Cy-induced tumor antigen release and host immune milieu change both contribute to optimal effector differentiation of the donor cells. Following the experimental procedure depicted in Figure 2, we treated or did not treat mice with established A20-WT tumors with Cy 1 day before receiving HA-specific CD4⁺ T-cell transfer. For comparison, a third group of mice with A20-HA tumors were treated with Cy followed by adoptive CD4⁺ T-cell transfer. (A) Profiles of donor cell division, PD-1 and Foxp3 expression. At 7 days after T-cell transfer, mouse spleen cells were subjected to FACS analysis. The histograms are gated on the donor CD4⁺ T cells. Numbers in histograms represent the percentage of divided donor cells. The expression profiles of PD-1 vs CFSE and Foxp3 vs CFSE of the donor CD4⁺ T cells are shown in dot plots. Numbers in plots represent the percentage of PD-1^hi or Foxp3⁺ cells in the divided donor cells (gated) or undivided donor cells for the group with A20-WT tumors but without Cy treatment. Data shown are representative of 2 independent experiments with similar results. (B) Summary of the results shown in panel A. Horizontal bars represent mean values. (C) Phenotype comparison of donor CD4⁺ T cells in response to vaccinia virus immunization and Cy-conditioning in tumor-bearing mice. Mice with established A20-HA tumors were either immunized with vacHA, or treated with Cy, on day 17 after tumor inoculation, followed by adoptive CD4⁺ T-cell transfer the next day. At 7 days after T-cell transfer, mouse spleen cells were subjected to the same FACS analyses as in panel A. (D) Cytokine intracellular staining for donor CD4⁺ T cells from vacHA immunized vs Cy-conditioned A20-HA–bearing mice. Purified CD4⁺ T cells were stimulated with HA peptide-pulsed fresh splenocytes for 5 hours before ICS. Plots shown are gated on the divided donor CD4⁺ T cells. The percentage of cells in each quadrant is shown. Results shown in panels C and D are representative of 2 independent experiments with 5 mice per group.