Figure 3
Enhancement of systemic Reovirus therapy by checkpoint blockade is dependent on CD8 cells. (a, b) C57Bl/6 mice (7 per group) bearing 5-day established B16 tumours were treated ±2 cycles of GM-CSF/Reovirus beginning on days 5 and 12, then three injections of anti-PD-1 (250 μg) or control IgG on days 19, 21, 23. (a) Tumours were measured 3x per week and mice euthanised when tumours reached 1.0 cm diameter. *P<0.05 log-rank test. (b) S/LN were harvested at time of sacrifice (as indicated). Single cell suspension cultures of S/LN were re-stimulated with B16 F/T lysate every 24 h. Supernatants were harvested after 72 h and tested for IFN-γ by ELISA. Bars on graphs show values +s.d. (triplicate wells) for individual mice. *P<0.05 two-tailed t-test. (c) Mice with 6-day established s.c. B16 tumours were treated with two rounds of PBS/PBS, PBS/Reo, GM-CSF/PBS or GM-CSF/Reovirus (days 6–10 and 13–17). On day 22 tumours were excised and analysed by intracellular staining for CD3+ CD8+ IFN-γ+, and CD3+ CD4+ IFN-γ+, T cells. The mean percentage of CD3+ CD4+ or CD3+ CD8+ cells, which were also IFN-γ positive, in tumours from each group is shown. Standard deviations represent values from four mice per group (except GM-CSF/PBS where n=3). *P<0.05, **P<0.01 two-tailed t-test. (d, e) C57Bl/6 mice (5 per group) bearing 5-day established B16 tumours, received three cycles of GM-CSF/Reovirus with co-injection of anti-CD4 or anti-CD8 depleting antibodies along with the GM-CSF, beginning on days 5, 12, 19. Anti-PD-1 (250 μg) or control IgG was administered on days 19, 21, 23. Tumours were measured 3x per week and mice euthanised when tumours reached 1.0 cm diameter. (d) Depletion of CD4 or CD8 cells on GM-CSF/Reovirus therapy; (e) Depletion of CD4 or CD8 cells on GM-CSF/Reo/anti-PD-1 therapy. *P<0.05 log-rank test. (d, e) are results from the same experiment.
