Total RNA was isolated from activated HD and HNSCC CD8+ T cells using the E.Z.N.A. total RNA isolation Kit (Omega Bio-tek) as per the manufacturer’s instructions. Six hundred and fifty nanograms of RNA was used to synthesize complementary DNA (cDNA) using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific) as per the manufacturer’s instructions. Predesigned primers for RT-qPCR were obtained using TaqMan Gene Expression Assays (Applied Biosystems, Thermo Fisher Scientific) to detect the expression of ADORA2A (assay ID: Hs001169123_m1) and GAPDH (assay ID: HS03929097_g1). The RT-qPCR was set up in a 96-well plate by adding 30 ng of cDNA, 1× TaqMan Gene Expression Master Mix (Applied Biosystems), and 1 μl of TaqMan Gene Expression Assay primers. All samples were run in quadruplicate. GAPDH was used as an internal control. RT-qPCR was cycled in Applied Biosystems StepOne Real-Time PCR System (Applied Biosystems). CT values were measured using StepOne software version 2.1 (Applied Biosystems). CT values for ADORA2A were normalized against measured CT values for GAPDH, and the ΔΔCT values were calculated as described previously (55). Relative quantity values, representing the fold change in ADORA2A gene expression in HNSCC CD8+ T cells compared to HD CD8+ T cells, were calculated as the 2−ΔΔCT values.

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