Anti-Tumor Immune Response Figure 3. Role of CD8 T cells and type-I IFN in CY-driven anti-tumor efficacy. (A) Anti-tumor responses depend on CD8+ T cells. Tumor- bearing mice were treated with CY at day 10 after tumor inoculation (day 0) and anti-CD8 mAbs (150 mg) were injected at days 21, 0, 2, 4, 6 and 8 with respect to CY treatment. Data shown are mean6SEM (n = 5) from one representative experiment from a total of three experiments with a total of 15 mice. * P,0.05 when CY is compared with CY + anti-CD8. (B) In vivo IFN-a/b neutralization marginally affects tumor growth in CY-treated mice. BALB/c mice bearing AB1-HA tumors were given IFN-a/b blocking antibody on days 21, +2, +4 with respect to CY treatment. Tumors were inoculated at day 0 and were treated with CY at day 9. Data shown are mean6SEM (n = 5) from one experiment. ns = not-significant. doi:10.1371/journal.pone.0006982.g003 Kd-restricted epitope from the influenza virus HA protein which is expressed as a tumor neo-antigen in AB1-HA cells [15]. Ten million CFSE-labeled tumor-specific transgenic CD8 T cells were adoptively transferred into mice at day 7 after tumor cell inoculation. Mice were treated with CY (150 mg/kg) at day 9 and lymphocytes were isolated from the tumor-draining and non- draining lymph nodes at day 14 (i.e., 5 days after CY injection and 7 days after adoptive transfer). Confirming previous work employing this same assay [1], we observed robust proliferation of tumor-specific CD8 T cells in the tumor-draining lymph nodes of untreated control tumor-bearing mice, as can be inferred from the serial dilution of CFSE dye intensity (Figure 4B). However, both the recovery of tumor-specific transgenic T cells as well as proliferation of these cells (assessed by serial CFSE dilution) were significantly reduced in the draining lymph nodes from CY-treated mice (Figure 4B). These findings are consistent with the notion that CY has a negative effect on T cell proliferation. Third, to further define the specificity of CD8 T cell responses after CY d treatment we used a MHC class I pentamer specific for the K - restricted HA epitope to visualize the endogenous response to the HA epitope. Thus, these experiments were done without adoptive T cell transfer. Since CY-induced lymphopenia is transient, with splenic cellularity recovering within 10 days after treatment (data not shown), we reasoned that early inhibition of T cell proliferation could be followed by recovery of the anti-tumor CD8 T cell response. Therefore, we measured CD8 T cell responses at day 10 after CY injection. Low frequencies of pentamer-positive HA-specific CD8 T cells were measured in the draining lymph nodes of both untreated and CY-treated tumor- bearing mice (Figure 4C) and there were no significant differences, although there was a trend towards lower frequencies of pentamer-positive cells after CY treatment (P = 0.06). Pheno- typic characterization of pentamer+ cells using the cytotoxicity marker CD43 [33] revealed that in untreated tumor-bearing mice, both CD43hi and CD43lo pentamer+ tumor specific CD8 T cells were present (Figure 4D), whereas the small populations of pentamer+ CD8 T cells in CY-treated mice appeared to be CD43lo. Overall, these data suggests that these T cells do not possess a highly activated and cytotoxic phenotype. However, despite this lack of CD8 T cell expansion, we found that mice that were cured through CY treatment invariably resisted rechallenge with AB1-HA tumor cells (n = 20, data not shown), which indicates that the CD8 T cell response that was mobilized by CY treatment eventually generated tumor-specific memory. PLoS ONE | www.plosone.org 4 September 2009 | Volume 4 | Issue 9 | e6982