Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 1 RT-PCR analysis of CXCL10, CXCL9, and CXCL11 gene expression in freshly isolated Langerhans cells (fLC), 48 h cultured LC (cLC), interferon-c (IFN-c)-stimulated cLC (c-cLC), freshly isolated splenic dendritic cells (fDC), 48 h cultured splenic DC (cDC), and IFN-c- stimulated cDC (c-cDC). LC and splenic DC were purified and cul- tured for 48 h in the absence or presence of 100 ng per mL of IFN-g. Samples from fresh and cultured LC and splenic DC were collected and mRNA expression was analyzed using specific primers for each chemokine. CXCL10, CXCL9, and CXCL11 mRNA expression was hardly detectable in fLC. In cLC, mRNA expression of CXCL10 and CXCL11, but not CXCL9, was induced. CXCL9 mRNA was strongly expressed only in g-cLC. In addition, mRNA for CXCL10 and CXCL11 was also strongly expressed in g-cLC. In splenic DC, mRNA for these T helper 1(Th1)-type chemokines was almost undetectable both in fDC and cDC. When stimulated with IFN-g, CXCL10, CXCL9, and CXCL11 mRNA expression was induced. Data are representative of three inde- pendent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Isolation, Cell Culture, Expressing

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 2 Production of T helper 1-type chemokines during culture of Langerhans cells (LC) and splenic dendritic cells (DC). Purified LC and splenic DC were cultured with or without interferon-g (IFN-g), and the concentration of CXCL10, CXCL9, and CXCL11 was measured at different time points (0, 12, 24, 36, 48 h) in the supernatants by ELISA. Representative data of three independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 3 Chemotaxis of mCXCR3-transfected 2B4 T cells. Langerhans cells (LC) and splenic dendritic cells (DC) were cultured for 48 h with 100 ng per mL of interferon-g (IFN-g) and the supernatants were collected. The culture supernatants were preincubated with or without neutralizing anti-chemokine monoclonal antibody (mAb) indicated in the figure for 30 min, and assessed for chemotactic activity to CXCR3 transfectant. RPMI 10 medium alone served as a negative control. RPMI 10 medium containing recombinant chemokine served as a positive control. The supernatants of IFN-g-stimulated LC and splenic DC exhibited chemo- tactic activity to CXCR3 transfectant, which is mediated at least by CXCL10 and CXCL9. Mean (SD) (n ¼ 3). Data are representative of three independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Chemotaxis Assay, Transfection, Cell Culture, Activity Assay, Negative Control, Recombinant, Positive Control

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 7 Regulation of T helper 1-type chemokines produced by La- ngerhans cells (LC) and splenic dendritic cells (DC). LC (&) and splenic DC (’) were purified and cultured (1.5 106 cells per mL per 200 mL in each well) for 48 h with or without various stimuli. The supe- rnatants were collected and the concentration of CXCL10 (a), CXCL9 (b), and CXCL11 (c) was measured by ELISA. CXCL10 production by LC was induced by interferon-g (IFN-g), interleukin (IL)-12, lipopoly- saccharide (LPS), Staphylococcus aureus Cowen 1 (SAC), and poly- inosinic–polycytidylic acid (Poly(I:C)). In the case of CXCL9 and CXCL11, only IFN-g induced their production by LC. In the case of splenic DC, the production of CXCL10, CXCL9, and CXCL11 was in- duced by IFN-g, IL-18, LPS, and Poly(I:C). Results are the mean (SD) (n ¼ 4). Significant increase (po0.05) compared with the unstimulated group. Data are representative of four independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Produced, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) CXCL9, 10 and 11 can all bind and signal through the chemokine receptor CXCR3 that is typically found on T cells. (B) The EMBL-ELI expression atlas (human) was analysed for relatedness in expression of CXCR3 and its’ ligands CXCL9, 10 and 11 across all tissues or (C) in distinct tissues and diseases.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Expressing

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) Schematic of the in vivo air pouch leukocyte recruitment model. (B) Analysis of chemokine concentration in the carrageenan inflamed air pouch. (C) Representative tSNE of all murine cells gated on live, single, CD45 + and built on CD4, CD8, F4/80, Ly6C, Ter119, CD3, TCRβ, CXCR3, Ly6G, CD11c, B220, CD11b, CD64, Siglec F, NK1.1 and TCRγδ. FlowSOM clusters are illustrated by gates. (D) tSNE analysis of air pouches injected with equimolar amounts of CXCL9, 10 and 11. (E) Quantification of all leukocytes (CD45 + ) and T cells within the air pouch following injection of CXCL9, 10 or 11. E analysed using a one-way ANOVA.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: In Vivo, Concentration Assay, Injection

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) Schematic of HS GAG structure, including sulphation points and the enzymes that produce them. (B) Normalised (relative to wild type) binding of labelled CXCL9, 10 or 11 to genetically modified CHO cells. (C) Normalised and absolute binding of CXCL9, 10 and 11 to CHO cells in which KS^ST1/2/3 have been genetically removed. (D and E) Normalised binding of CXCL9, 10 and 11 to CHO cells genetically engineered to express the enzymes regulating 3-O GAG sulphation. (F) EMBL-ELI expression atlas analysis of relatednessCXCL9, 10 and 11 and GAG sulphation gene expression. B and D, data plotted as mean from three separate pooled experiments. C and E data plotted as mean ± SEM from three separate pooled experiments and analysed using a one-way ANOVA.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Binding Assay, Genetically Modified, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) CXCL9, 10 and 11 all bind to the same receptor with different affinities and biased signalling outcomes and are found in over-lapping expression patterns during inflammation and disease. (B) Differential GAG interactions means that CXCL9 is more likely to be retained on GAGs on the cell surface or within the ECM, with CXCL10 and CXCL11 being more likely to be present in their soluble state.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Expressing

Journal: Journal of Clinical Investigation

Article Title: NLRC4 suppresses melanoma tumor progression independently of inflammasome activation

doi: 10.1172/jci86953

Figure Lengend Snippet: Figure 5. Absence of NLRC4 in macrophages alters the tumor cytokine and chemokine milieu. (A–F) WT and Nlrc4–/– mice were injected s.c. with 1 × 105 B16F10 cells. On day 12 after inoculation, total RNA was isolated from homogenized tumors and used to determine cytokine and chemokine expression via quantitative qPCR utilizing a PCR array. Selected genes from the array are displayed; data are pooled from 3 separate experiments (n = 3 mice per group). (G and H) WT and Nlrc4–/– mice were injected s.c. with 1 × 105 B16F10 cells; 14 days after inoculation, tumors were harvested, pooled, and FACS sorted based on CD45.2 and F4/80 staining. RNA was isolated from CD45.2- and CD45.2+F4/80+ cells and used to determine Cxcl9, Cxcl10, Cxcl13, and Cxcl16 expression by qPCR; data are representative of 2 independent experiments with n ≥ 5 pooled tumors per group. (I) WT and Nlrc4–/– BMDMs were challenged for 9 hours with B16F10 whole tumor homogenate. Cxcl9, Cxcl10, and Cxcl13 expression was determined by qPCR. Data are pooled from 3 independent experi- ments, and fold change in gene expression is relative to unstimulated samples. (J and K) WT and Nlrc4–/– BMDMs were challenged with 50 ng/ml LPS, 50 μg/ml LTA, 100 ng/ml FSL-1, and 1 μg/ml Pam3CSK4. Twenty hours later, supernatants were collected and levels of IL-6 (J) and IL-12p40 (K) determined by ELISA; data are representative of 3 independent experiments. (A–F and I) Error bars represent SEM. (J and K) Error bars represent SD. (I–K) *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, unpaired 2-tailed Student’s t test.

Article Snippet: Ifng, Cxcl9, Cxcl10, Cxcl13, and Cxcl16 primer pairs were all from Origene.

Techniques: Injection, Isolation, Expressing, Staining, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of insulin-like growth factors increases production of CXCL9/10 by macrophages and fibroblasts and facilitates CD8 + cytotoxic T cell recruitment to pancreatic tumours

doi: 10.3389/fimmu.2024.1382538

Figure Lengend Snippet: IGF blockade reverses phosphorylation of STAT3 in TAMs and CAFs to facilitate STAT1 induction of Cxcl9 and Cxcl10 genes (A) Top, immunofluorescent staining of F480 (green), pSTAT3 (red) and nuclei (blue) in formalin fixed paraffin embedded tissues from orthotopic murine PDAC tumours treated with IgG2 (control) antibody or IGF blocking antibody MEDI-573. Bottom, i mmunofluorescent staining of F480 (green), pSTAT1(red) and nuclei (blue) in formalin fixed paraffin embedded tissues from orthotopic murine PDAC tumours treated with IgG2 (control) antibody or IGF blocking antibody MEDI-573. Scale bar 50 µm. (B) Quantification of the number of F480 + macrophages displaying active pSTAT3 signalling in IgG control treated and anti-IGF treated orthotopic murine pancreatic tumours. Data displayed as percentage of pSTAT3 + /F480 + macrophages among all F480 + macrophages. (C) Quantification of the number of F480 + macrophages displaying active pSTAT1 signalling in IgG control treated and anti-IGF treated orthotopic murine pancreatic tumours. Data displayed as percentage of pSTAT1 + /F480 + macrophages among all F480 + macrophages. A total of 5-8 fields of view counted/mouse tumour, n= 5 mice per treatment group, *P ≤ 0.05, **P ≤ 0.01 using Mann-Whitney U test. (D) Top, immunofluorescent staining of αSMA (green), pSTAT3 (red) and nuclei (blue) in formalin fixed paraffin embedded tissues from orthotopic murine PDAC tumours treated with IgG2 (control) antibody or IGF blocking antibody MEDI-573. Bottom, immunofluorescent staining of PDGFRβ (green), pSTAT1 (red) and nuclei (blue) in formalin fixed paraffin embedded tissues from orthotopic murine PDAC tumours treated IgG2 (control) antibody or IGF blocking antibody MEDI-573. Scale bar 50 µm. (E) Quantification of the number of αSMA + fibroblasts displaying active pSTAT3 signalling in IgG control treated and anti-IGF treated orthotopic murine pancreatic tumours. Data displayed as percentage of pSTAT3 + /αSMA + fibroblasts among all αSMA + fibroblasts. (F) Quantification of the number of PDGFRβ + fibroblasts displaying active pSTAT1 signalling in IgG control treated and anti-IGF treated orthotopic murine pancreatic tumours. Data displayed as percentage of pSTAT1 + /PDGFRβ + fibroblasts among all PDGFRβ + fibroblasts. A total of 5-8 fields of view counted/mouse tumour, n= 5 mice per treatment group, *P ≤ 0.05, **P ≤ 0.01 using Mann-Whitney U test. (G) Schematic to display experimental design of mechanistic study assessing the role of STAT signalling in controlling response to IGF blockade in BMDMS/Fibroblasts. (H) Immunoblotting analysis of primary murine fibroblasts and (I) primary murine bone-marrow derived macrophages in response to IGF blockade. Whole cell lysates were probed for both total and phosphorylated AKT, total and phosphorylated STAT3 as well as GAPDH loading control. (J) Quantification of Stat1 mRNA expression levels in primary murine fibroblasts and treated with fibroblast conditioned media supplemented with either IgG control antibody (100 µg/ml), IGF-blocking antibody MEDI-573 (100 µg/ml) or recombinant IFNγ (50 ng/ml) for 1, 2, 4 or 6 hrs. n=3, ** P ≤ 0.01, **** P ≤ 0.0001 using two-way ANOVA with Dunnett’s multiple comparisons test. (K) Quantification of Icam1, Irf1 and Oas2 mRNA expression levels in primary murine fibroblasts and treated with fibroblast conditioned media supplemented with either IgG control antibody (100 µg/ml), IGF-blocking antibody MEDI-573 (100 µg/ml) or recombinant IFNγ (50 ng/ml) for 6 hrs. n=3, **** P ≤ 0.0001 using two-way ANOVA with Dunnett’s multiple comparisons test. (L) Immunocytochemistry staining of STAT1 (red) and nuclei (blue) in primary murine fibroblasts and treated with fibroblast conditioned media supplemented with either IgG control antibody (100 µg/ml), IGF-blocking antibody MEDI-573 (100 µg/ml) or recombinant IFNγ (50 ng/ml) for 6 hrs. Bottom right, quantification of STAT1 + area/total cell number in IgG control, anti-IGF and IFNγ treated fibroblasts. Data displayed as fold change compared to IgG control treatment. n=3, ** P ≤ 0.01,*** P ≤ 0.001 using one-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: Tissue sections were incubated overnight at 4°C with the following antibodies: CD8a (eBioscience, 53-6.7 clone, 14-0081-82, 1:50); CD8 (Dako, C8/144B clone, M7103, 1:100), Granzyme B (Abcam, ab4059, 1:100); PD-1 (Abcam, ab214421, 1:500); Ki67 (Abcam, ab15580, 1:1000); CC3 (Cell Signaling Technology, #9661, 1:200); CD4 (Invitrogen, 14-9766-80, 4SM95 clone, 1:50); FoxP3 (Cell Signaling Technology, #12653, 1:100); F4/80 (Biolegend, BM8 clone, 123101 1:50); F4/80 (Cell Signaling Technology, #70076, 1:100), CD206 (Abcam, ab64693, 1:1000); MHC II (Novus Biologicals, NBP1-43312, 1:100); CXCL9 (R&D Systems, AF-492-NA, 1:50); phospho-Stat3 (Tyr705) (Cell Signaling Technology, #4093, 1:100); phospho-Stat1 (Tyr701) (Cell Signaling Technology, #9167, 1:300); PDGFRβ (R&D Systems, AF1042, 1:50); αSMA (Abcam, ab5694, 1:200).

Techniques: Phospho-proteomics, Staining, Formalin-fixed Paraffin-Embedded, Control, Blocking Assay, MANN-WHITNEY, Western Blot, Derivative Assay, Expressing, Recombinant, Immunocytochemistry

Journal: Frontiers in Immunology

Article Title: Inhibition of insulin-like growth factors increases production of CXCL9/10 by macrophages and fibroblasts and facilitates CD8 + cytotoxic T cell recruitment to pancreatic tumours

doi: 10.3389/fimmu.2024.1382538

Figure Lengend Snippet: IGF blockade leads to increased CD8 + T cell recruitment towards human PDAC conditioned media utilising the precision cut tumour slice model (A) Schematic detailing the workflow for each fresh PDAC sample and generation of 250 µm precision cut tumour slices. (B) Left, densitometry data displaying expression of CXCL9 in conditioned media of PCTS tissue treated with IgG2 (control) antibody or IGF blocking antibody MEDI-573 for 72 hours, analysed by immunoblotting. Data displayed as fold change compared to the IgG2 control antibody of CXCL9/Ponceau loading control. n=3, *P ≤ 0.05 using one-sample t test. Right, representative immunoblotting analysis of PCTS CM, using ponceau as loading control. (C) Migration of Jurkat T cells through a 5 µm transwell insert towards PCTS conditioned media was measured after 15 hours using a haemocytometer. Conditioned media was generated from PCTS samples treated with IgG control antibody (100 µg/ml) or IGF-blocking antibody MEDI-573 (100 µg/ml) for 72 hours. (D) Data are presented separately for each patient displaying the number of migratory Jurkat T cells as a fold change compared to the IgG control treated PCTS conditioned media AFTER 15 hr. n=5. (E) Immunofluorescent staining of CD8 (green) and nuclei (blue) in formalin fixed paraffin embedded tissues from day 0 control PCTS samples, or PCTS samples treated with IgG2 (control) antibody or IGF blocking antibody MEDI-573 for 72 hours. Scale bar 50 µm. (F) Immunofluorescent staining of CD8 (green), cleaved caspase 3 (red) and nuclei (blue) in formalin fixed paraffin embedded tissues from PCTS samples treated with IgG2 (control) antibody or IGF blocking antibody MEDI-573 for 72 hours. Scale bar 50 µm. (G) Immunofluorescent staining of CD8 (green), ki67 (red) and nuclei (blue) in formalin fixed paraffin embedded tissues from PCTS samples treated with IgG2 (control) antibody or IGF blocking antibody MEDI-573 for 72 hours. Scale bar 50 µm. (H) Quantification of CD8 + T cells in Day 0 control, IgG control treated and anti-IGF treated PCTS samples. Data displayed as fold change of CD8 + T cells among all cells compared to day 0 control slices. A total of 3-4 fields of view counted/slice, n= 4 slices per treatment group, ns; P > 0.05, **P ≤ 0.01 one-way ANOVA Bonferroni’s multiple comparison post hoc test. (I) Quantification of CC3 + CD8 + T cells and Ki67 + CD8 + T cells in IgG control treated and anti-IGF treated PCTS samples. Data displayed as fold change of either CC3 + CD8 + T cells or Ki67 + CD8 + among all CD8 + T cells compared to IgG control treatment. A total of 3-4 fields of view counted/slice, n= 6 slices per treatment group, ns; P > 0.05 using two-way ANOVA with Bonferroni’s multiple comparisons test. (J) Picrosirius red staining of collagen fibres in formalin fixed paraffin embedded tissues from PCTS samples treated with IgG2 (control) antibody (top) or IGF blocking antibody MEDI-573 (bottom) for 72 hours. (K) Quantification of picrosirius red staining in PCTS samples. Data displayed as fold change in picrosirius red area over total area stained compared to IgG control treatment. n=4, *P ≤ 0.05 using one-sample t test.

Article Snippet: Tissue sections were incubated overnight at 4°C with the following antibodies: CD8a (eBioscience, 53-6.7 clone, 14-0081-82, 1:50); CD8 (Dako, C8/144B clone, M7103, 1:100), Granzyme B (Abcam, ab4059, 1:100); PD-1 (Abcam, ab214421, 1:500); Ki67 (Abcam, ab15580, 1:1000); CC3 (Cell Signaling Technology, #9661, 1:200); CD4 (Invitrogen, 14-9766-80, 4SM95 clone, 1:50); FoxP3 (Cell Signaling Technology, #12653, 1:100); F4/80 (Biolegend, BM8 clone, 123101 1:50); F4/80 (Cell Signaling Technology, #70076, 1:100), CD206 (Abcam, ab64693, 1:1000); MHC II (Novus Biologicals, NBP1-43312, 1:100); CXCL9 (R&D Systems, AF-492-NA, 1:50); phospho-Stat3 (Tyr705) (Cell Signaling Technology, #4093, 1:100); phospho-Stat1 (Tyr701) (Cell Signaling Technology, #9167, 1:300); PDGFRβ (R&D Systems, AF1042, 1:50); αSMA (Abcam, ab5694, 1:200).

Techniques: Expressing, Control, Blocking Assay, Western Blot, Migration, Generated, Staining, Formalin-fixed Paraffin-Embedded, Comparison

Journal: Frontiers in Immunology

Article Title: Inhibition of insulin-like growth factors increases production of CXCL9/10 by macrophages and fibroblasts and facilitates CD8 + cytotoxic T cell recruitment to pancreatic tumours

doi: 10.3389/fimmu.2024.1382538

Figure Lengend Snippet: TAM and CAF derived chemokines CXCL9 and CXCL10 facilitate CD8 + T cell recruitment to PDAC tumours upon IGF blockade. (A) Immunohistochemical staining of αSMA in formalin fixed paraffin embedded tissues from orthotopic murine PDAC tumours treated with IgG2 control antibody or IGF-blocking antibody MEDI-573. Scale bar; 50 µm. (B) Quantification of αSMA staining. Data displayed as total αSMA + area/total tumour area. A total of 5-8 fields of view counted/mouse tumour, n = 5 mice per treatment group, *P ≤ 0.05 using Mann-Whitney U test. (C) Quantification of F480 staining. Data displayed as % F480 + cells among all cells. A total of 5-8 fields of view counted/mouse tumour, n = 5 mice per treatment group, ns; P > 0.05 using Mann-Whitney U test. (D) Immunofluorescent staining of F480 (green), MHCII (red) and nuclei (blue) in formalin fixed paraffin embedded tissues from orthotopic murine PDAC tumours treated with IgG2 (control) antibody or IGF blocking antibody MEDI-573. Scale bar 50 µm. White arrows denote cells which are positive for both F480 and MHCII. (E) Immunofluorescent staining of F480 (green), CD206 (red) and nuclei (blue) in formalin fixed paraffin embedded tissues from orthotopic murine PDAC tumours treated with IgG2 (control) antibody or IGF blocking antibody MEDI-573. Scale bar 50 µm. White arrows denote cells which are positive for both F480 and CD206. (F) Quantification of MHCII + /F480 + macrophages in IgG control treated and anti-IGF treated orthotopic murine pancreatic tumours. Data displayed as percentage of MHCII + /F480 + cells among all cells. A total of 5-8 fields of view counted/mouse tumour, n= 5 mice per treatment group, * P ≤ 0.05 using Mann-Whitney U test. (G) Quantification of CD206 + /F480 + macrophages in IgG control treated and anti-IGF treated orthotopic murine pancreatic tumours. Data displayed as percentage of CD206 + /F480 + cells among all cells. A total of 5-8 fields of view counted/mouse tumour, n= 5 mice per treatment group, ns; P > 0.05 using Mann-Whitney U test. (H) LSL-Kras G12D/+; LSL-Trp53 R172H/+; Pdx-1-Cre (KPC) derived FC1242 cells were orthotopically implanted into the pancreas of syngeneic recipient (C57BL/6J) mice. Mice were treated with IgG2 control antibody or IGF-blocking antibody MEDI-573 at days 23. Tumours were harvested and digested at day 25 post implantation with TAMs (CD45 + /F4/80 + cells) and non-immune stromal cells (CD45 - /zsGreen -a ) being sorted by flow cytometry and subsequently subjected to transcriptional analysis. (I) Quantification of Il10, Tgfb, IL6, Tnfa, Cxcl9, Cxcl10 AND Cxcl12 mRNA expression levels in zsGreen - /CD45 + /F4/80 + tumour associated macrophages isolated from murine PDAC tumours treated with IgG2 control antibody or IGF-blocking antibody MEDI-573 (n=3). **P ≤ 0.01 using unpaired t tests (J) Quantification of Col1a1, Col1a2, Fn1, Il6, Cxcl9, Cxcl10 and Cxcl12 mRNA expression levels in zsGreen - /CD45 - stromal fibroblasts isolated from murine PDAC tumours treated with IgG2 control antibody or IGF-blocking antibody MEDI-573 (n=3). **P ≤ 0.01 using unpaired t tests. (K) Quantification of Cxcl9 and (L) Cxcl10 mRNA expression levels in primary murine fibroblasts isolated from the pancreata of wild-type C57BL/6J mice and treated with scrambled control siRNA (5 µM) Igfr1 siRNA (5 µM) Igf1r (5 µM) or a combination of both Igf1r and Insr siRNAs (5 µM). Expression data displayed as fold change compared to scrambled control siRNA treatment *, P ≤ 0.05; ** P ≤ 0.01 using one-way ANOVA with Tukey’s multiple comparisons post hoc test. (M) Left, Immunofluorescent staining of CXCL9 (red) and nuclei (blue) in formalin fixed paraffin embedded tissues from orthotopic murine PDAC tumours treated with IgG2 (control) antibody or IGF blocking antibody MEDI-573. White arrows denote CXCL9 + cells. Right, Immunohistochemical staining of CXCL10 in formalin fixed paraffin embedded tissues from orthotopic murine PDAC tumours treated with IgG2 control antibody or IGF-blocking antibody MEDI-573. Black arrows denote CXCL10 + cells. Scale bar 50 µm. (N) Quantification of CXCL9 + cells in IgG control treated and anti-IGF treated orthotopic murine pancreatic tumours. Data displayed as percentage of CXCL9 + cells among all cells. A total of 5-8 fields of view counted/mouse tumour, n= 5 mice per treatment group, *P ≤ 0.05 using Mann-Whitney U test. (O) Quantification of CXCL10 + cells in IgG control treated and anti-IGF treated orthotopic murine pancreatic tumours. Data displayed as percentage of CXCL10 + cells among all cells. A total of 5-8 fields of view counted/mouse tumour, n= 5 mice per treatment group, **P ≤ 0.01 using Mann-Whitney U test. (P) Summary schematic for CD8 + T cell chemotaxis assay. Fibroblast conditioned media was generated from primary murine fibroblasts isolated from the pancreata of wild-type C57BL/6J mice and treated with IgG control antibody (100 µg/ml) or IGF-blocking antibody MEDI-573 (100 µg/ml). Fresh primary murine fibroblasts were cultured in treated fibroblast conditioned media for 48 hours and conditioned media collected for use in CD8 + T cell chemotaxis assays. Migration of primary murine CD8 + T cells through a 5 µm transwell insert towards fibroblast conditioned media was measured after 15 hours using a haemocytometer. CD8 + T cells were treated with or without the CXCR3 antagonist SCH 546738 (10 nM) before inclusion in migration assay. (Q) Data are presented as the number of migratory T cells as a fold change compared to the IgG control treated fibroblast conditioned media AFTER 15 hr. n=3, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 using one-way ANOVA with Tukey’s multiple comparisons test. (R) Quantification of Cxcl9 and (S) Cxcl10 mRNA expression levels in primary murine fibroblasts isolated from the pancreata of wild-type C57BL/6J mice and treated with IgG control antibody (100 µg/ml) or IGF-blocking antibody MEDI-573 (100 µg/ml) for 24 hours. Expression data displayed as fold change compared to IgG control treatment. n=3, *** P ≤ 0.001 using Mann-Whitney U test.

Article Snippet: Tissue sections were incubated overnight at 4°C with the following antibodies: CD8a (eBioscience, 53-6.7 clone, 14-0081-82, 1:50); CD8 (Dako, C8/144B clone, M7103, 1:100), Granzyme B (Abcam, ab4059, 1:100); PD-1 (Abcam, ab214421, 1:500); Ki67 (Abcam, ab15580, 1:1000); CC3 (Cell Signaling Technology, #9661, 1:200); CD4 (Invitrogen, 14-9766-80, 4SM95 clone, 1:50); FoxP3 (Cell Signaling Technology, #12653, 1:100); F4/80 (Biolegend, BM8 clone, 123101 1:50); F4/80 (Cell Signaling Technology, #70076, 1:100), CD206 (Abcam, ab64693, 1:1000); MHC II (Novus Biologicals, NBP1-43312, 1:100); CXCL9 (R&D Systems, AF-492-NA, 1:50); phospho-Stat3 (Tyr705) (Cell Signaling Technology, #4093, 1:100); phospho-Stat1 (Tyr701) (Cell Signaling Technology, #9167, 1:300); PDGFRβ (R&D Systems, AF1042, 1:50); αSMA (Abcam, ab5694, 1:200).

Techniques: Derivative Assay, Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Control, Blocking Assay, MANN-WHITNEY, Flow Cytometry, Expressing, Isolation, Chemotaxis Assay, Generated, Cell Culture, Migration

Journal: Frontiers in Immunology

Article Title: Inhibition of insulin-like growth factors increases production of CXCL9/10 by macrophages and fibroblasts and facilitates CD8 + cytotoxic T cell recruitment to pancreatic tumours

doi: 10.3389/fimmu.2024.1382538

Figure Lengend Snippet: Inhibition of the IGF signalling axis facilitates T cell recruitment towards the PDAC TME. Summary schematic detailing the proposed mechanism through which IGF blockade facilitates CD8 + T cell recruitment towards the PDAC TME. IGF blockade inhibits STAT3 signalling in both TAMs and CAFs driving STAT1 mediated transcription of the T cell chemokines Cxcl9/10. Concomitantly, IGF blockade leads to a reduction in collagen deposition which may further facilitate CD8 + T cell infiltration into and through the PDAC TME.

Article Snippet: Tissue sections were incubated overnight at 4°C with the following antibodies: CD8a (eBioscience, 53-6.7 clone, 14-0081-82, 1:50); CD8 (Dako, C8/144B clone, M7103, 1:100), Granzyme B (Abcam, ab4059, 1:100); PD-1 (Abcam, ab214421, 1:500); Ki67 (Abcam, ab15580, 1:1000); CC3 (Cell Signaling Technology, #9661, 1:200); CD4 (Invitrogen, 14-9766-80, 4SM95 clone, 1:50); FoxP3 (Cell Signaling Technology, #12653, 1:100); F4/80 (Biolegend, BM8 clone, 123101 1:50); F4/80 (Cell Signaling Technology, #70076, 1:100), CD206 (Abcam, ab64693, 1:1000); MHC II (Novus Biologicals, NBP1-43312, 1:100); CXCL9 (R&D Systems, AF-492-NA, 1:50); phospho-Stat3 (Tyr705) (Cell Signaling Technology, #4093, 1:100); phospho-Stat1 (Tyr701) (Cell Signaling Technology, #9167, 1:300); PDGFRβ (R&D Systems, AF1042, 1:50); αSMA (Abcam, ab5694, 1:200).

Techniques: Inhibition

Journal: OncoImmunology

Article Title: Tumor-intrinsic RGS1 potentiates checkpoint blockade response via ATF3-IFNGR1 axis

doi: 10.1080/2162402x.2023.2279800

Figure Lengend Snippet: Figure 2. RGS1 is a modulator of IFNγ-JAK-STAT1 signaling. a Differentially expressed cancer-related gene sets (H_Hallmarks) with high RGS1 expression in the KIRC cohort, LUAD cohort, LUSC cohort, and SKCM cohort from TCGA. NES, normalized enrichment score. FDR q, false discovery rate q value. b – d GSEA output of genes in the HALLMARK_INTERFERON_GAMMA_RESPONSE by RGS1 high and low expression groups from the KIRC cohort (b), LUAD cohort (c) and LUSC cohort (d) in the TCGA database. ES, enrichment score. e – l Analysis of IFNγ-STAT1 signaling in 786O and Renca cells. Cell lysates of 786O and Renca cells stably expressing nc or shRGS1 (e, g) and Vector or RGS1-OE (i, k) were analyzed by western blotting using RGS1, STAT1, P-STAT1 (Y701), IFNGR1, and IRF1 antibodies. Tublin was used as an internal control. f, j IFNγ-inducible gene expression in 786O cells. mRNA expression of IRF1, IRF9, STAT1, and IFNGR1 were detected by real-time qPCR. Actin was used as an internal control. h, l IFNγ-inducible gene expression in Renca cells. mRNA expression of Irf1, Irf9, and Stat1 were detected by real-time qPCR. Actin was used as an internal control. Cells in e, f, i, j were stimulated with 10 ng/ml human recombinant IFNγ or 0.1% BSA negative control for 2 h. Cells in g, h, k, l were stimulated with 5 ng/ml mouse recombinant IFNγ or 0.1% BSA negative control for 2 h. m, n IFNγ-induced CXCL9 secretion. Renca (m) and LLC (n) cells were cultured in serum-free medium and treated with 5 ng/ml IFNγ for 24 h. The concentration of CXCL9 was analyzed using an ELISA kit. o, p Cell surface levels of IFNGR1 in nc or shRGS1 (o) and Vector or RGS1-OE (p) 786O cells (pre-gated with FSC-A vs. SSC-A, and FSC-A vs. FSC-H). Cells were treated with 10 ng/ml IFNγ for 2 h. Right, quantification of the mean fluorescence intensity (MFI). Unpaired t-test was performed with GraphPad Prism 9. All data are representative of three independent experiments. Data in the bar graphs represent mean ± S.D., n = 3. *p < .05, **p < .01, ***p < .001.

Article Snippet: The concentrations of mouse CXCL9/MIG, human cAMP level and the activity of PKA in the supernatants were analyzed using CXCL9 ELISA kit (R&D Systems, MCX900), cAMP ELISA Kit (Elabscience, E-EL-0056c) and PKA Colorimetric Activity Kit (Thermo Fisher scientific, EIAPKA) respectively, according to the manufacturer’s protocols.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Western Blot, Control, Gene Expression, Recombinant, Negative Control, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: OncoImmunology

Article Title: Tumor-intrinsic RGS1 potentiates checkpoint blockade response via ATF3-IFNGR1 axis

doi: 10.1080/2162402x.2023.2279800

Figure Lengend Snippet: Figure 5. RGS1 is associated with T cell infiltration in RCC and NSCLC mouse models. a, d Representative images and quantification (right) of immunohistochemistry staining of IFNGR1, ATF3 and CXCL9 expression in harvested CTRL and ShRgs1 Renca (a) or LLC (d) subcutaneous tumor sections. The percentages of positively stained area (right) were analyzed using Image J software. Scale bar, 100 µm. b, e T cell infiltration and quantification (below) of Renca (b) or LLC (e) subcutaneous tumor. Paraffin-embedded tissue sections of murine tumors were immunohistochemically stained with antibodies against CD3, CD4, CD8 and PD1. The percentages of positively stained cells (below) were analyzed using Image J software. Scale bar, 100 µm. c, f Representative immunofluorescence images of CD8 and PD1 staining in ShRgs1 or CTRL Renca (c) or LLC (f) tumor sections. White arrows indicate merge of CD8 and PD1 fluorescence signals. Scale bar, 50 µm. Data in the graphs represent mean ± S.D, n = 5. *p < .05, **p < .01, ***p < .001.

Article Snippet: The concentrations of mouse CXCL9/MIG, human cAMP level and the activity of PKA in the supernatants were analyzed using CXCL9 ELISA kit (R&D Systems, MCX900), cAMP ELISA Kit (Elabscience, E-EL-0056c) and PKA Colorimetric Activity Kit (Thermo Fisher scientific, EIAPKA) respectively, according to the manufacturer’s protocols.

Techniques: Immunohistochemistry, Staining, Expressing, Software, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: CD11c+ Tbet+ B cells constrain obesity- and vaccination-induced germinal center B cells and T helper cells

doi: 10.1101/2025.09.01.673552

Figure Lengend Snippet: Representative flow cytometry contour plots of PD-1 x CXCR5 expression within CD25-CD4+ T cells isolated from NCD (left) and HFD (right) mouse spleen (top) and liver (bottom) ( A ). Blue gates represent PD-1 HI CXCR5+ cells, yellow gates represent PD-1 HI CXCR5-cells, and black gates represent PD-1-cells ( A ). Frequency and absolute number of PD-1 HI CXCR5+ T FH cells ( B , blue), or PD-1 HI CXCR5-T PH cells ( C , yellow) within the CD25-CD4+ T cell compartments of spleen (top) and liver (bottom) of NCD (grey) and HFD (blue/yellow) mice. Quantification of Bcl-6 ( D ), ICOS ( E ), and T-bet ( F ) protein expression (gMFI) in splenic (left) or hepatic (right) CD25-CD4+ T cell subsets. Representative flow cytometry contour plots depicting relative expression of IL-21 and IFNg by PD-1 HI CXCR5+ T FH cells or PD-1 HI CXCR5-T PH cells from spleen (top) or liver (bottom) that are also CXCR3+ (blue/red) or CXCR3-(black) ( G ). Summary of frequency of IL-21 ( H,J; left) or IFNg ( I,K ; right) producing CD4+ PD-1 HI CXCR5+ T FH (blue) or PD-1 HI CXCR5-T PH (red) cells categorized by CXCR3+ expression as noted in spleen ( H, I ) or liver ( J,K ) from HFD-fed mice. Data pooled from 2 independent experiments with 4 female mice/group, Bar ± SEM; [ Student’s t-test (two-tailed) (B,C); one-way ANOVA (D-F; H-K)]; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: C57BL/6J WT female mice were injected IP with 0.25 mg of anti-mouse CXCR3 (CD183) and anti-mouse CXCL9 (MIG) or hamster IgG (BioXCell) every other day for 14 days after completing 12 weeks on HFD.

Techniques: Flow Cytometry, Expressing, Isolation, Two Tailed Test