Mab coated beads

Primary human CD4+ and CD8+ T cells, purchased from the Human Immunology Core at University of Pennsylvania, were isolated from healthy volunteer donors following leukapheresis by negative selection. All specimens were collected under a protocol approved by a University Institutional Review Board, and written informed consent was obtained from each donor. T cells were cultured in R10 medium and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies (mAb)-coated beads (Invitrogen). Eighteen to 24 hours after activation, human T cells were transduced using a spinoculation procedure. Briefly, 0.5 × 10⁶ T cells were infected with a multiplicity of infection (MOI) of 2 and 5 of concentrated C4-27z and MOv19-27z vector, respectively. Mixtures of cells and vectors were centrifuged at room temperature for 90min (2,500 rpm) in a table-top centrifuge (Sorvall ST 40). Human recombinant interleukin-2 (IL-2; Novartis) was added every 2–3 days to a 100 IU/mL final concentration and a cell density of 0.5 × 10⁶ to 1 × 10⁶ cells/mL was maintained. Once engineered T-cell cultures appeared to rest down, as determined by both decreased growth kinetics and cell-sizing determined using the Multisizer 3 Coulter Counter (Beckman Coulter), the T cells were used for functional analysis.

Primary human T cells, purchased from the Human Immunology Core at University of Pennsylvania, were isolated from healthy, normal donors following leukapheresis by negative selection. All T cell samples were collected under a protocol approved by a University Institutional Review Board, and written informed consent was obtained from each healthy, normal donor. T cells were cultured in R10 medium and stimulated with anti-CD3 and anti-CD28 monoclonal antibody (mAb)-coated beads (Invitrogen). Approximately 18 to 24 h after activation, human T cells were transduced using a spinoculation procedure. Briefly, 0.5 × 10⁶ T cells were infected with a multiplicity of infection (MOI) of 5 of the MOv19-27z vector. A mixture of cells and vectors were centrifuged at room temperature for 90 min (2500 rpm) in a table-top centrifuge (Sorvall ST 40). After the engineered T cells were rested, as determined by decreased growth kinetics and cell size which is measured using the Multisizer 3 Coulter Counter (Beckman Coulter), the rested T cells were then used for functional analysis.

Primary human T cells, purchased from the Human Immunology Core at University of Pennsylvania, were isolated from healthy, normal donors following leukapheresis by negative selection. All T cell samples were collected under a protocol approved by a University Institutional Review Board, and written informed consent was obtained from each healthy, normal donor. T cells were cultured in R10 medium and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies (mAb)-coated beads (Invitrogen). Approximately 18 to 24 h after activation, human T cells were transduced. Briefly, 0.5 × 10⁶ T cells were infected with a multiplicity of infection (MOI) 2 of the NKG2D receptor lentiviral vector and expanded for 2 weeks. Human recombinant interleukin-2 (IL-2; Novartis) was added every 2–3 days to a 50-IU/ml final concentration, and a cell density of 0.5 × 10⁶ to 1 × 10⁶ cells/ml was maintained. Engineered CAR T cells were rested in cytokine-free medium for 24 h and were then used for functional analysis.