Antigen-specific CD4⁺ T cells express increasing levels of Foxp3 and PD-1 during tumor progression. The timeline of the procedure is outlined. A total of approximately 2.5 to 3.0 × 10⁶ HA-specific naive CD4⁺ T cells (Thy1.1⁺) were labeled with CFSE and adoptively transferred into BALB/c recipients with established A20-HA tumors. (A) At the indicated time points, spleen cells were subjected to fluorescence-activated cell-sorting (FACS) analysis to evaluate the frequency and cell-division status of the donor CD4⁺ T cells (Thy1.1 vs CFSE). Numbers represent percentage of donor CD4⁺ T cells in total spleen cells. Foxp3 and PD-1 expression profiles relative to cell division of the gated donor CD4⁺ T cells are shown. Numbers indicate the percentage of cells in the corresponding quadrant. (B) Costaining of PD-1 and Foxp3 is shown for divided donor CD4⁺ T cells. Numbers represent the percentage of each subpopulation in divided donor CD4⁺ T cells. Plots shown are representative of 2 independent experiments, with 4 to 6 mice per group. (C) OVA-specific CD4⁺ T cells up-regulate the expression of Foxp3 and PD-1 during tumor progression. Identical experiments were conducted with the use of OVA-specific CD4⁺ T cells purified from DO11.10 Tg mice and A20-OVA tumors. Tumor-free mice receiving T-cell transfer were included as control. At 15 days after T-cell transfer, spleen cells were subjected to analyses as described in panel A. Plots shown are representative of 2 independent experiments with 5 mice per group.