Pd l1 cd274

Cells were labelled with combinations of PE-, FITC-, PerCP-, APC-, PE-Cy7-, V450- and Pacific Orange-conjugated mouse anti-human CD14, CD80, CD86, DC-SIGN (CD209) (all from BD Biosciences, Franklin Lakes, NJ, USA), PD-L1 (CD274) (Biolegend) and HLA-DR (Invitrogen) antibodies and appropriate isotype controls, using 0.25 μg of each antibody per staining reaction. Acquisition was performed on a BD FACSCanto II flow cytometer (BD Biosciences) and analysis of flow data was performed using FlowJo 7.6.4 (Tree Star, Inc., Ashland, OR, USA). For viability staining, cells were labelled with a Near-IR dead cell stain kit (Invitrogen) according to the manufacturer’s instructions and analysed by flow cytometry.

The treated and control cells were collected, washed once with PBS, centrifuged, and then blocked with 10% NGS on ice for 10 mins. The cells were then incubated with primary anti-human CD47 antibody (1 : 500) (BioLegend, San Diego, CA) and PD-L1 (CD274) (1 : 500) (BioLegend) on ice for 30 mins followed by secondary goat anti-mouse IgG (H+L) Alexa Fluor 488 antibody (1 : 500) (Invitrogen) for 30 mins on ice in the dark. The cells were washed with PBS, resuspended in cold 10% NGS, filtered with a 40 μm strainer, and analyzed with a BD LSRFortessa™ analyzer (BD Bioscience, Heidelberg, Germany).

Treatment conditions for flow cytometry analysis were conducted as follows, unless specified otherwise: Breast cancer cell lines were grown in the presence of E2 10 nmol/L (E2), in hormone-deprived (HD) conditions or treated with either vehicle, fulvestrant, birinapant, or the combination for a total of 72 hours. For IFNγ stimulation studies, after 48 hours, treatments were refreshed with or without IFNγ (10 ng/mL) for last 24 hours prior to flow cytometry analysis. Before the analysis, tumor cells or T cells were suspended in FACS staining buffer [1% BSA, 1 mmol/L EDTA in phosphate-buffered saline (PBS)] and stained with fluorochrome-conjugated antibodies against combinations of the following surface human antigens: HLA-ABC (BioLegend; cat. #311415, RRID:AB_493134), PD-L1/CD274 (BioLegend; cat. #329707, RRID:AB_940358), and HA-tag (BioLegend; cat. #901509, RRID:AB_2565072). Cell viability was determined using propidium iodide exclusion (Life Technologies, P3566) or DAPI (Thermo Fisher, 62248). Flow-cytometric data were acquired using an LSRFortessa cytometer (BD) and analyzed with FlowJo software version 10 (FlowJo LLC).