Ah1 peptide spsyvyhqf

CT26 (CRL-2638) and B16-F10 (CRL-6475) were from ATCC and cultured in RPMI (Lonza or Gibco) or DMEM (Lonza), respectively, containing 10 % FBS (Gemini Bio) and 100 U/mL penicillin plus 100 mg/mL streptomycin (Lonza). Cells were tested for mycoplasma. AZD6738 (ATRi) was provided by AstraZeneca and dosed at 75 mg/kg by oral gavage as described previously (15 (link), 17 (link)). FTY720 (Fingolimod hydrochloride, MilliporeSigma) was dissolved in sterile normal saline (0.9% sodium chloride) and dosed at 1 mg/kg via intraperitoneal injection. InVivoPlus anti–mouse PD-L1 antibody (clone 10F.9G2, Bio X Cell) was diluted to 1 mg/mL in InVivoPure pH 6.5 Dilution Buffer (Bio X Cell) and dosed at 10 mg/kg via intraperitoneal injection. AH1 peptide (SPSYVYHQF, Anaspec) was dissolved in DMSO at 20 mg/mL and diluted to a final concentration of 10 μg/mL in T Cell Media (RPMI, 10 % FBS, 100 U/mL penicillin plus 100 mg/mL streptomycin, and the following [all from Gibco]: 1× MEM–nonessential amino acids, 1 mM sodium pyruvate, 5 mM HEPES pH 8.0, 50 mM β-mercaptoethanol).

To evaluate CD8⁺ T cell effector functions, TILs were re-stimulated in vitro for 1 h at 37 ^oC, 5% CO₂ in RPMI medium with 1 μg/ml AH1 peptide (SPSYVYHQF, AnaSpec Inc.) and CD107a antibodies; 1x BrefA (BioLegend, 420601) was added for additional 4 h. Alternatively, TILs or blood-derived cells were stimulated with PMA (50 ng/ml) plus ionomycin (1 µg/ml) plus 1x BrefA for 4 h. Thereafter, intracellular cytokine staining was performed for flow cytometry analysis.