Murine il 2

10⁵ splenic CD44^-CD62L⁺CD4⁺ T cells from naive mice were cultured with either 3000 CD81⁺migcDC1s or 3000 CD81^-migDCs (CD81^-migcDC1s and migcDC2s) from Flt3L-treated or vehicle-treated tumors for 5 days in RPMI 1640 (Gibco) supplemented with 10% (v/v) heat-inactivated FCS (Gibco), 300 μg mL⁻¹ L-glutamine (Gibco), 100 units mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin (Gibco), 1 mM non-essential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen) and 0.02 mM 2-mercapto ethanol (Invitrogen). T-cell differentiation was initiated in respective wells with 5 ng ml^-1 murine IL-2 (Biolegend) and 1 µg mL^-1 anti-CD3 (clone 145-2C11, BD Biosciences) on day 2 and day 4. To inhibit CCL22, anti-mouse CCL22/MDC polyclonal antibody (2 μg mL⁻¹) (R&D systems) was administered to the respective wells.

To produce ecotropic retrovirus for murine T cell transduction, Phoenix-ECO cells were transfected with a 1:3 ratio of pCL-Eco and transfer plasmid using a CalPhos Mammalian Transfection Kit (Takara Bio). Primary murine T cells were isolated from spleens of WT or CD45.1⁺ C57BL/6 mice using the EasySep Mouse CD8⁺ T Cell Isolation Kit or EasySep Mouse T Cell Isolation Kit (StemCell Technologies) and activated for 48 h on six-well non-tissue culture (TC)-treated plates precoated with 0.5 mg ml⁻¹ anti-CD3 (BioXCell, Clone 2C11) and 5 mg ml⁻¹ anti-CD28 (BioXCell, Clone 37.51) at 10⁶ cells ml⁻¹ with 5 ml per well. T cells were cultured in complete RPMI with 1 mM sodium pyruvate, 0.05 mM β-mercaptoethanol and 1× minimal essential medium non-essential amino acids (ThermoFisher) supplemented with 10 ng ml⁻¹ murine IL-2 (BioLegend). Following activation, T cells were transduced by spinfection at 3 × 10⁶ cells per well in complete T cell medium for 90 min at 1,100g and 32 °C with 10 ng ml⁻¹ polybrene (Sigma) on non-TC-treated plates pre-coated with 15 mg ml⁻¹ RetroNectin (Takara Bio). CAR expression was assayed via flow cytometry 24 h post-transduction; T cells were maintained at a cell density of 10⁶ cells ml⁻¹, then used for adoptive transfer or in vitro assays at 48 h post-transduction.