Tumor-reactive CD8⁺ T lymphocytes obtained from tumor-bearing donors were expanded and functional following transplantation into syngeneic HSCT recipients and vaccination with tumor cells secreting gp96-Ig. Conditioned (9.5 Gy) B6 recipients received B6-CD45.1⁺ TCD-BM cells supplemented with 2.0 × 10⁶ B6-CD90.1⁺ CD4⁺ and CD8⁺ T lymphocytes obtained from E.G7 lymphoma-bearing donors, containing ∼1000 tumor-reactive CD8⁺ T cells (OT-I). Recipients were vaccinated intraperitoneally with irradiated (40 Gy) E.G7 cells secreting gp96-Ig 2 days after HSCT and repeated every 3 days for a total of 5 vaccinations. (A) CD4⁺ and CD8⁺ splenic and LN T cells from a typical tumor-bearing B6-CD90.1⁺ donor mouse. These animals were injected 3 weeks before HSCT with 5.0 × 10⁵ CD8⁺ OT-I T lymphocytes intravenously and 4.0 × 10⁶ E.G7 lymphoma cells intraperitoneally and bear progressively growing tumors. Tumor was only detectable at the site of injection (peritoneal cavity) at this time. Total events analyzed represented 2.5 × 10⁶ viable cells, and CD8⁺ OT-I T lymphocytes were clearly identified (1000 CD8⁺ CD90.1⁻ V_α2⁺ V_β5⁺/1.0 × 10⁶ CD8⁺ CD90.1⁺). (B) Multiple vaccinations with gp96-Ig-secreting tumor cells induced expansion of tumor-reactive CD8⁺ T cells obtained from tumor bearing donors; n = 4; ♦, E.G7; ●, E.G7-gp96-Ig. (C) Tumor-reactive CD8⁺ T lymphocytes expanded at the vaccine site and other lymphoid tissues 5 days following 3 vaccinations with tumor cells secreting gp96-Ig; n = 2. (D) Vaccination with gp96-Ig-secreting tumor cells induced highly IFN-γ⁺ and TNF-α⁺ tumor-reactive CD8⁺ T cells 5 days following 3 vaccinations; representative dot plots from n = 2.