mouse il17r  (R&D Systems)

Journal: The Journal of Experimental Medicine

Article Title: Interleukin-17–induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer

doi: 10.1084/jem.20190354

Figure Lengend Snippet: IL17 recruits neutrophils to the pancreatic tumor microenvironment. (A) Experimental protocol for orthotopic implantation of KPC cells into syngeneic WT mice followed by treatment with anti-IL17 and anti-IL17R mAb (aIL17/aIL17R) versus control isotype IgG. CyTOF, RNA sequencing (RNAseq), IHC, and multiplex IF were performed at 14 d after treatment initiation. (B) Heat map showing distribution of tumor-infiltrating immune cells as identified by CyTOF analysis of tumors from A, represented as a percentage of total CD45 + cells ( n = 5/group). DC, dendritic cell. (C) Ingenuity Pathway Analysis showing the top five cellular functions predicted using genes significantly downregulated in tumors from A. As indicated on the x axis, biological functions with P < 0.05 are sorted based on Z scores. (D) Quantification of Gr1 + cells measured in tumors from A by flow cytometry (left panel) or IHC (middle panel) and Ly6G + cells measured by IHC (right panel). Results are expressed as the relative percentage of total gated CD45 + cells for flow cytometry and total number of cells/mm 2 for IHC. (E) Representative images of neutrophils infiltrating human PDAC tissue versus normal adjacent tissue by CD15 staining performed by IHC. Scale bars represent 50 µm. (F) Quantification of CD15 staining in E. Results are expressed as the number of CD15 + cells per high-power field (hpf). *, P < 0.05.

Article Snippet: Targeted knockout of IL17RA in KPC cells was performed using a CRISPR/Cas9-mediated genome editing kit (Santa Cruz Biotechnology) with mouse IL17R CRISPR/Cas9 plasmid (sc-421093) and mouse IL17R HDR plasmid (sc-421093-HDR) following the manufacturer’s instructions.

Techniques: Control, RNA Sequencing, Multiplex Assay, Flow Cytometry, Staining

Journal: The Journal of Experimental Medicine

Article Title: Interleukin-17–induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer

doi: 10.1084/jem.20190354

Figure Lengend Snippet: Pharmacological and genetic blockade of IL17 signaling overcomes resistance to immune checkpoint inhibition. (A) Tumor growth curves for subcutaneously implanted KPC cells treated with anti-IL17/IL17R/PD-1 antibodies as described in ( n = 10 mice/group). (B) Tumor volumes of orthotopically implanted KPC cells treated with anti-IL17/IL17R/PD-1 antibodies as described in ( n = 10 mice/group). (C) Kaplan-Meier curves for syngeneic mice orthotopically implanted with KPC cells and treated with the indicated antibodies as described in ( n = 10 mice/group). (D) Tumor volumes of orthotopically implanted mT3 cells treated with anti-IL17/IL17R/PD-1 antibodies as described in ( n = 8 mice/group). (E) Tumor volumes of orthotopically implanted KPC cells (with genetic deletion of IL17R by CRISPR/Cas9 versus scramble control) into syngeneic mice in presence/absence of aPD-1 ( n = 5 mice/group). (F) Tumor volumes of orthotopically implanted KPC cells into syngeneic mice treated with anti-IL17/CTLA4 antibodies as described in ( n = 7 mice/group). *, P < 0.05; ****, P < 0.0001.

Article Snippet: Targeted knockout of IL17RA in KPC cells was performed using a CRISPR/Cas9-mediated genome editing kit (Santa Cruz Biotechnology) with mouse IL17R CRISPR/Cas9 plasmid (sc-421093) and mouse IL17R HDR plasmid (sc-421093-HDR) following the manufacturer’s instructions.

Techniques: Inhibition, CRISPR, Control

Journal: The Journal of Experimental Medicine

Article Title: Interleukin-17–induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer

doi: 10.1084/jem.20190354

Figure Lengend Snippet: The antitumoral effect of combinatorial IL17 and PD-1 blockade is CD8 + T cell dependent. (A) Flow cytometry–based analysis of tumor-infiltrating CD8 + and CD8 + IFNγ + cells. Tumors were obtained from syngeneic mice orthotopically implanted with KPC cells and treated with isotype IgG, aPD-1, aIL17/aIL17R, or aIL17/aIL17R/aPD-1 antibodies ( n = 10 mice/group). Results are expressed as the percentage of total CD45 + gated viable cells. (B) IHC-based quantification of tumor-infiltrating cells expressing granzyme B (GzmB + ) in tumors from A. Results are expressed as the total number of cells/mm 2 . (C) Tumor volumes of orthotopically implanted KPC cells into WT syngeneic mice treated with isotype IgG, anti-IL17/IL17R/PD-1, or anti-CD8 antibodies (aCD8; n = 10). (D) Tumor volumes of orthotopically implanted KPC cells into CD8-deficient (CD8 −/− ) syngeneic mice treated with isotype IgG or anti-IL17/IL17R/PD-1 antibodies ( n = 6–7 mice/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: Targeted knockout of IL17RA in KPC cells was performed using a CRISPR/Cas9-mediated genome editing kit (Santa Cruz Biotechnology) with mouse IL17R CRISPR/Cas9 plasmid (sc-421093) and mouse IL17R HDR plasmid (sc-421093-HDR) following the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Interleukin-17–induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer

doi: 10.1084/jem.20190354

Figure Lengend Snippet: IL17 recruits neutrophils to the pancreatic tumor microenvironment. (A) Experimental protocol for orthotopic implantation of KPC cells into syngeneic WT mice followed by treatment with anti-IL17 and anti-IL17R mAb (aIL17/aIL17R) versus control isotype IgG. CyTOF, RNA sequencing (RNAseq), IHC, and multiplex IF were performed at 14 d after treatment initiation. (B) Heat map showing distribution of tumor-infiltrating immune cells as identified by CyTOF analysis of tumors from A, represented as a percentage of total CD45 + cells ( n = 5/group). DC, dendritic cell. (C) Ingenuity Pathway Analysis showing the top five cellular functions predicted using genes significantly downregulated in tumors from A. As indicated on the x axis, biological functions with P < 0.05 are sorted based on Z scores. (D) Quantification of Gr1 + cells measured in tumors from A by flow cytometry (left panel) or IHC (middle panel) and Ly6G + cells measured by IHC (right panel). Results are expressed as the relative percentage of total gated CD45 + cells for flow cytometry and total number of cells/mm 2 for IHC. (E) Representative images of neutrophils infiltrating human PDAC tissue versus normal adjacent tissue by CD15 staining performed by IHC. Scale bars represent 50 µm. (F) Quantification of CD15 staining in E. Results are expressed as the number of CD15 + cells per high-power field (hpf). *, P < 0.05.

Article Snippet: Targeted knockout of IL17RA in KPC cells was performed using a CRISPR/Cas9-mediated genome editing kit (Santa Cruz Biotechnology) with mouse IL17R CRISPR/Cas9 plasmid (sc-421093) and mouse IL17R HDR plasmid (sc-421093-HDR) following the manufacturer’s instructions.

Techniques: Control, RNA Sequencing, Multiplex Assay, Flow Cytometry, Staining

Journal: The Journal of Experimental Medicine

Article Title: Interleukin-17–induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer

doi: 10.1084/jem.20190354

Figure Lengend Snippet: Pharmacological and genetic blockade of IL17 signaling overcomes resistance to immune checkpoint inhibition. (A) Tumor growth curves for subcutaneously implanted KPC cells treated with anti-IL17/IL17R/PD-1 antibodies as described in ( n = 10 mice/group). (B) Tumor volumes of orthotopically implanted KPC cells treated with anti-IL17/IL17R/PD-1 antibodies as described in ( n = 10 mice/group). (C) Kaplan-Meier curves for syngeneic mice orthotopically implanted with KPC cells and treated with the indicated antibodies as described in ( n = 10 mice/group). (D) Tumor volumes of orthotopically implanted mT3 cells treated with anti-IL17/IL17R/PD-1 antibodies as described in ( n = 8 mice/group). (E) Tumor volumes of orthotopically implanted KPC cells (with genetic deletion of IL17R by CRISPR/Cas9 versus scramble control) into syngeneic mice in presence/absence of aPD-1 ( n = 5 mice/group). (F) Tumor volumes of orthotopically implanted KPC cells into syngeneic mice treated with anti-IL17/CTLA4 antibodies as described in ( n = 7 mice/group). *, P < 0.05; ****, P < 0.0001.

Article Snippet: Targeted knockout of IL17RA in KPC cells was performed using a CRISPR/Cas9-mediated genome editing kit (Santa Cruz Biotechnology) with mouse IL17R CRISPR/Cas9 plasmid (sc-421093) and mouse IL17R HDR plasmid (sc-421093-HDR) following the manufacturer’s instructions.

Techniques: Inhibition, CRISPR, Control

Journal: The Journal of Experimental Medicine

Article Title: Interleukin-17–induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer

doi: 10.1084/jem.20190354

Figure Lengend Snippet: The antitumoral effect of combinatorial IL17 and PD-1 blockade is CD8 + T cell dependent. (A) Flow cytometry–based analysis of tumor-infiltrating CD8 + and CD8 + IFNγ + cells. Tumors were obtained from syngeneic mice orthotopically implanted with KPC cells and treated with isotype IgG, aPD-1, aIL17/aIL17R, or aIL17/aIL17R/aPD-1 antibodies ( n = 10 mice/group). Results are expressed as the percentage of total CD45 + gated viable cells. (B) IHC-based quantification of tumor-infiltrating cells expressing granzyme B (GzmB + ) in tumors from A. Results are expressed as the total number of cells/mm 2 . (C) Tumor volumes of orthotopically implanted KPC cells into WT syngeneic mice treated with isotype IgG, anti-IL17/IL17R/PD-1, or anti-CD8 antibodies (aCD8; n = 10). (D) Tumor volumes of orthotopically implanted KPC cells into CD8-deficient (CD8 −/− ) syngeneic mice treated with isotype IgG or anti-IL17/IL17R/PD-1 antibodies ( n = 6–7 mice/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: Targeted knockout of IL17RA in KPC cells was performed using a CRISPR/Cas9-mediated genome editing kit (Santa Cruz Biotechnology) with mouse IL17R CRISPR/Cas9 plasmid (sc-421093) and mouse IL17R HDR plasmid (sc-421093-HDR) following the manufacturer’s instructions.

Techniques: Flow Cytometry, Expressing

Journal: Aging (Albany NY)

Article Title: Unrepaired DNA damage in macrophages causes elevation of particulate matter- induced airway inflammatory response

doi: 10.18632/aging.101412

Figure Lengend Snippet: RAD50 flox/flox -Lys Mcre mice display exacerbated airway inflammation in response to particulate matter (PM) exposure. RAD50 flox/flox -LysM cre male mice and their wildtype littermates (n =5, 6, or 7 per group) were instilled intratracheally with PM at 100 μg·d-1 or the equivalent volume of normal saline (NS) as control for 2 days, and after 24 hours. In the bronchoalveolar lavage fluid, ( A ) the total number of inflammatory cells was quantified and the number of neutrophils was calculated. ( B ) Expressions of Il6, Cxcl1, Ifn-γ and Il17 levels in the lung tissue were determined using real-time PCR and ELISA. ( C ) Representative images of lung sections stained with hematoxylin and eosin (H & E). ( D ) Semiquantified inflammation score of the H & E staining (n =10 images per group). *p < 0.05, **p < 0.01,***p < 0.001.

Article Snippet: ELISA kits for mouse Cxcl1 (MKC00B), mouse Cxcl2 (MM200), mouse Il6 (M6000B), mouse Il17(R&D, M1700) and mouse Ifn-γ (R&D, MIF00) were purchased from R&D systems.

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Staining