Vaccination with tumor cells secreting the heat shock protein fusion gp96-Ig led to the activation, expansion, and functional competence of tumor-reactive CD8⁺ T lymphocytes in syngeneic HSCT recipients. Conditioned (9.5 Gy) B6 mice were transplanted with 5.0 × 10⁶ B6-CD45.1⁺ TCD-BM cells and adoptively transferred with 1.0 × 10⁶ CD8⁺ T cells specific for OVA_257-264 (OT-I) 5 days later. After a 2-day resting period, recipients were vaccinated intraperitoneally with irradiated (120 Gy) EL-4 lymphoma cells expressing OVA (E.G7) engineered to secrete gp96-Ig (E.G7-gp96-Ig). (A) Macrophages (F4/80^hi CD11b^hi Gr-1^lo) and inflammatory monocytes (Gr-1^hi CD11b^mid F4/80^lo) infiltrated the peritoneal cavity 2 days following vaccination with gp96-Ig-secreting tumor cells; n = 9 from a pool of 3 experiments. (B) Tumor-reactive CD8⁺ T lymphocytes (OT-I: CD8⁺ CD45.1⁻ V_α2⁺ V_β5⁺) expanded in the peritoneal cavity 5 days following vaccination with tumor cells secreting gp96-Ig; n = 8 from a pool of 3 experiments. (C) Tumor-reactive CD8⁺ T cells accumulated in secondary lymphoid organs 5 days following a second vaccination (13 days after HSCT) with gp96-Ig-secreting tumor cells; n = 3. (D) Vaccination with gp96-Ig-secreting tumor cells up to 2 weeks after HSCT resulted in expansion of cotransplanted tumor-reactive CD8⁺ T cells 5 days later; n = 3 to 8 from 3 independent experiments. (E) Tumor-reactive CD8⁺ T lymphocytes transitioned from central-memory (T_CM, CD62L⁺ CD44⁺) to effector cells (T_eff, CD62L⁻ CD44⁺) following vaccination with tumor cells secreting gp96-Ig: (left) representative dot plots; (right) n = 5 to 6 from a pool of 2 experiments. (F) Vaccination with gp96-Ig-secreting tumor cells induced highly IFN-γ⁺ tumor-reactive CD8⁺ T cells: (left) representative dot plots; (right) n = 4 from a representative of 3 experiments.