CD19-specific lysis of tumor targets by genetically modified UCB-derived T cells. (A) Killing of tumor CD19⁺ target cells (HLA I^neg Daudi and genetically modified U251T) by CD8⁺ CD19R⁺ T-cell clone in 4-hour CRA. Background lysis of CD19^– (parental K562 and U251T) cells is shown. (B) Killing of HLA class I/II^– K562 cells transfected to express CD19 by CD8⁺ T-cell clone. (C) Killing of 4 primary B-ALL samples by UCB-derived genetically modified CD19-specific T-cell line (i) and T-cell clone (ii). (Flow cytometry on the B-ALL samples established that the lymphoid-gated cells were 68% to 83% CD19⁺CD10⁺ (versus 96% for Daudi cells) and 92% to 98% CD19⁺ (versus 100%), while the total population was 44% to 72% CD19⁺CD10⁺ (versus 91%) and 70% to 94% CD19⁺ (versus 99%). Background lysis of CD19^–BE2 neuroblastoma cells is shown. CRA results of mean ± SD specific lysis of triplicate wells at E/T cell ratios of 50:1 to 1:1 are shown. (D) Intracellular multiparameter flow cytometry evaluating intracellular perforin and granzyme A expression, gating on CD8α⁺ lymphocytes, and cell-surface expression of Fas and FasL, gating on CD3⁺ lymphocytes, obtained from cryopreserved unmanipulated UCB (i and iii) and genetically modified UCB-derived T-cell clone (ii and iv). Crosshairs were established using isotype-matched nonspecific control mAbs.