α cd3e clone 145 2c11

Female BALB/c and C57BL/6 mice were purchased from Charles River Breeding Laboratory (Wilmington, MA). All of our investigations have been approved by the McMaster Animal Research Ethics Board. To generate murine T lymphoctes for retroviral transduction, 3 × 10⁶ freshly isolated splenocytes were cultured in 1 mL T cell media (RPMI supplemented with 10 % FBS, 2 mM L-glutamine, 10 mM HEPES, 0.5 mM sodium pyruvate, 0.1 mM non-essential amino acids, 55 μM β-mercaptoethanol, and 0.1 mg/mL normocin or 100U/mL penicillin + 100 μg/mL streptomycin) supplemented with 0.3 μg/mL α-CD3e (clone 145-2C11, Cat No. 553057, BD Pharmingen, San Diego, CA) and 400U/mL rhIL-2 (Cat No. 200–02, Peprotech, Rocky Hill, NJ). Twenty-four hours after activation, 600 μL of media were removed from T cell cultures and 100 μL of the concentrated retroviral supernatant was added, along with 2 μg/mL Lipofectamine 2000 and 1.6 μg/mL Polybrene (Sigma-Aldrich). Cultures were spun at 2000 rpm, 32 °C for 90 min, allowed to rest for 1–4 h, and were supplemented with 0.5 mL T cell media + 400U/mL rhIL-2. This process was repeated at 48 h after activation with the 72 h retroviral concentrates. Seventy-two hours after activation, retrovirally transduced T cell cultures were expanded into 30 mL of DC media + 400U/mL rhIL-2. Six to eight days after activation, resultant CAR-T cells were enumerated for use in vitro.