Type I interferon signaling induces melanoma cell-intrinsic PD-1 and its inhibition antagonizes immune checkpoint therapy
This study focuses on cancer cell-intrinsic programmed cell death (PD-1) functional regulation by examining induction of PD-1 in melanoma cells via type I interferon receptor signaling. Treatment of mouse melanoma cells with IFN-alpha or IFN-beta led to chromatin opening at and STAT1 and IFN regulatory factor 9 (IRF9) binding to a PDCD1 enhancer motif. Overall design: Serum-starved murine YUMM4.1 melanoma cells were treated with or without recombinant mouse IFN-alpha (60 ng/ml) or IFN-beta (60 ng/ml) for 3 hrs. Cells were harvested, washed twice in ice-cold PBS, centrifuged at 700 g for 5 min at 4°C, and cell suspensions (~1 x 106, > 90% viability) cryopreserved according to GENEWIZ ATAC-seq sample preparation guidelines (Azenta Life Sciences, South Plainfield, NJ). ATAC-seq library preparation and DNA sequencing were conducted at Azenta Life Sciences. In brief, cell samples were thawed, washed, and genomic DNA contamination removed by DNase I (Thermo Fisher Scientific) treatment. Cell number and viability were quantified on a Countess Automated Cell Counter (Thermo Fisher Scientific). Cells were lysed, cytosolic fractions removed, nuclei treated with Tn5 transposase (Illumina, Hayward, CA) for 30 min at 37°C, and tagmented DNA was purified using the MinElute PCR Purification Kit (Qiagen). Subsequently, tagmented DNA was barcoded using the Nextera Index Kit v2 (Illumina), PCR-amplified, and cleaned using solid-phase reversible immobilization (SPRI) beads to generated purified DNA libraries. Sequencing libraries were clustered on a flow cell, loaded onto an Illumina HiSeq instrument (model 4000 or equivalent) according to the manufacturer's instructions and samples sequenced using a 2x150 bp Paired End (PE) configuration. Image analysis and base calling were conducted using Control Software (CS). Raw sequence bcl data files were converted to fastq files and de-multiplexed using bcl2fastq v2.17 conversion software (Illumina). One mismatch was allowed for index sequence identification.