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
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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

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Figure 1

Antigen-specific tumor regression upon adoptive transfer of in vitro primed CTLs.

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Visualization of intratumoral CTL dissemination during adoptive T cell t...
C57BL/6 mice were injected s.c. with 2 × 106 EL4 tumor cells or with the OVA-expressing variant EG7 expressing mCFP or mYFP, respectively. On day 5, mice were adoptively transferred with 5 × 106 OT-I CD8+ T cells that were activated in vitro for 48 hours. A second group of recipient mice were adoptively transferred with 5 × 106 naive OT-I CD8+ T cells. Naive OT-I CD8+ T cells were transferred on day 3 to allow additional time for in vivo activation. (A) Tumor growth was followed over time. In vitro, but not in vivo, primed OT-I CD8+ T cells induced complete regression of EG7 tumors. (B) Confocal images of tumor frozen sections 5–7 days following adoptive transfer of naive or in vitro activated OT-I T cells. Most EG7-mYFP tumor cells were rapidly eliminated after transfer of activated OT-I CD8+ T cells but not after transfer of naive OT-I CD8+ T cells. Scale bars: 100 μm.