Timing of i.v. CpG injections determines antitumor immunity.

<p>Established B16OVA tumors were treated with cryo ablation alone or in combination with i.v. CpG administration concurrent with ablation, or 1 day before or after ablation. Forty days later, naïve and treated tumor-free mice (7–10 mice per group) received a s.c. re-challenge with tumor cells (25.000–50.000 B16OVA cells) on the contra-lateral flank and survival was monitored. Representative of 2 experiments is shown.</p

Route of CpG administration determines induction of tumor-specific CTL.

<p>(<b>A</b>) Representative dot plots of CD8⁺OVA-Kb tetramer⁺ cells of individual mice. Mice bearing B16OVA tumors (6–9 mm) were subjected to cryo ablation alone or received additional CpG injections via the indicated routes. Ten days after tumor destruction, cells from spleen and tumor draining lymph nodes were isolated and re-stimulated with IFN-γ-treated γ-irradiated B16OVA tumor cells. Cultures were cleaned by a Ficoll step after 3–4 days of culture and at day 8–9 cells were analyzed for the presence of tumor-specific CTL using APC-labeled OVA-K_b tetramers. Cells were gated on CD8⁺ T cells. Data from one representative mouse per group is shown. (<b>B</b>) Quantitative analyses of collective data as shown in (A) (mean levels of 2 separate experiments (4–6 mice per group/experiment)). * indicates significant differences (p<0.05) of the indicated group compared to all other groups.</p

Route of CpG administration affects T cell activation.

<p>(<b>A</b>) Percentages of IFN-γ⁺ T cells within the CD4⁺ or CD8⁺ populations after cryo ablation +/− CpG. B16-OVA tumor-bearing mice were treated with cryo ablation combined with CpG administration via the indicated routes. Seven days after ablation, spleen, tumor-draining lymph nodes and non-draining lymph nodes were isolated. Cells were stimulated with PMA/ionomycin and stained for CD8, CD4 and IFN-γ. * indicate significant differences (p<0.05) between the indicated CpG-treated group and the Naïve and Cryo group. (<b>B</b>) IFN-γ production after cryo ablation +/− CpG. Cells were stimulated with OVA for 96 hours and IFN-γ was measured in supernatant by ELISA. The mean levels of the Cryo+CpG i.v. groups in both organs are significantly different from all other groups (n = 6/group, 2 experiments).</p

Quantity and quality of lymph node DC.

<p>(<b>A</b>) Absolute numbers of CD11c⁺ cells in the draining lymph nodes 2 days after ablation. Tumor-bearing mice were left untreated (NT) or were subjected to cryo ablation (cryo). In addition, some groups of mice received CpG via the indicated routes. Brackets indicate significant differences (p<0.05) between indicated groups (n = 4–6/group, 2 similar experiments). (<b>B</b>) Uptake of antigen after cryo ablation. OVA-Alexa488 was injected in B16OVA tumors (6–9 mm) just prior to cryo ablation to monitor the fate of antigen after in situ tumor destruction. Mice were additionally treated with CpG via the indicated routes. Two days after ablation, the uptake of antigen was analyzed in CD11c⁺ cells enriched from pools of tumor-draining lymph nodes. Numbers in the panels indicate the percentage of OVA⁺ of all CD11c⁺ cells. Data from one representative mouse is shown per group. (<b>C</b>) Quantitative analyses of replicates of experiments as shown in (B) (5 mice/group, representative of 2 experiments). (<b>D</b>) Uptake of antigen and CpG by CD11c⁺ cells monitored after p.t. and i.v. CpG-Cy5 administration and intra-tumoral injection of OVA-Alexa488. Data from one representative mouse is shown (n = 4/group). (<b>E</b>) The expression of CD80 on CD11c⁺ antigen-loaded cells. * indicates significantly different (p<0.05) from all other groups (n = 5/group, 2 similar experiments).</p

Route of CpG administration determines efficacy of antitumor immunity.

<p>(<b>A</b>) Kaplan-Meier survival curves of naïve mice versus mice that have been treated with cryo ablation alone, or in combination with concurrent CpG administration via the indicated routes. Established B16OVA tumors on the right femur were treated with cryo ablation alone or in combination with CpG administrations via different routes: p.t., i.v., or s.c. contra lateral of the tumor. Forty days later, naïve and tumor-free mice (8–13 mice per group) received a s.c. re-challenge with tumor cells (25.000 B16OVA cells). (<b>B</b>) Tumor size determined every 2–3 days after treatment. Data is representative of two independent experiments.</p