Effects of vaccination on tumor infiltrating CTL and T_reg.

Infiltration of tumor tissue by CD8+ and Treg cells was analyzed by flow cytometry. Representative histogram (A; CD8+) and dot plots (B and C; gating for Treg) are shown. CD8+ and CD4+ fraction and CD25+FoxP3+ fraction were gated on lining cells and CD4+ cells, respectively. After preparation of a single cell suspension from resected tumors, cells were stained either with an anti-CD8 (A and D) or Treg antibody pannel (CD4+, CD25+, FoxP3+; B, C and E). Cell subsets were calculated as a percent of positive cells (CD8+ or CD4+CD25+FoxP3+) within the living cell fraction. Values given are means ± SEM, n = 4.