducted to estimate the independent prognostic performance of the gene signature. The Chinese Glioma Genome Atlas (CGGA) dataset was used for external validation. Finally, we investigated the differences in the immune microenvironment between different prognostic groups and predicted potential compounds targeting each group. RESULTS: A 16-gene cell death index (CDI) was established. Patients were clustered into either the high risk or the low risk groups according to the CDI score, and those in the low risk group presented significantly longer OS and PFS than the high CDI group. ROC curves demonstrated outstanding performance of the gene signature in both the training and validation groups. Furthermore, immune cell analysis identified higher infiltration of neutrophils, macrophages, Treg, T helper cells, and aDCs, and lower infiltration of B cells in the high CDI group. Interestingly, this group also showed lower expression levels of immune checkpoint molecules PDCD1 and CD200, and higher expression levels of PDCD1LG2, CD86, CD48 and IDO1. CONCLUSION: Our study proposes that the CDI signature can be utilized as a prognostic predictor and may guide patients' selection for preferential use of immunotherapy in GBM.

SLAMF7 engagement superactivates macrophages in acute and chronic inflammation.

Macrophages regulate protective immune responses to infectious microbes, but aberrant macrophage activation frequently drives pathological inflammation. To identify regulators of vigorous macrophage activation, we analyzed RNA-seq data from synovial macrophages and identified SLAMF7 as a receptor associated with a superactivated macrophage state in rheumatoid arthritis. We implicated IFN-γ as a key regulator of SLAMF7 expression and engaging SLAMF7 drove a strong wave of inflammatory cytokine expression. Induction of TNF-α after SLAMF7 engagement amplified inflammation through an autocrine signaling loop. We observed SLAMF7-induced gene programs not only in macrophages from rheumatoid arthritis patients but also in gut macrophages from patients with active Crohn's disease and in lung macrophages from patients with severe COVID-19. This suggests a central role for SLAMF7 in macrophage superactivation with broad implications in human disease pathology.

Competitive Endogenous RNA Network Activates Host Immune Response in SARS-CoV-2-, panH1N1 (A/California/07/2009)-, and H7N9 (A/Shanghai/1/2013)-Infected Cells.

The global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still ongoing, as is research on the molecular mechanisms underlying cellular infection by coronaviruses, with the hope of developing therapeutic agents against this pandemic. Other important respiratory viruses such as 2009 pandemic H1N1 and H7N9 avian influenza virus (AIV), influenza A viruses, are also responsible for a possible outbreak due to their respiratory susceptibility. However, the interaction of these viruses with host cells and the regulation of post-transcriptional genes remains unclear. In this study, we detected and analyzed the comparative transcriptome profiling of SARS-CoV-2, panH1N1 (A/California/07/2009), and H7N9 (A/Shanghai/1/2013) infected cells. The results showed that the commonly upregulated genes among the three groups were mainly involved in autophagy, pertussis, and tuberculosis, which indicated that autophagy plays an important role in viral pathogenicity. There are three groups of commonly downregulated genes involved in metabolic pathways. Notably, unlike panH1N1 and H7N9, SARS-CoV-2 infection can inhibit the m-TOR pathway and activate the p53 signaling pathway, which may be responsible for unique autophagy induction and cell apoptosis. Particularly, upregulated expression of IRF1 was found in SARS-CoV-2, panH1N1, and H7N9 infection. Further analysis showed SARS-CoV-2, panH1N1, and H7N9 infection-induced upregulation of lncRNA-34087.27 could serve as a competitive endogenous RNA to stabilize IRF1 mRNA by competitively binding with miR-302b-3p. This study provides new insights into the molecular mechanisms of influenza A virus and SARS-CoV-2 infection.

Comparison of transcriptome profiles of nucleated red blood cells in cord blood between preterm and full-term neonates.

BACKGROUND: The reactivation of fetal γ-globin expression is an effective strategy for ameliorating the clinical symptoms of ß-hemoglobinopathies. However, the mechanism of globin switching, especially the roles of long non-coding RNAs (lncRNAs) in this process, remains elusive. METHODS: We compared the in vivo transcriptome profiles of nucleated red blood cells (NRBCs) isolated from the umbilical cord blood of preterm and full-term newborns. We collected 75 umbilical cord blood samples and performed qPCR of the candidate genes. RESULTS: In this study, we identified 7,166 differentially expressed protein-coding genes, 3,243 differentially expressed lncRNAs, and 79 differentially expressed microRNAs. Our data show that the Fanconi anemia pathway and the H19/let-7/LIN28B axis may be involved in γ- to ß-globin gene switching. Moreover, we constructed the hub gene network of the differentially expressed transcription factors. Based on qPCR, we found that BCL11A was differentially expressed based on biological sex. We also confirmed that H19 is differentially expressed and established the H19-related network to reveal the potential regulatory mechanisms. CONCLUSION: We present the profiles of the in vivo transcriptome differences of NRBCs between preterm and full-term neonates for the first time, and provide novel research targets for ß-hemoglobinopathies.

Cell specific peripheral immune responses predict survival in critical COVID-19 patients.

SARS-CoV-2 triggers a complex systemic immune response in circulating blood mononuclear cells. The relationship between immune cell activation of the peripheral compartment and survival in critical COVID-19 remains to be established. Here we use single-cell RNA sequencing and Cellular Indexing of Transcriptomes and Epitomes by sequence mapping to elucidate cell type specific transcriptional signatures that associate with and predict survival in critical COVID-19. Patients who survive infection display activation of antibody processing, early activation response, and cell cycle regulation pathways most prominent within B-, T-, and NK-cell subsets. We further leverage cell specific differential gene expression and machine learning to predict mortality using single cell transcriptomes. We identify interferon signaling and antigen presentation pathways within cDC2 cells, CD14 monocytes, and CD16 monocytes as predictors of mortality with 90% accuracy. Finally, we validate our findings in an independent transcriptomics dataset and provide a framework to elucidate mechanisms that promote survival in critically ill COVID-19 patients. Identifying prognostic indicators among critical COVID-19 patients holds tremendous value in risk stratification and clinical management.

Single-cell transcriptomics reveals a low CD8+ T cell infiltrating state mediated by fibroblasts in recurrent renal cell carcinoma.

PURPOSE: Recurrent renal cell carcinoma(reRCC) is associated with poor prognosis and the underlying mechanism is not yet clear. A comprehensive understanding of tumor microenvironment (TME) of reRCC may aid in designing effective anticancer therapies, including immunotherapies. Single-cell transcriptomics holds great promise for investigating the TME, however, this technique has not been used in reRCC. Here, we aimed to explore the difference in the TME and gene expression pattern between primary RCC (pRCC) and reRCC at single-cell level. EXPERIMENTAL DESIGN: We performed single-cell RNA sequencing analyses of 32,073 cells from 2 pRCC, 2 reRCC, and 3 adjacent normal kidney samples. 41 pairs of pRCC and reRCC samples were collected as a validation cohort to assess differences observed in single-cell sequencing. The prognostic significance of related cells and markers were studied in 47 RCC patients underwent immunotherapy. The function of related cells and markers were validated via in vitro and in vivo experiments. RESULTS: reRCC had reduced CD8+ T cells but increased cancer-associated fibroblasts (CAFs) infiltration compared with pRCC. Reduced CD8+ T cells and increased CAFs infiltration were significantly associated with a worse response from immunotherapy. Remarkably, CAFs showed substantial expression of LGALS1 (Gal1). In vitro, CAFs could induce CD8+ T cells apoptosis via Gal1. In vivo, knockdown of Gal1 in CAFs suppressed tumor growth, increased CD8+ T cells infiltration, reduced the proportion of apoptotic CD8+ T cells and enhanced the efficacy of immunotherapy. CONCLUSIONS: We delineated the heterogeneity of reRCC and highlighted an innovative mechanism that CAFs acted as a suppressor of CD8+ T cells via Gal1. Targeting Gal1 combined with anti-PD1 showed promising efficacy in treating RCC.

Blood transcriptome analysis revealed the immune changes and immunological adaptation of wildness training giant pandas.

The giant panda (Ailuropoda melanoleuca) is a global flagship species for biodiversity conservation. As the time for captive giant pandas to be released into the wild matures, wildness training is provided to allow adaptation to their natural environment. It is assumed that changes in the immune system would be integral in this adaptation from captive to wild, where many more pathogens would be encountered in their natural habitats. Therefore, this study aims to determine the expression changes of immune-related genes and their potential as immunoassay markers for adaptation monitoring in wildness training giant pandas, and then to understand the adaptation strategy of wildness training giant pandas to the wild environment, thereby improving the success rate of panda reintroduction. We obtained 300 differentially expressed genes (DEGs) by RNA-seq, with 239 up-regulated and 61 down-regulated DEGs in wildness training giant pandas compared to captive pandas. Functional enrichment analysis indicated that up-regulated DEGs were enriched in several immune-related terms and pathways. There were 21 immune-related DEGs, in which most of them were up-regulated in wildness training giant pandas, including several critical innate and cellular immune genes. IL1R2 was the most significantly up-regulated gene and is a signature of homeostasis within the immune system. In the protein-protein interaction (PPI) analysis, CXCL8, CXCL10, and CCL5 were identified as the hub immune genes. Our results suggested that wildness training giant pandas have stronger innate and cellular immunity than captive giant pandas, and we proposed that a gene set of CXCL8, CXCL10, CCL5, CD3D, NFKBIA, TBX21, IL12RB2, and IL1R2 may serve as potential immunoassay markers to monitor and assess the immune status of wildness training giant pandas. Our study offers the first insight into immune alterations of wildness training giant pandas, paving the way for monitoring and evaluating the immune status of giant pandas when reintroducing them into the wild.

Transcriptional correlates of malaria in RTS,S/AS01-vaccinated African children: a matched case-control study.

Background: In a phase 3 trial in African infants and children, the RTS,S/AS01 vaccine (GSK) showed moderate efficacy against clinical malaria. We sought to further understand RTS,S/AS01-induced immune responses associated with vaccine protection. Methods: Applying the blood transcriptional module (BTM) framework, we characterized the transcriptomic response to RTS,S/AS01 vaccination in antigen-stimulated (and vehicle control) peripheral blood mononuclear cells sampled from a subset of trial participants at baseline and month 3 (1-month post-third dose). Using a matched case-control study design, we evaluated which of these 'RTS,S/AS01 signature BTMs' associated with malaria case status in RTS,S/AS01 vaccinees. Antigen-specific T-cell responses were analyzed by flow cytometry. We also performed a cross-study correlates analysis where we assessed the generalizability of our findings across three controlled human malaria infection studies of healthy, malaria-naive adult RTS,S/AS01 recipients. Results: RTS,S/AS01 vaccination was associated with downregulation of B-cell and monocyte-related BTMs and upregulation of T-cell-related BTMs, as well as higher month 3 (vs. baseline) circumsporozoite protein-specific CD4+ T-cell responses. There were few RTS,S/AS01-associated BTMs whose month 3 levels correlated with malaria risk. In contrast, baseline levels of BTMs associated with dendritic cells and with monocytes (among others) correlated with malaria risk. The baseline dendritic cell- and monocyte-related BTM correlations with malaria risk appeared to generalize to healthy, malaria-naive adults. Conclusions: A prevaccination transcriptomic signature associates with malaria in RTS,S/AS01-vaccinated African children, and elements of this signature may be broadly generalizable. The consistent presence of monocyte-related modules suggests that certain monocyte subsets may inhibit protective RTS,S/AS01-induced responses. Funding: Funding was obtained from the NIH-NIAID (R01AI095789), NIH-NIAID (U19AI128914), PATH Malaria Vaccine Initiative (MVI), and Ministerio de Economía y Competitividad (Instituto de Salud Carlos III, PI11/00423 and PI14/01422). The RNA-seq project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under grant number U19AI110818 to the Broad Institute. This study was also supported by the Vaccine Statistical Support (Bill and Melinda Gates Foundation award INV-008576/OPP1154739 to R.G.). C.D. was the recipient of a Ramon y Cajal Contract from the Ministerio de Economía y Competitividad (RYC-2008-02631). G.M. was the recipient of a Sara Borrell-ISCIII fellowship (CD010/00156) and work was performed with the support of Department of Health, Catalan Government grant (SLT006/17/00109). This research is part of the ISGlobal's Program on the Molecular Mechanisms of Malaria which is partially supported by the Fundación Ramón Areces and we acknowledge support from the Spanish Ministry of Science and Innovation through the 'Centro de Excelencia Severo Ochoa 2019-2023' Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program.

Digital gene expression analysis of NSCLC-patients reveals strong immune pressure, resulting in an immune escape under immunotherapy.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are currently one of the most promising therapy options in the field of oncology. Although the first pivotal ICI trial results were published in 2011, few biomarkers exist to predict their therapy outcome. PD-L1 expression and tumor mutational burden (TMB) were proven to be sometimes-unreliable biomarkers. We have previously suggested the analysis of processing escapes, a qualitative measurement of epitope structure alterations under immune system pressure, to provide predictive information on ICI response. Here, we sought to further validate this approach and characterize interactions with different forms of immune pressure. METHODS: We identified a cohort consisting of 48 patients with advanced non-small cell lung cancer (NSCLC) treated with nivolumab as ICI monotherapy. Tumor samples were subjected to targeted amplicon-based sequencing using a panel of 22 cancer-associated genes covering 98 mutational hotspots. Altered antigen processing was predicted by NetChop, and MHC binding verified by NetMHC. The NanoString nCounter® platform was utilized to provide gene expression data of 770 immune-related genes. Patient data from 408 patients with NSCLC were retrieved from The Cancer Genome Atlas (TCGA) as a validation cohort. RESULTS: The two immune escape mechanisms of PD-L1 expression (TPS score) (n = 18) and presence of altered antigen processing (n = 10) are mutually non-exclusive and can occur in the same patient (n = 6). Both mechanisms have exclusive influence on different genes and pathways, according to differential gene expression analysis and gene set enrichment analysis, respectively. Interestingly, gene expression patterns associated with altered processing were enriched in T cell and NK cell immune activity. Though both mechanisms influence different genes, they are similarly linked to increased immune activity. CONCLUSION: Pressure from the immune system will lay the foundations for escape mechanisms, leading to acquisition of resistance under therapy. Both PD-L1 expression and altered antigen processing are induced similarly by pronounced immunoactivity but in different context. The present data help to deepen our understanding of the underlying mechanisms behind those immune escapes.