Two-photon imaging of intratumoral CD8⁺ T cell cytotoxic activity during adoptive T cell therapy in mice
J. Clin. Invest. Béatrice Breart, et al. 118:1390 doi:10.1172/JCI34388 [Go to this article.]

Figure 5
Dynamics of CTL-mediated tumor cell apoptosis in vivo. (A) Intravital 2-photon imaging of mice bearing EG7-DEVD tumors and transferred with activated GFP-expressing OT-I CTLs showed a close juxtaposition of CTLs (pseudocolored in red) and apoptotic tumor cells (green). (B) The apoptosis index (reflecting FRET disruption) was calculated for individual tumor cells together with the number of CTLs in contact. The percentage of tumor cells undergoing apoptosis is shown. (C) The apoptosis index of individual tumor cells was tracked over time. Representative tumor cells with a constant low (live→live), a high (apoptotic→apoptotic), or an increasing (live→apoptotic) apoptosis index are shown. (D) Examples of tumor cells undergoing apoptosis while establishing interaction with CTLs. (E) Tumor cells initiated apoptosis during interactions with CTLs. Individual tumor cells were divided into 3 categories on the basis of the evolution of their apoptosis index over time. The percentage of tumor cells engaged by CTLs is shown for each category. (F) The killing of 1 tumor cell by an individual CTL took an average of 6 hours. A total of 129 individual stable interactions between a CTL and a live tumor cell were recorded (for an average of 35 minutes each), which represented a cumulative time of imaging of 74 hours and 41 minutes. The number of killing events (as detected by FRET loss in individual tumor cells) was expressed as a function of the elapsed cumulative time of imaging. The rate of cell killing was estimated to be 1 tumor cell every 6 hours per CTL.