Tumor-induced suppression of CTL expansion and subjugation by gp96-Ig vaccination.

Established tumors suppress antitumor immune responses and induce tolerance by incompletely characterized mechanisms, and this phenomenon is an important barrier to tumor immunotherapy. Single vaccination with tumor cells expressing gp96-Ig stimulates robust expansion of tumor-specific CTLs in tumor-naïve mice and this expansion is inhibited by established tumors. Interestingly, frequent vaccinations restore antitumor immune responses in the presence of established tumors. Syngeneic EG7 tumor-bearing mice have heterogeneous responses to frequent vaccination with EG7-gp96-Ig, with 32% complete responders and 68% partial responders. Comparison of responders to nonresponders revealed an inverse correlation between tumor-specific CTL expansion in the peripheral blood and tumor size. To identify immune cells and molecules associated with effective antitumor immune responses, reverse transcription-PCR arrays were performed using cells isolated from the vaccination site. ELISAs, cellular phenotyping, and tumor immunohistochemistry were also performed comparing vaccine responders to nonresponders. These data show that up-regulation of T-bet, RORgammat, IFNgamma, CCL8, CXCL9, and CXCL10 at the vaccination site are associated with vaccine-induced antitumor immunity. These data correlate with increased CTL expansion in the peripheral blood of responders, increased infiltration of responder tumors by CD8+ cells and interleukin-17+ cells, and decreased infiltration of responder tumors by CD11b+Gr-1+ cells and FoxP3+ cells. Furthermore, serum ELISAs revealed a significant elevation of transforming growth factor-beta in nonresponders as compared with responders. Interestingly, CD8+ T cells isolated from responders and nonresponders have equivalent cytotoxic activity in vitro. Taken together, our data suggest that established tumors may escape immunosurveillance by preventing clonal expansion of tumor-specific CTL without inducing anergy.