Figure 2. Type-I IFN production assessed by Ly6AE upregula- tion on CD8 T cells. Data shown are representative histograms displaying the shift in staining intensity from isotype control to Ly6AEhi CD8 T cells from one of three tumor-bearing mice per treatment group. Poly(I:C) was used as a positive control for Ly6AE expression. (A) From top to bottom: Ly6AE on CD8 T cells in PBS-treated mice, CY-treated mice, CY + anti-IFN-a/b treated mice, poly-I:C treated mice and isotype control staining (tinted curve), in PBL samples, day 3 after i.p. CY injection. (B) Type-I IFN responses are long-lived and systemic. Ly6AE expression in tumor draining and non-draining lymph nodes analyzed at day 10 post CY injection. CY-treated mice and untreated mice are compared. doi:10.1371/journal.pone.0006982.g002 confirming our previous finding that upregulation of Ly6AE expression is a bona fide marker for systemic type-I IFN release [5,23]. To evaluate the longevity of the IFN-a/b response, we analyzed Ly6AE expression on day 10 after CY injection in the tumor draining and non-draining lymph nodes. The results clearly Anti-Tumor Immune Response show sustained Ly6AE upregulation on CD8 T cells in both draining and non-draining lymph nodes (Figure 2B), demon- strating that CY injection results in long-lasting and systemic type- I IFN release. Therefore, the combined data suggest that CY- injection in tumor-bearing mice results in systemic immune activation, consistent with the concept of immunogenic cell death [7,18] and consistent with previously reported data [17,29]. Anti-tumor responses depend on CD8 T cells but not on IFN-ab CY has a strong CD8 T cell-dependent anti-tumor effect in AB1-HA tumor-bearing mice, routinely leading to 75–100% cure rate when small (i.e., just palpable) tumors are treated (Figure 3A), confirming our previous data [18], whereas CD8 depletion alone had a weak effect accelarating tumor growth (P = 0.07, data not shown). As we demonstrated previously, CY treatment in tumor- bearing athymic nude mice (which lack functional T cells) only had a transient anti-tumor effect and did not result in any curative anti-tumor responses [18]. Thus, T cells play an essential role in achieving cures. Since CY treatment is associated with an IFN-a/ b response and because primary CD8 T cell responses to apoptotic cells and cross-presented antigens are type-I IFN-dependent [8,30], we hypothesized that the type-I IFN response could be responsible for priming the anti-tumor CD8 T cell response. This was tested by in vivo neutralization of IFN-a/b. We found that the anti-tumor efficacy of CY was only marginally affected by type-I IFN neutralization (Figure 3B), indicating that the CY-induced anti-tumor CD8 T cell response was not dependent on IFN-a/b. As a positive control for the efficiency of in vivo IFN-a/b- neutralization, we have shown that poly-I:C-mediated anti-tumor responses were abrogated using this anti-IFN-a/b treatment [5]. Thus, our data suggest that the CY-induced anti-tumor T cell response is type-I IFN independent and may therefore not be a de novo response because such a response would be type-I IFN- dependent [8]. Instead, we reasoned that CY could make existing responses more effective, either by expanding tumor-specific CD8 T cells or by increasing the sensitivity of tumor cells to T cell- mediated apoptosis. To test this, we further studied anti-tumor CD8 T cell responses after CY treatment. Impaired CD8 T cell responses after CY treatment CY is a cytotoxic and lymphodepleting drug. A single dose of CY (150 mg/kg) resulted in a 75% reduction of splenic cellularity [18] as well as of total CD8 T cell numbers (data not shown) Thus, expansion of anti-tumor CD8 T cells after CY chemother- apy seems paradoxical. To study this, we characterized total and tumor-specific CD8 T cell responses after CY treatment, using three different experimental approaches. First, we assessed CD8 T cell proliferation in the tumor-draining lymph nodes after CY treatment by intracellular Ki-67 staining. Ki-67 is a nuclear marker that is upregulated in cycling cells [18,31,32]. Interestingly, CY treatment selectively depleted cycling cells, as shown by the almost complete absence of Ki-67hi CD8 T cells three days after a single CY injection (Figure 4A). We have recently reported similar findings for CD4 T cells and foxp3+ CD4+ regulatory T cells [18]. This specific loss of Ki-67hi T cells was not seen with a different cytotoxic drug, gemcitabine (GEM) (Figure 4A), despite the fact that GEM causes lymphodepletion in mice [14]. Since both drugs are cytotoxic, the difference between CY and GEM may be one of kinetics, in which the CY-mediated T cell proliferation arrest is more long-lived. Second, to assess to the impact of CY on the tumor-specific CD8 T cell response, we adoptively transferred TCR-transgenic CD8 T cells into CY treated mice. Clone 4 transgenic CD8 T cells recognize a PLoS ONE | www.plosone.org 3 September 2009 | Volume 4 | Issue 9 | e6982