Anti cd3 clone 145 2c1

Ovalbumin (OVA) and human CD19-expressing MC38 colon adenocarcinoma (MC38^OVA and MC38^hCD19) cells were grown at 37 °C and 5% CO₂ in standard DMEM medium supplemented with 10% FBS (Sigma), 2 mM glutamine, 1 mM sodium pyruvate, 1% penicillin/streptomycin, 2 mM Hepes and 0.1 mM non-essential amino acids (NEAA, all Gibco). 5 x 10⁵ MC38^OVA cells were subcutaneously (s.c.) injected into the flanks of 8-12 weeks old C57BL/6 mice. Six days after tumor inoculation, mice were irradiated sublethally (6 Gy). The following day, mice were injected intravenously (i.v.) with 2 x 10⁶ OT-I CD8⁺ T cells that were isolated from the spleen and peripheral lymph nodes of OT-1 mice and stimulated with 0.5 μg/ml anti-CD3 (clone 145-2C1), 1 μg/ml anti-CD28 antibodies (clone 37.51, both Bio X Cell) and 10 ng/ml rhIL-2 (Peprotech) for 72 h with or without 1 mM 2-desoxyglucose (2-DG, Sigma). Tumor size was assessed every day using a caliper. For some experiments, tumor tissue was harvested and digested with 1 mg/ml collagenase type I and 100 μg/ml DNase I (both Roche) and T-cell infiltration analyzed by flow cytometry. Alternatively, Rag1^–/– mice were s.c. injected with 1 × 10⁶ MC38^hCD19. After 7 days, 2 x 10⁶ anti-hCD19 CAR T cells^{33 (link)} treated with and without 2 mM 2-DG for 2 days were adoptively transferred i.v.

To activate and differentiate murine CD8⁺ T cells into cytotoxic T cells (CTLs), delta-surface plates (Nunc) were pre-coated with 12 μg/ml polyclonal anti-hamster IgG (MP Biomedicals) for 2 h and washed once with PBS. In 12-well plates, 2 x 10⁶ cells were activated with 0.5 μg/ml of anti-CD3 (clone 145-2C1) together with 1 μg/ml anti-CD28 (clone 37.51, both Bio X Cell). After 2 days of activation, T cells were re-plated with fresh medium containing 10 ng/ml rhIL-2 or a combination of 10 ng/ml IL-7 and IL-15 (all Peprotech) and differentiated at a density of 5 x 10⁵ cells/ml for additional 4 days. For chronic antigenic stimulation in vitro, 2 x 10⁶ cells were activated with 0.5 μg/ml of anti-CD3 and 1 μg/ml anti-CD28 for 2 days followed by anti-CD3 stimulation. A total of 5 x 10⁵ cells/ml were restimulated every other day using 0.5 μg/ml of plate bound anti-CD3 over 6 days. In some experiments, 10 µM N-acetylcysteine (NAC) or 2 mM 2-desoxyglucose (2-DG, both Sigma) were added during cultivation. T cell differentiation was performed in normoxia (21% oxygen) or in hypoxia (2% oxygen) as indicated in the figure legends.

Purified P14 CD8⁺ T cells were transferred i.v. in sterile PBS into recipient mice. Depending on the experiment, a total of 3–6 x 10³ P14 T cells were transferred 1 d before infection. In competitive co-transfer experiments, GFP and tdTomato-expressing WT and transgenic P14 T cells were mixed in a 1:1 ratio before transfer. In some cases, GFP⁺ andTomato⁺ P14 T cells were also labeled with Cell Trace Violet (Thermo Fischer Scientific) before transfer. 2-DG-treated and control P14 T cells were activated with 0.5 μg/ml of anti-CD3 (clone 145-2C1) and 1 μg/ml anti-CD28 (clone 37.51, both Bio X Cell) in presence of 10 ng/ml rhIL-2 (Peprotech) and cultivated for 3–4 days in vitro. A total of 1 × 10⁶ control and 1 × 10⁶ 2-DG-treated P14 T cell were co-transferred i.v. into LCMV^CL13 infected mice 7 days post-infection (d.p.i.).