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: 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