Journal: Nature Communications

Article Title: Type I interferon signaling induces melanoma cell-intrinsic PD-1 and its inhibition antagonizes immune checkpoint blockade

doi: 10.1038/s41467-024-51496-2

Figure Lengend Snippet: Single-cell RNA-seq analysis of a type I interferon-α/β receptor (IFNAR) and type II interferon-γ receptor (IFNGR) subunit gene expression shown in units of transcript per million (TPM)/10 (black bars; mean ± SEM) in patient melanoma cells (left) versus tumor-infiltrating T-cells (right). Gene expression of additional cytokine and growth factor receptor subunits is also shown (gray bars). b Violin plots of IFNAR1 and IFNAR2 gene expression (TPM/10; median, bold white line; top and bottom quartiles, thin white lines) in patient melanoma (MM) versus tumor-infiltrating T-cells generated from the single-cell RNA-seq dataset above. c IFNAR1 and IFNAR2 surface protein expression (percent positivity, mean ± SEM, left) by patient tumor cells and tumor-infiltrating lymphocytes (TILs), with representative flow cytometric histograms shown (right). d Relative IFNAR1 and IFNAR2 gene expression (fold change, mean ± SEM) in human melanoma cell lines (black bars) versus human activated CD3 + T-cells (gray bars) as determined by RT-qPCR. e IFNAR1 and IFNAR2 surface protein expression (percent positivity, mean ± SEM, left) by human A2058 and A375 melanoma cells, with representative flow cytometric histograms shown for A2058 melanoma cells (right). f Relative Ifnar1 and Ifnar2 gene expression (fold change, mean ± SEM) in murine melanoma cell lines (black bars) versus murine activated CD3 + T-cells (gray bars) as determined by RT-qPCR. g IFNAR1 and IFNAR2 surface protein expression (percent positivity, mean ± SEM, left) by murine melanoma cells, with representative flow cytometric histograms shown for B16-F10 and YUMM1.7 melanoma cells (right). IFNAR1 (left) and IFNAR2 (right) surface protein expression (percent positivity, mean ± SEM) by PD-1 + versus PD-1 - h , human A2058 and A375 and i , murine melanoma cell subsets, with representative flow cytometric histograms shown for A2058 cells. Results represent biologically independent samples of a , b n = 1252 MM cells (left panel) and 2040 T-cells (right panel), c n = 4, and biologically independent experiments of d , f , i n = 3, e n = 12, g n = 6, and h n = 7 (A2058) and n = 6 (A375). Statistical analyses included b Mann-Whitney test, two-sided, c paired t -test, one-sided, and h , i unpaired t -test, two-sided. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant. See also Supplementary Figs. – . Source data, including exact p -values, are provided as a Source Data file.

Article Snippet: The following antibodies (abs) were used for flow cytometric analyses of human cells: Alexa Fluor 647-conjugated (BD Biosciences) or PerCP-eFluor 710-conjugated anti-human PD-1 (clone MIH4, 20 μg/mL, Thermo Fisher Scientific, Waltham, MA) and Alexa Fluor 647-conjugated or PerCP-eFluor 710-conjugated mouse IgG1 isotype controls (clone MOPC-31C, 20 μg/mL, BD Biosciences; clone P3.6.2.8.1, 20 μg/mL, Thermo Fisher Scientific), unconjugated anti-human PD-1 clinical ab, nivolumab (100 μg/mL, Bristol Myers Squibb, Cambridge, MA) was obtained from the BWH Pharmacy, and Ultra-LEAF unconjugated human IgG4 isotype control (clone QA16A15, 100 μg/mL, BioLegend), FITC-conjugated or PE-conjugated anti-human IgG4 (clone HP6023, 1:50, Abcam, Waltham, MA), PE-conjugated anti-human PD-L1 (clone 29E.2A3, 15 μg/mL, BioLegend) and PE-conjugated mouse IgG2b isotype control (clone MPC-11, 15 μg/mL, BioLegend), APC-conjugated anti-human PD-L2 (clone 24 F.10C12, 15 μg/mL, BioLegend) and APC-conjugated mouse IgG2a isotype control (clone MOPC-173, 15 μg/mL, BioLegend), PE-conjugated anti-human IFNAR1 (clone 85228, 20 μg/mL, Thermo Fisher Scientific) and PE-conjugated mouse IgG1 isotype control (clone P3.6.2.8.1, 20 μg/mL, Thermo Fisher Scientific) or PE-conjugated mouse IgG1 isotype control (clone MOPC-21, 20 μg/mL, BioLegend), Alexa Fluor 750-conjugated anti-human IFNAR1 (clone 85228, 20 μg/mL, R&D Systems, Minneapolis, MN) and Alexa Fluor 750-conjugated mouse IgG1 isotype control (clone 11711, 20 μg/mL, R&D Systems), APC-conjugated or APC-Vio770-conjugated anti-human IFNAR2 (clone REA124, 20 μg/mL, Miltenyi Biotec, Gaithersburg, MD) and APC-conjugated or APC-Vio770-conjugated human IgG1 isotype control (clone REA293, 20 μg/mL, Miltenyi Biotec), FITC-conjugated anti-human IFN-β (clone A1(IFNb)), 5 μg/mL, and FITC-conjugated mouse IgG1 (clone MOPC-21, 5 μg/mL, Thermo Fisher Scientific), PE-conjugated anti-human IFN-α[2b] (clone 7N4-1, 1.5 μg/mL) and PE-conjugated mouse IgG1 isotype control (clone MOPC-21, 1.5 μg/mL, BD Biosciences), FITC-conjugated anti-mouse Interferon alpha (clone RMMA-1, 50 μg/mL, PBL Assay Science, Piscataway, NJ) and FITC-conjugated rat IgG1 (clone eBRG1, 50 μg/mL, Thermo Fisher Scientific), anti-mouse Interferon-beta 1 (clone D2J1D, 75 ng/mL), rabbit IgG isotype control (clone DA1E, 75 ng/mL Cell Signaling Technology, Danvers, MA), and PE-conjugated donkey anti-rabbit IgG (clone Poly4064, 1.25 μg/mL BioLegend).

Techniques: RNA Sequencing, Gene Expression, Generated, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal:

Article Title: Activation of PKR by Bunyamwera Virus Is Independent of the Viral Interferon Antagonist NSs

doi: 10.1128/JVI.77.9.5507-5511.2003

Figure Lengend Snippet: Effect of IFN and PKR in cell culture. (A) Vero cells were grown to 80% confluence and incubated overnight with various concentrations of human interferon alpha A/D (BglII) (PBL Biomedical Laboratories). After 24 h of incubation, cells were infected with 0.01 PFU/cell of wt BUNV or BUNdelNSs virus and incubated in Dulbecco's modified Eagle medium-10% fetal calf serum, again containing IFN. Virus yields were measured 18 h postinfection in plaque assays, as described previously (22). (B) IFN sensitivity of viruses in wt cells (columns 1 to 4) or PKR(−/−) MEF cells (columns 5 to 8). Cells were treated either with 1,000 neutralizing units of sheep polyclonal anti-mouse IFN-α/β (PBL Biomedical Laboratories) or with 1,000 units of IFN-α and -β for 24 h, infected with 0.01 PFU/cell of wt BUNV (columns 1, 2, 5, and 6) or BUNdelNSs virus (columns 3, 4, 7, and 8), and incubated in Dulbecco's modified Eagle medium-10% fetal calf serum, again containing IFN. Virus yields were measured 18 h postinfection with MEFs derived from IFN-α/β receptor knockout mice. Averages and standard deviations of results of three independent experiments are shown.

Article Snippet: Cells were treated either with 1,000 neutralizing units of sheep polyclonal anti-mouse IFN-α/β (PBL Biomedical Laboratories) or with 1,000 units of IFN-α and -β for 24 h, infected with 0.01 PFU/cell of wt BUNV (columns 1, 2, 5, and 6) or BUNdelNSs virus (columns 3, 4, 7, and 8), and incubated in Dulbecco's modified Eagle medium-10% fetal calf serum, again containing IFN.

Techniques: Cell Culture, Incubation, Infection, Virus, Modification, Derivative Assay, Knock-Out

Journal:

Article Title: Stromal Cell-Mediated Suppression of Human T-Cell Leukemia Virus Type 1 Expression In Vitro and In Vivo by Type I Interferon ▿

doi: 10.1128/JVI.02564-08

Figure Lengend Snippet: Involvement of type I IFNs in suppression of HTLV-1 expression by stromal cells. (A and B) ILT-Hod cells were cultured in the presence of rhIFN-α, rhIFN-β, or rhIL-10 at the indicated concentrations for 72 h, and the concentrations of HTLV-1 p19 in the supernatants (A) and viable cell numbers (B) were evaluated by ELISA or using Cell Counting Kit-8, respectively. The results indicate means and SD of duplicate wells. (C) The HTLV-1 gag mRNA expression levels in ILT-Hod cells were measured after a 24-h coculture without (open bars) or with (closed bars) NIH 3T3 cells in the presence of anti-mouse IFN-α or IFN-β neutralizing antibodies (4 × 103 neutralizing units/ml). (D) ILT-Hod cells were pretreated with or without 1 μg of anti-human IFN α/β receptor monoclonal antibody or control mouse IgG2a/ml for 1 h prior to coculture with HEK293T cells for 2 h, and then the HTLV-1 gag mRNA expression levels in ILT-Hod cells were evaluated. The data in panels C and D are the fold changes compared to control cells cultured in medium alone and represent the means and SD of duplicate wells. (E) Reporter 293T cells containing an IFN-β reporter plasmid (293T/IFN-β-luc) were cultured with or without ILT-Hod cells or ILT-Hod cells fixed with 1% formaldehyde, and the luciferase activities in the whole-cell lysates were measured after coculture for 24 h. The results are means and SD of duplicate samples. (F) The MxA (○) and HTLV-1 gag (•) mRNA levels in ILT-Hod cells were measured after coculture with HEK293T cells for the indicated periods. The data represent the fold changes compared to control ILT-Hod cells cultured alone for 1 h.

Article Snippet: Mouse monoclonal antibodies against human IFN-α/β receptor (PBL Biomedical Laboratories, New Brunswick, NJ) and control mouse IgG2a,k (BioLegend) were added to cocultures of ILT-Hod and HEK293T cells.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Cell Counting, Control, Plasmid Preparation, Luciferase

Journal: eLife

Article Title: Type I interferon underlies severe disease associated with Junín virus infection in mice

doi: 10.7554/eLife.55352

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-mouse IFNAR-1 (anti-IFN-α/βR) monoclonal antibody (MAR1-5A3) , Bio X Cell , Cat# BE0241 RRID: AB_2687723 , (500 μg primary dose; 250 μg maintenance dose).

Techniques: Knock-In, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Software