Anti-Tumor Immune Response Figure 6. Role of TRAIL-mediated tumor cell apoptosis. (A) DR5 expression on AB1-HA tumor cells in vitro. (B) Tumor growth curves in CY- treated and untreated TRAIL-deficient mice, compared with immunocompetent mice and nude mice. Data shown are mean6SEM (n = 10) from two combined experiments. Tumor cells were inoculated at day 0 and treated with CY at day 10. ** P,0.005 when CY in BALB/c is compared with CY in TRAIL-deficient mice. doi:10.1371/journal.pone.0006982.g006 It is not entirely clear how CY has such a strong effect on proliferating T cells, although several studies suggest that it may be linked to increased numbers of NO-producing myeloid-derived suppressor cells [42,43]. Whereas depletion of proliferating regulatory T cell is beneficial [18,19,44], the impact of CY on the CD8 T cell response could be negative and seems inconsistent with the key role of CD8 T cells in tumor resolution. Our explanation is that the importance of CY-triggered CD8 T cell responses lies not so much in their total numbers, but rather in their capacity to kill tumor cells. In this model, CY kills the majority of tumor cells and sensitizes the remaining tumor cells for T cell-mediated apoptosis, allowing the tumor-specific CD8 T cell pool to become effective without the need for further expansion. The benefit of regulatory T cell depletion [18,19,45,46] may be that existing anti-tumor CD8 T cells are liberated from suppression, allowing them to kill the sensitized tumor cells. A tumor cell sensitization model [34] is supported by the key role of TRAIL. Since no curative responses were ever observed in athymic nude mice, it is evident that CY does not kill all tumor cells. Therefore we propose that a subset of tumor cells that receives a sublethal hit from the drug is sensitized for apoptosis by agonistic anti-TRAIL-R (DR5) antibodies or tumor-specific CD8 T cells and most likely also NK cells. TRAIL sensitization of tumor cells, including human mesothelioma cells, by chemother- apeutic drugs has been well characterized [47,48] and has been translated into therapeutic approaches involving soluble TRAIL or other TRAIL-receptor agonists [35,49,50]. It has been proposed that the synergy of TRAIL-ligation and chemotherapy can be explained by integration of the intrinsic (through chemotherapy) and alternative (death receptor) pathways of apoptosis [47]. Recently, Johnstone and colleagues showed that the histone diacetylase inhibitor vorinostat augmented the anti- tumor effects of anti-DR5 antibodies in vivo and in vitro through downregulation of the apoptosis inhibitor cellular-FLIP [51]. Similar in vitro data were shown earlier with cycloheximide and sensitivity to TRAIL apoptosis [52]. TRAIL sensitivity can also be increased through the proteasome inhibitor bortezomib [53]. The liver toxicity that we observed in our current study seems consistent with recent work showing that the drug 5-azacytidine sensitizes hepatocytes for TRAIL apoptosis [54] and with a recent study showing that DR5-mediated apoptosis of cholangiocytes contributes to liver disease [37]. Increased TRAIL sensitivity explains why massive T cell expansion is not necessary and why type-I IFNs are not required for the anti-tumor effect in our model. Our data suggest that CD8 T cells are a major source of TRAIL, since these cells are essential for tumor resolution. NK cells may also be required [55]. A more precise determination of the relative roles of NK cells and CD8 T cells and of the source(s) of TRAIL will be the subject of further studies, which should include measuring the in vitro sensitivity of maphosphamide- treated tumor cells to DR5 ligation. The role of IFN-c could be related to TRAIL, since both type-I and type-II IFNs have the capacity to induce TRAIL expression [56,57] and anti-tumor function [58]. The importance of TRAIL suggests that TRAIL PLoS ONE | www.plosone.org 8 September 2009 | Volume 4 | Issue 9 | e6982