Journal: Frontiers in Immunology

Article Title: Single cell analysis revealed that two distinct, unique CD4 + T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice

doi: 10.3389/fimmu.2024.1340048

Figure Lengend Snippet: Analysis of anti-tumor cytotoxic function of bulk-old IEL CD4 + T cells. To investigate the role of old IEL CD4 + T cells in intestinal tumor immunity, we examined their anti-tumor cellular immunity in vitro . Pooled data from 2 similar independent experiments were analyzed (n=6 mice/group). “Org” means organoid in this figure. (A) Experimental design of this experiment. Organoids were generated from intestinal tumors of APC min mice, and co-cultured with CD4 + T cells and CD3 + T cells from SP and IEL of young and old mice. After 7 days of co-culture, they were harvested, and the numbers tumor cells and effector T cells were assessed by flow cytometry. (B) Representative images of organoids and IELs after 7 days of co-culture. (C) The number of live epithelial cells (7-AAD - EpCAM + CD45 - cells) collected from organoids in each group. Graphs show mean ± SEM. ∗P < 0.05. (D) Number of IELs (7-AAD - EpCAM - CD45 + cells) in each group (n = 6). Graphs show mean ± SEM. ∗P < 0.05.

Article Snippet: CD45 + cells were enriched from each group using CD45 microbeads (Miltenyi Biotec), followed by cell hashing with TotalSeq-C anti-mouse hashtag antibodies (hashtag1-young IEL, hashtag2-old IEL, hashtag3-young SP, hashtag4-old SP).

Techniques: In Vitro, Generated, Cell Culture, Co-Culture Assay, Flow Cytometry

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

Article Title: Universal Surface Biotinylation: a simple, versatile and cost-effective sample multiplexing method for single-cell RNA-seq analysis

doi: 10.1093/dnares/dsac017

Figure Lengend Snippet: Principle of USB method and comparison of USB and Ab labelling methods. (A) Principle of the USB and Ab methods (top). In the USB method, cell surface proteins (represented by small rectangles on the cell) are universally biotinylated with S-NHS-biotin and treated with hashtag DNA-tagged streptavidin. In the Ab method, cells are treated with biotin-conjugated antibody against specific cell surface protein (small rectangles on the cell) followed by the hashtag DNA-tagged streptavidin. If the specific protein is not expressed on some cells, those cells are not labelled by the Ab method (bottom right), whereas the USB method can label all the cells (bottom left). (B) Efficiency of cell labelling examined by flow cytometry. The USB labelling is highly efficient, with 99.6% of undifferentiated (top left) and 96.4% of differentiated cells (top right) being PE positive. In contrast, using the Ab method, 87.8% of undifferentiated cells (bottom left) and 9.9% of differentiated cells (bottom right) were PE positive. (C) Cell labelling by the USB method (top panel) and the Ab method (bottom panel) confirmed by fluorescence microscopy. In this case, the Ab method uses an antibody against mouse CDH1. CDH1 is known to be expressed in the undifferentiated ES cells, while the expression is limited to some cells in differentiated states. (Left) Undifferentiated ES cells; (right) differentiated cells (12 days after induction of differentiation). ph, phase-contrast images; PE, fluorescence images of TotalSeq-PE-streptavidin; arrowhead, PE negative cells in left panel; arrow, PE positive cells in right panel.

Article Snippet: The Cell-Hashtag antibody is a cocktail of antibodies intended to universally label cells of adult tissue origin (e.g. TotalSeq anti-mouse Hashtag Antibody, BioLegend).

Techniques: Comparison, Flow Cytometry, Fluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: Homeostatic, repertoire and transcriptional relationships between colon T regulatory cell subsets

doi: 10.1101/2023.05.17.541199

Figure Lengend Snippet: (A) UMAP projection of colonic CD4 T cells clusters from a representative scRNAseq analysis (left), color-coded for expression of activated CD4 T cell and Foxp3+ Treg gene signatures (right) (genes in Materials and Methods) (B) UMAP projection of colonic Treg cell clusters from a representative scRNAseq analysis of colonic Tregs (repeat of experiment in ), color-coded for expression of RORγ+ and Helios+ Treg gene signatures (genes in Materials and Methods) (top). UMAP projection of colonic Treg cells split between hashtags from different mice. Each hashtag represents an individual mouse of the listed condition.

Article Snippet: Before sorting, cells were hashtagged with mouse TotalSeqA DNA-barcoded hashtag antibodies at the same time as staining with fluorophore-conjugated antibodies (BioLegend).

Techniques: Expressing

Journal: bioRxiv

Article Title: Homeostatic, repertoire and transcriptional relationships between colon T regulatory cell subsets

doi: 10.1101/2023.05.17.541199

Figure Lengend Snippet: Helios+ Tregs and Rorγ+ Tregs have unique transcriptional signatures that are conserved across responses to individual microbes. (A) UMAP projection of colonic Treg cells from a representative scRNAseq analysis of colonic Tregs in GF (germ-free) and monocolonized mice, color-coded for expression of RORγ+ and Helios+ Treg gene signatures (genes in Materials and Methods). Repeat in . (B) UMAP projection of colonic Treg cells as in (A), split between hash tagged cells from different mice. Each hashtag represents an individual mouse: GF control, and GF mice monocolonized with C.ram ( C. ramosum ), EcN ( E. coli Nissle), and P.mag ( P. magnus ). (C) Heatmap of top differentially expressed genes from each cluster (Helios+, Rorγ+ or DN) across the different Treg subsets from GF or monocolonized mice. (D) String analysis and dotplot of top differentially expressed genes in Helios+ Tregs pooled from all GF and monocolonized mice. (E) String analysis and dotplot of top differentially expressed genes in Rorγ+ Tregs pooled from all GF and monocolonized mice. (F) 2D representation of Tregs based on gene-signature scores calculated for Helios+ Tregs and Rorγ+ Tregs (genes used to calculate scores in Materials and Methods).

Article Snippet: Before sorting, cells were hashtagged with mouse TotalSeqA DNA-barcoded hashtag antibodies at the same time as staining with fluorophore-conjugated antibodies (BioLegend).

Techniques: Expressing, Control

Journal: Cancer Research Communications

Article Title: LYVE-1–expressing Macrophages Modulate the Hyaluronan-containing Extracellular Matrix in the Mammary Stroma and Contribute to Mammary Tumor Growth

doi: 10.1158/2767-9764.CRC-24-0205

Figure Lengend Snippet: LYVE-1 + macrophages localize near stromal HA in the nulliparous mouse mammary gland and are associated with ECM regulation. A, Representative images from mammary gland capsular regions of 5-week Csf1r fl/fl (top) and Lyve1 Cre Csf1r fl/fl (bottom) mice immunostained for LYVE-1 (green), CD206 (red), HABP (magenta), and DAPI (blue; n = 4 per genotype). B, Representative images from mammary gland stroma regions of 5-week Csf1r fl/fl (top) and Lyve1 Cre Csf1r fl/fl (bottom) mice immunostained for LYVE-1 (green), F4/80 (red), HABP (magenta), and DAPI (blue; n = 4 per genotype). C, Mammary glands from 5-week Csf1r fl/fl (gray) and Lyve1 Cre Csf1r fl/fl (blue) female mice were assessed for CD45 − LYVE-1 + frequency by flow cytometry. D, Mammary glands from 5-week Csf1r fl/fl (gray) and Lyve1 Cre Csf1r fl/fl (blue) female mice were assessed for CD45 + F4/80 + CD11b + LYVE-1 + macrophage count by flow cytometry. Fold change of Lyve1 ( E ), Hyal1 , and Hyal2 ( F ) RNA expression from 15-week Csf1r fl/fl (gray) and Lyve1 Cre Csf1r fl/fl (blue) female mice. G, MFI of HABP in mammary glands assessing CD45 + F4/80 + CD11b + LYVE-1 − and CD45 + F4/80 + CD11b + LYVE-1 + macrophages. H, Mammary gland from 5-week and 15-week Csf1r fl/fl (gray) and Lyve1 Cre Csf1r fl/fl (blue) female mice assessed for HA by ELISA and normalized by weight. I, Female mammary glands assessed by ECM qRT-PCR array with differentially downregulated genes associated with biological process Gene Ontology (GO) terms (black) and molecular function GO terms (white; n = 3 per genotype). *, P < 0.05; **, P < 0.01; ****, P < 0.0001, using Student t test. Scale bars, 100 µm. Each dot represents one mouse.

Article Snippet: Following red blood cell removal and blocking, cells were stained at room temperature with fixable viability dye (eBiosciences), CD45 (BD Biosciences, 30-F11), and respective hashtagging antibodies (TotalSeq A0301 anti-mouse Hashtag 1 or TotalSeq A0302 anti-mouse Hashtag 2).

Techniques: Flow Cytometry, RNA Expression, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Cancer Research Communications

Article Title: LYVE-1–expressing Macrophages Modulate the Hyaluronan-containing Extracellular Matrix in the Mammary Stroma and Contribute to Mammary Tumor Growth

doi: 10.1158/2767-9764.CRC-24-0205

Figure Lengend Snippet: Identification and localization of LYVE-1 + macrophages in mammary tumors. A, Representative images from formalin-fixed and paraffin-embedded (FFPE) sections of EO771 tumors from Csf1r fl/fl mice stained by H&E, and immunostained for LYVE-1 (green), HABP (magenta), F4/80 (red), and DAPI (blue; n = 5). CD45 + F4/80 + CD11b + LYVE-1 + macrophage count, CD45 + F4/80 + CD11b + LYVE-1 + macrophage count normalized to tumor weight ( B ), and frequency of CD206, PDPN, and TIM4 from CD45 + F4/80 + CD11b + LYVE-1 + macrophage subset in EO771 tumors from C57BL/6 mice ( C ). FlowJo generated tSNE plots from CD45 + F4/80 + CD11b + LYVE-1 + subset in 5-week female mammary glands ( D ), and EO771 C57BL/6 tumors ( E ). **, P < 0.01. Scale bars, 100 µm. Each dot represents one mouse.

Article Snippet: Following red blood cell removal and blocking, cells were stained at room temperature with fixable viability dye (eBiosciences), CD45 (BD Biosciences, 30-F11), and respective hashtagging antibodies (TotalSeq A0301 anti-mouse Hashtag 1 or TotalSeq A0302 anti-mouse Hashtag 2).

Techniques: Staining, Generated

Journal: Cancer Research Communications

Article Title: LYVE-1–expressing Macrophages Modulate the Hyaluronan-containing Extracellular Matrix in the Mammary Stroma and Contribute to Mammary Tumor Growth

doi: 10.1158/2767-9764.CRC-24-0205

Figure Lengend Snippet: scRNA-seq identifies macrophage subsets in the tumor microenvironment. A, UMAP projection of 17 distinct clusters from the total population of CD45 + cells from EO771 tumors from Csf1r fl/fl and Lyve1 Cre Csf1r fl/fl mice ( n = 2 per genotype) with cell-specific labels. B, Dot plot of macrophage-associated genes from cells derived from Csf1r fl/fl tumors. C, Dot plot of enriched genes in macrophage clusters. D, Intercluster GSEA from Csf1r fl/fl macrophage clusters [normalized enrichment scale (NES) adjusted P -value < 0.05] with detailed gene set names in . E, Feature plot of Folr2 from tumors from Csf1r fl/fl and Lyve1 Cre Csf1r fl/fl mice. F, Average cell counts per cluster from tumors from Csf1r fl/fl and Lyve1 Cre Csf1r fl/fl mice. G, Waterfall plots of relative enrichment in cluster 7 Lyve1 Cre Csf1r fl/fl relative to Csf1r fl/fl of genes associated with glycosaminoglycan binding.

Article Snippet: Following red blood cell removal and blocking, cells were stained at room temperature with fixable viability dye (eBiosciences), CD45 (BD Biosciences, 30-F11), and respective hashtagging antibodies (TotalSeq A0301 anti-mouse Hashtag 1 or TotalSeq A0302 anti-mouse Hashtag 2).

Techniques: Derivative Assay, Binding Assay