Measuring T-cell/CAR T-cell bystander tumor killing in vitro and in vivo

In the era of T cell-mediated immunotherapies, a central and growing problem is the recurrence of tumors lacking target antigen (Ag). Strategies that can prevent outgrowth of antigen-loss cells may improve response to therapy more effectively than those that rely on identifying multiple Ag targets after resistance arises. In addition to a T cell's direct killing response to binding cognate Ag, upregulation of death-receptor ligands and secretion of pro-inflammatory cytokines contribute to the indirect killing of surrounding, antigen-negative (bystander) cell populations, in a process termed "bystander killing". To investigate the mechanism and scope of T cell bystander killing, we describe methods of in vitro killing assays with flow cytometry and live microwell imaging, as well as in vivo tumor models with bioluminescent imaging and multiphoton live imaging, to observe this process in real time. The approaches can be easily adapted to investigate many other tumor types, T cell therapies, and targeting strategies.