Journal: Cellular and Molecular Immunology

Article Title: IFN-γ upregulates membranous and soluble PD-L1 in mesothelioma cells: potential implications for the clinical response to PD-1/PD-L1 blockade

doi: 10.1038/s41423-019-0245-x

Figure Lengend Snippet: IFN-γ induces the upregulation and secretion of PD-L1 in mesothelioma cell lines. All analyses were performed on the mesothelioma cell lines MPP89, IST-MES1 and IST-MES2, untreated or treated with 10 ng/ml IFN-γ for 48 h. Cell lines were obtained from the Interlab Cell Line Collection (ICLC, IRCCS Ospedale Policlinico San Martino, Genoa, Italy), a member of the ICLAC (International Cell Line Authentication Committee), on 05 July 2017 and kept frozen until use from 27 December 2018 to 12 April 2019. ICLC verified the authenticity of the cell lines by using STR loci plus amelogenin (Cell IDTM System, Promega, Madison, WI, USA) and tested the cell lines for mycoplasma contamination by using bisbenzimide (Hoechst 33258) DNA fluorescence staining and PCR analysis with oligonucleotide primer pairs (TIB Molbiol S.r.l., Genoa, Italy). a Cytofluorimetric analysis of PD-L1 expression on the surface of mesothelioma cell lines performed with the antibody MIH1 (eBioscience Thermo Fisher Scientific, Milan, Italy). Green histograms: basal expression of PD-L1; blue histograms: PD-L1 expression upon incubation with IFN-γ; red histograms: negative control. Data are shown as the number of cells vs. log far red fluorescence intensity in arbitrary units. The numbers inside the panels indicate the mean fluorescence intensity (MFI) of each histogram. The MFI of the negative control (cells stained with the second reagent alone) is shown for comparison (red histograms). The black horizontal bar indicates the gate of each histogram used to calculate the PD-L1-positive cells. b Western blot analysis of PD-L1 total cell protein from lysates of the three cell lines upon incubation with IFN-γ (+), or the basal content of PD-L1 (−). Western blotting was performed with the anti-PD-L1 antibody E1L3N (Cell Signaling Technology, Milan, Italy) also used for PD-L1 immunohistochemistry in mesothelioma tissues (not shown). The β-Actin content of each lysate is shown for comparison. c mRNA levels of transcripts coding for the full-length PD-L1 molecule in the indicated mesothelioma cell lines incubated with IFN-γ. Data are shown as the expression fold change (log scale) relative to the basal mRNA levels of PD-L1 in each cell line. d ELISA of culture supernatants harvested from cultures of the mesothelioma cell lines incubated with IFN-γ (black columns) or in the absence of IFN-γ (white columns, CTR)

Article Snippet: ICLC verified the authenticity of the cell lines by using STR loci plus amelogenin (Cell IDTM System, Promega, Madison, WI, USA) and tested the cell lines for mycoplasma contamination by using bisbenzimide (Hoechst 33258) DNA fluorescence staining and PCR analysis with oligonucleotide primer pairs (TIB Molbiol S.r.l., Genoa, Italy). a Cytofluorimetric analysis of PD-L1 expression on the surface of mesothelioma cell lines performed with the antibody MIH1 (eBioscience Thermo Fisher Scientific, Milan, Italy).

Techniques: Fluorescence, Staining, Expressing, Incubation, Negative Control, Western Blot, Immunohistochemistry, Enzyme-linked Immunosorbent Assay