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Single-Cell RNA-Seq Analysis Uncovers Distinct Functional Human NKT Cell Sub-Populations in Peripheral Blood.

ABSTRACT: V?24-invariant human natural killer T (NKT) cells comprise a unique subset of CD1d-restricted T cells with potent immune regulatory function and are involved in the development of a variety of human diseases. However, the lack of comprehensive molecular subset identities limits their objective classification and clinical application. Using unbiased single-cell RNA sequencing (scRNA-seq) of over 4000 unstimulated and 7000 stimulated human peripheral blood NKT cells, we identified four and five clusters of NKT cells from each NKT group, respectively. Our study uncovers multiple previously unrecognized NKT subsets with potential functional specificities, including a cluster of NKT cells with regulatory T cell property. Flow cytometry and Ingenuity Pathway Analysis confirmed the existence of these NKT populations and indicated the related functional capacities. Our study provides the unbiased and more comprehensive molecular identities of human NKT subsets, which will eventually lead the way to tailored therapies targeting selected NKT subsets.

SUBMITTER: Zhou L

PROVIDER: S-EPMC7264113 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Single-Cell RNA-Seq Analysis Uncovers Distinct Functional Human NKT Cell Sub-Populations in Peripheral Blood.

Zhou Li L Adrianto Indra I Wang Jie J Wu Xiaojun X Datta Indrani I Mi Qing-Sheng QS

Frontiers in cell and developmental biology 20200526

Vα24-invariant human natural killer T (NKT) cells comprise a unique subset of CD1d-restricted T cells with potent immune regulatory function and are involved in the development of a variety of human diseases. However, the lack of comprehensive molecular subset identities limits their objective classification and clinical application. Using unbiased single-cell RNA sequencing (scRNA-seq) of over 4000 unstimulated and 7000 stimulated human peripheral blood NKT cells, we identified four and five cl ...[more]

PMID: 32528956

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Project description:BackgroundSingle-cell transcriptomics provides means to study cell populations at the level of individual cells. In leukocyte biology this approach could potentially aid the identification of subpopulations and functions without the need to develop species-specific reagents. The present study aimed to evaluate single-cell RNA-seq as a tool for identification of chicken peripheral blood leukocytes. For this purpose, purified and thrombocyte depleted leukocytes from 4 clinically healthy hens were subjected to single-cell 3' RNA-seq. Bioinformatic analysis of data comprised unsupervised clustering of the cells, and annotation of clusters based on expression profiles. Immunofluorescence phenotyping of the cell preparations used was also performed.ResultsComputational analysis identified 31 initial cell clusters and based on expression of defined marker genes 28 cluster were identified as comprising mainly B-cells, T-cells, monocytes, thrombocytes and red blood cells. Of the remaining clusters, two were putatively identified as basophils and eosinophils, and one as proliferating cells of mixed origin. In depth analysis on gene expression profiles within and between the initial cell clusters allowed further identification of cell identity and possible functions for some of them. For example, analysis of the group of monocyte clusters revealed subclusters comprising heterophils, as well as putative monocyte subtypes. Also, novel aspects of TCRγ/δ + T-cell subpopulations could be inferred such as evidence of at least two subtypes based on e.g., different expression of transcription factors MAF, SOX13 and GATA3. Moreover, a novel subpopulation of chicken peripheral B-cells with high SOX5 expression was identified. An overall good correlation between mRNA and cell surface phenotypic cell identification was shown.ConclusionsTaken together, we were able to identify and infer functional aspects of both previously well known as well as novel chicken leukocyte populations although some cell types. e.g., T-cell subtypes, proved more challenging to decipher. Although this methodology to some extent is limited by incomplete annotation of the chicken genome, it definitively has benefits in chicken immunology by expanding the options to distinguish identity and functions of immune cells also without access to species specific reagents.

Project description:Background: Atherosclerosis (AS) is a chronic inflammatory disease involving various cell types, cytokines, and adhesion molecules. Herein, we aimed to uncover its key molecular mechanisms by single-cell RNA-seq (scRNA-seq) analysis. Methods: ScRNA-seq data of cells from atherosclerotic human coronary arteries were analyzed using the Seurat package. Cell types were clustered, and differentially expressed genes (DEGs) were screened. GSVA (Gene Set Variation Analysis) scores of hub pathways were compared among different cell clusters. DEGs in endothelial cells between apolipoprotein-E (ApoE)-/- mice and specific TGFbR1/2 KO ApoE-/- mice fed with high-fat diet were overlapped with those from human AS coronary arteries. In fluid shear stress and AS, hub genes were determined based on the protein-protein interaction (PPI) network, which were verified in ApoE-/- mice. Finally, hub genes were validated in three pairs of AS coronary arteries and normal tissues by histopathological examination. Results: ScRNA-seq identified nine cell clusters in human coronary arteries, namely, fibroblasts, endothelial cells, macrophages, B cells, adipocytes, HSCs, NK cells, CD8+ T cells, and monocytes. Among them, endothelial cells had the lowest fluid shear stress and AS and TGF-beta signaling pathway scores. Compared to ApoE-/- mice fed with normal diet, fluid shear stress and AS and TGF-beta scores were both significantly lower in endothelial cells from TGFbR1/2 KO ApoE-/- mice fed with normal or high-fat diet. Furthermore, the two hub pathways had a positive correlation. Three hub genes (ICAM1, KLF2, and VCAM1) were identified, and their expression was distinctly downregulated in endothelial cells from TGFbR1/2 KO ApoE-/- mice fed with normal or high-fat diet than in those from ApoE-/- mice fed with a normal diet, which were confirmed in human AS coronary artery. Conclusion: Our findings clarified the pivotal impacts of pathways (fluid shear stress and AS and TGF-beta) and genes (ICAM1, KLF2, and VCAM1) in endothelial cells on AS progression.

Project description:BackgroundRheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease with a multifactorial etiology. Peripheral blood is the main channel of the immune system, and peripheral blood mononuclear cells (PBMCs) are the immune cells that initiate the autoimmune inflammatory process. However, there are few reports on the mechanisms of peripheral blood immunity in RA.MethodsScRNA-seq was performed on four RA samples and integrated with single-cell transcriptome data from four healthy control samples downloaded from publicly available databases for analysis.ResultsA total of 52,073 cells were used for descending clustering analysis to map RA peripheral blood immune cells at single-cell resolution. Redimensional clustering analysis of four major immune cells (T cells, monocytes, B cells, and natural killer cells) revealed that double-negative T (DNT) cells were significantly altered in abundance and function. And a number of genes (including SOCS3, cAMP-responsive element modulator (CREM), B2M, MTFP1, RSRP1, and YWHAB) were specifically downregulated in DNT cells. RA T cells, especially DNT cells, exhibit significant metabolic defects and dysfunction, mainly in the form of inhibition of oxidative phosphorylation, ATP synthesis, and major histocompatibility complex (MHC)-I-mediated antigen presentation. In addition, cellular communication networks were established, and it was evident that RA is significantly attenuated in the number and intensity of cellular communication. Monocytes and T cells play key roles in the process of the immune inflammatory response through CCL and MHC-related pathways.ConclusionsThis study describes the landscape of the peripheral blood immune system and cell communication in RA, characterizes the abundance of PBMCs, gene expression profiles, and changes in signaling pathways in RA patients, and identifies several key cell subpopulations (DNT and classic monocytes) and specific genes (SOCS3, CREM, B2M, MTFP1, RSRP1, and YWHAB). Meanwhile, we propose that classic monocytes in peripheral blood may migrate to sites of inflammation in synovial tissue under the chemotaxis of the chemokines CCL3 and CCL3L1, differentiate into macrophages, secrete proinflammatory cytokines, and thus participate in the inflammatory response. These findings provide new insights for the future elucidation of the peripheral blood immune mechanisms of RA and the search for new clinical therapeutic targets.

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Single-Cell RNA-Seq Analysis Uncovers Distinct Functional Human NKT Cell Sub-Populations in Peripheral Blood.

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Single-Cell RNA-Seq Analysis Uncovers Distinct Functional Human NKT Cell Sub-Populations in Peripheral Blood.

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