Sample processing and single cell RNA-sequencing of peripheral blood immune cells from COVID-19 patients

Abstract Background Endometriosis is a refractory and recurrent disease and it affects nearly 10% of reproductive-aged women and 40% of infertile patients. The commonly accepted theory for endometriosis is retrograde menstruation where endometrial tissues invade into peritoneal cavity and fail to be cleared due to immune dysfunction. Therefore, the comprehensive understanding of immunologic microenvironment of peritoneal cavity deserves further investigation for the previous studies mainly focus on one or several immune cells. Results High-quality transcriptomes were from peritoneal fluid samples of patients with endometriosis and control, and firstly subjected to 10 × genomics single-cell RNA-sequencing. We acquired the single-cell transcriptomes of 10,280 cells from endometriosis sample and 7250 cells from control sample with an average of approximately 63,000 reads per cell. A comprehensive map of overall cells in peritoneal fluid was first exhibited. We unveiled the heterogeneity of immune cells and discovered new cell subtypes including T cell receptor positive (TCR+) macrophages, proliferating macrophages and natural killer dendritic cells in peritoneal fluid, which was further verified by double immunofluorescence staining and flow cytometry. Pseudo-time analysis showed that the response of macrophages to the menstrual debris might follow the certain differentiation trajectory after endometrial tissues invaded into the peritoneal cavity, that is, from antigen presentation to pro-inflammation, then to chemotaxis and phagocytosis. Our analyses also mirrored the dysfunctions of immune cells including decreased phagocytosis and cytotoxic activity and elevated pro-inflammatory and chemotactic effects in endometriosis. Conclusion TCR+ macrophages, proliferating macrophages and natural killer dendritic cells are firstly reported in human peritoneal fluid. Our results also revealed that immune dysfunction happens in peritoneal fluid of endometriosis, which may be responsible for the residues of invaded menstrual debris. It provided a large-scale and high-dimensional characterization of peritoneal microenvironment and offered a useful resource for future development of immunotherapy.

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Multi tissue processing for single cell sequencing of human immune cells v1

10.17504/protocols.io.bz4qp8vw ◽

2021 ◽

Author(s):

Daniel Rainbow ◽

Sarah Howlett ◽

Lorna Jarvis ◽

Joanne Jones

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Immune Cells ◽

Human Tissues ◽

Single Cell Sequencing ◽

Tissue Processing ◽

Simultaneous Processing ◽

Single Cell Rna Sequencing ◽

Human Immune

This protocol has been developed for the simultaneous processing of multiple human tissues to extract immune cells for single cell RNA sequencing using the 10X platform, and ideal for atlasing projects. Included in this protocol are the steps needed to go from tissue to loading the 10X Chromium for single cell RNA sequencing and includes the hashtag and CiteSeq labelling of cells as well as the details needed to stimulate cells with PMA+I.

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Predicting bacterial infection outcomes using single cell RNA-sequencing analysis of human immune cells

Nature Communications ◽

10.1038/s41467-019-11257-y ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 11

Author(s):

Noa Bossel Ben-Moshe ◽

Shelly Hen-Avivi ◽

Natalia Levitin ◽

Dror Yehezkel ◽

Marije Oosting ◽

...

Keyword(s):

Bacterial Infection ◽

Single Cell ◽

Rna Sequencing ◽

Immune Cells ◽

Sequencing Analysis ◽

Single Cell Rna Sequencing ◽

Human Immune

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Single-cell RNA sequencing of immune cells in gastric cancer patients

Aging ◽

10.18632/aging.102774 ◽

2020 ◽

Vol 12 (3) ◽

pp. 2747-2763 ◽

Cited By ~ 2

Author(s):

Kai Fu ◽

Bingqing Hui ◽

Qian Wang ◽

Chen Lu ◽

Weihong Shi ◽

...

Keyword(s):

Gastric Cancer ◽

Single Cell ◽

Cancer Patients ◽

Rna Sequencing ◽

Immune Cells ◽

Single Cell Rna Sequencing ◽

Gastric Cancer Patients

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Clonal Hematopoiesis-Driver DNMT3A Mutations Alter Immune Cells in Heart Failure

Circulation Research ◽

10.1161/circresaha.120.317104 ◽

2020 ◽

Cited By ~ 1

Author(s):

Wesley T Abplanalp ◽

Sebastian Cremer ◽

David John ◽

Jedrzej Hoffmann ◽

Bianca Schuhmacher ◽

...

Keyword(s):

Heart Failure ◽

T Cell ◽

Chronic Heart Failure ◽

Single Cell ◽

Rna Sequencing ◽

Immune Cells ◽

Driver Mutations ◽

Clonal Hematopoiesis ◽

Mutation Carriers ◽

Single Cell Rna Sequencing

Rationale: Clonal hematopoiesis (CH) driven by mutations of DNA methyltransferase 3a (DNMT3A) is associated with increased incidence of cardiovascular disease and poor prognosis of patients with chronic heart failure (HF) and aortic stenosis. Although experimental studies suggest that DNMT3A CH-driver mutations may enhance inflammation, specific signatures of inflammatory cells in humans are missing. Objective: To define subsets of immune cells mediating inflammation in humans using single-cell RNA-sequencing. Methods and Results: Transcriptomic profiles of peripheral blood mononuclear cells were analysed in N=6 HF patients harboring DNMT3A CH-driver mutations and N=4 patients with HF and no DNMT3A mutations by single-cell RNA-sequencing. Monocytes of HF patients carrying DNMT3A mutations demonstrated a significantly increased expression of inflammatory genes compared to monocytes derived from HF patients without DNMT3A mutations. Among the specific up-regulated genes were the prototypic inflammatory interleukin (IL) IL1B, IL6, IL8, the inflammasome NLRP3, and the macrophage inflammatory proteins CCL3 and CCL4 as well as resistin, which augments monocyte-endothelial adhesion. Silencing of DNMT3A in monocytes induced a paracrine pro-inflammatory activation and increased adhesion to endothelial cells. Furthermore, the classical monocyte subset of DNMT3A mutation carriers showed increased expression of T-cell stimulating immunoglobulin superfamily members CD300LB, CD83, SIGLEC12, as well as the CD2 ligand and cell adhesion molecule CD58, all of which may be involved in monocyte-T cell interactions. DNMT3A mutation carriers were further characterized by increased expression of the T-cell alpha receptor constant chain and Th1, Th2, Th17, CD8+ effector, CD4+ memory and Treg specific signatures. Conclusions: This study demonstrates that circulating monocytes and T-cells of HF patients harboring CH-driver mutations in DNMT3A exhibit a highly inflamed transcriptome, which may contribute to the aggravation of chronic heart failure.

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Single-Cell RNA Sequencing Reveals Expanded Clones of Islet Antigen-Reactive CD4+ T Cells in Peripheral Blood of Subjects with Type 1 Diabetes

The Journal of Immunology ◽

10.4049/jimmunol.1700172 ◽

2017 ◽

Vol 199 (1) ◽

pp. 323-335 ◽

Cited By ~ 22

Author(s):

Karen Cerosaletti ◽

Fariba Barahmand-pour-Whitman ◽

Junbao Yang ◽

Hannah A. DeBerg ◽

Matthew J. Dufort ◽

...

Keyword(s):

T Cells ◽

Type 1 Diabetes ◽

Single Cell ◽

Rna Sequencing ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Single Cell Rna Sequencing ◽

Islet Antigen

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Single-cell RNA-sequencing reveals profound changes in circulating immune cells in patients with heart failure

Cardiovascular Research ◽

10.1093/cvr/cvaa101 ◽

2020 ◽

Cited By ~ 3

Author(s):

Wesley T Abplanalp ◽

David John ◽

Sebastian Cremer ◽

Birgit Assmus ◽

Lena Dorsheimer ◽

...

Keyword(s):

Heart Failure ◽

Flow Cytometry ◽

Single Cell ◽

Rna Sequencing ◽

Immune Cells ◽

Healthy Subjects ◽

Patients With Heart Failure ◽

Single Cell Rna Sequencing ◽

The Impact ◽

Classical Monocytes

Abstract Aims Identification of signatures of immune cells at single-cell level may provide novel insights into changes of immune-related disorders. Therefore, we used single-cell RNA-sequencing to determine the impact of heart failure on circulating immune cells. Methods and results We demonstrate a significant change in monocyte to T-cell ratio in patients with heart failure, compared to healthy subjects, which were validated by flow cytometry analysis. Subclustering of monocytes and stratification of the clusters according to relative CD14 and FCGR3A (CD16) expression allowed annotation of classical, intermediate, and non-classical monocytes. Heart failure had a specific impact on the gene expression patterns in these subpopulations. Metabolically active genes such as FABP5 were highly enriched in classical monocytes of heart failure patients, whereas β-catenin expression was significantly higher in intermediate monocytes. The selective regulation of signatures in the monocyte subpopulations was validated by classical and multifactor dimensionality reduction flow cytometry analyses. Conclusion Together this study shows that circulating cells derived from patients with heart failure have altered phenotypes. These data provide a rich source for identification of signatures of immune cells in heart failure compared to healthy subjects. The observed increase in FABP5 and signatures of Wnt signalling may contribute to enhanced monocyte activation.

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Single-cell RNA sequencing shows the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma

Nature Communications ◽

10.1038/s41467-021-24010-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Daniel Wai-Hung Ho ◽

Yu-Man Tsui ◽

Lo-Kong Chan ◽

Karen Man-Fong Sze ◽

Xin Zhang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Single Cell ◽

Tumor Cells ◽

Rna Sequencing ◽

Immune Cells ◽

Tumor Heterogeneity ◽

Sequencing Analysis ◽

T Cell Infiltration ◽

Single Cell Rna Sequencing ◽

Immune Oncology

AbstractInteraction between tumor cells and immune cells in the tumor microenvironment is important in cancer development. Immune cells interact with the tumor cells to shape this process. Here, we use single-cell RNA sequencing analysis to delineate the immune landscape and tumor heterogeneity in a cohort of patients with HBV-associated human hepatocellular carcinoma (HCC). We found that tumor-associated macrophages suppress tumor T cell infiltration and TIGIT-NECTIN2 interaction regulates the immunosuppressive environment. The cell state transition of immune cells towards a more immunosuppressive and exhaustive status exemplifies the overall cancer-promoting immunocellular landscape. Furthermore, the heterogeneity of global molecular profiles reveals co-existence of intra-tumoral and inter-tumoral heterogeneity, but is more apparent in the latter. This analysis of the immunosuppressive landscape and intercellular interactions provides mechanistic information for the design of efficacious immune-oncology treatments in hepatocellular carcinoma.

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Reference Transcriptomes of Porcine Peripheral Immune Cells Created Through Bulk and Single-Cell RNA Sequencing

Frontiers in Genetics ◽

10.3389/fgene.2021.689406 ◽

2021 ◽

Vol 12 ◽

Author(s):

Juber Herrera-Uribe ◽

Jayne E. Wiarda ◽

Sathesh K. Sivasankaran ◽

Lance Daharsh ◽

Haibo Liu ◽

...

Keyword(s):

T Cells ◽

Single Cell ◽

Immune Cells ◽

Peripheral Blood ◽

Cell Types ◽

Cell Populations ◽

Rna Seq ◽

Cell Type ◽

Transcriptomic Data ◽

Single Cell Rna Sequencing

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B-cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B-cell, conventional CD4 and CD8 αβ T-cells, NK cells, and γδ T-cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T-cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology.

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Chicken peripheral blood lymphocyte response to ALV-J infection assessed by single-cell RNA sequencing

10.1101/2021.01.12.426350 ◽

2021 ◽

Author(s):

Manman Dai ◽

Min Feng ◽

Ziwei Li ◽

Weisan Chen ◽

Ming Liao

Keyword(s):

T Cells ◽

T Cell ◽

Viral Infection ◽

Single Cell ◽

Rna Sequencing ◽

Peripheral Blood ◽

Cell Populations ◽

Control Group ◽

Marker Genes ◽

Single Cell Rna Sequencing

ABSTRACTChicken peripheral blood lymphocytes (PBLs) exhibit wide-ranging cell types, but current understanding of their subclasses, immune cell classification, and function is limited and incomplete. Previously, we found that viremia caused by avian leukosis virus subgroup J (ALV‐J) was eliminated by 21 days post infection (DPI), accompanied by increased CD8+ T cell ratio in PBLs and low antibody levels. Here we performed single-cell RNA sequencing (scRNA-seq) of PBLs in ALV-J infected and control chickens at 21 DPI to determine chicken PBL subsets and their specific molecular and cellular characteristics, before and after viral infection. Eight cell clusters and their potential marker genes were identified in chicken PBLs. T cell populations (clusters 6 and 7) had the strongest response to ALV-J infection at 21 DPI, based on detection of the largest number of differentially expressed genes (DEGs). T cell populations of clusters 6 and 7 could be further divided into four subsets: activated CD4+ T cells (cluster A0), Th1-like cells (cluster A2), Th2-like cells (cluster A1), and cytotoxic CD8+ T cells. Hallmark genes for each T cell subset response to viral infection were initially identified. Furthermore, pseudotime analysis results suggested that chicken CD4+ T cells could potentially differentiate into Th1-like and Th2-like cells. Moreover, ALV-J infection probably induced CD4+ T cell differentiation into Th1-like cells in which the most immune related DEGs were detected. With respect to the control group, ALV-J infection also had an obvious impact on PBL cell composition. B cells showed inconspicuous response and their numbers decreased in PBLs of the ALV-J infected chickens at 21 DPI. Percentages of cytotoxic Th1-like cells and CD8+ T cells were increased in the T cell population of PBLs from ALV-J infected chicken, which were potentially key mitigating factors against ALV-J infection. More importantly, our results provided a rich resource of gene expression profiles of chicken PBL subsets for a systems-level understanding of their function in homeostatic condition as well as in response to viral infection.

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