Analysis of intracellular signaling after stimulation through the virus-specific TCRs and CD19-CARs. Intracellular phospho-specific staining of CD3ζ, ZAP70 (pY292 and pY319), p38, ERK, and JNK. Bi-specific T cells were either stimulated with K562 (gray), the indicated K562 transfectants at a 1:5 ratio, or PMA/ionomycin for 2 minutes (CD3ζ and ZAP70), or 10 minutes (p38, ERK, and JNK), permeabilized and stained with phospho-specific mAbs, and analyzed by flow cytometry. The flow plots for CD3ζ, ZAP70, and p38 were performed on a FACSCanto II (BD Biosciences), and for ERK and JNK on a FACSCalibur (BD Biosciences). Data are representative of B*0702_CMVpp65 bi-specific T cells and representative of 4 independent experiments with bi-specific T cells from 4 donors.