induced AB1 tumor cell line [16] with the influenza virus HA gene [15]. The tumor-expressed HA protein allows us to monitor the anti-tumor T cell response [5,15,22], but it does not impact on tumor immunogenicity, as evidenced by the fact that AB1-HA cured mice are also protected against re-challenge with the parental AB1 line [23]. The immuno-stimulatory properties of CY (CytoxanTM) have been known for decades. In the 1980s, it was shown that CY depleted cycling suppressor T cells, now known as regulatory T cells, and thereby activated anti-tumor CD8 T cells [24]. However, we have recently shown that the anti-tumor efficacy of CY in the AB1-HA model cannot be explained by regulatory T cell depletion alone [18]. Here, we show that CY kills tumor cells by apoptosis and that it has a CD8 T cell- and NK cell-dependent anti-tumor effect in the AB1-HA tumor model. At the same time, CY has a strong negative effect on T cell proliferation, limiting the potential expansion of anti-tumor CD8 T cells, raising the question how a CD8 T cell-dependent anti-tumor response functions without T cell expansion. We found that the anti-tumor immune response depended on different effector molecules to eliminate the tumor: IFN-c and TRAIL. The role of TRAIL was supported by data showing that agonistic anti-TRAIL-receptor (DR5) antibodies enhanced the effects of CY in athymic nude mice. Thus, a DR5-agonist compensates for the lack of functional T cell in nude mice. The role of TRAIL suggests that the efficacy of CY can be explained by tumor cell sensitization for T cell and/ or NK cell apoptosis. A model in which CY sensitizes tumor cells for TRAIL-mediated death may help explain the chemotherapy paradox, since such a model emphasizes tumor cell susceptibility rather than expansion of anti-tumor CD8 T cells. Results The objective of this study was to investigate the potential discrepancy between lymphodepleting anti-tumor chemotherapy and the anti-tumor immune responses that are linked to successful chemotherapy [11,25]. To test this, we used a murine mesothe- lioma model (AB1-HA) that is sensitive to both chemotherapy and immunotherapy [1,4,18], in combination with the DNA cross- linker cyclophosphamide (CY). Apoptotic cell death after maphosphamide treatment of tumor cells As we have recently shown [18], AB1-HA murine mesothelioma cells are killed by maphosphamide, which is the active metabolite of CY. To further determine how CY kills tumor cells, AB1-HA Anti-Tumor Immune Response cells were treated with the active metabolite maphosphamide at different concentrations for 24 hours. Induction of apoptosis was evaluated using the fluorescent FAM-VAD-fmk pan-caspase staining reagent (FLIVO), which binds activated caspases [26]. In vitro exposure of tumor cells to maphosphamide increased the proportion of FAM-VAD-fmk reactive cells in a dose-dependent manner, suggesting that cell death was associated with pan-caspase activation and hence apoptosis (Figure 1). Similarly, FAM-VAD- fmk staining cells were observed after in vivo treatment of tumor- bearing mice with cyclophosphamide (data not shown). Thus, as we show here, the CY metabolite maphosphamide induces apoptosis in AB1-HA tumor cells, providing a plausible explana- tion for the in vitro cytotoxic effect of maphosphamide and the in vivo anti-tumor effect of CY in athymic nude mice, as previously demonstrated [18]. Immunogenicity of CY chemotherapy Apoptotic tumor cell death can be immune-stimulatory [7]. Thus, to evaluate the immune effects of CY-induced tumor cell apoptosis, we analyzed changes in gene expression within CY- treated tumors using real time PCR SuperArrays, allowing simultaneous analysis of 84 cytokine genes using real-time PCR. Preliminary data indicate that several pro-inflammatory genes were upregulated in CY-treated tumors, including type-I IFNs (IFN-a4 and IFN-b1) but also IL-1a, IL-19, IL-2 and OX40L (Van der Most, unpublished data). Type-I IFN production after CY treatment was further analyzed by measuring the expression levels of Ly6AE on CD8 T cells. Ly6AE upregulation on T cells is a very early non-antigen-specific (bystander) response of T cells to IFN-exposure [27,28] and can therefore serve as a surrogate for type-I IFN (IFN-a/b) production. Indeed, we have recently confirmed that Ly6AE-upregulation on CD8 T cells occurs in a strictly IFN-a/b-dependent manner after AB1-HA tumor treat- ment with the TLR3 and TLR7 agonists poly-I:C and imiquimod [5,23]. Therefore, to assess IFN-a/b production, we measured Ly6AE expression on peripheral blood CD8 T cells harvested three days after administration of CY (150 mg/kg) in AB1-HA tumor-bearing mice. We found that CY treatment resulted in an upregulation of Ly6AE expression on the majority of CD8 T cells in the PBL pool when compared to untreated tumor-bearing mice (Figure 2A), consistent with systemic exposure of the CD8 T cells to IFN-a/b. The level of upregulation was similar to that observed with (intratumoral) poly-I:C injection (Figure 2A). Neutralization of type-I IFNs, using an anti-IFN-a/b serum, completely inhibited Ly6AE upregulation on peripheral blood CD8 T cells (Figure 2A), Figure 1. The CY metabolite maphosphamide kills by inducing apoptosis. AB1-HA tumor cells were treated with different doses of maphosphamide for 24 h and stained with FLIVO reagent. doi:10.1371/journal.pone.0006982.g001 PLoS ONE | www.plosone.org 2 September 2009 | Volume 4 | Issue 9 | e6982