In conclusion, the combined data shown here reveal a discrepancy between the key role of CD8 T cells in tumor resolution and the strong anti-proliferative affects of CY on CD8 T cells. Antitumor responses depend on IFN-c and perforin To address the question how CD8 T cells can be the key mediators for tumor resolution under conditions that limit T cell proliferation, we set out to delineate the effector mechanisms responsible for the CY-induced anti-tumor immune responses. We evaluated the roles of different CD8 T cell effector mechanisms using gene-targeted mice lacking specific CD8 T cell effector functions, i.e., IFN-c and/or perforin. To define the role of IFN-c, we analyzed tumor growth in IFN-c knock-out mice and found that IFN-c deficiency prevented the anti-tumor response in 7/10 mice (total for two experiments) (Figure 5A) (P,0.05 in the growth curve and P,0.05 in the survival curve). To determine whether direct CD8 T cell cytotoxicity was required for the CY- induced anti-tumor response, we assessed the efficacy of CY in perforin (pfp)-deficient mice. Perforin-deficiency did not abolish the anti-tumor efficacy of CY (Figure 5B), and there was no significant difference in survival (Figure 5B). It is important to note that slightly larger tumors were treated in these experiments (4–6 mm2 instead of 1 mm2 in other experiments), which has a negative impact on the efficacy of CY. This explains why CY is less efficacious in these experiments. Finally, we evaluated the efficacy of CY in IFN-c/perforin double knock-out mice and found that combined deficiency of IFN-c and perforin completely abrogated the CY-induced anti-tumor effects (9/9 mice, two experiments), consistent with the results from the IFN-c-knock-out mice and highlighting the key role of this effector molecule (Figure 5C). In fact, these tumors grew with similar kinetics as in T-cell deficient nude mice (Figure 6B), consistent with our previous data [18]. Role of TRAIL suggests a tumor cell sensitization model As several chemotherapeutic drugs sensitize tumor cells for death ligand-mediated cell apoptosis (TRAIL) [34], we analyzed expression of the TRAIL receptor DR5 on AB1-HA tumor cells. We could detect expression of DR5 on tumor cells (Figure 6A) suggesting that TRAIL-mediated apoptosis could be involved in the anti-tumor effects of CY. To evaluate the in vivo relevance of TRAIL-mediated killing, tumor-bearing TRAIL-deficient mice were treated with CY and tumor growth in these mice was compared with immunocompetent mice. Analysis of tumor growth curves revealed that TRAIL-deficiency indeed affected the anti- tumor efficacy of CY (Figure 6B, P,0.005), suggesting that TRAIL could be an important effector molecule for anti-tumor CD8 T cells. Athymic nude mice were used as a control to assess the tumor growth rate in the absence of T cells. Agonistic anti-DR5 antibodies rescue anti-tumor efficacy in nude mice A TRAIL-dependent tumor-resolution mechanism predicts that the partial anti-tumor response of CY in athymic nude mice (Figure 6B) [18] could potentially be rescued by mimicking TRAIL-mediated tumor cell apoptosis. We tested this by combining CY-treatment in nude mice with agonistic anti-DR5 antibodies (clone MD5-1) [35,36], reasoning that CY-sensitized tumor cells would then be killed by antibody-mediated DR5 ligation, compensating for the lack of T cell derived TRAIL. Thus, nude mice received a single dose of CY at day 9 post tumor cell inoculation (tumor size 4–6 mm2) and were treated with anti-DR5 Anti-Tumor Immune Response on days 8, 12 and 16 post tumor cell inoculation. The results clearly show that DR5-treatment alone was completely ineffective and that CY alone had a very transient effect (Figure 7A). Note that the single CY treatment in nude mice was suboptimal when compared to earlier data (Figure 6B) [18], which can be explained by the fact that these experiments were done with CY injection at day 9 rather than day 7. Indeed, the CY efficacy in athymic nude mice is very limited when larger (.161 mm) tumors are treated, emphasizing the role of T cells. Indeed, repetition of the experiment with CY-treatment done at day 7 and anti-DR5 on days 6, 10 and 14 (Figure 7B) confirmed the benefit of anti-DR5 antibodies in combination with CY and also increased efficcy of earlier CY treatment (day 7 instead of 9). Combined, the data clearly show that combination of CY with the agonistic anti- DR5 antibody led to prolonged control of tumor growth (Figure 7). Unfortunately, the CY/anti-DR5 combination treatment was also associated with liver toxicity, preventing us from studying long-term control of tumor growth [37]. TRAIL blockade alone did not significantly affect the efficacy of CY in nude mice, suggesting that the transient anti-tumor effect did not depend on this mechanism (Figure 7B). Discussion The objective of the current study was to address the chemo/ immunotherapy paradox: how can CD8 T cells eradicate the tumor when the chemotherapeutic drug antagonizes their proliferation? We used CY in our AB1-HA malignant mesothe- lioma model to address this issue because CY eliminates AB1-HA tumors in a CD8 T cell-dependent fashion but depletes cycling T cells at the same time. Our study makes four points. First, the immune system (i.e., CD8 T cells and NK cells) is essential to achieve curative responses after chemotherapy. This could suggest that T cells and NK cells are required to prevent a small number of chemotherapy-surviving tumor cells from re-establishing a new tumor [38]. Second, the CY analogue maphosphamide induces apoptosis in tumor cells, consistent with its direct cytotoxic effects on AB1-HA tumor cells [18]. In vivo, the result of chemotherapy is a systemic and long-lived type-I IFN response. Third, CY depleted cycling Ki-67hi T cells, blocked proliferation of CFSE-labeled tumor-specific CD8 T cells and did not expand the endogenous tumor-specific CD8 T cell response. Thus, the anti-tumor CD8 response after CY injection does not depend on proliferation for its mode of action. Fourth, multiple effector functions were important for tumor resolution: IFN-c and TRAIL. The role of TRAIL suggests that CY sensitizes tumor cells for TRAIL-mediated apoptosis. Thus, it is possible that anti-tumor CD8 T cells use IFN- c and TRAIL to kill tumor cells. An intriguing possibility is that TRAIL-mediated tumor cell killing contributes to immunogenic cell death. CY kills cells by inducing apoptosis and this seems to result in a systemic type-I IFN response, confirming earlier studies [17,29,39] and consistent with the notion of ‘immunogenic cell death’. Similar data have been reported by Schiavoni and coworkers who used expression of Ly6C on T cells (similar to Ly6AE) and PCR to detect type-I IFN production [17]. Thus, we now confirm and extend these observations. There may be two possible sources for IFN production: the tumor cells themselves [29] and the monocytes that phagocytose apoptotic tumor cells. There is direct evidence for the former hypothesis [29], but the fact that cells exposed to the CY-related drug melphalan activate phagocytosing DCs [40] indicates that the latter may also occur. Irrespective of the mechanism, type-I IFNs are essential to generate de novo CD8 T cell responses to apoptotic cells [8] and to cross-presented PLoS ONE | www.plosone.org 6 September 2009 | Volume 4 | Issue 9 | e6982