(AB1-HA) has been described [15,16]. AB1-HA cells (16106 in PBS) were injected s.c. into the right flank of recipient mice and tumor growth monitored using microcalipers. Mice were eutha- nized when tumors reached 10610 mm as per Animal Ethics guidelines. Pentamer staining For pentamer (ProImmune) staining, cells were blocked with 20 ml FCS (109, RT) after which 5 ml pentamer was added (309, 4uC). Cells were stained with mAbs against CD8 and TCRb and anti-CD19 were included to reduce background. Lyons-Parish analysis of Ag presentation For CFSE (Molecular Probes) labeling, lymph node cells from TCR-transgenic CL4 mice were incubated with 2.5 mM CFSE (109 RT) and then centrifuged through a FCS cushion. A total of 16107 cells were injected i.v. into recipient mice. CFSE-labeled cells were recovered 7 days after adoptive transfer and analyzed by FACS. In vivo antibody treatments The IFN-a/b neutralizing sheep Ig and matching normal sheep Ig [17,61] were used as described [5]. Mice were i.v. injected with 0.2 ml of immunoglobulins on day –1, +2, and +4 with respect to CY administration. Monoclonal anti-IFN-c antibodies (clone XMG 1.2) were purified from hybridoma supernatant and were injected from day 7 after tumor cell inoculation onwards (q3dx4, References 1. Nowak AK, Lake RA, Marzo AL, Scott B, Heath WR, et al. (2003) Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation, cross- priming rather than cross-tolerizing host tumor-specific CD8 T cells. J Immunol 170: 4905–4913. 2. Marzo AL, Lake RA, Lo D, Sherman L, McWilliam A, et al. (1999) Tumor antigens are constitutively presented in the draining lymph nodes. J Immunol 162: 5838–5845. 3. Thomas AM, Santarsiero LM, Lutz ER, Armstrong TD, Chen YC, et al. (2004) Mesothelin-specific CD8(+) T cell responses provide evidence of in vivo cross- priming by antigen-presenting cells in vaccinated pancreatic cancer patients. J Exp Med 200: 297–306. 4. 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Immunity 24: 787–799. 9. van der Most RG, Currie AJ, Robinson BW, Lake RA (2008) Decoding dangerous death: how cytotoxic chemotherapy invokes inflammation, immunity or nothing at all. Cell Death Differ 15: 13–20. 10. Suzuki E, Sun J, Kapoor V, Jassar AS, Albelda SM (2007) Gemcitabine has significant immunomodulatory activity in murine tumor models independent of its cytotoxic effects. Cancer Biol Ther 6: 880–885. 11. Lake RA, Robinson BWS (2005) Immunotherapy and chemotherapy - a practical partnership. Nature Reviews Cancer 5: 397–405. 12. Zitvogel L, Casares N, Pequignot MO, Chaput N, Albert ML, et al. (2004) Immune response against dying tumor cells. Adv Immunol 84: 131–179. 13. Haynes NM, van der Most RG, Lake RA, Smyth MJ (2008) Immunogenic anti- cancer chemotherapy as an emerging concept. Curr Opin Immunol. 14. Nowak AK, Robinson BW, Lake RA (2002) Gemcitabine exerts a selective effect on the humoral immune response: implications for combination chemo- immunotherapy. Cancer Res 62: 2353–2358. 15. Marzo AL, Lake RA, Robinson BW, Scott B (1999) T-cell receptor transgenic analysis of tumor-specific CD8 and CD4 responses in the eradication of solid tumors. Cancer Res 59: 1071–1079. Anti-Tumor Immune Response 100 mg per injection). CD8a T cell depletion was performed using purified YTS.169 monoclonal antibody (Dr Kathy Davern, Monoclonal Antibody Facility, Western Australian Institute for Medical Research) as previously described [5]. CD8 depletion (.95%) was verified by FACS analysis of PBL. Agonistic anti- DR5 antibody (clone MD5-1) [36] was injected i.p. at days -1, 3 and 7 relative to CY treatment (50 mg/injection). Neutralizing TRAIL antibodies (clone N2B2) [36] were injected i.p. at days 21, 2, 5 and 8 relative to CY treatment (200 mg/injection). Statistics Data were statistically evaluated using Prism software (Graph- Pad). Survival responses were analyzed by Kaplan-Meyer using log-rank test. 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(2008) Immunogenic cancer cell death: a key-lock paradigm. Curr Opin Immunol. 26. Pozarowski P, Grabarek J, Darzynkiewicz Z (2004) Flow cytometry of apoptosis. Curr Protoc Cell Biol;Chapter 18: Unit 18 18. 27. Dumont FJ, Coker LZ (1986) Interferon-alpha/beta enhances the expression of Ly-6 antigens on T cells in vivo and in vitro. Eur J Immunol 16: 735–740. 28. Khodadoust MM, Khan KD, Park EH, Bothwell AL (1998) Distinct regulatory mechanisms for interferon-alpha/beta (IFN-alpha/beta)- and IFN-gamma- mediated induction of Ly-6E gene in B cells. Blood 92: 2399–2409. 29. Jovasevic VM, Mokyr MB (2001) Melphalan-induced expression of IFN-beta in MOPC-315 tumor-bearing mice and its importance for the up-regulation of TNF-alpha expression. J Immunol 167: 4895–4901. PLoS ONE | www.plosone.org 10 September 2009 | Volume 4 | Issue 9 | e6982