Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice
Béatrice Breart, Fabrice Lemaître, Susanna Celli, Philippe Bousso
Béatrice Breart, Fabrice Lemaître, Susanna Celli, Philippe Bousso
View: Text | PDF
Research Article Oncology

Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice

Abstract

CTLs have the potential to attack tumors, and adoptive transfer of CTLs can lead to tumor regression in mouse models and human clinical settings. However, the dynamics of tumor cell elimination during efficient T cell therapy is unknown, and it is unclear whether CTLs act directly by destroying tumor cells or indirectly by initiating the recruitment of innate immune cells that mediate tumor damage. To address these questions, we report real-time imaging of tumor cell apoptosis in vivo using intravital 2-photon microscopy and a Förster resonance energy transfer–based (FRET-based) reporter of caspase 3 activity. In a mouse model of solid tumor, we found that tumor regression after transfer of in vitro–activated CTLs occurred primarily through the direct action of CTLs on each individual tumor cell, with a minimal bystander effect. Surprisingly, the killing of 1 target cell by an individual CTL took an extended period of time, 6 hours on average, which suggested that the slow rate of killing intrinsically limits the efficiency of antitumor T cell responses. The ability to visualize when, where, and how tumor cells are killed in vivo offers new perspectives for understanding how immune effectors survey cancer cells and how local tumor microenvironments may subvert immune responses.

Authors

Béatrice Breart, Fabrice Lemaître, Susanna Celli, Philippe Bousso

×

Figure 2

Visualization of intratumoral CTL dissemination during adoptive T cell therapy.

Options: View larger image (or click on image) Download as PowerPoint
Antigen-specific tumor regression upon adoptive transfer of in vitro pri...
(A and B) In vitro, but not in vivo, primed CTLs massively infiltrated the EG7 tumors. The distribution of intratumoral CTLs (white) was visualized and quantified on frozen sections of EG7 tumor after adoptive transfer of naive or in vitro activated OT-I CD8+ T cells. (A) CTLs were counted in multiple individual areas of the tumor encompassing a fixed volume of 500 × 500 × 10 μm. The percentage of tumor areas containing the indicated number of CTLs is shown. The response mounted by in vivo primed OT-I CD8+ T cells resulted in a lower and less homogenous CTL infiltrate than did that mounted by in vitro activated CD8+ T cells at all time points analyzed. (B) Representative images showing CTL infiltration after transfer of naive or activated OT-I CD8+ T cells. (C) Dissemination of in vitro primed CTLs occurred concomitantly with tumor cell elimination. Two days after adoptive transfer of in vitro activated OT-I CD8+ T cells, CTLs (red) accumulated in the vicinity of tumor microvessels (white, stained for PECAM). On day 3, CTLs were more evenly distributed, and many EG7 tumor cells had been eliminated. Note that CTL-rich areas tend to have a lower density of EG7 tumor cells (yellow). Scale bars: 100 μm.