anti ifnγr antibody  (R&D Systems)

Journal: iScience

Article Title: An interleukin-27-centered cytokine circuit regulates macrophage and T cell interactions in autoimmune diabetes

doi: 10.1016/j.isci.2025.113537

Figure Lengend Snippet: T cells stimulate macrophage IL-27 production by IFNγ signaling and CD40-CD40L interaction (A) CD40L blockade suppresses IL-27 expression in islet macrophages. NOD. Il27p28-eGFP females (6–9 weeks old) were injected with anti-CD40L or control antibodies weekly for 3 weeks. Islet-infiltrating cells were analyzed at 1 week after the last injection. The frequencies of GFP (IL-27p28) + cells among CD11c + F4/80 + and CD11c low F4/80 + macrophages are pooled from four independent experiments ( n = 11–12 per group). ∗ p < 0.05 by unpaired t test. (B) IFNγ signaling is important for islet macrophage production of IL-27. Islet-infiltrating cells were analyzed for GFP (IL-27p28) in NOD. Ifngr1 +/+ and NOD. Ifngr1 +/− female littermates (9–11 weeks old). The frequencies of GFP (IL-27p28) + cells among CD11c + F4/80 + and CD11c low F4/80 + macrophages are pooled from six independent experiments ( n = 10–13 per group). ∗ p < 0.05 by unpaired t test. (C) IFNγR and CD40L blockade reduces IL-27p28 production in the coculture of wild-type (WT) CD4 T cells and BMDMs. WT splenic CD4 T cells (1 × 10 6 ) were cocultured with WT BMDMs (2 × 10 5 ) in the presence or absence of anti-CD3 (2 μg/mL) with the addition of anti-IFNγR and/or anti-CD40L (10 μg/mL) or the corresponding control antibodies for 3 days. Culture media were collected and analyzed for IL-27p28 by ELISA. Summarized results are from four independent experiments as indicated by different symbols. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, one-way ANOVA followed by Tukey’s multiple comparison. (D) IFNγR blockade reduces IL-27p28 production in the coculture of WT CD8 T cells and BMDMs. WT CD8 T cells and WT BMDMs were cocultured as described in (C). Culture media were collected and analyzed for IL-27p28 by ELISA. Summarized results are from two independent experiments with two biological replicates each time. ∗∗ p < 0.01, one-way ANOVA followed by Tukey’s multiple comparison. (E) The ability of macrophages to respond to IFNγ is important for CD4 T cell-induced IL-27p28 production. WT splenic CD4 T cells (1 × 10 6 ) were cocultured with WT or Ifngr1 −/− BMDMs (2 × 10 5 ) in the presence or absence of anti-CD3 (2 μg/mL) for 3 days. Culture media were collected and analyzed for IL-27p28 by ELISA. Results were pooled from two independent experiments with two biological replicates each time. ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison. (F) IFNγ and CD40 signaling synergistically enhances macrophage IL-27p28 expression. BMDMs were stimulated with IFNγ (10 ng/mL), anti-CD40 (5 μg/mL), or both for 3 days. Culture media were collected and analyzed for IL-27p28 by ELISA. Results were pooled from three independent experiments with one to two biological replicates each time. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison. (G) IFNγR and CD40L blockade reduces IL-27 production in the coculture of WT CD4 T cells and BMDMs. Culture media from the same cultures shown in (C) were analyzed for IL-27p28/EBI3 heterodimers by ELISA. ∗∗ p < 0.01, ∗∗∗ p < 0.001, one-way ANOVA followed by Tukey’s multiple comparison. (H) Activated human T cells induce IL-27 p28/EBI3 heterodimer production in the PBMC culture. PBMCs (7.5 × 10 5 ) were cultured in the presence or absence of anti-CD3 (2 μg/mL) with the addition of anti-IFNγRα and/or anti-CD40L (10 μg/mL) or the corresponding control antibodies for 3 days. Culture media were collected and analyzed for IL-27p28/EBI3 heterodimers by ELISA. Summarized results are from eight subjects as indicated by different symbols. ∗Adjusted p < 0.05 by Wilcoxon signed-rank test.

Article Snippet: Purified anti-mouse IFNγR , BioXCell , Clone GR-20, cat#BE0029; RRID: AB_1107576.

Techniques: Expressing, Injection, Control, Enzyme-linked Immunosorbent Assay, Comparison, Cell Culture

Journal: bioRxiv

Article Title: Inflammation-associated monocytes express ACOD1 to curtail inflammatory behaviour in IBD and experimental colitis

doi: 10.1101/2024.11.15.623347

Figure Lengend Snippet: a-b . Gene set enrichment analysis (GSEA) performed on a) CXCL9/10 + cells (IAM hu ; ) or b) Cxcl9/10 + cells (IAM mo ; ) with bar plots of top 20 pathways by normalised enrichment score (left) and enrichment plots (right) for IFNα/β and IFNγ signalling. Heatmaps show average expression of top enriched “leading edge” genes within each pathway. c. RT-PCR mRNA levels of indicated genes, normalised to Actb, by CD115 + Ly6C hi BM monocytes FACS purified from Ms4a3 Cre/+ .Rosa26 LSL-CAG-tdTomato/+ . Cx3cr1 +/gfp reporter mice, cultured with CSF-1 for 2days, then stimulated for 24 hours with rIFNα, rIFNβ, rIFNγ, LPS or the supernatant “secretome” from day6 DSS treated colon (colon explant). Symbols represent individual mice. Data are from five mice from one of two independent experiment performed, analysed by paired Students t test versus control. d. RT-PCR mRNA levels of indicated genes, normalised to Actb, by FACS purified Ly6C + colonic monocytes from day 5, 2% DSS treated Myd88 −/− mice relative to expression by FACS purified colonic Ly6C + monocytes from colitic C57BL/6J mice. Symbols represent individual mice (n=4-5) from one experiment analysed by unpaired t test with multiple comparison (5% False discovery rate, two-stage step-up method). e. RT-PCR mRNA levels of indicated genes, normalised to Actb, by FACS purified Ly6C + colonic monocytes from day 6, 2% DSS treated C57BL/6J mice treated with anti-IFNα/βR (clone MAR1-5A3) or anti-IFNγR1 (clone GR-20) or isotype control on day 2, 3, 4 and 5 of DSS administration. Symbols represent individual mice (n=4-5 from one experiment analysed by unpaired t test with multiple comparison (5% False discovery rate, two-stage step-up method). f. RT-PCR mRNA levels of indicated genes, normalised to Actb, by FACS purified Ly6C + colonic monocytes from day 6, 2% DSS treated Rag2 −/− mice relative to expression by FACS purified colonic Ly6C + monocytes from colitic C57BL/6J mice. Symbols represent individual mice (n=7) pooled from two experiments, analysed by unpaired t test with multiple comparison (5% False discovery rate, two-stage step-up method). g. RT-PCR mRNA levels of indicated genes, normalised to Actb, by FACS purified Ly6C + colonic monocytes from day 6, 2% DSS treated NSG mice relative to expression by FACS purified Ly6C + colonic monocytes from colitic C57BL/6J mice. Symbols represent individual mice (n=5*) from two experiments analysed by unpaired t test with multiple comparison (5% False discovery rate, two-stage step-up method). *one sample was beneath the limit of detection for Cxcl9

Article Snippet: Anti-mouse IFNgR1 (clone GR-20), anti-mouse IFNAR1 (clone MAR1-5A3) and mouse IgG1 isotype control were obtained from BioXcell, diluted in sterile PBS and administered at 250mg/mL intraperitoneally per mouse per day on days 2, 3, 4 and 5 of acute DSS colitis.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Purification, Cell Culture, Control, Comparison