Figure 6.

Kinetics of CD19-specific CAR T cells after encounter with a TKI-resistant Ph⁺ALL cell line (SU/SR). CAR T cells (1 × 10⁵) were exposed to SU/SR cells (5×10⁵) once on day 0 or twice on days 0 and 10 at an E:T ratio of 1:5 in the absence of cytokines. CAR T cells or Ph⁺ALL cells were planted alone as controls. Data are presented as mean ± standard deviation of experiments from three donors. (A, B) Change of cell numbers of CAR T cells (A) and SU/SR cells (B) were determined by use of a combination with trypan blue exclusion test and flow cytometric analysis with the use of CD3-APC and CD19-PE mAbs. CD3⁺ cells represent CART cells and CD19⁺ cells represent SU/SR cells. Open circles (○) indicate the cell numbers after the first SU/SR exposure; closed triangles (▲) indicate cell numbers after the second SU/SR exposure. (C) Change of CAR expression on CAR T cells. The intensity of CAR expression is shown as RFI, determined by calculating the ratio of mean fluorescence intensity for specific staining to that for control staining. Open circles (○) indicate RFI after the first SU/SR exposure; closed triangles (▲) indicate RFI after the second SU/SR exposure. (D, E) Cytokine release by CAR T cells. The concentrations of IFN-γ and IL-2 in the supernatant of the co-cultures were determined by means of ELISA, as shown in (D) and (E), respectively. Open circles (○) indicate the cytokine concentrations after the first SU/SR exposure; closed triangles (▲) after the second SU/SR exposure; open squares (□) after no SU/SR exposure (CAR T cells alone).