Mouse spleen dissociation kit

Unstimulated T cells were used as a negative control. Naïve CD8⁺ T were isolated using a mouse Spleen Dissociation Kit (130-095-926, Miltenyi Biotec) and mouse CD8a (Ly-2) MicroBeads (130-117-044, Miltenyi Biotec). CD8⁺ T cells were used immediately after isolation. Naïve CD8⁺ T cells were stained with 5 mmol/L of carboxyfluorescein succinimidyl ester (CFSE) by incubation at room temperature for 5 minutes in the dark. The CFSE surplus was removed by two washing steps with 1×PBS. Enriched naïve CD8⁺ T cells (1 × 10⁵) were stimulated in U-bottomed 96-well plates that were precoated with 2 μg/mL anti-CD3 (130-097-621, Miltenyi Biotec) and anti-CD28 (130-093-182, Miltenyi Biotec). After being stimulated for 48 hours, the cells were transferred to new wells and rested for 24 hours. For the conditioned medium, tumor cells were seeded at a density of 50,000 cells/cm² and cultured in the presence or absence of KYNU (1 μmol/L) or vehicle control (10% DMSO in PBS) for 48 hours. Supernatants of each group of tumor cells were centrifuged at 1,000 × g for 5 minutes. The CFSE-labeled CD8⁺ T cells were exposed to a conditioned medium or media supplemented with 5% FBS. Then, the CD8⁺ T cells were collected, washed twice in 1×PBS, and resuspended in 500 μL of 1×PBS before analysis by flow cytometry. Unstimulated T cells were used as a negative control.