Illumina NovaSeq 6000 paired end sequencing; Restoring PD-1 immune checkpoint inhibitor response following IFNγ-driven adaptive resistance
Restoring PD-1 immune checkpoint inhibitor response following IFNγ-driven adaptive resistance
Once treatment was completed, mice were sacrificed and tumours were harvested and processed into RNA for sequencing. YUMM2.1 cells (provided by Richard Marais, The University of Manchester) were maintained in RPMI-1640 (Sigma-Aldrich) supplemented with 10% foetal bovine serum (FBS; Life Technologies) and 1% penicillin-streptomycin (P/S; Sigma-Aldrich). All cells were maintained under standard conditions at 37°C in a 5% CO2 humidified incubator and passaged before reaching confluency. For IFN-γ-pre-treatment, IFN-γ 50 IU/ml was added to the medium for at least 2 weeks. IFN-γ-naïve or IFN-γ-pre-treated YUMM2.1 cells were implanted subcutaneously into 8-week-old C57BL/6J female mice. There were 4 experimental treatments.  Control - After tumour establishment, IFN-γ-naïve YUMM2.1 tumour-bearing mice received two doses of IgG2a antibodies (300ug per dose) three days apart.  PD-1 resistance - After tumour establishment, IFN-γ-naïve YUMM2.1 tumour-bearing mice received two doses of α-PD-1 antibodies (300ug per dose) three days apart. Following the final dose of α-PD-1, tumours were permitted to regrow to their pre-treatment size before further treatment commenced. At this point, we initiated one week of treatment with vehicle.  PD-1 responding - After tumour establishment, IFN-γ-naïve YUMM2.1 tumour-bearing mice received two doses of α-PD-1 antibodies (300ug per dose) three days apart.  IFN-γ-pre-treated - IFN-γ-pre-treated YUMM2.1 tumour-bearing mice received two doses of IgG2a antibodies (300ug per dose) three days apart. Total RNA was extracted using QIAzol Lysis Reagent and isolated using RNAeasy mini kit (both from QIAGEN). Illumina stranded RNA library