Journal: European journal of immunology

Article Title: CCL17 transgenic mice show an enhanced Th2-type response to both allergic and non-allergic stimuli.

doi: 10.1002/eji.200535564

Figure Lengend Snippet: Fig. 1. Increased production and secretion of CCL17 protein with biological and functional activity by KC of Tg mice. (A) Epidermal KC in ear skin were strongly stained by anti-CCL17 antibody in Tg mice compared to non-Tg mice. (B) KC from Tg mice produced a significantly higher level of CCL17 than those from non-Tg mice. (C) A significantly higher level of CCL17 was detected in the serum from Tg mice than that from non-Tg mice. The results are presented as the mean SD.*p <0.05. (D) The chemoattractant activity of supernatants of cultured KC from Tg mice and recombinant murine CCL17 was determined by chemotaxis assay. CCL17 secreted by KC from Tg mice showed chemotactic activity for CCR4+ cells. The number of migrating cells was expressed as a percentage of the number of cells loaded in the upper chamber (mean SD). Supernatant and CCR4+ cells: open circles, supernatant and control cells: open squares, recombinant murine CCL17 and CCR4+ cells: closed circles, recombinant murine CCL17 and control cells: closed squares.

Article Snippet: The antibodies used were polyclonal goat anti- murine CCL17 antibody (AF529, R & D systems) at 0.2 lg/mL as a primary antibody and horseradish peroxidase conjugated donkey anti-goat IgG (sc-2056, Santa Cruz Biotechnology, CA) at 0.2 lg/mL as a secondary antibody.

Techniques: Functional Assay, Activity Assay, Staining, Produced, Cell Culture, Recombinant, Chemotaxis Assay, Control

Journal: Science Advances

Article Title: Human interleukin-12α and EBI3 are cytokines with anti-inflammatory functions

doi: 10.1126/sciadv.adg6874

Figure Lengend Snippet: ( A ) Percentage of CD25 hi Foxp3 + T reg cells [fluorescence-activated cell sorting (FACS)] in human PBMC cultures treated with IL-12a C96S (20 ng/ml) and EBI3 (20 ng/ml) or the combination of both IL-12a C96S and EBI3 ( n = 8). Data are presented as means + SEM. Statistical significance was determined by Friedman test. * P < 0.05 and ** P < 0.01. ( B ) Concentrations of IL-4 (ELISA) in culture supernatants from SEA-stimulated human PBMCs alone or in combination with IL-12 C96S or EBI3 ( n = 8 donors). The dotted line indicates mean secretion of IL-4 from PBS-treated PBMCs. ( C ) Concentrations of CCL17 (ELISA) in culture supernatants from IL-4–stimulated human PBMCs alone or in combination with IL-12 C96S or EBI3 ( n = 3 donors). (B and C) Data are presented as individual values. Donor-dependent effect is shown by the connecting line. Statistical significance was determined by Wilcoxon test. * P < 0.05 and ** P < 0.01.

Article Snippet: PBMC supernatants were analyzed for IL-4 using the IL-4 Human ELISA Kit from Thermo Fisher Scientific (KHC0041) and for CCL17 using the human CCL17/TARC DuoSet ELISA Kit from R&D systems (DY364).

Techniques: Fluorescence, FACS, Enzyme-linked Immunosorbent Assay

Journal: Mucosal immunology

Article Title: Short ragweed pollen promotes M2 macrophage polarization via TSLP/TSLPR/OX40L signaling in allergic inflammation

doi: 10.1038/s41385-019-0187-8

Figure Lengend Snippet: Antibodies used for immunohistochemical and immunofluorescent staining

Article Snippet: The results were photographed with an epifluorescence microscope (Eclipse 400; Nikon, Garden City, NY) using a digital camera (DMX 1200; Nikon). table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Antibodies Company Cat.# Dilution Arg1 Abcam Ab60176 1:50 Ym1 Abcam Ab93034 1:100 FIZZ1 Abcam Ab39626 1:100 F4/80 Biolegend B226028 1:100 TSLP ProSci 4023 1:200 TSLPR Prosci 4209 1:100 OX40L Santa Cruz sc-10952 1:100 TARC Bioss BS-2453R 1:200 MDC Bioss BS-1761R 1:200 IL-4 BioLegend 504101 1:100 IL-5 BioLegend 504301 1:100 IL-13 Santa Cruz sc-1776 1:100 Open in a separate window Antibodies used for immunohistochemical and immunofluorescent staining Statistical analysis Student’s t -test was used to compare differences between two groups.

Techniques: Immunohistochemical staining

Journal: bioRxiv

Article Title: Cancer cell genetics shaping of the tumor microenvironment reveals myeloid cell-centric exploitable vulnerabilities in hepatocellular carcinoma

doi: 10.1101/2023.10.29.564350

Figure Lengend Snippet: A. PCA plot depicting the Nras G12D / Pten KO and Nras G12V / Pten KO HCC cell line transcriptome (n=3 per cell line). B. Barplot depicting the signaling pathways enriched in the down-regulated genes shown in (blue colored dots). C-D. Proteomic profile analyses of Myc OE /Trp53 KO , Myc OE /Pten KO , Nras G12D / Pten KO and Nras G12V / Pten KO bulk tumors ( C ) or conditioned media collected from HCC cancer cell lines ( D ), relative to control livers or AML12 cell line, respectively. Indicated values are shown as fold change of the pixel density compared to control for each assessed protein. E. Barplot depicting the quantification of GM-CSF secretion in the conditioned media of Nras G12D / Pten KO (n=6, from ) and pT3-Nras G12V / Pten KO cell lines (n=3). F-G. Barplot depicting the Csf2 gene expression levels extracted from RNA-seq datasets of control livers (n=3), bulk Nras G12D / Pten KO (n=4) and Nras G12V / Pten KO tumors (n=3) ( F ) or Nras G12D / Pten KO (n=3) and Nras G12V / Pten KO HCC cell lines ( G ) (n=3 per cell line). H. Barplot depicting the Csf2 gene expression levels assessed by RT-qPCR in Myc OE /Trp53 KO , Myc OE /Pten KO , Nras G12D / Pten KO and Nras G12V / Pten KO HCC cell lines (n=3 for each cell line). One Myc OE /Trp53 KO sample is set as baseline level. I. Barplot depicting the GM-CSF protein levels assessed by Luminex assay in control livers (n=6), Myc OE /Trp53 KO (n=3), Myc OE /Pten KO (n=3), Nras G12D / Pten KO (n=3) and Nras G12V / Pten KO (n=3) bulk tumor lysates. J. Barplots depicting the Csf2 , Ccl6 and Ccl17 gene expression levels assessed by RT-qPCR in control (n=4), Myc OE /Trp53 KO (n=5), Myc OE /Pten KO (n=5), Nras G12D / Pten KO (n=5) and Nras G12V / Pten KO (n=4) end-stage bulk tumors. One control liver sample is set as the baseline level for all genes. K. Barplot depicting the relative pixel density of GM-CSF levels determined by proteomic profile analyses of control mouse and genetically-distinct HCC-bearing mice sera. Control sample is set as the baseline level. L. UMAP representation of Csf2 expression in the scRNA-seq from . M. UMAP representation of Csf2ra, Csf2rb and Ccl6 expression in the ‘Monocytic cell’ clusters identified by scRNA-seq . N. Barplot depicting the enrichment of the GM-CSF signature in each of the indicated subpopulations of the ‘Monocytic cells’ cluster identified by scRNA-seq (presented in ). O. Boxplot depicting the enrichment of GM-CSF negatively regulated genes (n=709) from the GM-CSF signature in TCGA: LIHC patients segregated according to their high correlation with the Nras G12D / Pten KO or Nras G12V / Pten KO transcriptional signatures (same sample size as ). P. Kaplan-Meier survival curves of TCGA: LIHC patients segregated according to their high/low enrichment of GM-CSF negatively regulated genes (n=709) from the GM- CSF signature (High n=62, Low n=103). Graphs show mean ± SEM ( E-J ). Statistical significance was determined by differential expression analyses ( F, G ) one-way ANOVA ( H-J ), unpaired Student’s T-test ( E, O ) and log-rank test ( P ). Vertical lines at -log10(FDR)=2.5 are used as threshold for ( B, N ). *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: The following Taqman probes (Thermo Fisher) were used for qPCR: Ubc (Mm01201237_m1), Il6 (Mm00446190_m1), Il1b (Mm00434228_m1), Il1a (Mm00439620_m1), Ccl6 (Mm01302419_m1), Ccl17 (Mm00516136_m1), Csf2 (Mm01290062_m1), Nlrp3 (Mm00840904_m1), Arg1 (Mm00475988_m1) and Sp1 (Mm00489039_m1).

Techniques: Protein-Protein interactions, Control, Gene Expression, RNA Sequencing, Quantitative RT-PCR, Luminex, Expressing, Quantitative Proteomics

Journal: bioRxiv

Article Title: Cancer cell genetics shaping of the tumor microenvironment reveals myeloid cell-centric exploitable vulnerabilities in hepatocellular carcinoma

doi: 10.1101/2023.10.29.564350

Figure Lengend Snippet: A. Representative IHC images of H&E and Masson Trichrome staining performed on liver sections from HDTVi- and LOI-induced Nras G12D / Pten KO HCC-bearing mice at end-stage. B. Barplot depicting the quantification of intratumoral GM-CSF levels present in end-stage HDTVi- (n=7) and LOI-induced (n=4) Nras G12D / Pten KO HCC. C. Quantification of the content of myeloid (CD45 + CD11b + ) and lymphoid (CD45 + CD11b - ) cells relative to total CD45 + leukocytes in tumors collected from end-stage HDTVi- (n=7) and LOI-induced (n=5) Nras G12D / Pten KO HCC. HDTVi-induced end-stage Nras G12D / Pten KO HCC-bearing mice shown in are included in this graph. D. Barplots depicting the percentage of intratumoral Ly6C high monocytes, neutrophils and Ly6C low subsets relative to total CD45 + leukocytes in end-stage HDTVi- (n=7) and LOI-induced (n=3) Nras G12D / Pten KO HCC. E-F. Barplot depicting the relative mRNA expression of Ccl6 ( E ) and Ccl17 ( F ) genes in end-stage LOI-induced Nras G12D / Pten KO HCC post-preclinical trial treatment with a- IgG2a (n=5) or a-GM-CSF (n=7). Values represent relative fold changes compared to one IgG2a-treated sample. Ubc was used as a housekeeping gene. G. Barplot depicting the relative tumor growth of LOI-induced Nras G12D / Pten KO HCC at 2 weeks post-treatment initiation with either a-IgG2a (n = 10) or a-GM-CSF (n = 16). H. Barplot depicting the percentage of intratumoral Ly6C low F4/80 low cells relative to total CD45 + leukocytes in LOI-induced Nras G12D / Pten KO HCC post-preclinical trial treatment with a-IgG2a (n = 8) or a-GM-CSF (n = 9). I. Barplot depicting the percentage of CD45 - Ki67 + cells in LOI-induced Nras G12D / Pten KO HCC post-preclinical trial treatment with a-IgG2a (n = 8) or a-GM-CSF (n = 9). J. Barplot depicting the percentage of Ki67 + Ly6C low F4/80 low cells in LOI-induced Nras G12D / Pten KO HCC post-preclinical trial treatment with a-IgG2a (n = 8) or a-GM- CSF (n = 9). K. Barplot depicting the percentage of intratumoral Ly6C low F4/80 low cells relative to total CD45 + leukocytes in LOI-induced Nras G12D / Pten KO HCC post-preclinical trial treatment with a-IgG2a (n = 9), a-GM-CSF (n = 12), a-GM-CSF+a-PD-L1 (n =4), a- GM-CSF- + a-VEGF (4), and a-VEGF + a-PD-L1 (n = 6). LOI-induced Nras G12D / Pten KO HCC-bearing mice treated with a-IgG2a (n=8) and a-GM-CSF (n=9) shown in Fig. S8H are included in this graph. L. 1. Cancer cell genetics shape distinct histopathological features and TME contexture in genetically-distinct murine HCC, which overall predict the prognostic rates of distinct human HCC subclasses 2. Nras G12D / Pten KO secretome fosters a myeloid dominant, pro-inflammatory/immunosuppressive TME wherein Nras G12D driven, ERK1/2-SP1-dependent GM-CSF expression drives the accumulation of pro-tumoral Ly6C low monocyte-derived cells. 3. Neutralizing GM-CSF and VEGF in Nras G12D /Pten KO HCC reprograms the TME, promotes cancer cell death and significantly extends animal survival. Graph shows mean ± SEM ( B, D-K ). Statistical significance was determined by unpaired Student’s T ( E- K ). *p < 0.05; **p<0.01; ***p < 0.001.

Article Snippet: The following Taqman probes (Thermo Fisher) were used for qPCR: Ubc (Mm01201237_m1), Il6 (Mm00446190_m1), Il1b (Mm00434228_m1), Il1a (Mm00439620_m1), Ccl6 (Mm01302419_m1), Ccl17 (Mm00516136_m1), Csf2 (Mm01290062_m1), Nlrp3 (Mm00840904_m1), Arg1 (Mm00475988_m1) and Sp1 (Mm00489039_m1).

Techniques: Staining, Expressing, Derivative Assay