Apoptosis is often considered as an immunologically silent or even tolerogenic death modality (8
). Challenging this viewpoint, it has been shown that gemcitabine-induced apoptosis of hemagglutinin-expressing AB1 mesothelioma cells can prime CD8 T cells in vivo, thus eliciting an effective antitumor immune response (38
). Similarly, local γ irradiation of OVA-transfected B16F10 tumors can increase both the generation of antitumor immune effector cells and their trafficking to the tumor site (39
). The present data suggest that some but not all regimens of chemotherapy-induced apoptosis can elicit an effective antitumor immune response. Although anthracyclins (and in particular DX, which we used as a model agent) elicited immunogenic apoptosis in vitro (– and –), ex vivo ( A) and in vivo () MC (, , C, 6 A, 7 A, 8 A, and 9 B) was relatively inefficient in eliciting immunogenic cell death. The incorporation of DX a posteriori into MC-killed cells could not substitute for the DX treatment ab initio (), underscoring the contribution of the host cell (rather than the chemical nature of anthracyclins) to the immunogenic phenotype. Preparations of DX- or MC-treated cells that contained a similar percentage of dying cells ( A) were injected, and the morphological aspect of cell death elicited by DX and MC was similar (). Both were accompanied by caspase activation ( B) without any difference in the expression of some major endogenous adjuvants, such as HSP70 and HMGB-1 ( B), and the only difference resided in the degree of DNA fragmentation, which was more advanced with MC ( C). Moreover, DCs phagocytosed MC-treated cells less efficiently than DX-treated cells ( A), thus underscoring a hitherto unsuspected heterogeneity in apoptotic cells with respect to their uptake by DCs (40
). Importantly, inhibition of caspase activation by Z-VAD-fmk or transfected p35 greatly reduced the immunogenic potential of DX-treated cells, an effect that was observed in a variety of different tumor models in vitro and in vivo (–) and also affected DC-mediated recognition and phagocytosis ( A). However, caspase activation is not sufficient to elicit an immunogenic cell death because MC-treated cells, which do exhibit caspase activation ( B), are inefficient antitumor vaccines (). Thus, a specific combination of factors, caspase activation, and a yet-to-be defined antigenic property of anthracyclin-treated cells have to cooperate to elicit an antitumor immune response. Disruption of the membrane integrity of DX-treated tumor cells by freeze-thawing is sufficient to abolish their immunogenic property ( F, 7, A–C, and 8 A), meaning that intact apoptotic cells or bodies are required for optimal antigenicity. Induction of necrotic cell death is usually achieved by several cycles (usually three or more) of freeze-thawing, whereas we performed one single freeze-thaw cycle, a procedure that avoids a gross liquefaction of cellular structures. Thus, in contrast with other reports (41
), freeze thawing as performed here did not result in the uptake of cells by DCs ( A) nor in immunogenic cell death ( C). Regardless of these mechanistic details, it appears that the particular protocol of apoptosis induction determines the immunogenicity of cell death. Apparently, two different subcategories of apoptosis exist, one that is highly immunogenic and another that is comparatively less immunogenic.
DX-treated dying tumor cells are efficiently phagocytosed by myeloid and plasmacytoid DCs () and are highly efficient in eliciting antigen-specific CTLs ( A and 7), which are required for the antitumor immune response (). In contrast, NK cells ( C) and NKT cells (Fig. S3) are not stringently required for mounting an antitumor immune response, at least in this experimental setting. Thus, in contrast to other experimental systems, such as viral infection (43
), NK cells are dispensable for the priming of DCs by DX-treated tumor cells. Of note, intratumoral DX injection could elicit a chemotherapeutic response whose positive outcome requires an intact cellular immune system (). Again, these properties (phagocytosis of DX-treated cells by DCs, induction of antigen-specific CTLs, and elicitation of an immune response by intratumoral injection of DX) were largely reduced by simultaneous treatment with the caspase inhibitor Z-VAD-fmk, correlating with a reduced immunogenicity. To our knowledge, this is the first report indicating that caspase activation is broadly required for the elicitation of immunogenic tumor cell death. Previous reports have described that caspase activation rather reduces the immunogenic potential of tumor cells. Thus, a PROb clone (REGb) that fails to activate caspases is much more immunogenic than the parental PROb line (44
). Similarly, a PROb clone manipulated to stably express an anti-sense cytochrome c
cDNA undergoes atypical apoptosis without caspase activation, yet has an increased immunogenic potential (27
). As another example, caspases have been documented to destroy immunodominant epitopes, thus blunting the immune response against lymphoma cells (45
). In NOD mice, which are prone to develop autoimmune diabetes, systemic caspase inhibition reduced the priming of T cells by β cells undergoing spontaneous apoptosis (46
) and, conversely, local caspase inhibition abolished the tolerogenic effect of β cell apoptosis induced by streptozotocin (37
). The data presented here, which address the importance of caspase activation in a direct fashion, suggest that caspase activation ameliorates the antigenic properties of dying tumor cells. This points to an interesting problem. The expression of caspase inhibitors has negative prognostic value for some tumors (47
). It is possible that failure to activate caspases not only increases the resistance of tumor cells to chemotherapeutic agents, in a cell-autonomous fashion, but also reduces the probability of immunogenic death, thus blunting the host's antitumor defense system.
It is important to underscore the fact that the immunogenic effects of anthracyclin-treated tumor cells were observed in the absence of any adjuvant or costimulus. Hence, coapplication of adjuvants might be expected to improve the efficacy of the vaccination schedule. As a fascinating possibility, our data suggest a strategy of inducing immunogenic cell death in established tumors either ex vivo or in vivo. Whether such a simple strategy might be efficient in treating patients has to be addressed in the future.