Expansion of CD4⁺CD25⁺Foxp3⁺ T cells after treatment of tumor-bearing mice with HSC transplantation and posttransplantation vaccination. Irradiated tumor-bearing mice received a transplantation of bone marrow supplemented with 3 × 10⁶ T cells from tumor-bearing donors. The recipients were given 3 vaccines with irradiated AGN2a-4P cells as shown in Figure 1. Spleens were harvested on day 8, 11, 18, or 25 after transplantation and analyzed by flow cytometry. (A) The percentages of splenic CD25⁺Foxp3⁺ CD4 T cells were determined at each time point. (B) Foxp3⁺ and Foxp3⁻ CD4 T cells were analyzed for Ki-67 expression on day 8 after transplantation. Nontumor-bearing (naive) nontransplanted mice were included as controls. The numbers indicate percentages of cells in the respective quadrants. (C) Fluorescence histograms depicting cytolytic T lymphocyte–associated antigen-4 and GITR expression on electronically gated CD4⁺Foxp3⁺ splenocytes harvested on day 8 after transplantation. Naive nontransplanted mice were included as controls. (D) Expression of L-selectin and CD103 on gated CD4⁺Foxp3⁺ splenocytes harvested from HSC recipients on day 8 after transplantation or naive nontransplanted mice. The data are representative of at least 2 separate experiments with similar results.