Fig. 5.

Kinetics of IS formation in live Kappa-CAR T cells under total internal reflection fluorescence microscopy (TIRF) to evaluate the dynamics of CAR IS quality. All cells were imaged by TIRF microscopy on lipid bilayers carrying Kappa protein conjugated with Alexa Fluor 488 at the indicated time points. a Kappa-41BB cells were imaged at 1 min, 3 min, 5 min, 10 min, 20 min, and 30 min. Time-lapsed differential interference contrast (DIC) (top panel) and TIRF (bottom panel) images are shown. b Kappa-CD28 cells were imaged at 1min, 3min, 5min, 10min, 20min, and 30min. Time-lapsed DIC (top panel) and TIRF (bottom panel) images are shown. c Schematic representation of recombinant retroviral vectors encoding kappa-CAR. Both Kappa-CD28-CAR and Kappa-4-1BB-CAR constructs (CD28 and 4-1BB) contain CD28 transmembrane domain and intracellular domain of CD3 zeta. The difference between Kappa-CD28-CAR and Kappa-4-1BB-CAR constructs is that Kappa-4-1BB-CAR construct contains intracellular domain of 4-1BB molecule