Experimental design: naive OT-I cells isolated from NR4a1fl/flNR4a2fl/fl, OT-I+, tdTomato-reporter+ mice were transduced with CreERT2 construct and then transferred to B16-OVA-tumor-bearing Rag2 mice (first transfer), which B16-OVA tumor cells were inoculated into 10 days before. Ten days later, TILs were isolated and sorted into the CD8+PD1+TIM3+LAG3+tdTomato fraction, and then 1 x 104 cells were transferred to Rag2 mice. The next day, B16-OVA tumor cells (1 x 106) were subcutaneously injected. Mice were treated with tamoxifen (TAM) or vehicle (corn oil) for 5 days to delete NR4a1/2, and 21 days after second transfer, TILs were isolated from tumors and analyzed by flow cytometry.
