Figure 3.

Ex vivo OT-I proliferation following T-HIFU or M-HIFU. (A) Diagram shows the treatment schedule for assessing OT-I proliferation following HIFU ablation. Mice bearing EG7 tumors were treated with mock, T-HIFU or M-HIFU ablation and received a peritumoral injection of 50 µg CpG within 30 minutes after HIFU ablation, divided over two injections. 16-24 hours after HIFU treatment, inguinal LNs were excised and single cell LN suspensions (0.75 × 10⁶ cells) were co-cultured with 0.5 × 10⁵ CFSE labelled CD8⁺ OT-I T cells for 72 h. (B) Graph shows OT-I proliferation upon co-culture of NdLN and TdLN cells following mock, T-HIFU and M-HIFU treatment plus CpG administration. Differences in OT-I proliferation between treatments were analyzed using ordinary two-way ANOVA followed by Tukey’s multiple comparison test. Differences between LNs were analyzed using repeated measures two-way ANOVA followed by Bonferroni’s multiple comparison test (control n = 7, T-HIFU n = 7, M-HIFU n = 7 animals). Shown are pooled results from three independent experiments. CFSE, carboxyfluorescein succinimidyl ester; CpG, cytidyl guanosyl; LN, lymph node; M-HIFU, mechanical-high intensity focused ultrasound; NdLN, non-draining lymph node; T-HIFU, thermal-high intensity focused ultrasound; TdLN, tumor-draining lymph node.