T-cell activation and division after stimulation through the virus-specific TCRs and CD19-CARs. (A) Up-regulation of CD25 after CD19 or TCR stimulation. Bi-specific T cells were stimulated with an equal number of either K562/CD19 or K562/HLA-B*0702 cells pulsed with CMVpp65 peptide or left unstimulated and examined for CD25 expression 24 hours later. Expression of CD25 on unstimulated cells is shown in the gray histograms and expression of CD25 on stimulated T cells is shown in the white histograms. (B) CFSE dilution assay after CD19 or TCR stimulation. Bi-specific T cells were labeled with CFSE, stimulated with an equal number of the indicated K562 transfectants or with CD3/28 beads, and CFSE staining intensity was analyzed 72 hours after stimulation. CFSE staining of unstimulated T cells is shown in the gray histograms and those of stimulated in the white histograms. Data are representative of 3 independent experiments. (C) Cell growth of bi-specific T cells after CD19 or TCR stimulation without exogenous IL-2. A total of 2 × 106 bi-specific T cells were stimulated with either K562/CD19 transfectants or K562/HLA transfectants pulsed with CMV peptide, and the absolute cell count was determined by the standard trypan blue dye exclusion method 4 days after stimulation. Data are pooled from 4 independent experiments with bi-specific T cells from 4 donors. The mean and SD of the absolute cell number is shown.
