Sample processing and single cell RNA-sequencing of peripheral blood immune cells from COVID-19 patients.
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ABSTRACT:
SUBMITTER: Yao C
PROVIDER: S-EPMC8114807 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Understanding the mechanism that leads to immune dysfunction in severe coronavirus disease 2019 (COVID-19) is crucial for the development of effective treatment. Here, using single-cell RNA sequencing, we characterized the peripheral blood mononuclear cells (PBMCs) from uninfected controls and COVID-19 patients and cells in paired broncho-alveolar lavage fluid (BALF). We found a close association of decreased dendritic cells (DCs) and increased monocytes resembling myeloid-derived suppressor cells (MDSCs), which correlated with lymphopenia and inflammation in the blood of severe COVID-19 patients. Those MDSC-like monocytes were immune-paralyzed. In contrast, monocyte-macrophages in BALFs of COVID-19 patients produced massive amounts of cytokines and chemokines, but secreted little interferons. The frequencies of peripheral T cells and NK cells were significantly decreased in severe COVID-19 patients, especially for innate-like T and various CD8+ T cell subsets, compared to healthy controls. In contrast, the proportions of various activated CD4+ T cell subsets among the T cell compartment, including Th1, Th2, and Th17-like cells were increased and more clonally expanded in severe COVID-19 patients. Patients' peripheral T cells showed no sign of exhaustion or augmented cell death, whereas T cells in BALFs produced higher levels of IFNG, TNF, CCL4, CCL5, etc. Paired TCR tracking indicated abundant recruitment of peripheral T cells to the severe patients' lung. Together, this study comprehensively depicts how the immune cell landscape is perturbed in severe COVID-19.
| S-EPMC7574992 | biostudies-literature
Project description:The peripheral immune system is thought to affect the pathology of the central nervous system in Alzheimer's disease (AD). However, current knowledge is inadequate for understanding the characteristics of peripheral immune cells in AD. This study aimed to explore the molecular basis of peripheral immune cells and the features of adaptive immune repertoire at a single cell level. We profiled 36,849 peripheral blood mononuclear cells from AD patients with amyloid-positive status and normal controls with amyloid-negative status by 5' single-cell transcriptome and immune repertoire sequencing using the cell ranger standard analysis procedure. We revealed five immune cell subsets: CD4+ T cells, CD8+ T cells, B cells, natural killer cells, and monocytes-macrophages cells, and disentangled the characteristic alterations of cell subset proportion and gene expression patterns in AD. Thirty-one cell type-specific key genes, comprising abundant human leukocyte antigen genes, and multiple immune-related pathways were identified by protein-protein interaction network and pathway enrichment analysis. We also found high-frequency amplification clonotypes in T and B cells and decreased diversity in T cells in AD. As clone amplification suggested the activation of an adaptive immune response against specific antigens, we speculated that the peripheral adaptive immune response, especially mediated by T cells, may have a role in the pathogenesis of AD. This finding may also contribute to further research regarding disease mechanism and the development of immune-related biomarkers or therapy.
| S-EPMC8382575 | biostudies-literature
Project description:The COVID-19 pandemic caused by SARS-CoV-2 has reached 5.5 million deaths worldwide, causing a huge impact globally. This highly contagious viral infection produces a severe acute respiratory syndrome that includes cough, mucus, fever and pneumonia. Likewise, many hospitalized patients develop severe pneumonia associated with acute respiratory distress syndrome (ARDS), besides an exacerbated and uncontrolled systemic inflammation which in some cases induce lethal cytokine storm. Although vaccines have clearly had a beneficial effect on disease development, there is still a high percentage of patients who develop pathology related to ineffective immune system response. Therefore, a thorough understanding of the modulatory mechanisms that regulate the response to SARS-CoV-2 is crucial to find effective therapeutic alternatives. Previous studies describe the relevance of Neddylation in immune system activation further its implications in viral infection. In this context, the present study postulates Neddylation, a reversible ubiquitin-like post-translational modification of proteins and controls their stability, localization and activity, as a key regulator in the immune response against SARS-CoV-2. For the first time, we describe an increase of serum global neddylation levels of COVID-19 patients particularly associated in the early response of the infection. In addition, the results showed that overactivation of neddylation control activation, proliferation, and response of peripheral blood mononuclear cells (PBMCs) derived from COVID-19 patients. Inhibition of neddylation and subsequent avoidance of activation of PBMCs, which reduces cytokine production and proteome modulation, may therefore be a critical mechanism and an efficient therapeutic approach to immunomodulate COVID -19 patients and avoid the much-feared cytokine storm.
2022-08-02 | PXD033673 | Pride
Project description:We carried out sex-balanced sampling of peripheral blood mononuclear cells (PBMCs) from confirmed COVID-19 inpatients (hospitalized) and outpatients (infected), uninfected close contacts (exposed), and healthy controls.
2021-04-15 | GSE171555 | GEO
Project description:To explore gene expression profiles that is predictive of COVID-19 deterioration, PBMC were collected from early stage non-severe COVID-19 patients and analyzed by grouping patients into 2 groups, deteriorated and non-deteriorated, based on the subsequent clinical courses.
2024-07-01 | GSE207625 | GEO
Project description:BackgroundUlcerative colitis (UC) is a complicated disease caused by the interaction between genetic and environmental factors that affects mucosal homeostasis and triggers an inappropriate immune response. Single-cell RNA sequencing (scRNA-seq) can be used to rapidly obtain the precise gene expression patterns of thousands of cells in the intestine, analyze the characteristics of cells with the same phenotype, and provide new insights into the growth and development of intestinal organs, the clonal evolution of cells, and immune cell changes. These findings can provide new ideas for the diagnosis and treatment of intestinal diseases.AimTo identify clinical phenotypes and biomarkers that can predict the response of UC patients to specific therapeutic drugs and thus aid the diagnosis and treatment of UC.MethodsUsing the Gene Expression Omnibus (GEO) database, we analyzed peripheral blood cell subtypes of patients with UC by scRNA-seq combined with bulk RNA sequencing (RNA-seq) to reveal the core genes of UC. We then combined weighted gene correlation network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) analysis to reveal diagnostic markers of UC.ResultsAfter processing the scRNA-seq data, we obtained data from approximately 24340 cells and identified 17 cell types. Through intercellular communication analysis, we selected monocyte marker genes as the candidate gene set for the prediction model. Construction of a WGCNA coexpression network identified RhoB, cathepsin D (CTSD) and zyxin (ZYX) as core genes. Immune infiltration analysis showed that these three core genes were strongly correlated with immune cells. Functional enrichment analysis showed that the differentially expressed genes were closely related to immune and inflammatory responses, which are associated with many challenges in the diagnosis and treatment of UC.ConclusionThrough scRNA-seq analysis, LASSO diagnostic model building and WGCNA, we identified RhoB, CTSD and ZYX as core genes of UC that are closely related to monocyte infiltration that may serve as diagnostic markers and molecular targets for UC therapeutic intervention.
| S-EPMC9724533 | biostudies-literature
Project description:The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global crisis; however, our current understanding of the host immune response to SARS-CoV-2 infection remains limited. Herein, we performed RNA sequencing using peripheral blood from acute and convalescent patients and interrogated the dynamic changes of adaptive immune response to SARS-CoV-2 infection over time. Our results revealed numerous alterations in these cohorts in terms of gene expression profiles and the features of immune repertoire. Moreover, a machine learning method was developed and resulted in the identification of five independent biomarkers and a collection of biomarkers that could accurately differentiate and predict the development of COVID-19. Interestingly, the increased expression of one of these biomarkers, UCHL1, a molecule related to nervous system damage, was associated with the clustering of severe symptoms. Importantly, analyses on immune repertoire metrics revealed the distinct kinetics of T-cell and B-cell responses to SARS-CoV-2 infection, with B-cell response plateaued in the acute phase and declined thereafter, whereas T-cell response can be maintained for up to 6 months post-infection onset and T-cell clonality was positively correlated with the serum level of anti-SARS-CoV-2 IgG. Together, the significantly altered genes or biomarkers, as well as the abnormally high levels of B-cell response in acute infection, may contribute to the pathogenesis of COVID-19 through mediating inflammation and immune responses, whereas prolonged T-cell response in the convalescents might help these patients in preventing reinfection. Thus, our findings could provide insight into the underlying molecular mechanism of host immune response to COVID-19 and facilitate the development of novel therapeutic strategies and effective vaccines.
| S-EPMC8421539 | biostudies-literature
Project description:Background: The advent of single-cell RNA sequencing (scRNAseq) and additional single-cell omics technologies have provided scientists with unprecedented tools to explore biology at cellular resolution. However, reaching an appropriate number of good quality reads per cell and reasonable numbers of cells within each of the populations of interest are key to infer relevant conclusions about the underlying biology of the dataset. For these reasons, scRNAseq studies are constantly increasing the number of cells analysed and the granularity of the resultant transcriptomics analyses. Methods: We aimed to identify previously described fibroblast subpopulations in healthy adult human skin by using the largest dataset published to date (528,253 sequenced cells) and an unsupervised population-matching algorithm. Results: Our reanalysis of this landmark resource demonstrates that a substantial proportion of cell transcriptomic signatures may be biased by cellular stress and response to hypoxic conditions. Conclusions: We postulate that careful design of experimental conditions is needed to avoid long processing times of biological samples. Additionally, computation of large datasets might undermine the extent of the analysis, possibly due to long processing times.
| S-EPMC8984215 | biostudies-literature
Project description:BackgroundSingle-cell RNA sequencing (scRNA-seq) provides high-dimensional measurements of transcript counts in individual cells. However, high assay costs and artifacts associated with analyzing samples across multiple sequencing runs limit the study of large numbers of samples. Sample multiplexing technologies such as MULTI-seq and antibody hashing using single-cell multiplexing kit (SCMK) reagents (BD Biosciences) use sample-specific sequence tags to enable individual samples to be sequenced in a pooled format, markedly lowering per-sample processing and sequencing costs while minimizing technical artifacts. Critically, however, pooling samples could introduce new artifacts, partially negating the benefits of sample multiplexing. In particular, no study to date has evaluated whether pooling peripheral blood mononuclear cells (PBMCs) from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30?min co-incubation at 4?°C) results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures.ResultsHere, we applied the 10x Genomics scRNA-seq platform to MULTI-seq and/or SCMK-labeled PBMCs from a single donor with and without pooling with PBMCs from unrelated donors for 30?min at 4?°C. We did not detect any alloreactivity signal between mixed and unmixed PBMCs across a variety of metrics, including alloreactivity marker gene expression in CD4+ T cells, cell type proportion shifts, and global gene expression profile comparisons using Gene Set Enrichment Analysis and Jensen-Shannon Divergence. These results were additionally mirrored in publicly-available scRNA-seq data generated using a similar experimental design. Moreover, we identified confounding gene expression signatures linked to PBMC preparation method (e.g., Trima apheresis), as well as SCMK sample classification biases against activated CD4+ T cells which were recapitulated in two other SCMK-incorporating scRNA-seq datasets.ConclusionsWe demonstrate that (i) mixing PBMCs from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30?min co-incubation at 4?°C) does not cause an allogeneic response, and (ii) that Trima apheresis and PBMC sample multiplexing using SCMK reagents can introduce undesirable technical artifacts into scRNA-seq data. Collectively, these observations establish important benchmarks for future cross-sectional immunological scRNA-seq experiments.
| S-EPMC7816397 | biostudies-literature
Project description:Human natural killer (NK) cells in peripheral blood perform many functions, and classification of specific subsets has been a longstanding goal. We report single-cell RNA sequencing of NK cells, comparing gene expression in unstimulated and interleukin (IL)-2-activated cells from healthy cytomegalovirus (CMV)-negative donors. Three NK cell subsets resembled well-described populations; CD56brightCD16-, CD56dimCD16+CD57-, and CD56dimCD16+CD57+. CD56dimCD16+CD57- cells subdivided to include a population with higher chemokine mRNA and increased frequency of killer-cell immunoglobulin-like receptor expression. Three novel human blood NK cell populations were identified: a population of type I interferon-responding NK cells that were CD56neg; a population exhibiting a cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2; and finally, a small population, with low ribosomal expression, downregulation of oxidative phosphorylation, and high levels of immediate early response genes indicative of cellular activation. Analysis of CMV+ donors established that CMV altered the proportion of NK cells in each subset, especially an increase in adaptive NK cells, as well as gene regulation within each subset. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analyzing NK cell responses in health and disease.
| S-EPMC7160259 | biostudies-literature