The ratio of effector to regulatory T cells within the tumor microenvironment is considered indicative of the effect of adaptive immune responses on tumor progression and metastasis 45 . We investigated the numbers of tumor infiltrating T cell populations in subcutaneously growing B16 tumors treated locally for 2 weeks with CpG-Stat3 siRNA, CpG-scrambled RNA control or PBS only. Although CpG-Stat3 siRNA treatment did not induce significant changes in overall CD4 + T cell numbers within the tumors, as shown by flow cytometric analysis (Fig. 6a) , the percentage of CD4 + /FoxP3 + Treg cells within all CD4 + T cells dropped from approximately 60% to 25% after peritumoral injections of CpG-Stat3 siRNA (Fig. 6b) . We observed an increase in the infiltration of total CD8 + T cells in the tumor stroma, although CpG-scrambled RNA control treatment also led to some recruitment of CD8 + T cells (Fig. 6c) . These effects probably result from TLR9-mediated immunostimulation of tumor-infiltrating antigen presenting cells. Fluorescent immunostaining of frozen tumor tissues with anti-CD8 antibody confirmed data generated by the flow cytometric analysis that CpG-Stat3 siRNA treatment caused increased CD8 + T cell infiltration in tumors (data not shown). Activation of tumor antigen-specific CD8 + T cells is believed to be critical for immune-mediated antitumor effects. We therefore examined the ability of CpG-Stat3 siRNA treatment to generate CD8 + T cells specific for the B16 tumor antigen, TRP2. ELISPOT assays to determine IFNγ production by T cells isolated from TDLNs in response to recall stimulation with TRP2 peptide antigen indicated that in vivo CpG-Stat3 siRNA administration indeed induced antigen-specific CD8 + T cells (Fig. 6d ).