Cyclophosphamide Chemotherapy Sensitizes Tumor Cells to TRAIL-Dependent CD8 T Cell-Mediated Immune Attack Resulting in Suppression of Tumor Growth Robbert G. van der Most1*, Andrew J. Currie1, Amanda L. Cleaver1, Joanne Salmons1, Anna K. Nowak1, Sathish Mahendran1, Irma Larma 1 , Amy Prosser1, Bruce W. S. Robinson1,2, Mark J. Smyth3, Anthony A. Scalzo4, Mariapia A. Degli-Esposti4, Richard A. Lake1* 1 National Research Centre for Asbestos Related Diseases, University of Western Australia, Perth, Australia, 2 School of Medicine and Pharmacology, University of Western Australia, Perth, Australia, 3 Cancer Immunology Program, Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Australia, 4 Centre for Experimental Immunology, Lions Eye Institute, and Immunology and Virology Program, Centre for Ophthalmology and Visual Science, The University of Western Australia, Nedlands, Australia Abstract Background: Anti-cancer chemotherapy can be simultaneously lymphodepleting and immunostimulatory. Pre-clinical models clearly demonstrate that chemotherapy can synergize with immunotherapy, raising the question how the immune system can be mobilized to generate anti-tumor immune responses in the context of chemotherapy. Methods and Findings: We used a mouse model of malignant mesothelioma, AB1-HA, to investigate T cell-dependent tumor resolution after chemotherapy. Established AB1-HA tumors were cured by a single dose of cyclophosphamide in a CD8 T cell- and NK cell-dependent manner. This treatment was associated with an IFN-a/b response and a profound negative impact on the anti-tumor and total CD8 T cell responses. Despite this negative effect, CD8 T cells were essential for curative responses. The important effector molecules used by the anti-tumor immune response included IFN-c and TRAIL. The importance of TRAIL was supported by experiments in nude mice where the lack of functional T cells could be compensated by agonistic anti-TRAIL-receptor (DR5) antibodies. Conclusion: The data support a model in which chemotherapy sensitizes tumor cells for T cell-, and possibly NK cell-, mediated apoptosis. A key role of tumor cell sensitization to immune attack is supported by the role of TRAIL in tumor resolution and explains the paradox of successful CD8 T cell-dependent anti-tumor responses in the absence of CD8 T cell expansion. Citation: van der Most RG, Currie AJ, Cleaver AL, Salmons J, Nowak AK, et al. (2009) Cyclophosphamide Chemotherapy Sensitizes Tumor Cells to TRAIL- Dependent CD8 T Cell-Mediated Immune Attack Resulting in Suppression of Tumor Growth. PLoS ONE 4(9): e6982. doi:10.1371/journal.pone.0006982 Editor: Derya Unutmaz, New York University School of Medicine, United States of America Received February 28, 2009; Accepted August 19, 2009; Published September 10, 2009 Copyright: 2009 van der Most et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research grants from the NH&MRC (www.nhmrc.gov.au; grant number 572493) and the Raine Medical Research Foundation (www.raine.uwa.edu.au), Australia. M. D.-E., M.J.S., and A.A.S. are supported by Fellowships from the NH&MRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: robbertvdm@gmail.com (RGvdM); rlake@cyllene.uwa.edu.au (RAL) Introduction Tumor antigens are cross-presented to the immune system [1,2,3]. However, the ensuing anti-tumor CD8 T cell response is usually not effective and fails to control tumor growth. Indeed, the immunological outcome of antigen cross-presentation is deter- mined by the context in which tumor antigens are presented. Altering that context is an important goal for anti-cancer immunotherapy [4,5,6]. Cytotoxic chemotherapy can play a role in this process since apoptotic tumor cell death can be an immunostimulatory event (‘immunogenic cell death’) [7,8,9], potentially adding an immunostimulatory signal to cross-presented antigens. An immune priming effect has now been shown for several chemotherapeutic drugs, including gemcitabine [4,10] and doxorubicin [7]. As a result, chemo- and immunotherapy are no longer considered to be a priori antagonistic [11] and the concept of combined chemo-immuno therapy is receiving more attention [11,12,13]. However, the notion that chemotherapy and anti- tumor T cell responses can be synergistic must be reconciled with the fact that many chemotherapeutic drugs deplete lymphocytes [14]. In fact, lymphodepletion after chemotherapy was the main reason why chemo- and immunotherapy were seen as antagonis- tic. The growing insight that chemotherapy can be immunosti- mulatory presents a paradox: how are effective anti-tumor T cell responses generated under lymphodepleting conditions? The present study aims to address the paradoxical relationship between immunogenicity and lymphodepletion. To study this, we have used a mouse model of malignant mesothelioma (AB1-HA), which is sensitive to both chemotherapy and immunotherapy [4,15,16], in combination with the chemotherapeutic drug cyclophospha- mide (CY), since CY treatment is associated with innate and adaptive immune activation [17,18,19,20,21]. The AB1-HA tumor cell line was generated by transfection of the asbestos- PLoS ONE | www.plosone.org 1 September 2009 | Volume 4 | Issue 9 | e6982