Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice (original) (raw)
Antigen-specific tumor regression upon adoptive transfer of in vitro primed CTLs. To visualize CTL responses against a solid tumor, we used the well-characterized EL4/EG7 subcutaneous tumor model (10, 21). C57BL/6 mice injected s.c. with EL4 tumor cells or with the OVA-expressing variant EG7 developed solid tumors that failed to be rejected in the absence of additional manipulation. Similar tumor growth patterns were observed with EL4 or EG7 tumor cells transfected with a membrane-targeted cyan fluorescent protein (mCFP) or membrane-targeted yellow fluorescent protein (mYFP), respectively. As shown in Figure 1, adoptive T cell therapy through the transfer of in vitro activated OT-I CTLs resulted in the complete regression of EG7-mYFP tumors, but had no detectable effect on the growth of EL4-mCFP tumors. Interestingly, transfer of equivalent numbers of naive OT-I T cells (even at early time points) failed to induce tumor regression with little to no tumor cell elimination being detected in tumor frozen sections (Figure 1).
Antigen-specific tumor regression upon adoptive transfer of in vitro primed CTLs. 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.
CTLs enhance their effector functions upon antigen recognition in the tumor environment. Nevertheless, both in vitro and in vivo primed CD8+ T cells were found to infiltrate the tumor and displayed enhanced effector functions at the tumor site (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI34388DS1). These hallmarks of effector activity were highly reduced or absent in OT-I T cells found in the draining lymph node or within intratumoral OT-I CTL infiltrating the nonantigen bearing EL4-mCFP tumors (Supplemental Figure 1). These results strongly suggest that transferred CTLs enhanced their effector functions at the tumor site as a result of antigen recognition. Intratumoral CTL density, however, was different. After transfer of activated OT-I T cells, the CTL density was 1.3 ± 0.2 × 104 T cells/mm3 on day 2 and reached 2.5 ± 0.3 × 104 T cells/mm3 on day 3. CTL infiltration was significantly lower and less homogenous at all time points after transfer of naive OT-I T cells, with only 0.2 ± 0.03 × 104 OT-I T cells/mm3 on day 7 (Figure 2, A and B).
Visualization of intratumoral CTL dissemination during adoptive T cell therapy. (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.
Topography of tumor destruction upon adoptive T cell therapy. We next focused on the efficient antitumor response mounted by in vitro activated CTLs by visualizing the distribution of intratumoral OT-I CTLs during the course of tumor destruction. Two days after being transferred, CTLs accumulated in multiple discrete areas of EG7 tumors found in the vicinity of tumor microvessels (Figure 2C), their likely port of entry. On day 3, CTLs were found more evenly distributed, and most of the tumor cells had been eliminated. Tumor areas containing low numbers of CTLs usually tended to have a higher density of tumor cells (Figure 2C). On day 5, the residual mass harvested at the tumor injection site did not show any viable EG7-mYFP. These observations suggest that intratumoral CTL infiltration is initiated in multiple regions of the tumor that progressively extend and ultimately merge. Intravital 2-photon imaging performed in tumor-bearing mice 2–3 days after CTL adoptive transfer revealed that CTL located inside regions where tumor cells had been eliminated were usually motile (Supplemental Movie 1). In contrast, many CTLs were sequestered at the border of these regions, being engaged in long-lasting interactions with tumor cells (Supplemental Movie 1). Thus, intratumoral CTL dissemination occurred concomitantly with tumor cell elimination. Noteworthy, many CTLs in the vicinity of tumor cells had a high granzyme B content (Supplemental Figure 2).
The local activity of CTLs on individual tumor cells drives tumor regression. The close relationship between the topography of tumor elimination and that of CTL infiltration suggested that CTLs were playing a continuous role during tumor elimination. However, it was still unclear whether CTLs were acting by eliminating tumor cells directly or by recruiting innate effector cells that were responsible for most of the tumor destruction. To discriminate between these possibilities, we took advantage of the observation that mice injected with a mixture of EL4-mCFP and EG7-mYFP cells develop chimeric tumors (Figure 3), which are composed of small individual patches of EL4-mCFP and EG7-mYFP cells. We reasoned that if the primary effectors of tumor cell killing were the CTLs themselves, then EG7-mYFP cells should be solely targeted. In contrast, if innate effectors were largely involved in tumor destruction, both EG7-mYFP and EL4-mCFP should be destroyed. Strikingly, 3 days after injection of OT-I CTLs, virtually all EG7-mYFP patches were cleared, whereas EL4-mCFP patches appeared minimally, if at all, affected (Figure 3A). This observation was further confirmed by analyzing the tumor cell composition as a percentage (Figure 3, B and C) in absolute numbers (Supplemental Figure 3). Tumor cell mixtures containing as little as 10% EL4 tumor cells were not rejected following adoptive transfer of OT-I CTLs, which confirmed that tumor destruction proceeded with minimal nonspecific lysis. Thus, elimination of each individual tumor cell required the local action of antigen-specific CTLs, and tumor elimination occurred with little to no bystander effect.
Direct action of CTLs on individual tumor cells drives tumor regression. Mice were injected with a mixture of EL4-mCFP and EG7-mYFP tumor cells. On day 5, some mice were adoptively transferred with 5 × 106 in vitro activated OT-I CD8+ T cells. Three days after transfer, tumors were harvested, and confocal imaging was performed on frozen sections. (A) Mice injected with the tumor cell mixture developed chimeric tumors composed of small individual patches of EL4-mCFP and EG7-mYFP cells (left). The transfer of OT-I CTLs resulted in the clearance of EG7-mYFP patches, whereas EL4-mCFP patches appeared minimally affected (middle and right). Scale bars: 100 μm (left and middle); 10 μm (right). (B) The contribution of EG7-mYFP, EL4-mCFP, or other cells (i.e., nonfluorescent cells) to the overall tumor volume was determined from confocal images of frozen tumor sections. Each dot represents the value derived from an individual section. (C) Single-cell suspensions of tumor were analyzed by flow cytometry. The relative percentage of EG7-mYFP and EL4-mCFP cells is shown. Data are gated on tumor cells.
Real-time imaging of tumor cell apoptosis in vivo. To characterize the dynamics of tumor cell lysis by intratumoral CTLs, we transfected EG7 tumor cells with a fluorescent probe containing the CFP and YFP molecules linked by a peptide containing the caspase 3 cleavage motif DEVD (referred to as EG7-DEVD) (22). Apoptosis-induced caspase 3 activation resulted in substrate cleavage and subsequent Förster resonance energy transfer (FRET) disruption. As shown in Figure 4, FRET loss, which translated into a higher apoptosis index (see Methods), was readily detected in cultured EG7-DEVD tumor cells subjected to UVB irradiation or that cocultured with activated OT-I CTLs (Figure 4). Importantly, FRET loss correlated with Annexin V staining on tumor cells cocultured with OT-I CTLs (Supplemental Figure 4). No FRET changes were detected when EG7 tumor cells were transfected with a control substrate containing a non-cleavable peptide DEVG (Figure 4).
A fluorescent probe to track tumor cell apoptosis. (A) EG7 tumor cells were stably transfected with a FRET-based fluorescent probe monitoring caspase 3 activity. Briefly, CFP and YFP molecules are linked by a peptide containing the sequence DEVD, which is cleaved by activated caspase 3. EG7 cells were also transfected with a control probe (noncleavable by caspase 3) bearing a mutation in the cleavage motif (DEVG). Cleavage of the probe upon caspase 3 activation resulted in FRET disruption. Tumor cell apoptosis was monitored by 2-photon imaging by calculating the ratio of CFP to YFP emission (for the sake of clarity, this is referred to as the apoptosis index). (B and C) EG7-DEVG or EG7-DEVD tumor cells were subjected to UVB irradiation for 1 minute. Eight hours later, cells were visualized by 2-photon imaging. UVB irradiation resulted in FRET disruption in EG7-DEVD but not in control EG7-DEVG tumors cells. Scale bars: 10 μm. The apoptosis index plotted for individual tumor cells is shown. Tumor cells with a ratio greater than 1.7 were considered to be undergoing apoptosis. (D and E) Flow cytometric analysis of FRET loss in EG7-DEVD and EG7-DEVG tumor cells subjected to UVB irradiation or cocultured with activated OT-I CTLs for 5 hours. The population of EG7 tumor cells displaying FRET loss is shown in green, and the corresponding percentage is indicated.
Intravital 2-photon imaging of mice bearing an EG7-DEVD tumor and adoptively transferred with OT-I CTL revealed a close juxtaposition of CTLs and apoptotic tumor cells (Figure 5 and Supplemental Movie 2). As shown in Figure 5, tumor cells found in contact with CTLs were much more likely to have undergone apoptosis than were tumor cells with no CTL in their vicinity. By tracking the apoptosis index of individual tumor cells over time, we found that, of the tumor cells that remained alive during the imaging period (n = 869), only 13% were associated with CTLs (Figure 5 and Supplemental Movie 3). In contrast, 92% (n = 13) of the tumor cells that underwent apoptosis during the imaging period (as detected by FRET loss) were stably engaged by at least 1 CTL (Figure 5 and Supplemental Movie 4). This observation provides additional evidence that CD8+ T cell–mediated cytotoxicity accounted for the bulk of tumor cell destruction during T cell adoptive therapy. Finally, we tracked 129 stable interactions between CTLs and live tumor cells and enumerated the number of killing events (as detected by FRET loss in individual tumor cells) as a function of the cumulated time of imaging. Using this approach, we estimated the rate of cell killing to be 1 tumor cell every 6 hours per CTL (Figure 5 and Movie 4). In summary, these results demonstrate that CTLs directly mediate tumor elimination, but require, on average, several hours to kill 1 target cell.
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.
In vivo primed CTLs failed to control tumor growth despite displaying effective cytotoxic activity in situ. Finally, we asked whether an impaired T cell cytotoxic activity accounted for the inefficient antitumor response mounted by naive antigen-specific CD8+ T cells. To this end, we visualized the response of adoptively transferred naive OT-I T cells in EG7/EL4 chimeric tumors. Although CTL infiltration was quite variable in the different regions of the tumor (Figure 6A), EG7 patches were eliminated in CTL-rich areas, which was evidence that in vivo primed CTLs were not grossly impaired in their ability to kill target cells (Figure 6A, right). A similar conclusion was reached when we visualized the apoptosis of EG7-DEVD tumors induced by in vivo primed OT-I T cells, because we detected a close association between OT-I CTLs and apoptotic tumor cells (Figure 6B). Thus, the low level of CD8+ T cell infiltration, rather than a defect in the cytotoxic activity, appeared to be responsible for the inefficient response mounted by in vivo primed OT-I T cells.
In vivo primed CTLs failed to control tumor growth despite exhibiting effective cytotoxic activity at the tumor site. (A) Naive OT-I CD8+ T cells (5 × 106 cells) were adoptively transferred 3 days after the mice were injected with a mixture of EL4-mCFP and EG7-mYFP tumor cells. Representative confocal images of tumor sections indicate a distinct level of CTL infiltration. EG7 tumor cells were eliminated in the CTL-rich area, which indicated in situ cytotoxic activity (right). Little to no EG7 killing was detected in regions of the tumor with little CTL infiltration (left). Scale bars: 100 μm. (B) Naive OT-I CD8+ T cells were adoptively transferred in mice injected with EG7-DEVD tumor cells. Representative images indicate that in vivo primed CTLs (red) were closely associated with apoptotic tumor cells (green).