GM-CSF conditioning followed by systemic reovirus is an effective therapy in mice preimmunized with reovirus. (a) C57Bl/6 mice were vaccinated i.p. with either PBS (virus naive) or reovirus (2 × 107 TCID50) (preimmune). After 14 days, mice were seeded with s.c. B16 tumors and 3 days later were treated with once cycle of PBS/PBS, GM-CSF/PBS, PBS/reovirus, or GM-CSF/reovirus treatment (three daily injections of GM-CSF i.p. followed by two daily injections of reovirus i.v.) (eight total groups, three treatments virus naive or five treatments preimmune). Survival (tumor <1.0 cm diameter) with time is shown. (b) C57Bl/6 mice, vaccinated 14 days earlier with PBS, reovirus (2 × 107 TCID50) (preimmune) or VSV (106 pfu i.v.) (VSV immune), were seeded with s.c B16 tumors. After 5 days, these tumor bearing mice were treated with three cycles of GM-CSF/reovirus. Additional groups of mice were vaccinated with reovirus and then treated with three cycles of PBS/reovirus, GM-CSF/PBS, or PBS/PBS. Survival (tumor <1.0 cm diameter) with time is shown. (c) C57Bl/6 mice were vaccinated i.p. with either PBS (two mice) or reovirus (2 × 107 TCID50) (2–3 per group). After 5, 7, 9, or 14 days, levels of anti-reovirus NAb were measured as described in Materials and Methods (preincubation of serum from PBS-treated mice with reovirus was not able to reduce the toxicity to L929 cells to any greater extent than DMEM). (d) C57Bl/6 mice were vaccinated i.p. with either PBS (PBS(VAC)), VSV (2 × 107 pfu VSV-GFP) (VSV(VAC)) or reovirus (2 × 107 TCID50) (REO(VAC)). After 14 days, mice were seeded with s.c. B16 tumors and 5 days later (compared with treatment of 3-day established tumors in a) were treated with one cycle of GM-CSF/reovirus treatment (three daily injections of GM-CSF i.p. followed by two daily injections of reovirus i.v.). Survival (tumor <1.0 cm diameter) with time is shown. (e) C57Bl/6 mice bearing 10-day established s.c. B16 tumors were treated with three cycles of PBS/PBS, GM-CSF/PBS, PBS/reovirus, or GM-CSF/reovirus. Survival (tumor <1.0 cm diameter) with time is shown.