gp96 vaccines induce CD4⁺ and CD8⁺ tumor-specific T-cell responses. Balb/c mice (3 per group) were subcutaneously vaccinated twice with either normal gp96N or tumor-derived gp96A, gp96BC, or gp96BCD followed by challenge with tumor A. One week later, splenocytes were isolated, pooled, and restimulated with irradiated tumor-A cells for 5 days. Shown are the results of T cells from mice immunized with different gp96 vaccines. (A) Expression of CD25, CD69, CD62L, and CD44 by gated CD8⁺ T cells measured by flow cytometric analysis. Numbers inside represent mean fluorescence index (MFI). (B) Percentages of proliferative T cells measured by CFSE dilution assay on gated CD4⁺ and CD8⁺ T cells. (C) Percentages of IFN-γ or IL-4–positive T cells on gated CD4⁺ and CD8⁺ T cells. (D) Cytotoxicity of CTLs against tumor-A cells. Representative results of 3 independent experiments are shown. The error bars in panel D represent SD of 3 independent experiments.