Journal: Cell Reports Medicine

Article Title: A PD-1-targeted, receptor-masked IL-2 immunocytokine that engages IL-2Rα strengthens T cell-mediated anti-tumor therapies

doi: 10.1016/j.xcrm.2024.101747

Figure Lengend Snippet:

Article Snippet: CD45 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat# 130-110-618.

Techniques: Purification, Blocking Assay, Control, Recombinant, Staining, Luciferase, Isolation, Cell Isolation, Biomarker Discovery, Amplification, Knock-In, Plasmid Preparation, Expressing, Software

Journal: Cell

Article Title: A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions

doi: 10.1016/j.cell.2020.07.025

Figure Lengend Snippet:

Article Snippet: Flow cell 1 was left empty to use as a reference flow cell for on-line subtraction of bulk solution refractive index and for evaluation of non-specific binding of analyte to the chip surface using Biacore T200 Control Software (version 3.2) (GE Healthcare).

Techniques: Recombinant, Western Blot, Staining, Protease Inhibitor, Expressing, Gel Extraction, Sequencing, Software, Chromatography

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Thoracic cavity of control mice and β-cat gain-of-function (GOF) mice at E15.5. Dotted lines show the outline of the thymic primordium. In many cases, blood clots were observed in the central core of the thymic primordium in β-cat GOF mice. Representative data from three independent experiments are shown. TH: thymus, H: heart. Bar: 1 mm. ( B ) Labeling of β5t-iCre-activated cell progenies with tdTomato fluorescence in the thymus of control mice and β-cat GOF mice at E15.5. Mouse sections were nuclear counterstained with TO-PRO3. Bottom panels are magnifications of white-boxed areas in the top panels. Representative data from three independent experiments are shown. Bars: indicated in figures. ( C ) Hematoxylin and eosin staining (top) and immunofluorescence staining for K5 and K8 (middle) and β-catenin and β5t (bottom) on sagittal sections of thymic primordium in control mice and β-cat GOF mice at E15.5. Representative results from three independent experiments are shown. Bar: 100 μm. ( D ) Intracellular staining of β-catenin in CD45 − EpCAM + TECs isolated from control mice and β-cat GOF mice at E15.5. Histograms show β-catenin expression in control TECs (blue line) and β-cat GOF TECs (red line). Shaded area and black line represent the fluorescence in the absence of anti-β-catenin antibody in control TECs and β-cat GOF TECs, respectively. Plots on the right show net median fluorescence intensity (MFI) values (means and standard error of the means [SEMs], n = 4–5). The numbers in parentheses indicate percentage of control value. *p < 0.05.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Control, Labeling, Fluorescence, Staining, Immunofluorescence, Isolation, Expressing

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Immunofluorescence staining for CD45 and Foxn1 on sagittal sections of thymic primordium in control mice and β-cat GOF mice at E11.5–E15.5. The sections were nuclear counterstained with TO-PRO3. Anterior–posterior (A–P) and dorsal–ventral (D–V) orientations of the images are indicated. Representative data from three independent experiments are shown. Bar: 100 μm. ( B ) Quantitative RT-PCR analysis of mRNA expression levels (means and standard error of the means [SEMs], n = 3–4) of indicated genes relative to Gapdh levels in CD45 − EpCAM + thymic epithelial cells (TECs) isolated from the thymus of control mice and β-cat GOF mice at E15.5. ( C ) The numbers of CD45 + thymocytes were analyzed by flow cytometry. Plots show the numbers (means and SEMs, n = 3–4) of CD45 + thymocytes in the thymus of control mice and β-cat GOF mice at E15.5. ( D ) Flow cytometric analysis of double negative (DN) thymocytes from control mice and β-cat GOF mice at E15.5. Shown are profiles of CD44 and CD25 expression. The numbers in dot plots indicate the frequency of cells within indicated area. ( E ) Cell numbers (means and SEMs, n = 3–4) of indicated DN thymocyte subpopulations from control mice and β-cat GOF mice at E15.5 are plotted. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., not significant.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Immunofluorescence, Staining, Control, Quantitative RT-PCR, Expressing, Isolation, Flow Cytometry

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Immunofluorescence staining for CD45 and Foxn1 on sagittal sections of the thymic primordium in control mice and β-cat gain-of-function (GOF) mice at E11.5–E15.5. Shown are merged images with nuclear counterstaining (TO-PRO3) (left) and images obtained in each channel (middle, right) for the thymus from control mice and β-cat GOF mice at the indicated stages. Representative data from three independent experiments are shown. Bar: 100 μm. ( B ) Flow cytometric analysis of thymic epithelial cells (TECs) from indicated mice at E15.5, showing the purity of the isolated TECs for quantitative RT-PCR analysis. Shown are representative profiles of CD45, PI, and EpCAM expression in total cells (left) and in isolated CD45 − EpCAM + TECs (right) from control mice and β-cat loss-of-function (LOF) mice. The numbers indicate the frequency of cells within indicated areas.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Immunofluorescence, Staining, Control, Isolation, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Flow cytometric analysis of splenocytes from control mice and β-cat gain-of-function (GOF) mice at 11 wk. Shown are representative dot plot profiles of CD3 and TCRβ expression (left) and CD3 and TCRδ expression (right) in PI − viable cells. The numbers in dot plots indicate the frequency of cells within indicated area. ( B ) Cell numbers (means and standard error of the means [SEMs], n = 4–6) of indicated subpopulations in the spleen (left) and the iLN (right) from control and β-cat GOF mice at 8 wk are plotted. ( C ) Shown are representative dot plots of TCRδ and Vγ5 expression in CD45 + cells in the thymus from control mice and β-cat GOF mice at E15.5. The frequency of TCRδ + Vγ5 + cells is plotted (mean and SEMs; n = 4–6). ( D ) Flow cytometric analysis of dendritic epidermal T cells (DETCs) in the skin epidermis from control mice and β-cat GOF mice at 8 wk. Shown are representative dot plot profiles of CD3 and Vγ5 expression in epidermal cells. The numbers in dot plots indicate the frequency of cells within indicated area. The frequency of CD3 + Vγ5 + cells in epidermal cells is plotted (means and SEMs; n = 3). ( E ) Histograms for Vγ4 and Vγ1 expression in TCRβ - δ + cells in the iLN from control mice and β-cat GOF mice at 8 wk are shown. The frequency of Vγ4 + and Vγ1 + cells is plotted (means and SEMs; n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; N.S., not significant.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Control, Expressing

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Thoracic cavity of control mice and β-cat LOF mice at E15.5. Dotted lines show the outline of the thymic primordium. Representative data from three independent experiments are shown. TH: thymus, H: heart. Bar: 1 mm. ( B ) Immunofluorescence staining for β-catenin and Foxn1 (left) or K5 and K8 (right) on sagittal sections of the thymus from control mice and β-cat LOF mice at E15.5. Shown are merged images with nuclear counterstaining (TO-PRO3) (top) and images obtained in each channel (middle, bottom). Representative data from three independent experiments are shown. Bar: 100 μm. ( C ) Intracellular staining of β-catenin in CD45 − EpCAM + thymic epithelial cells (TECs) from control mice and β-cat LOF mice at E15.5. Histograms show β-catenin expression in control TECs (blue line) and β-cat LOF TECs (red line). Shaded area and black line represent the fluorescence in the absence of anti-β-catenin antibody in control TECs and β-cat LOF TECs, respectively. Plots show net median fluorescence intensity (MFI) values for β-catenin (means and standard error of the means [SEMs], n = 4). The numbers in parentheses indicate percentage of control value. ( D ) Flow cytometric analysis of enzyme-digested thymic cells from indicated mice at E15.5. Shown are profiles of EpCAM and CD45 expression in PI − viable cells (left) and UEA1 reactivity and Ly51 expression in CD45 − EpCAM + cells (right). The numbers in dot plots indicate the frequency of cells within indicated area. ( E ) Plots show the number of total thymic cells (left) and the frequency and the number of total TECs (middle) and cortical thymic epithelial cell (cTECs; right) from control mice and β-cat LOF mice at E15.5 (means and SEMs, n = 4). **p < 0.01; N.S., not significant.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Control, Immunofluorescence, Staining, Expressing, Fluorescence

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Intracellular staining of β-catenin in UEA1 − Ly51 + cortical thymic epithelial cells (cTECs; left) and UEA1 + Ly51 − medullary thymic epithelial cells (mTECs; right) from control mice and β-cat LOF mice at 2 wk. Histograms show β-catenin expression in cTECs and mTECs from control mice (blue line) and β-cat LOF mice (red line). Shaded area and black line represent the fluorescence in the absence of anti-β-catenin antibody in control TECs and β-cat LOF TECs, respectively. Plots show net MFI values for β-catenin in cTECs and mTECs (means and standard error of the means [SEMs], n = 3). The numbers in parentheses indicate percentage of control value. ( B ) Quantitative RT-PCR analysis of mRNA expression levels (means and SEMs, n = 5) of indicated genes relative to Gapdh levels in UEA1 − Ly51 + cTECs (top) and UEA1 + Ly51 − mTECs (bottom) in the thymus of control mice and β-cat LOF mice at 2 wk. ( C ) Flow cytometric analysis of enzyme-digested thymic cells from control mice and β-cat LOF mice at 2 wk. Shown are representative profiles of EpCAM and CD45 expression in PI − viable cells (left) and UEA1 reactivity and Ly51 expression in CD45 − EpCAM + viable cells (right). The numbers in dot plots indicate the frequency of cells within indicated area. ( D ) Plots show the number (means and SEMs, n = 6) of cTECs and mTECs in the thymus from control mice and β-cat LOF mice at 2 wk. ( E ) Immunofluorescence analysis of β5t (green), CCL21 (red), and Aire (cyan) on transverse sections of thymus from control mice and β-cat LOF mice at 2 wk. Representative data from three independent experiments are shown. Bar: 100 μm. Ctrl: Control, LOF: β-cat LOF. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., not significant.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Staining, Control, Expressing, Fluorescence, Quantitative RT-PCR, Immunofluorescence

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: Flow cytometric analysis of cTECs and mTECs from indicated mice at 2 wk. Shown are representative profiles of CD45, PI, and EpCAM expression of total cells after magnetic depletion of CD45 + cells, UEA1 reactivity and Ly51 expression in CD45 − EpCAM + cells, and isolated cTECs and isolated mTECs from control mice and β-cat loss-of-function (LOF) mice. The numbers indicate the frequency of cells within indicated areas.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Expressing, Isolation, Control

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet: ( A ) Bars show body weight (left) and thymus weight (right) at 6 mo in control female mice and β-cat LOF female mice (means and standard error of the means [SEMs], n = 4). ( B ) Appearance of thymus from control mice and β-cat LOF mice at 6 mo. Representative data from four independent experiments are shown. Bar: 1 mm. ( C ) Plots show the number (means and SEMs, n = 4) of total thymocytes in the thymus from control mice and β-cat LOF mice at 6 mo. ( D ) Flow cytometric analysis of enzyme-digested thymic cells from control mice and β-cat LOF mice at 6 mo. Shown are representative profiles of EpCAM and CD45 expression in PI − viable cells (left) and UEA1 reactivity and Ly51 expression in CD45 − EpCAM + viable cells (right). The numbers in dot plots indicate the frequency of cells within indicated area. ( E ) Plots show the number (means and SEMs, n = 4) of cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) in the thymus from control mice and β-cat LOF mice at 6 mo. ( F ) Immunofluorescence analysis of K5 (green) and K8 (magenta) on transverse sections of thymus from control mice and β-cat LOF mice at 6 mo. Representative data from three independent experiments are shown. Bar: 100 μm. Ctrl: Control, LOF: β-cat LOF. *p < 0.05; N.S., not significant.

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Control, Expressing, Immunofluorescence

Journal: eLife

Article Title: Fine-tuning of β-catenin in mouse thymic epithelial cells is required for postnatal T-cell development

doi: 10.7554/eLife.69088

Figure Lengend Snippet:

Article Snippet: For the isolation of TECs, CD45 − cells were enriched with magnetic-bead-conjugated anti-CD45 antibody (Miltenyi Biotec) before multicolor staining for flow cytometric cell sorting.

Techniques: Transduction, Plasmid Preparation, Recombinant, Sequencing, DNA Library Preparation, Software

Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: APC-Vio770 Recombinant human anti B220(clone REA755) , Miltenyi Biotec , Cat# 130–110-849; RRID:AB_2658286.

Techniques: Negative Control, Recombinant, Purification, Staining, Gene Expression, Lysis, Immunoprecipitation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Cell Isolation, Software, Microscopy

Journal: Nature Communications

Article Title: Adipose stem cell niche reprograms the colorectal cancer stem cell metastatic machinery

doi: 10.1038/s41467-021-25333-9

Figure Lengend Snippet: a Forest plot of survival changes in high (>30, obesity) versus low (≥18.5 and <25, lean) BMI CRC patients. Data represent the risk ratio ± 95% CI. Statistical significance was calculated by a Random-effect meta-analysis model. b Kaplan–Meier of progression-free survival (PFS) curve in a cohort of 511 CRC patients, based on BMI status. Healthy weight indicates 18,5 30. Statistical significance was calculated using the log-rank (Mantel–Cox) test. c H&E analysis and CDX2 expression on primary and liver metastasis in CRC patients with healthy weight or affected by obesity. Black arrow heads indicate tumor-infiltrating adipose cells. Li: liver; T = tumor. d Immunohistochemical analysis of CD34 (brown color), CD31 (green color), and CD45 (red color) in tissues as in c . For c , d one representative of 9 independent experiments is shown. e Phase-contrast analysis of CMS2 cells (CSphC #9) treated with medium or V-ASC CM. For ( c–e ) scale bars, 100 µm. One representative of three independent experiments is shown. f ELDA software analysis of the clonogenic activity in CMS2 CR-CSphCs following treatment with medium or V-ASC CM. g Clonogenic assay of CMS2 CR-CSphC lines TOP–GFP high and TOP–GFP low (15% highest/lowest TOP–GFP levels) treated with medium or V-ASC CM. For ( f–g ) statistical significance was calculated using the two-tailed t test and data are mean ± standard error of three independent experiments performed with CR-CSphCs isolated from three different CRC patients (CSphC #8, #9). h Percentage of TOP–GFP positive cells, in CMS2 cells treated with medium or V-ASC CM (left panel) . Box plots show min-to-max values, with line indicating the mean value. Flow cytometry analysis of TOP–GFP (black color indicates Wnt - cells; green color scale indicates low, intermediate, and high Wnt + cells) (right panel). Statistical significance was calculated using the paired two-tailed t test. Data are mean ± standard error of independent experiments performed with different CR-CSphCs (#1, #4, #5, #8, #9, #11, #21). i Number of mouse tumor xenografts generated by subrenal capsule injection of 10, 100, 1000, or 10,000 CR-CSphCs, alone or in combination with 50,000 V-ASCs (upper panel). Percentage of cancer-initiating cell (CIC) and its fold increase of cells (lower panels). Data are mean ± standard error (95% confidence interval) of 12 independent experiments performed with CR-CSphCs injected as described above. Statistical significance was calculated by ELDA software ( http://bioinf.wehi.edu.au/software/elda/ ). j In vivo imaging and CK20 immunohistochemistry analysis of xenograft tumor formation obtained by subrenal capsule injection of 100 CR-CSphCs alone or together with V-ASCs at the indicted time points. Photon signal of all metastatic sites (kidney, liver, and lungs) at 12 weeks. A yellow dotted line indicates a tumor xenograt lesion. Tumor (T), kidney (K), liver (Li), and lung (Lu) are indicated. One representative of 12 independent experiments is shown. Scale bars, 100 µm.

Article Snippet: Triple staining was performed using antibodies against CD34 (ICO115, mouse IgG 1 , CST, 1:50 dilution), CD31 (JC70A, mouse IgG 1k , Dako, 1:50 dilution), and CD45 (D9M8I, rabbit IgG, CST, 1:200 dilution), revealed by specific secondary antibodies, and detected by DAB, Vina Green and Vulcan Fast Red chromogen, respectively.

Techniques: Expressing, Immunohistochemical staining, Software, Activity Assay, Clonogenic Assay, Two Tailed Test, Isolation, Flow Cytometry, Generated, Injection, In Vivo Imaging, Immunohistochemistry

Journal: Nature Communications

Article Title: Adipose stem cell niche reprograms the colorectal cancer stem cell metastatic machinery

doi: 10.1038/s41467-021-25333-9

Figure Lengend Snippet: a Clustergram of tumor microenvironment-related genes in CR-CSphCs (CSphC #1, #8, #9, #21) and CD44v6 − or CD44v6 + enriched cells. Data are presented as normalized expression values. b VEGF production in cells as indicated. Data are mean ± SD of 4 independent experiments. Box and whiskers show min-to-max values, with line indicating the mean value. c , Gating strategy of CD271/VEGFR expression on ASCs (upper panels) . Dot-plots of CD271/VEGFR staining with or without the indicated antibody (FMO-APC control, minus CD271-PE-Cy7) (middle panel) . Flow cytometry analysis of CD271 and VEGFR in ASCs. Data are representative of 3 independent experiments performed with 10 different ASC lines (lower panel) . d , Percentage of CD31 positivity, by flow cytometry analysis, on CD34 + /CD31 - /CD45 - enriched ASCs exposed to vehicle (Medium), CD44v6 + CR-CSCs CM (CSC #1, #8, #9, #21), in presence or absence of VEGF neutralizing antibody, or VEGF for 14 days. Data are mean ± SD of three independent experiments using 3 different ASC cultures. e Phase-contrast micrographs of capillary-like tubular structures of Huvec cells treated as indicated for 16 h. Scale bars, 500 µm. One representative of 3 independent experiments is shown. f Immunohistochemical analysis of CD31 (red) and CD44v6 (green) on tumor xenografts generated by subcutaneous injection of CR-CSphCs alone or in combination with S-ASCs or V-ASCs (upper panel). Percentage of vascular surface area, based on CD31 positivity, in tumor xenografts (lower panel). Scale bars, 200 µm. Data are representative of 3 independent experiments. For b, d, and f statistical significance was calculated using the unpaired two-tailed t test. g Transcriptomic profile correlation between CMS2 CR-CSphCs (CSphC #8, #9) treated with S-ASCs or V-ASC conditioned medium (CM) and CMS4-associated gene signature. h GSEA of CMS4-associated gene signature in CMS2 CR-CSphCs (CSphC #8, #9) treated with V-ASC CM ( upper panel ). Top ten significantly up- and downregulated CMS4 signature genes in treated cells ( lower panel ). Statistical significance between two groups was determined by unpaired Student’s t test (2-tailed). i , Kinetics and whole-body in vivo imaging analysis of mice ( n = 6) intrasplenically injected with LUC-GFP CMS4, or CMS2 CR-CSphCs alone or co-injected with V-ASCs and treated as indicated. Data are mean ± S.D. of independent experiments performed with CR-CSphCs isolated from two different CMS2 (CSphC #8, #9) and CMS4 (#1, #21) CRC patients.

Article Snippet: Triple staining was performed using antibodies against CD34 (ICO115, mouse IgG 1 , CST, 1:50 dilution), CD31 (JC70A, mouse IgG 1k , Dako, 1:50 dilution), and CD45 (D9M8I, rabbit IgG, CST, 1:200 dilution), revealed by specific secondary antibodies, and detected by DAB, Vina Green and Vulcan Fast Red chromogen, respectively.

Techniques: Expressing, Staining, Control, Flow Cytometry, Immunohistochemical staining, Generated, Injection, Two Tailed Test, In Vivo Imaging, Isolation

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A) Experimental setup for (A)–(F), n = 3 individual mice/group. (B) Volcano plot from RNA-seq identifying differentially expressed genes (DEGs), with at least log 2 4-fold change at FDR < 0.05, between virus-induced lung and splenic CD4 + T cells. (C) Induced genes from (B) ranked by fold induction. (D) Heatmap of gene expression induction of all induced enzymes (orange) with Arg1 position indicated (red). (E and F) Representative (E) RNA-seq tracks of the Arg1 locus and (F) Arg1 expression (RNA-seq) in splenic and lung CD4 + T cells after influenza infection. (G) Experimental setup for (H)–(K). (H) Representative fluorescence-activated cell sorting (FACS) plots of ARG1 protein expression in WT and Arg1 CKO mice. (I) Viral titers in the lung at days 7 and 9 p.i. Representative experiment (of two independent experiments) shown with n = 4–5 mice per group per time point. (J) Representative hematoxylin and eosin (H&E) histology staining and (K) pathology score of the lungs at day 9 p.i. Combined data from two independent experiments, n = 11–13. *p < 0.05 (two-tailed Student’s t test). Each dot represents a sample from an individual mouse. See also .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: RNA Sequencing Assay, Virus, Expressing, Infection, Fluorescence, FACS, Staining, Two Tailed Test

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A) Experimental setup for (B)–(L). (B and C) Numbers of (B) total lung mononuclear cells and (C) lung CD4 + T cells at days 7 and 9 p.i., n = 12–17. (D and E) Representative FACS plots showing (D) influenza-specific (NP311–325 tetramer + ) lung CD4 + T cells and their CD11a and CD49d expression (in red) and (E) percentages of lung CD11a + CD49d + cells. (F–H) Numbers of lung (F) CD11a + CD49d + CD4 + T cells, (G) Ki67 + CD4 + T cells, and (H) NP311–235 tetramer + CD4 + T cells at days 7 and 9 p.i. in WT and Arg1 CKO mice, n = 4–6. (I and J) Representative FACS plots of (I) lung NP311–235 tetramer + CD4 + T cells and (J) T-bet expression by CD11a lo CD49d lo , CD11a + CD49d + , and NP311–235 tetramer + CD4 + T cells at day 9 p.i. (K and L) Numbers of (K) intracellular IFN-γ + , IL-2 + , and IL-10 + -producing lung CD4 + T cells (at day 7 p.i. and after in vitro restimulation with PR8-infected or non-infected dendritic cells), with (L) a representative FACS plot of data in (K), n = 14–15. *p < 0.05, **p < 0.01. (B and C) Kruskal-Wallis test; (F–I and L) Mann-Whitney test. ns, no statistically significant difference. (B, C, K, and L) Combined data from three individual experiments. (F–H) One representative of three independent experiments shown. Each dot represents a sample from an individual mouse. See also .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Expressing, In Vitro, Infection, MANN-WHITNEY

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A) Experimental setup for (B)–(D). (B) Representative FACS plot showing transferred WT and Arg1 CKO CD4 + T cells. (C and D) Numbers of CD4 + T cells recovered from (C) spleens and (D) lymph nodes 7 days post transfer, n = 4. (E) Experimental setup for (F)–(H). (F) Representative FACS plot of lung WT and Arg1 CKO CD11a + CD49d + cells at day 7 p.i. (G and H) Frequency of lung (G) CD11a + CD49d + CD4 + T cells and (H) Ki67 + CD4 + T cells at day 7 p.i., n = 5. (I) Experimental setup for (J)–(R). (J) Body weight of mice receiving no cells, n = 1, or WT or Arg1 CKO cells, n = 8–10. (K) Percentage of CD4 + T cells in the spleens, n = 8–10. (L and M) Numbers of CD4 + T cells in (L) spleens and (M) colons, n = 8–10. Representative of two individual experiments. (N and O) Weight of (N) spleens and (O) colons of mice injected with WT or Arg1 CKO CD4 + T cells, n = 15. (P–R) Colon pathology of mice injected with WT or Arg1 CKO based on (P) severity and (Q) inflammation, assessed via H&E histology staining. (R) Representative H&E staining of the colons. (N–Q) Combined data from two individual experiments, n = 15 individual mice/group. Each dot represents a sample from a single mouse. *p < 0.05, **p < 0.01, ***p < 0.001. (C, D, G, and H) paired Student’s t test; (J–Q) two-tailed Student’s t test.

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Injection, Staining, Two Tailed Test

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A) Experimental setup for (B)–(G). (B) Splenic naive CD4 + T cells in uninfected WT and Arg1 CKO animals, n = 14–16 (data from four combined experiments shown). (C and D) Representative (C) histogram of cell trace violet dilution at day 3 post activation and (D) division index (n = 3–4, one representative of two individual experiments shown). (E) 5-ethynyl-2′-deoxyuridine (EdU) incorporation at days 2.5 (n = 7–8) and 5 (n = 5) post activation (data from two combined individual experiments). (F) IFN-γ and IL-10 secretion at day 3 post activation (n = 5–7, data from two combined individual experiments shown). (G) Representative FACS plots showing intracellular IFN-γ and IL-10 staining at 5 days post activation. (H) Simplified schematic of ARG1 and ARG2 subcellular localization to cytoplasm (cyto) or mitochondria (mito). (I) Splenic naive CD4 + T cells in naive WT and Arg2 KO mice, n = 12–14 (data derived from four individual experiments). (J and K) CD4 + T cells from WT and Arg2 KO mice were activated in vitro for 3 days and (J) IFN-γ, IL-10, and (K) IL-17A measured (IFN-γ, n = 3; IL-10, n = 6, data from two combined individual experiments; IL-17A, n = 3–4). (L and M) RNA-seq analyses of CD4 + T cells from WT, Arg1 C KO, or global Arg2 KO mice at 22 h post in vitro activation (n = 3 individual mice/group) with (L) numbers of differentially expressed genes (DEGs) and (M) venn diagram and list of overlapping enriched biological pathways derived from DEGs. Each dot represents a sample from a single mouse. *p < 0.05, **p < 0.01. (D–F and I–K) Two-tailed Student’s t test. ns, no statistically significant difference. See also .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Activation Assay, Staining, Derivative Assay, In Vitro, RNA Sequencing Assay, Two Tailed Test

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A) Simplified diagram of the classical arginase pathway. (B–D) CD4 + T cells were isolated from the spleens of WT and Arg1 CKO mice and stimulated in vitro with anti-CD3 and anti-CD28 antibodies for 22–24 h. (B) Ornithine, n = 3 samples from individual mice, done in triplicate; (C) polyamine, n = 3 samples from individual mice; and (D) arginine abundancy in the CD4 + T cells, as determined by liquid chromatography-mass spectrometry, n = 3 samples from individual mice. (E–G) (E) Glycolysis (ECAR) and oxidative phosphorylation (OCR), as determined by Seahorse metabolomic profiling in response to oligomycin (oligo), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (fccp), and rotenone (rot) are shown with (F) accompanying statistical evaluation. ECAR, extracellular acidification rate; OCR, oxygen consumption rate. (E–G) n = 4, each dot represents individual mouse, one representative of three total experiments shown. *p < 0.05. (B, D, F, and G) Mann-Whitney test; (C) Kruskal-Wallis test. ns, no statistically significant difference. See also .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Isolation, In Vitro, Liquid Chromatography, Mass Spectrometry, MANN-WHITNEY

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A and B) Volcano plot of (A) differential metabolite abundance in in vitro -activated (22–24 h) CD4 + T cells from WT and Arg1 CKO mice, n = 4–6. Positive and negative ionization mode features (gray), unannotated features that exceeded log 2 FC > 0.26 (~20% change) and adjusted p value < 0.05 (black), annotated features which exceeded log 2 FC > 0.26 and adjusted p value < 0.05 (red- and blue-highlighted and labeled for clarity), and (B) glutamine abundancy. (C) Simplified schematic of intersecting arginine and glutamine pathways. (D and E) Abundance of (D) glutamate and (E) α-ketoglutarate (αKG). For αKG, n = 3 individual mice shown with technical replicates. (F) Schematic of Gpt2 activity in glutamine metabolism. (G and H) Representative FACS plots showing glutamate pyruvate transaminase 2 (GPT2) protein expression in WT, (G) Arg1 KO, and (H) Arg2 KO CD4 + T cells on day 3 post in vitro activation. (I) Percentage of IFN-γ + , IL-10 + , and IFN-γ-IL-10 double-positive T cells assessed via flow cytometry after in vitro stimulation with or without AOA treatment, n = 2–4 (representative of two independent experiments). *p < 0.05, **p < 0.01, ****p < 0.0001. (B, D, E, G, and I) Two-tailed Student’s t test; (L) one-way ANOVA. ns, no statistically significant difference, TCA cycle, tricarboxylic acid cycle. See also .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: In Vitro, Labeling, Activity Assay, Expressing, Activation Assay, Flow Cytometry, Two Tailed Test

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: (A and B) Representative FACS plots showing (A) arginase 1 (ARG1) expression and (B) CAT-1 expression at days 2 and 3 in healthy donor CD4 + T cells after in vitro stimulation, n = 4. (C) Amount of IFN-γ (left), IL-10 (middle), or ratio of IL-10 to IFN-γ (IL-10/IFN-γ) (right) secreted by CD4 + T cells after CD3+CD46 stimulation in vitro for 36 h in the presence of Nω-hydroxy-nor-L-arginine (nor-NOHA) or vehicle, n = 4. (D–K) CD4 + T cells were isolated from the blood of patients with arginase-1 deficiency (designated as P) and age-matched healthy controls (designated as HC). (D–F) IFN-γ and IL-10 secretion by CD3+CD46-activated CD4 + T cells (36 h, primary activation) with (D) individual values and (E) cumulative data for the IL-10/IFN-γ ratio during primary activation, n = 4 (four individual patients with multiple blood samples taken over a 2-year period), and (F) after CD3+CD46 restimulation for ~18–20 h post resting (5 days), n = 4 (individual patients, some with multiple blood samples taken over a 2-year period). (G and H) 5-ethynyl-2′-deoxyuridine (EdU) incorporation at day 5 post primary stimulation with (G) a representative FACS plot and (H) cumulative data, n = 2 healthy controls with technical duplicates, and n = 3 patients. (I) Percent of live CD4 + T cells after restimulation, as described under (F), n = 2 healthy controls done in duplicate and n = 3 patients, 1 with a technical duplicate. (J) Polyamine abundancy in resting CD4 + T cells. (K) Glycolysis (ECAR) and oxidative phosphorylation (OCR) as determined by Seahorse metabolomic profiling of CD4 + T cells after CD3+CD46 stimulation for 24 h. (L–O) ARG1 was over expressed in isolated CD4 + T cells from three healthy donors (n = 3) by electroporation of ARG1 into the cells with (L) a representative FACS plot showing ARG1 expression in control (CTRL) electroporated versus ARG1 electroporated CD4 + T cells prior to activation. ARG1 overexpressing or control CD4 + T cells were activated in vitro for 36 h. (M) Representative FACS plot, (N) cumulative data showing IFN-γ and IL-10 production, and (O) percent of cells that are Ki67 + . (P) Pathway analysis of DEGs derived from microarray analyses of CD3+CD46-activated CD4 + T cells (6 h) from patients 1 and 2 and two age-matched healthy control cells, n = 2. *p < 0.05, **p < 0.01. (C, N, and O) Paired Student’s t test; (E, F, H, and I) Mann-Whitney test. fccp, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; oligo, oligomycin; rot, rotenone. See also and .

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Expressing, In Vitro, Isolation, Activation Assay, Electroporation, Control, Derivative Assay, Microarray, MANN-WHITNEY

Journal: Immunity

Article Title: Loss of CD4 + T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection

doi: 10.1016/j.immuni.2023.07.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Naïve CD4 + T cells were isolated from WT CD45.1 + and Arg1 CKO (CD45.2 + ) mouse spleens using the manufacturer’s protocol (#130–104-453, Miltenyi Biotech) and combined at a 50:50% ratio (the ratio was assessed by flow cytometry before injection) and 3.2×10 6 combined cells/mouse were injected i.v. into Rag1 KO mice.

Techniques: Virus, Isolation, Recombinant, Staining, Cell Isolation, Flow Cytometry, RNA Sequencing Assay, In Vitro, Microarray, CRISPR, Control, Software

Journal: Cell Reports Medicine

Article Title: A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8 + T cells

doi: 10.1016/j.xcrm.2023.101154

Figure Lengend Snippet: BF10 repopulates infiltrated immune cells in tumor tissues and LNs (A) Experimental scheme. The HNSC/Q1-2 tumor cells (5 × 10 5 cells) were orthotopically inoculated into mice, followed by administrations of the indicated components (Ctrl-IgG, IL-10-Fc, αCSF1R, and BF10) with a 3 day interval for 3 doses. Tumor-infiltrated CD45 + immune cells were assessed by flow cytometry. (B–D) Population of CD8 + T cells (B), TAMs (C), and CD4 + T cells (D) in tumors from the indicated groups. (E) Representative plots (left) and population (right) of the granzyme B (GZMB)-producing tumor-infiltrating T cells from the indicated tumors. (F) Representative population of the TCF1 + Tim3 − T cells (Prog Tex) and the TCF1 − Tim3 + T cells (Term Tex) among total tumor-infiltrating CD44 + PD-1 + CD8 + T cells from the indicated mice. (G–H) Representative images of CD8 + T cell numbers from the indicated groups. (G) Multiplexed immunofluorescence (mIF) staining performed with Opal 7-Color IHC kit (PerkinElmer) for CD4 (green), CD8 (sky blue), GZMB (red), and nuclei (hyacinth). Representative composite images obtained and quantified by the Vectra Polaris Imaging System and Inform software. Scale bar, 100 μm. (H) Quantitative result of CD8 + T cells. (I–K) Representative image of Ki67 expression in tumor-draining LNs (tdLNs). (I) The isolated tdLN examined by immunohistochemistry (IHC). The brown color indicates Ki67 + cells. Scale bar, 300 μm. (J) Representative mIF images of tdLN. The isolated tdLNs were stained with DAPI (blue), CD8 (green), PD-1 (yellow), FoxP3, CD19 (pink), and Ki67 (red). Whole-tissue composite images were captured and analyzed with the Vectra Polaris Imaging System and Inform software. Scale bar, 400 μm. (K) Quantification of the cell number of CD8 + PD1 + , CD8 + PD1 + Ki67 + , and CD19 + cells in the BF10-treated versus control group. The data were presented as mean ± SD, and the statistics were calculated using unpaired Student’s t test. (two group comparison) and one-way ANOVA (more than 3 groups) with an appropriate test. ∗∗p < 0.01.

Article Snippet: CD45 (D3F8Q) Rabbit mAb , Cell Signaling , Cat#70527; RRID: AB_2799780.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Imaging, Software, Expressing, Isolation, Immunohistochemistry, Control, Comparison

Journal: Cell Reports Medicine

Article Title: A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8 + T cells

doi: 10.1016/j.xcrm.2023.101154

Figure Lengend Snippet: scRNA-seq analysis of syngeneic HNSCC treated with BF10 or its subcomponents (A) Experimental scheme for scRNA-seq. TILs were isolated. Single-cell libraries were prepared using a Chromium NextGEM Single Cell 3′ Reagent Kit (v.3.1 kit) and sequenced with Illumina NovaSeq 6000. Data were processed and analyzed with Cell Ranger pipeline (v.5.0.1, 10× Genomics) and Loupe browser (v.5.0). A detailed analysis of T cell subtype was assessed with ProjecTILs (v.1.0). CD45 − cells were harvested for bulk RNA-seq. (B) Top, uniform manifold approximation and projection (UMAP) plot of the total cells colored by the 7 major cell lineages. The cell counts of each cluster are indicated in brackets. Bottom, a heatmap to represent the top 10 upregulated genes of each cluster. (C) UMAP plots of markers for different types of immune cells. (D) UMAP plot of color-coded immune cell types as indicated (reference: CellKb, https://www.cellkb.com/immune .). (E) Left, UMAP plots of cell-type distributions in different treatment groups. Right, pie charts showing the immune cell compositions of different treatment groups. (F) Projection of T cells into a TIL reference atlas using ProjecTILs in tumors of different treatment groups. Colored cells represent the reference states defined by previous literature ; black cells represent projected cells from the treatment groups, and black contour lines represent the density of projected cells over the atlas. (G) T cell subtype composition and proportion of the four treatment conditions with subtypes defined by ProjecTILs classifier. (H) DEGs in exhausted T cells (Texs) between pairs of treatments.

Article Snippet: CD45 (D3F8Q) Rabbit mAb , Cell Signaling , Cat#70527; RRID: AB_2799780.

Techniques: Isolation, RNA Sequencing

Journal: Cell Reports Medicine

Article Title: A CSF-1R-blocking antibody/IL-10 fusion protein increases anti-tumor immunity by effectuating tumor-resident CD8 + T cells

doi: 10.1016/j.xcrm.2023.101154

Figure Lengend Snippet:

Article Snippet: CD45 (D3F8Q) Rabbit mAb , Cell Signaling , Cat#70527; RRID: AB_2799780.

Techniques: Control, Flow Cytometry, RNA Sequencing, Sequencing, Recombinant, Labeling, Enzyme-linked Immunosorbent Assay, Binding Assay, Polymer, Cell Isolation, Selection, Software

Journal: iScience

Article Title: C5aR1-positive adipocytes mediate non-shivering thermogenesis in neonatal mice

doi: 10.1016/j.isci.2024.111261

Figure Lengend Snippet: Identification of the CD45 + preadipocyte population in BAT of neonatal mice (A) Schematic diagram describing the experiment strategy to carry out single cell RNA seq of SVF from BAT from neonatal mice. (B) tSNE plot revealing SVF populations isolated from BAT of neonatal mice. (C) Normalized gene expression value as violin plots of selected cluster-specific genes from neonatal mice. (D) Volcano plot of the global gene expression profile of ASC1-3 and ASC4 (left) and tSNE plot showing colocalization of Itgb1 and Ptprc in the ASCs and ASC4 clusters (right). (E) FACS gating strategy for the isolation of CD45 + ASCs by FACS. (F) CD45 + ASCs were isolated from CD29 + SCA1 + cells with CD45 + . (G) CD45 − and CD45 + ASCs had adherent growth capacity, morphology of fibroblast-like cells. Scale bars, 10μm. (H) RT-qPCR of CD45 − and CD45 + ASCs sorted by FACS. CD45 + ASCs markers, Ptprc , adipose progenitor markers: Pdgfrα , Cd34 , and Itgb1 ( n = 3). Statistical significance was assessed by two-tailed Student’s t test. Data are represented as mean ± SEM. ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.005.

Article Snippet: CD45 monoclonal Antibody , Proteintech , Cat# 60287-1-Ig; RRID: AB_2881404.

Techniques: RNA Sequencing, Isolation, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: iScience

Article Title: C5aR1-positive adipocytes mediate non-shivering thermogenesis in neonatal mice

doi: 10.1016/j.isci.2024.111261

Figure Lengend Snippet: In vitro differentiated CD45 + adipocytes were distinct from classical brown adipocytes (A) Oil red O staining of adipogenic differentiation of isolated CD45 − and CD45 + ASCs in vitro . Scale bars, 20μm. (B) mRNA expression of the adipogenic differentiation of isolated CD45 − and CD45 + ASCs in vitro . ( n = 3). (C) mRNA expression of the indicated genes in differentiated adipogenic cells in (A) and white adipocytes ( n = 3). (D) Immunoblotting for UCP1 and PPARγ in differentiated cells in (A). (E) RNA-seq of CD45 − and CD45 + adipocytes. Volcano plot of the global gene expression profile ( n = 3). (F) Top 10 genes upregulated and downregulated. (G) (Top) upregulated KEGG pathways and typical genes. (Bottom) downregulated KEGG pathways and typical genes. (H) Expression of C5ar1 mRNA in CD45 − and CD45 + ASC and adipogenic differentiated CD45 − and CD45 + adipocytes. (I) Immunoblotting for C5aR1 in adipogenic differentiated CD45 − and CD45 + adipocytes. Statistical significance was assessed by two-tailed Student’s t test. Data are represented as mean ± SEM. ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.005.

Article Snippet: CD45 monoclonal Antibody , Proteintech , Cat# 60287-1-Ig; RRID: AB_2881404.

Techniques: In Vitro, Staining, Isolation, Expressing, Western Blot, RNA Sequencing, Gene Expression, Two Tailed Test

Journal: iScience

Article Title: C5aR1-positive adipocytes mediate non-shivering thermogenesis in neonatal mice

doi: 10.1016/j.isci.2024.111261

Figure Lengend Snippet: The loss of the C5ar1 gene in adipocytes during the perinatal period decreased the thermogenesis ability of newborn mice (A) Administration of shRNA- C5ar1 in CD45 + adipocytes significantly suppressed C5ar1 expression compared to the control group. (Left) RT-qPCR for C5ar1 in shNC and sh C5ar1 CD45 + adipocytes. (Right) Immunoblotting for C5aR1 in shNC and sh C5ar1 CD45 + adipocytes ( n = 3). (B) Oil red O staining of shNC and sh C5ar1 CD45 + adipocytes. Scale bars, 20μm. (C) The mRNA expression of the indicated genes of C5ar1 knockdown differentiated CD45 + adipocytes ( n = 3). (D) Immunoblotting for UCP1 and PPARγ of C5ar1 knockdown differentiated CD45 + adipocytes ( n = 3). (E) C5ar1 AKO mice ( C5ar1 flox/flox ; Adipoq Cre + ) were generated by breeding C5ar1 flox/flox mice with Adiponectin -Cre mice. (F) Immunofluorescence staining of UCP1 and C5aR1 in the BAT of the control and C5ar1 AKO neonatal mice. Scale bars, 50μm. (G) (left)Infrared thermo-imaging 5 min after the beginning of cold challenge. (Right) back skin temperature during cold challenge (22° C) (control, n = 8 AKO, n = 7). (H) Body weight and BAT/body weight ratio in control and C5ar1 AKO neonatal mice. (I) Gross appearance of the interscapular BAT of the control and C5ar1 AKO neonatal mice. Scale bars, 1mm. (J) H&E (Top) and UCP1 immunohistochemical staining (bottom). Scale bars, 20μm. BAT adipocyte sizes of control and C5ar1 AKO neonatal mice (right). (K) Relative mRNA expression of indicated genes of BAT from control and C5ar1 AKO neonatal mice ( n = 8). (L) Immunoblotting for UCP1 of BAT from control and C5ar1 AKO neonatal mice ( n = 6). (M) RNA-seq of BAT from control and C5ar1 AKO neonatal mice. Volcano plot of the global gene expression profile ( n = 3). (N) Upregulated KEGG pathways and downregulated KEGG pathways. Statistical significance was assessed by two-tailed Student’s t test. Data are represented as mean ± SEM ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.005.

Article Snippet: CD45 monoclonal Antibody , Proteintech , Cat# 60287-1-Ig; RRID: AB_2881404.

Techniques: shRNA, Expressing, Control, Quantitative RT-PCR, Western Blot, Staining, Knockdown, Generated, Immunofluorescence, Imaging, Immunohistochemical staining, RNA Sequencing, Gene Expression, Two Tailed Test

Journal: iScience

Article Title: C5aR1-positive adipocytes mediate non-shivering thermogenesis in neonatal mice

doi: 10.1016/j.isci.2024.111261

Figure Lengend Snippet: C5ar1 knockdown in CD45 + brown adipocytes promote PF4 release to inhibit brown adipocyte maturation (A) Relative mRNA expression of the cytokines genes ( Pf4 , Ccl3 , Ccl4 , Ccl12 ) of C5ar1 knockdown differentiated CD45 + adipocytes compared to the control group ( n = 6). (B) The concentration of PF4 in the supernatant of C5ar1 knockdown differentiated CD45 + adipocytes compared to the control group ( n = 3). (C) Pf4 mRNA expression in differentiated CD45 − and CD45 + adipocytes ( n = 6). (D) Concentration of PF4 in the supernatant of differentiated CD45 − and CD45 + adipocytes ( n = 3). (E) Pf4 mRNA expression of BAT from Control and C5ar1 AKO neonatal mice ( n = 8). (F) Concentration of PF4 in BAT of Control and C5ar1 AKO neonatal mice ( n = 6). (G) Relative mRNA expression of the indicated genes of adipocyte differentiation of CD45 − ASCs cultured without or with 20 ng/mL PF4 ( n = 6). (H) Immunoblotting for UCP1 and PPARγ of adipocyte differentiation of CD45 − ASCs cultured without or with 20 ng/mL PF4 ( n = 3). (I) Relative mRNA expression of the indicated genes from adipocyte differentiation of CD45 − ASCs cultured in conditioned media from shNC, sh C5ar1 , sh Pf4 or sh C5ar1 + shPf4 CD45 + adipocytes ( n = 3). (J) Immunoblotting for UCP1 and PPARγ of adipocyte differentiation of CD45 − ASCs cultured in conditioned media from shNC, sh C5ar1 , sh Pf4 or sh C5ar1 + shPf4 CD45 + adipocytes ( n = 3). (K) Graphical abstract of this study: The loss of C5ar1 in CD45 + adipocytes increased Pf4 mRNA level and increased the secretion of PF4. PF4 inhibited the maturity and thermogenesis ability of both CD45 + and CD45 − adipocytes. Statistical significance was assessed by two-tailed Student’s t test (A–G) or one-way ANOVA (I). Data are represented as mean ± SEM ∗ ≤0.05, ∗∗ ≤0.01, ∗∗∗ ≤0.005.

Article Snippet: CD45 monoclonal Antibody , Proteintech , Cat# 60287-1-Ig; RRID: AB_2881404.

Techniques: Knockdown, Expressing, Control, Concentration Assay, Cell Culture, Western Blot, Two Tailed Test

Journal: iScience

Article Title: C5aR1-positive adipocytes mediate non-shivering thermogenesis in neonatal mice

doi: 10.1016/j.isci.2024.111261

Figure Lengend Snippet:

Article Snippet: CD45 monoclonal Antibody , Proteintech , Cat# 60287-1-Ig; RRID: AB_2881404.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, SYBR Green Assay, Sequencing, Software

Journal: Journal of Alzheimer's Disease Reports

Article Title: Gliosis Precedes Amyloid-β Deposition and Pathological Tau Accumulation in the Neuronal Cell Cycle Re-Entry Mouse Model of Alzheimer’s Disease

doi: 10.3233/ADR-200170

Figure Lengend Snippet: Markers of neuroinflammatory microglia activation is present in the cortex of 12-month-old NCCR mice. A) 12-month-old NCCR mice show clusters of activated microglia compared to TRE-SV40T controls. Iba1-labeled microglia show CD45 co-labeling (arrows). There are also CD45 pos /Iba-1 neg cells, indicative of brain leukocyte infiltration (arrowheads). Iba-1 labeled microglia also show co-labeling with CD68 (arrows), and MHCII (arrows). Age-matched control brain sections from TRE-SV40T Tg mice littermates (TAg control) that were maintained on the same diet regimen did not show CD45, CD68, or MHCII labeling. B) Confocal imaging shows p16 labeling in a subset of microglia (arrows). The p16 co-labeled microglia also show blebbing, a morphological feature associated with senescent microglia (arrows). Age-matched control brain sections from TRE-SV40T Tg mice littermates (TAg control) that were maintained on the same diet regimen did not showp16 labeling (data not shown). C) Image J software was used to quantify the percent of area covered by MHCII immunofluorescence. MHCII-positive areas were increased by 770% in the NCCR mice compared to TAg control mice at 12 months of age (unpaired t-test with Welch’s correction for unequal variances, p = 0.033). n = 2 animals per group, with 2 sections per animal and 20 individual regions of interest evaluated per brain section, as shown in .

Article Snippet: The following antibodies were used in this study: Iba1 and p16 (ab178846 and ab108349, respectively, abcam, Cambridge, MA, USA); CD68 (MCAA1957GA, Bio-Rad, Hercules, CA, USA); MHCII (#556999, BD Biosciences, San Jose, CA, USA); CD45 (YW62.3, Thermo Fisher Scientific, Waltham, MA, USA); SV40T and PCNA (Pab 101 and PC10, respectively, Santa Cruz Biotechnology, Dallas, TX, USA); GFAP (MAB360) (Millipore, Billerica, MA, USA); Alexa Fluor conjugated goat-anti mouse 488, goat-anti rabbit 594, and goat-anti-rat 594 (Thermo Fisher Scientific).

Techniques: Activation Assay, Labeling, Control, Imaging, Software, Immunofluorescence

Journal: Cell reports

Article Title: Endothelial cell expression of a STING gain-of-function mutation initiates pulmonary lymphocytic infiltration

doi: 10.1016/j.celrep.2024.114114

Figure Lengend Snippet: (A) Diagram of the STING V154M conditional knockin (CKI). (B) Tail DNA from a STING CKI/WT mouse and a STING CKI/WT × CMV-Cre mouse was PCR amplified using primers indicated in (A). STING WT allele gives a 596-bp fragment, STING CKI allele gives a 774-bp fragment, and upon deletion of the gene trap from the CKI allele a 636-bp fragment is generated. (C) STING expression by CD45 + immune cells from the blood of mice inheriting the indicated STING alleles as assessed by flow cytometry. (D–H) Eight-week-old age-, sex-, and littermate-matched CKI ( n = 13–23, white) and CKI × CMV-Cre mice ( n = 16–29, red) and 12-week-old age-, sex-, and littermate-matched WT ( n = 6, gray) and VM mice ( n = 10, pink) were evaluated by the following measures. Data shown represent at least two independent experiments. Bar graphs represent mean ± SD. (D) Representative 43 field H&E-stained lungs. Images are representative of at least two independent experiments. (E) Immunofluorescence staining for DAPI (gray), CD3 (cyan), LYVE1 (yellow), and B220 (magenta) on CKI × CMV-Cre mouse lung. (D and E) Scale vars, 200 μm. Images are representative of at least two independent experiments. (F) Percentage of EV immune cells among live CD45 + lung cells, and total counts of CD45 + lung EV cells. (G) Percentage of CD69 + EV CD3 + T cells in the lung. (H) Body weight from mice, normalized as the fold change relative to the mean body weight of sex-matched CKI and WT controls, and spleen weight. See also and . ns, not significant; p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: CD45 microbeads, mouse , Miltenyi , Cat#130-052-301.

Techniques: Knock-In, Amplification, Generated, Expressing, Flow Cytometry, Staining, Immunofluorescence

Journal: Cell reports

Article Title: Endothelial cell expression of a STING gain-of-function mutation initiates pulmonary lymphocytic infiltration

doi: 10.1016/j.celrep.2024.114114

Figure Lengend Snippet: (A) CD45 + immune and CD45 − non-hematopoietic cells from the lungs of 12-week-old CKI × YFP ( n = 8, white), CKI × Nkx2.1-Cre × YFP ( n = 3, purple), CKI × PDGFRa-Cre × YFP ( n = 4, blue), CKI × Tie2-Cre × YFP ( n = 4, green), and CKI × CMV-Cre × YFP ( n = 4, red) mice were evaluated for the percentage of YFP + cells within the EPCAM + CD31 − CD140a − epithelial, CD140a + EPCAM − CD31 − fibroblast, and CD31 + EPCAM − CD140a − endothelial cell compartments. Data are from one experiment. (B–F) Eight-week-old CKI × Nkx2.1-Cre ( n = 9, purple), CKI × PDGFRa-Cre ( n = 6, blue), and CKI × Tie2-Cre ( n = 8, green) mice were compared to sex- and littermate-matched control CKI mice (no additional Cre genes) ( n = 3, n = 3, n = 8, respectively). Data shown represent at least two independent experiments. Bar graphs represent mean ± SD. (B) Percentage of lung EV immune cells within the total CD45 + lung population, and total number of CD45 + EV immune cells. (C) Percentage of CD69 + lung EV T cells. (D and E) Representative 10× field H&E stains on lungs from CKI and CKI × Tie2-Cre mice. Immunofluorescence staining of CKI × Tie2-Cre mouse lung: DAPI (gray), CD3 (cyan), LYVE1 (yellow), and B220 (magenta). Scale bars, 200 μm. Images are representative of at least two independent experiments. (F) Body weight normalized as the fold change compared to the mean body weight of sex-matched CKI control mice; spleen weight. See also . ns, not significant; p > 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: CD45 microbeads, mouse , Miltenyi , Cat#130-052-301.

Techniques: Control, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Endothelial cell expression of a STING gain-of-function mutation initiates pulmonary lymphocytic infiltration

doi: 10.1016/j.celrep.2024.114114

Figure Lengend Snippet: (A) CD45 + splenic immune cells from 12 week-old CKI × YFP ( n = 7, white), CKI × Rorc-Cre × YFP ( n = 3, teal), and CKI × LysM-Cre × YFP ( n = 3, brown) mice were evaluated for the percentage of YFP + cells within B220 + B cells, CD3 + T cells, CD11b + and/or CD11c + myeloid cells, CD11b + Ly6G + neutrophils, CD11b + Ly6G − Ly6C + monocytes, and CD11c + MHCII + dendritic cells. Data are from one experiment. (B–H) Eight-week-old CKI × Rorc-Cre ( n = 8, teal) and CKI × LysM-Cre ( n = 7, brown) mice were compared to age- and sex-matched control CKI mice ( n =5, n =5, respectively). Data shown represent at least two independent experiments. Bar graphs represent mean ± SD. (B) Percentage of CD3 + T cells and CD11b + and/or CD11c + myeloid cells within the total CD45 + splenocyte population. (C) Percentage of CD69 + activated T cells. (D) Percentage of Ly6G + neutrophils and Ly6C + Ly6G − monocytes within the splenic myeloid subset. (E) Percentage of lung EV immune cells within total number of CD45 + lung cells and total number of EV immune cells. (F) Mean number of inguinal lymph nodes in CKI controls compared to the indicated strains. An additional cohort of CKI × CMV-Cre mice ( n = 27, red) and their matched CKI controls ( n = 18, white) are included. (G) Percentage of CD69 + and PD-1 + cells within EV T cell compartment. (H) Percentage of CD11b + Ly6C hi inflammatory monocytes within lung EV myeloid compartment, and percentage of CD86 + cells within the lung EV monocyte compartment. ns, not significant; p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: CD45 microbeads, mouse , Miltenyi , Cat#130-052-301.

Techniques: Control

Journal: Cell reports

Article Title: Endothelial cell expression of a STING gain-of-function mutation initiates pulmonary lymphocytic infiltration

doi: 10.1016/j.celrep.2024.114114

Figure Lengend Snippet: (A) CKI × YFP ( n = 9, white), CKI × Cdh5-Cre ERT2 × YFP ( n = 11, yellow), and CKI × CAGG-Cre ERTM × YFP ( n = 8, orange) mice were treated with tamoxifen P0-P2 and CD31 + LECs and CD45 + lung cells were evaluated for YFP expression at 5–7 weeks of age. Eight-week-old sex- and littermate-controlled STING CKI × YFP ( n = 4, white) and STING CKI × Tie2-Cre × YFP ( n = 5, green) mice were similarly assessed. (B) Representative 4× field H&E histology of lung sections from CKI × Cdh5-Cre ERT2 (two mice top row: left shows modest immune aggregate formation, right shows more extensive immune aggregate formation), CKI controls, and CKI × CAGG-Cre ERTM mice. Images are representative of at least two independent experiments. (C) Immunofluorescence staining of mouse lungs from indicated strains: for DAPI (gray), CD3 (cyan), LYVE1 (yellow), and B220 (magenta). Images are representative of at least two independent experiments. (D) Percentage of lung EV immune cells within total CD45 + lung populations, and total number of EV immune cells. (E) Percentage of CD69 + EV T cells. (F) Body weight, normalized as the fold change compared to the mean body weight of sex-matched CKI control mice, and spleen weight. (A and D–F) Data shown represent at least two independent experiments. Bar graphs represent mean ± SD. See also , , and . ns, not significant; p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: CD45 microbeads, mouse , Miltenyi , Cat#130-052-301.

Techniques: Expressing, Immunofluorescence, Staining, Control

Journal: Cell reports

Article Title: Endothelial cell expression of a STING gain-of-function mutation initiates pulmonary lymphocytic infiltration

doi: 10.1016/j.celrep.2024.114114

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD45 microbeads, mouse , Miltenyi , Cat#130-052-301.

Techniques: Recombinant, Staining, Ligation, Expressing, High Throughput Screening Assay, RNA Sequencing, Knock-In, Software