Adoptive therapy with chimeric antigen receptor (CAR) T cells shows great promise clinically. However, there are important aspects of CAR-T cell biology which have not been explored particularly with respect to the kinetics of: activation, immune synapse formation and tumor cell killing. Moreover, the effects of signaling via the endogenous TCR or CAR on killing kinetics are unclear. To address these issues, we developed a novel transgenic mouse (designated CAR.OT-I), in which CD8+ T cells co-expressed the clonogenic OT-I T cell receptor, recognizing the H-2Kb presented ovalbumin peptide SIINFEKL, and a scFv specific for human HER2. Primed CAR.OT-I T cells were mixed with SIINFEKL-pulsed or HER2-expressing tumor cells and visualized in real time using time-lapse microscopy. We found that engagement via CAR or TCR did not affect cell death kinetics, except that the time from degranulation to CAR-T cell detachment was faster when CAR was engaged. We showed, for the first time, that individual CAR.OT-I cells can kill multiple tumor cells ('serial killing'), irrespective of the mode of recognition. At low E:T ratios, tumor cell killing rate was similar via TCR or CAR ligation over the first 20 hours of co-incubation. However, from 20-50 hours, tumor cell death mediated through CAR became attenuated, due to CAR downregulation throughout the timecourse. Our study provides important insights into CAR-T/tumor cell interactions, with implications for single- or dual-receptor-focused T cell therapy.
- Received February 14, 2015.
- Accepted February 17, 2015.
- Copyright © 2015, American Association for Cancer Research.