T Cell Receptor Signaling Activation Protocol

TCR signaling was activated by using CD3/CD28 antibody clusters^{47 (link)} (Fig. 4) or anti-TCR^{4 (link)} (clone C305, Sigma-Aldrich, Fig. 7) as described previously. Briefly, biotin-conjugated IgGs were mixed with CD3 antibody (10 μg/ml) and CD28 antibody (5 μg/ml) and were further clustered with streptavidin. Then the antibody clusters were used to stimulate the T cells cultured on nonspecific immunoglobulin G (IgG,10 μg/ml) coated glass-bottom dish. For co-expression of biosensor and CAR molecule, the wild-type or ITAM mutated (XX3) 1928ζ CAR was linked downstream of the gene of biosensor by a P2A linker in the lentiviral construct. The CD19 CAR-T cells expressing biosensors were generated by lentivirus transduction of Jurkat T cells. For imaging of CAR signaling upon antigen stimulation, CAR-T cells were dropped on the glass-bottom dishes that have been coated with the NIH-3T3 cells expressing CD19 antigens. The time-lapse fluorescence images were taken with a Nikon Eclipse Ti inverted microscope at an interval of 30 s. The W-VIEW GEMINI imaging splitting optics (Hamamatsu, Japan) with an iXon Ultra 897 camera was used to capture the ECFP (a 474/40 nm emission filter) and FRET (a 535/25 nm emission filter) fluorescent signals simultaneously. During Imaging, the cells were maintained with 5% CO₂ at 37 °C using the Tokai Hit ST Series Stage Top Incubator (Tokai Hit, Japan).