Preparation of PMEL, WT OT-I, MPC1-KO OT-I, Tcf7DTR-GFP P14 T cells, and human CD8+ T cells

Spleens from PMEL or OT-I mice were mechanically disrupted and grounded through a 70-μm strainer (Fisher Scientific). Red blood cells (RBC) were lysed with ACK lysis buffer (2 mL per spleen, Gibco / Thermo Fisher Scientific) for 5 min at 25°C. The splenocytes were washed twice with cold complete RPMI medium, which contained RPMI-1640 (Gibco), FBS (10% v/v), HEPES (pH 7.2~7.5, 1% v/v), penicillin/streptomycin (1% v/v), sodium pyruvate (1% v/v, Gibco / Thermo Fisher Scientific), and 2-mercaptoethanol (0.1% v/v, Gibco / Thermo Fisher Scientific). Splenocytes were then resuspended at a cell density of 2 × 10⁶ /mL in complete RPMI medium supplemented with mouse IL-2 (10 ng/mL) and IL-7 (1 ng/mL, PeproTech), as well as human gp100_25–33 or OVA_257–264 peptide (1 μM, GenScript) for PMEL or OT-I T cells, respectively. After a 3-day culture, live cells were enriched by density gradient centrifugation against Ficoll-Paque PLUS (GE Healthcare), followed by another 2-day culture at a cell density of 0.5~1.0 × 10⁶/mL in complete RPMI medium supplemented with mouse IL-2 (10 ng/mL) and IL-7 (10 ng/mL) to afford activated CD8⁺ T cells with purity > 95% (flow cytometry analyses). MPC1-KO OT-I T cells were prepared similarly as WT OT-I T cells. Peptide-primed CD8⁺ T cells were next used for ACT in mice. For in vitro experiments, the peptide-primed CD8⁺ T cells were cultured in complete RPMI medium supplemented with IL-2 (10 ng/mL) and IL-7 (10 ng/mL) for another 2 days to afford primed CD8⁺ T cells in resting phase (day 7). In the pyruvate feeding experiment, primed CD8⁺ T cells in resting phase were cultured in RPMI medium with low-glucose (1-g/L D-glucose, Thermo Fisher Scientific), FBS (10% v/v), HEPES (pH 7.2~7.5, 1% v/v), penicillin/streptomycin (1% v/v), and 2-mercaptoethanol (0.1% v/v), which was further supplemented with IL-2 (10 ng/mL) and sodium pyruvate at different concentrations for 2 days.

Tcf7 ^DTR-GFP P14 T cells were isolated from splenocytes of Tcf7 ^DTR-GFP P14 mice by mashing the spleen through a 100 μm nylon cell strainer (BD Falcon). Red blood cells were lysed with ACK lysis buffer. For Fig. 3e,​,ff and Extended Data Fig. 3h,​,i,i, CD8⁺ T cells isolated using mouse CD8⁺ T cell negative enrichment kit (StemCell Technologies) were activated on plates coated with anti-mouse CD3 antibody (17A2, eBioscience / Thermo Fisher Scientific) and anti-CD28 antibody (37.51, eBioscience / Thermo Fisher Scientific) plus in complete RPMI medium containing IL-2 (50 ng/ml) for 2 days before adoptive transfer. For Fig. 3g,​,h,h, Tcf7 ^DTR-GFP P14 T cells were primed by LCMV gp33-41 peptide (1 μM, GenScript) and prepared similarly as described above for PMEL T cells.

Human peripheral blood mononuclear cells (PBMCs) from anonymous healthy donors (prepared as buffycoats) were activated in vitro with anti-human CD3 (OKT3, BioLegend) and CD28 (CD28.2, BioLegend) antibodies in the presence of human IL-2 (10 ng/mL) for 2 days. Activated human CD8⁺ T cells were isolated by Ficoll density gradient separation and magnetic-activated cell sorting (MACS) using a human CD8⁺ T cell isolation kit (Miltenyi Biotec) for in vitro assays.