FIGURE 5.

Macrophages sabotage anti-tumor CTL activity during immunotherapy in elderly mice. Young (n = 6–9/group) and elderly (n = 7–9/group) C57BL/6J mice were inoculated s.c. with 5 × 10⁵ AE17-sOVA mesothelioma tumor cells. Macrophages were depleted using anti-F4/80 antibody 2 days prior to the start of IL-2/anti-CD40 immunotherapy. Anti-F4/80 antibody was injected daily, alternating between i.p. and i.t. (100 μg/dose in 100 μl PBS). When tumors reached 15–21 mm² (tumor size calculated as width × length measured using calipers) i.t. IL-2/anti-CD40 immunotherapy (20 μg IL-2 and 40 μg anti-CD40 Ab, 100 μl/dose in PBS) was administered every 2–3 days. In vivo CTL activity was assessed following two doses of i.t. IL-2/anti-CD40 immunotherapy when tumor size was similar between groups. Target cells pooled from LNs and spleen cells of naïve C57BL/6J mice were pulsed with SIINFEKL (SIIN) and labeled with a high concentration of CFSE. No peptide control cells (CTRL) were labeled with a low concentration of CFSE. These populations were pooled equally and injected i.v. into mice for flow cytometry analysis 16–18 h later (example DLN plots in A). CTL activity was calculated as a percentage, based on reduction of the SIINFEKL population relative to the no peptide control population in DLN (B), spleen (C) and tumor (D). Data is pooled from three experiments shown as mean ± SEM, with the average value above each bar graph. ^∗p < 0.05, ^∗∗p < 0.01, ^#p < 0.001.