Journal: The Journal of experimental medicine

Article Title: Landscape of mast cell populations across organs in mice and humans.

doi: 10.1084/jem.20230570

Figure Lengend Snippet: Figure 1. Mrgprb2+ and Mrgprb2neg MCs represent transcriptionally distinct MC populations. (A) UMAP plot of the scRNAseq performed on sorted peritoneal cavity. MCs. (B) UMAP plot of the scRNAseq performed on sorted immune cells from the back skin. Black arrowhead: MC population. (C). UMAP plot of the scRNAseq performed on sorted immune cells from the gut mucosa. Black arrowhead: MC population. (D) Violin plot of the expression of Mrgprb2, Mrgprb1, Mcpt4, Mcpt1, Kit, and Cpa3. (E) UMAP plot of all MC populations aggregated. (F) PCA showing the segregation between skin/peritoneal cavity and gut MCs. (G) Heatmap of 74 representative DEGs between MCs from gut, peritoneal cavity, and skin MC related. Characteristic genes and surface markers up- regulated in gut (green) or peritoneal cavity and skin MCs (red) are highlighted. (H and I) (H) Mrgprb2 and (I) Mcpt1 expression density on the aggregated populations. (J) Representative 3D confocal microscopy images of Avidin SRho (red), Mcpt1 (green), and DAPI (cyan) fluorescent signals of back skin, duo- denum, and colon. Arrows indicate gut mucosa and muscularis. Data are representative of three independent experiments. Bars = 100 (skin) and 70 (duodenum and colon) μm. DC, dendritic cell; NK, natural killer.

Article Snippet: Quality control numbers, ambient RNA decontamination, and analyses of scRNAseq samples Cell Ranger software (v.7.0.1; 10X Genomics) was used to demultiplex Illumina BCL files to FASTQ files (CellRanger mkfastq), to perform alignment (to mouse GRCm38/mm10 genome), filtering, UMI counting, and to produce gene–barcode matrices (CellRanger count).

Techniques: Expressing, Confocal Microscopy, Avidin-Biotin Assay

Journal: The Journal of experimental medicine

Article Title: Landscape of mast cell populations across organs in mice and humans.

doi: 10.1084/jem.20230570

Figure Lengend Snippet: Figure 3. MrgprB2+ and MrgprB2neg MCs have different hematopoietic origins and turnover kinetics. (A and B) Mrgprb2 (A) and Mcpt1 (B) expression density in MCs identified in the aggregated scRNAseq data of neonates (left) or adult (right) mice. (C) Representative 3D confocal microscopy images of Avidin

Article Snippet: Quality control numbers, ambient RNA decontamination, and analyses of scRNAseq samples Cell Ranger software (v.7.0.1; 10X Genomics) was used to demultiplex Illumina BCL files to FASTQ files (CellRanger mkfastq), to perform alignment (to mouse GRCm38/mm10 genome), filtering, UMI counting, and to produce gene–barcode matrices (CellRanger count).

Techniques: Expressing, Confocal Microscopy, Avidin-Biotin Assay

Journal: Nature immunology

Article Title: Adaptive plasticity of IL-10 + and IL-35 + T reg cells cooperatively promotes tumor T cell exhaustion

doi: 10.1038/s41590-019-0346-9

Figure Lengend Snippet: a, scRNAseq tSNE plots of bulk T reg cells from naive LNs or 14 days post inoculation B16 tumors (TIL T reg cells) from Foxp3 Cre-YFP mice. b, scRNAseq tSNE plot depicting the expression of Il10 and Ebi3 in individual T reg cells overlaid on the same tSNE plot as in ( a ). c, Heat maps contrasting the top 30 genes selected based on the differential expression analysis of T reg cells utilizing the two-sided Negative Binomial Exact test, demonstrating the lack of distinct transcriptional signatures. p-values were adjusted to control the false discovery rate (FDR) set at 0.05. Treg cells were first stratified into IL-10 − Ebi3 − , IL-10 + Ebi3 − , IL-10 − Ebi3 + , and IL-10 + Ebi3 + as described in ( b ). (Naive LN): IL-10 − Ebi3 − (n=717 cells), IL-10 + Ebi3 − (n=3 cells), IL-10 − Ebi3 + (n=83 cells), and IL-10 + Ebi3 + (n=3 cells). (TIL): IL-10 − Ebi3 − (n=491 cells), IL-10 + Ebi3 − (n=88 cells), IL-10 − Ebi3 + (n=491 cells), and IL-10 + Ebi3 + (n=111 cells). d, Bar graphs with overlaid scatter-dots depicting the TCR V β gene usage, comparing the T reg cells subpopulations and effector T cells. Three independent B16 tumor-bearing Il10 GFP . Ebi3 Tom . Foxp3 Cre-YFP mice were used to harvest T reg cell subpopulations for sequencing without pooling. Bars represent mean values. e, In vitro tracing of adaptive plasticity in cytokine expression by TCR-stimulation. ( top ): Naive T reg cells from LN and spleens were double-sort purified and stimulated with anti-CD3/CD28-coated beads in the presence of hIL2 and CD11c + cells for 72 hours, followed by FACS analysis. ( bottom ): Stacked bar graph summarizing four independent experiments. Statistical significance was determined by Two-way ANOVA with Holm-Sidak multiple comparisons ( # p=0.0073, *p=0.0016, **=P=0.0017, ***p=0.0002, ****p<0.0001). f, Diffusion pseudo-time analysis depicting the stochastic oscillation of IL-10 and IL-35 (Ebi3) expression based on the transcriptomic features of sequenced T reg cells from the scRNAseq experiment. g, Stacked-bar graph demonstrating the distribution of indicated T reg subpopulations along the Pseudotime projection as analyzed in ( f ). The Pseudotime projection was evenly divided into 10 fractions and the percent distribution of each T reg cell subpopulation was calculated.

Article Snippet: Except for RNAseq and scRNAseq data analysis, GraphPad Prism software was used to determine the statistical significance.

Techniques: Expressing, Quantitative Proteomics, Control, Sequencing, In Vitro, Purification, Diffusion-based Assay