Whether CTLs or other host effector cells are responsible for most of the killing during adoptive T cell therapy is controversial. Here, we used 2 novel and independent strategies to demonstrate that CTLs, but not innate effectors, were responsible for the bulk of tumor cell destruction in a model of T cell therapy (Figure and Figure ). Tumor killing occurred with minimal bystander activity (Figure ) — a feature that may play a role in the emergence of antigen-loss variants seen after T cell therapy in some clinical trials (23
). Thus, in our system, tumor regression occurred with each individual tumor cell being eliminated by CTLs. The primary role of CTLs as cytotoxic effectors does not preclude an indirect contribution of other immune cells locally. For example, evidence for intratumoral T cell–macrophage interactions have been reported (19
) and could possibly enhance CTL effector functions. In addition, other cells, such as stromal cells, could be targeted as well by CTLs during this process, as reported previously (9
In some cases, isolated CTLs were found in contact with 2 apoptotic tumor cells, which suggests that serial (24
) or simultaneous (25
) killing previously described in vitro may also occur in vivo. The strong enrichment for granzyme-positive CTLs at the tumor site (Figure and Supplemental Figure 1) suggests that the perforin/granzyme pathway may contribute to tumor cell killing. The important role of CTL cytotoxicity revealed here by imaging approaches was surprising given that the transfer of perforin–/–
OT-I CTLs was shown previously to efficiently induce regression of EG7 tumors (11
). This observation might be explained by the redundancy of CD8+
T cell cytotoxic pathways. Alternatively, a distinct mechanism for tumor regression may be involved when CD8+
T cells are experimentally prevented to kill target cells. Thus, although our approach does not discriminate between the various CTL killing pathways, it provides, to our knowledge, the first direct assessment of intratumoral CD8+
T cell cytotoxic activity.
Recent reports have used 2-photon microscopy to depict intratumoral T cell migration, and one study reported an example of tumor cell disintegration following interaction with a T cell. To extend these observations and characterize the dynamics of CTL killing in tumors, we combined the use of intravital 2-photon imaging and that of a fluorescent reporter of caspase 3 activity. Interestingly, we estimated the killing rate (per CTL) to be 1 tumor cell every 6 hours. Some heterogeneity in the killing ability of CTLs may exist and could not be fully appreciated here because imaging periods were limited to 1–2 hours. However, CTL-target cells almost always remained in interaction during the entire imaging period, confirming that during this process, CTLs are occupied for hours, not minutes. In addition, the period during which CTLs are sequestered by tumor cells often extended beyond the killing, because CTLs remained in contact with apoptotic tumor cells for up to several hours (Supplemental Movies 2 and 5).
The estimated length required for a CTL to kill a tumor cell was much longer than that described for CTLs to kill other target cells in vivo. Two-photon imaging in lymph nodes showed that CTL killing of peptide-pulsed B cells and subsequent CTL detachment from the target cell requires less than 25 minutes (26
). Consistent with a rapid killing kinetic in vivo, the half-life of adoptively transferred peptide-pulsed splenocytes was found to be approximately 1 hour in lymphocytic choriomeningitis virus–immune animals (27
). These differences suggest that factors such as the tumor microenvironment and/or the cell type of the target (tumor cell versus splenocytes) may influence the rate of killing. Defects in TCR proximal signaling among intratumoral CD8+
T cells have been documented and could also possibly account for these differences (28
). In the case of adoptive therapy with activated CD8+
T cells, the relatively slow rate of killing was compensated by an elevated density of infiltrating CTLs. In other contexts, however, the kinetics of CTL-tumor cell lysis could represent a limiting factor for tumor rejection and could account for the dose- and time-dependent efficiency of adoptive T cell therapy (29
). This view is further supported by a pattern of response mounted by naive CD8+
T cells. We found that naive antitumor CD8+
T cells were primed in vivo, infiltrated the tumor, and exerted cytotoxicity in situ. Yet, they failed to eliminate the tumor, most likely because their intratumoral density was too low, which left many areas of the tumor free of CTLs. Thus, whereas tumors use many mechanisms to prevent CTL-mediated cytotoxicity, our results provide evidence that, in some cases, functional CTLs may be present at the tumor site but in insufficient numbers to induce tumor regression. The presence of a low-to-moderate number of functional CTLs could possibly contribute to a temporary state of equilibrium between the tumor and the immune response (31
In summary, we provided direct evidence that CTLs can mediate most of the tumor cell killing during adoptive T cell therapy in situ, but at a relatively slow rate. Achieving a high and relatively uniform density of functional CTLs in the tumor could therefore be critical to counterbalance this limitation during T cell–based cancer immunotherapeutic strategies. As illustrated in the present study, real-time imaging tumor cell apoptosis in live animals should provide new opportunities for monitoring antitumor immune responses and for dissecting their modulation by the tumor microenvironment.