Mice.

NOD.Cg-Prkdc^scid IL2rg^tm1Wjl/SzJ (NSG) mice (Jackson Laboratories), bred in SPF conditions, both males and females at 7 weeks of age, were used for adoptive transfer experiments. Briefly, 1 × 10⁶ NAC-induced CD8⁺ Tscm or control CD8⁺ T cells, generated as described above, were cotransferred by retro-orbital injection with 5 × 10⁶ to 6 × 10⁶ autologous PBMCs, previously depleted of CD8⁺ T cells by incubation with anti-CD8 APC mAb (Supplemental Table 2), followed by detection of APC by anti-APC microbeads (Miltenyi) and negative magnetic separation with LS columns (Miltenyi), according to the manufacturer’s instructions. Depletion was >99%. Mice were sacrificed 15 days after transfer. For mononuclear cell isolation, tissues were minced and filtered through a 100-μm cell strainer, and stained with mAbs (Supplemental Table 2) as described above. To detect intracellular markers, cells were fixed and permeabilized with the FoxP3 transcription factor staining buffer kit (catalog BMS00-5523-00, eBioscience) according to the manufacture’s instructions and stained with anti-Ki-67 and anti-TCF-1/7 mAb (Supplemental Table 2).

For tumor experiments and ACT, female NSG mice were intravenously injected with 2.5 × 10⁵ NALM6-GL cells, followed 7 days later by 2.5 × 10⁵ CD19-CAR⁺CD8⁺ T cells expanded with Dynabeads and IL-2 and IL-12 in the presence or absence of 20 mM NAC, as described above. CAR lentiviral transduction was performed as previously described (21). Recombinant human IL-15 (obtained from the National Cancer Institute) (60) was injected intraperitoneally every other day (1 μg per mouse). Tumor burden was measured using the Xenogen IVIS Lumina (Caliper Life Sciences), as previously described (21).