Fig 3.

The effect of anti-TGF-β on vaccine efficacy is not due to blocking Treg cells. TC1 cells (2 × 10⁴) were injected s.c. in the right flank of C57BL/6 mice. Four days after tumor challenge, some mice were immunized with the peptide vaccine as described in Fig 1. Some immunized mice were also treated i.p with 1D11 (100 μg) every other day from the time of immunization for two weeks. A. Four days or 13 days after tumor injection, tumor draining lymph node cells were recovered and stained with anti-CD3, anti-CD4, anti-CD25 and anti-Foxp3. The proportions of CD3⁺CD4⁺CD25⁺Foxp3⁺ cells were determined by flow cytometry. Each symbol represents one data point. Median are shown as bars. B. Sixteen days after tumor challenge, tumor infiltrating Tregs were examined by flow cytometry. Tumor infiltrating lymphocytes were recovered as described in the Materials and Methods section, stained with anti-CD3, anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3. Presented density plots were gated on the CD3⁺CD4⁺ population (upper row). Presented pseudo dot plots represented entire population (lower row). C. Some mice were treated with anti-CD25 (1 mg) five and three days before tumor injection. The experiment was terminated on day40 after tumor challenge. Each data point represents mean ± SD. All groups had 5 mice each. D. Eleven days after tumor challenge, tumor draining lymph node cells were used to examine Treg suppressive activity. Fifty thousand CD4⁺CD25⁻ cells with 5 × 10⁴ accessory cells with/without 1.25 × 10⁴ CD4⁺CD25⁺ Treg cells were stimulated with 0.5 μg/ml anti-CD3 for 72 hr. Cell proliferation was measured by ³H-thymidine incorporation, The % suppression was determined as described in the Materials and Methods section. These experiments were repeated at least twice with comparable results.