Journal: Theranostics

Article Title: Nintedanib enhances the efficacy of PD-L1 blockade by upregulating MHC-I and PD-L1 expression in tumor cells

doi: 10.7150/thno.65828

Figure Lengend Snippet: Analysis of mechanisms related to immune activation. (A) Heat map of the expression of signature genes of interferon-gamma (IFN-γ) response (B) quantified by signature score. (C) Heat map of the expression of signature genes of IFN-α response (D) quantified by signature score. Gene Set Enrichment Analysis (GSEA) plot and gene sets for (E) RESPONSE TO INTERFERON-GAMMA and (F) ANTIGEN PROCESSING AND PRESENTATION. Flow cytometry analysis of (G) major histocompatibility complex class I (MHC-I) and (H) PD-L1 expression after different treatments in MC38 cells. The MHC-I expression levels were evaluated by MFI in (I) MC38, (J) LLC, (K) 4T1, and (O) B16F10 cells. The PD-L1 expression levels were evaluated by MFI in (L) MC38, (M) LLC, (N) 4T1, and (P) B16F10 cells. (Q) Representative western blot images of p-STAT3/STAT3, transporter associated with antigen processing 1 (TAP1), PD-L1 and beta-2-microglobulin (β2M).

Article Snippet: Recombinant mouse interferon-gamma (rMuIFN-γ; HY-P7071) and recombinant human interferon-gamma (rHuIFN-γ; HY-P7025) were purchased from MedChem Express (Monmouth Junction, NJ, USA).

Techniques: Activation Assay, Expressing, Flow Cytometry, Western Blot

Journal: STAR Protocols

Article Title: Multiparameter flow cytometry assay to analyze the pulmonary T cell profiles in the ovine model of respiratory syncytial virus infection

doi: 10.1016/j.xpro.2022.101688

Figure Lengend Snippet:

Article Snippet: Anti-IFN-γ-AF647 (Clone CC302; mouse IgG1; 1:160 dilution) , Bio-Rad , Cat# MCA1783A647.

Techniques: Virus, Recombinant, Sterility, Staining, Cell Culture, Hood, Flow Cytometry, Software

Journal: Vaccines

Article Title: Mannose-Modified Chitosan-Nanoparticle-Based Salmonella Subunit OralVaccine-Induced Immune Response and Efficacy in a Challenge Trial in Broilers

doi: 10.3390/vaccines8020299

Figure Lengend Snippet: Antibodies used for lymphocyte cell surface and intracellular IFNγ immunostaining.

Article Snippet: Rabbit anti-chicken IFNγ , Cat# AHP945Z; Bio-Rad; Hercules, CA, USA.

Techniques: Immunostaining

Journal: Journal of Veterinary Science

Article Title: Positive effects of porcine IL-2 and IL-4 on virus-specific immune responses induced by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 DNA vaccine in swine

doi: 10.4142/jvs.2014.15.1.99

Figure Lengend Snippet: Humoral immune responses in vaccinated piglets from the different groups detected with a commercial ELISA kit.

Article Snippet: To further evaluate cellular immune responses, IFN-γ levels in peripheral blood isolated from the vaccinated piglets at 35 dpi were measured using a commercial ELISA kit (Pig Interferon-γ, IFN-γ ELISA Kit; CUSABIO, USA) according to the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Reshaping tumor microenvironment by regulating local cytokines expression with a portable smart blue-light controlled device

doi: 10.1038/s42003-024-06566-y

Figure Lengend Snippet: a Schematics of light-controlled gene expression system and plasmids constructions. b Regulation of gene expression with light intensities from 0 to 1000 μW cm -2 with a customized 24-well plate light-controllable hardware, and c the intensity-response curve of mRuby expression in P815-M-ILs cells at 48 h (flow cytometry assay). d Time-dependent mRNA expression levels of Ifng and Cxcl10 (qPCR assay, normalized to time zero) in P815-IFNG cells and P815-M cells (control group). e Time-dependent production of murine IFNG and CXCL10 proteins (ELISA assay) under the continuous illuminating (On), dark control (Off) and alternative illuminating group (12/12 h light/dark cycles, 12 h O/N). Flow cytometry data is a representative of three independent experiments, each with similar results. Other data are shown as mean ± sem ( n = 3). All statistical significant were computed against the control group, with * P < 0.05; ** P < 0.01.

Article Snippet: Cell supernatants from in vitro studies were collected at designed time points (0, 12, 24, 48, and 72 h after illumination) to quantify cytokines and chemokines with murine CXCL10 and IFNG ELISA kit (BOSTER, China).

Techniques: Gene Expression, Expressing, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Reshaping tumor microenvironment by regulating local cytokines expression with a portable smart blue-light controlled device

doi: 10.1038/s42003-024-06566-y

Figure Lengend Snippet: a Timeline of in vivo experiment using DBA/2 mice with matched bilateral tumors (Left: P815-IFNG; Right: P815-M). b During illumination, representative BLI images of bilateral tumor-bearing mice at different times (Day 0, Day 3, and Day 6). c – d Representative BLI images of bilateral tumor-bearing mice were taken on different days (Day 0, Day 3, and Day 6) after illumination. Tumor size was quantified with average radiance value and compared with BLI signal at day 0 (the first day of illumination). e – g Blood analyses were performed after 6-day illumination, and the results show changes in the number of white blood cells (WBC) e , lymphocytes f , neutrophils g , monocytes ( h ) between light (2.5 mW cm -2 ), dark and control groups. i – l The concentration of murine INFG i , CXCL10 j , IL-6 k , and IL-10 ( l ) in plasma by the end of day 6 under illumination. Data are shown as mean ± s.e.m ( n = 4). Statistic significant was computed against the control group as * P < 0.05; ** P < 0.01.

Article Snippet: Cell supernatants from in vitro studies were collected at designed time points (0, 12, 24, 48, and 72 h after illumination) to quantify cytokines and chemokines with murine CXCL10 and IFNG ELISA kit (BOSTER, China).

Techniques: In Vivo, Control, Concentration Assay, Clinical Proteomics

Journal: Cell Reports Medicine

Article Title: MYC activation impairs cell-intrinsic IFNγ signaling and confers resistance to anti-PD1/PD-L1 therapy in lung cancer

doi: 10.1016/j.xcrm.2023.101006

Figure Lengend Snippet: Genetic inactivation and expression of IFNγ receptor 1 (IFNGR1) in LC (A) Chromatograms depicting, at the genomic level, the p.Q212X mutation at IFNGR1 in PDC11 cells and the matched normal DNA. The nucleotide change is indicated by an arrow. (B) Western blot of IFNGR1 and PD-L1 in the indicated LC cells. ACTIN and TUBULIN, protein-loading controls. (C) Western blot showing changes in levels of the indicated proteins in the PCD11 cells expressing ectopic wild-type IFNGR1 (IFNGR1-PDC11) on treatment with IFNγ (30 nM), as compared with the empty vector (EV-PDC11) control. Experiments were independently repeated at least twice with similar results. (D) mRNA levels assessed by real-time qPCR of the indicated transcripts (relative to ACTIN ) under basal conditions and after administering IFNγ (30 nM), in H596 cells, in PCD11 parental cells (PDC11), in cells expressing ectopic wild-type IFNGR1 and in EV-PDC11 controls. Results are presented as the median of independent biological triplicates. Asterisks denote statistical significance (∗p < 0.05; ∗∗∗p < 0.005; ∗∗∗∗p < 0.001) from two-tailed Student’s t tests. Data are presented as the mean ± SD. (E) Representative weak and strong immunostaining of IFNGR1 in lung primary tumors. Scale bars, 50 μm. (F) Distribution of the immunostaining of IFNGR1 among lung tumors, by histopathology and levels of HLA-I/B2M and PD-L1. Fisher’s exact test. ns, not significant, LuAD, lung adenocarcinoma; LuSCC, lung squamous cell carcinoma; ns, not significant.

Article Snippet: To determine IFNGR1 levels by immunohistochemistry we used the polyclonal anti-IFNGR1 antibody (10808-1-AP, Proteintech; 1:200 dilution.

Techniques: Expressing, Mutagenesis, Western Blot, Plasmid Preparation, Control, Two Tailed Test, Immunostaining, Histopathology

Journal: Cell Reports Medicine

Article Title: MYC activation impairs cell-intrinsic IFNγ signaling and confers resistance to anti-PD1/PD-L1 therapy in lung cancer

doi: 10.1016/j.xcrm.2023.101006

Figure Lengend Snippet: Oncogenic MYC and low level of constitutive expression of IFNγsign genes (A) Oncoplot of the presence of alterations in the indicated genes. Immunoresponse indicates the presence of mutations of genes involved in immunorecognition (e.g., B2M , TAP1/2 , CALR ) or response to IFNγ ( JAK2 and IFNGR1 ) , and CCLE at cBioPortal. (B) Graphs showing gene expression values in transcripts per million (TPMs) for the transcripts within the IFNγ signature from RNA-seq and at GEO: GSE109720) in (lo)MAX-, (hi)MAX-, and (hi)MYC/MAX-expressing cells. Bars indicate means; differences assessed by two-sided Student’s t test. ns, not significant; S, SCLC. (C) Gene set enrichment analysis (GSEA) showing an inverse correlation in gene expression between the IFNγsign and datasets, including from lungs of mice overexpressing Nmyc (GEO: GSE6077). FDR, false discovery rate; NES, normalized enrichment score.

Article Snippet: To determine IFNGR1 levels by immunohistochemistry we used the polyclonal anti-IFNGR1 antibody (10808-1-AP, Proteintech; 1:200 dilution.

Techniques: Expressing, Gene Expression, RNA Sequencing

Journal: Cell Reports Medicine

Article Title: MYC activation impairs cell-intrinsic IFNγ signaling and confers resistance to anti-PD1/PD-L1 therapy in lung cancer

doi: 10.1016/j.xcrm.2023.101006

Figure Lengend Snippet:

Article Snippet: To determine IFNGR1 levels by immunohistochemistry we used the polyclonal anti-IFNGR1 antibody (10808-1-AP, Proteintech; 1:200 dilution.

Techniques: Virus, Recombinant, Modification, DC Protein Assay, Plasmid Preparation, Sequencing, SYBR Green Assay, Magnetic Beads, Gene Expression, Software

Journal: Frontiers in Immunology

Article Title: IL-15 Harnesses Pro-inflammatory Function of TEMRA CD8 in Kidney-Transplant Recipients

doi: 10.3389/fimmu.2017.00778

Figure Lengend Snippet: IL-15-stimulated TEMRA CD8 from Kidney-Transplant (KT) Recipients induces the activation of endothelium through IFN-γ and TNF-α secretion. (A) Expression of CD25 or CD69 by CD8 T cell subsets purified from KT recipients stimulated for 24 h with plate-bound anti-CD3 (2 µg/mL) and IL-15 (10 ng/mL). Representative flow data are shown, and bars indicate mean ± SEM of pooled data ( n = 10–12). (B) Schematic overview of the strategy to assess the inflammation of human umbilical vein endothelial cell (HUVEC) by IL-15-activated CD8 T cell subsets from KT. (C) Expression of CX3CL1 by HUVEC after 6 h of culture in the presence of medium control or selective inhibitors [anti-IFN-γ (10 µg/mL) or anti-TNF-γ (10 µg/mL) mAb] and with supernatant (dilution of supernatant of 1/6) from CD8 T cell subsets from KT stimulated overnight with plate-bound anti-CD3 (2 µg/mL) and IL-15 (10 ng/mL). Bars indicate mean ± SEM of pooled data ( n = 4–6). Mann–Whitney test (A) or Kruskal–Wallis test followed by a Dunn’s Multiple Comparison Test (C) was performed (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: Antibodies against the following proteins were purchased from BD Biosciences: CD3 (HIT3a and UCHT1), CD69 (SK7), CD25 (M-A251), CD28 (CD28.2), pSTAT5 pY694 (clone 47), p38MAPK pT180/pY182 (38/p38), TNFα (Mab11), IFN-γ (B27), and Annexin V. Antibodies against the following proteins were purchased from Miltenyi Biotech: CD8 (BW135/80), CD45RA (T6D11), CCR7 (REA108), pAKT pS473 (REA359), and pERK1/2 pT202/pY204 (REA152).

Techniques: Activation Assay, Expressing, Purification, Control, MANN-WHITNEY, Comparison