Treatment of anti-ckit prevents the development of T-cell anergy in tumor-bearing mice. Thy1.2⁺ CD4 HA-specific TCR-transgenic T cells (5 × 10⁶/mouse) were injected via tail vein into congenic Thy1.1⁺ MCA26 or HA-MCA26 tumor-bearing mice 3 days after the first dose of anti-ckit or rat Ig injection (50 μg/mouse). At day 15 after transfer, Thy1.2⁺ splenocytes were recovered by sorting. (A) Proliferative responses of sorted Thy1.2⁺ CD4 HA-specific T cells to HA peptides. The culture was pulsed with [³H]-thymidine for the last 8 hours of a 72-hour culture. Stimulation index (SI) is calculated as the proliferation count (cpm) in the presence of HA peptide divided by that in the absence of HA peptide. Data shown are from a representative of 2 reproducible experiments (3 to 4 mice per group). *P <.001 (B) The residual tumor weight. The residual tumors were resected from the liver tissue and the tumor weight (gram) was measured. Error bars represent standard deviation. (C) The expression of Foxp3 in tumor (HA)–specific T cells. RNA was prepared from Thy1.2⁺ CD4 HA TCR transgenic T cells recovered from treated mice and Foxp3 expression was analyzed by one-step RT-PCR and real-time PCR. GAPDH expression was used as housekeeping gene control. (D) Intracellular staining of Foxp3 in tumor (HA)–specific T cells. Splenocytes were prepared from treated mice and stained with fluorochrome-conjugated anti-Thy1.2 (FITC) plus anti-CD4 (APC) plus anti-CD25 (PE-Cy7) plus anti-Foxp3 (PE). Thy1.2⁺CD4⁺ gated dot plots are presented. Naive mice with adoptively transferred T cells and rat Ig (left); tumor-bearing mice with adoptively transferred CD4 T cells and rat Ig control (middle); tumor-bearing mice with adoptively transferred T cells and anti-ckit (right). (E) Cytokine profile of tumor-specific T cells. Culture supernatants of recovered Thy1.2⁺ CD4 HA TCR transgenic T cells in the presence of HA peptide (5 μg/mL) and irradiated antigen-presenting cells (naive splenocytes) were collected. The naive CD4 HA TCR splenocytes cultured in the presence or absence of HA peptide were used as positive and negative controls, respectively. The cytokine concentrations were measured by ELISA kits (R&D Systems). The cytokine profile of supernatant collected from culture of tumor (HA)–specific T cells skewed toward a Th1 response with a higher concentration of IFN-γ and a lower concentration of IL-10 (*P < .05; Student t test).