Experimental model and treatment administration. (A) The 2-tumor site model. BALB/c mice were inoculated subcutaneously with 5 × 106 A20 tumor cells at 2 different sites: right and left abdomen. Intratumoral injections of CpG were performed in the right tumor while the left tumor served for evaluation of the systemic antitumor immune response. (B) Schema of treatment. Treatment started 5 to 7 days after tumor inoculation when the tumors reached 0.5 cm to 0.7 cm in largest diameter. Mice then received daily injections of intratumoral CpG over 5 days (100 μg per injection, red arrows) and 2 intraperitoneal injections of mAbs used for T-cell modulation (anti-OX40, anti-CTLA4, anti-GITR, anti-FR4) on the first and last day of treatment (blue arrows). (C) CpG effect on the injected (right) and noninjected (left) tumors. In mice bearing 2 tumors, intratumoral injections of CpG induced regression of the injected (right) tumor, but failed to cure the distant (left) tumor, unless combined with effective T-cell modulation (*anti-OX40 + anti-CTLA4 mAbs were used in this example for T-cell modulation). All tumor curves presented in the following figures will show tumor growth at the left (non-CpG injected) site, which reflects the systemic antitumor effect of the immunotherapy.
