Detection of cellular immune reactivity toward retrovirally transduced T cells requires 2-5 amplification cycles of coculture of posttreatment PBMCs with autologous CAR T cells. (A) Patient 6 PBMCs obtained before (Pre-Tx), during (days 16, 29, 36) and after treatment (days 52, 86, 122) with CAR T cells were cocultured as described (see “Methods”) with irradiated autologous CAR T cells in an autologous mixed lymphocyte culture. At 2-5 cycles of weekly stimulation, PBMCs were assayed for up-regulated CD107 expression by flow cytometry upon 2-hour stimulation with PHK67 fluorescein isothiocyanate-labeled autologous CAR T cells. Autologous NTD cultured T cells were used as negative control stimulator cells. The plots show CD107 versus CD8 expression within the CD3 T-cell population of PBMC cultures obtained from patient 6 after 2-5 cycles of coculture. Ellipsoids indicate CD107 positivity.