Immune competence of T cells produced de novo in tumor-bearing mice. RAG-2–deficient BALB/c mice were challenged with 5 × 10⁶ J558 tumor cells or PBS as control. Two days later, the mice received 500 rad irradiation and infusion of nu/nu bone marrow cells or a mixture of bone marrow cells from P1CTL⁺ nu/nu and nu/nu mice. The recipient mice were killed at the fourth week and tumor-specific T cells analyzed for their phenotype and response to P1A antigenic peptide. (A) High frequency of P1A-specific T cells among the TILs of nu/nu bone marrow recipients. Left panels show representative FACS profiles depicting highly expanded tumor-specific T cells among the TILs (top left panel) but not in the spleen (bottom left panel). Middle panels depict control stains. A summary graph of P1A-reactive CD8 T cells among the splenocytes and TIL of nu/nu recipients is shown in the right panel. (B) Enhanced activation of P1A-specific CD8 T cells. cell-surface phenotype of P1A-reactive and nonreactive CD8 T cells in the spleens of tumor-bearing mice reconstituted with a 4:3 mixture of bone marrow from nu/nu and P1CTL⁺nu/nu mice. (C) Cytokine response of spleen cells in tumor-bearing mice. Spleen cells from tumor-bearing mice reconstituted with a mixture of nu/nu and P1CTL⁺nu/nu bone marrow were stimulated with P1A (left panels) or control peptides (right panels) in the presence of Golgi blocker for 6 hours and stained with anti–IFN-γ antibodies after fixation and membrane permeablization. Data shown are gated CD8⁺ cells and are representative of 3 independent experiments. (D) Accumulation and immune competence of P1A-specific T cells in the tumors. Top panels show accumulation (left) and activation status (right) of tumor-reactive T cells; the bottom panels show cytokine response to tumor antigenic peptide P1A. Tumor single-cell suspension from chimera mice reconstituted with P1CTL⁺nu/nu bone marrow were stimulated with P1A or control peptides in the presence of Golgi blocker for 6 hours and stained with anti–IFN-γ antibodies after fixation and membrane permeablization. Cytokine response is shown for gated CD8⁺ T cells. T-depleted splenocytes from syngeneic WT mice were used as antigen-presenting cells (APCs). All data presented in this figure have been repeated 2 to 3 times.