Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors. PBMCs were activated with 50 ng/mL OKT-3 antibody (MACS) and 100 IU/mL IL-2 for two days prior to transduction and maintained in 100 IU/mL thereafter. Trandsuction was performed by centrifugation of activated T cells in media from retroviral producers at 2000× g at room temperature for 1 h on RetroNectin-coated plates (Takara Bio) for two consecutive days. Experiments were performed in compliance with all relevant ethical regulations and in accordance with MSK IRB Protocol 00009377.
Mouse T cells were engineered as previously described16 (link). Briefly, T-cells were isolated from spleens of naive mice by mechanical disruption using a 100 μm cell strainer. Splenocytes were collected and red blood cells were lysed with ACK (ammonium-chloride-potassium) lysing buffer (ThermoFisher A1049201). Splenocytes were activated overnight with CD3/CD28 Dynabeads (Life Technologies) and 50 IU/mL human IL-2. Activated T cells were transduced by centrifugation with retroviral supernatant from transduced Phoenix-Eco cells on RetroNectin-coated plates (TakaraBio) for 2 consecutive days.
Mouse T cells were engineered as previously described16 (link). Briefly, T-cells were isolated from spleens of naive mice by mechanical disruption using a 100 μm cell strainer. Splenocytes were collected and red blood cells were lysed with ACK (ammonium-chloride-potassium) lysing buffer (ThermoFisher A1049201). Splenocytes were activated overnight with CD3/CD28 Dynabeads (Life Technologies) and 50 IU/mL human IL-2. Activated T cells were transduced by centrifugation with retroviral supernatant from transduced Phoenix-Eco cells on RetroNectin-coated plates (TakaraBio) for 2 consecutive days.
