PD-1^hi effector cells that develop in tumor-bearing mice selectively down-regulate IL-7R and undergo apoptosis. Following the same experimental procedure depicted in Figure 2, we transferred Thy1.1⁺ CD4⁺ T cells derived from HA-TCR Foxp3^GFP double transgenic mice into BALB/c mice with established A20-HA tumors, with or without Cy preconditioning of the hosts. Donor cells were recovered 7 days after transfer and analyzed by FACS. (A) Expression of PD-1 and Foxp3(GFP) on donor CD4⁺ T cells. Costaining of PD-1 and Foxp3(GFP) on donor CD4⁺ T cells before transfer is included for comparison. Numbers represent the percentage of cells in each quadrant in donor CD4⁺ T cells. (B) Representative expression profiles of CD127 and annexin V on non-Treg donor cells. The Foxp3(GFP)-negative cells in each sample are gated and evaluated for expression of PD-1 vs CD127 and PD-1 vs annexin V. Numbers represent the percentage of cells in each quadrant in the Foxp3⁻ donor CD4⁺ T cells. The results of all samples are presented as percent of CD127^hi or percent of annexin V^hi cells in Foxp3⁻ donor CD4⁺ T cells and shown in scatter plots. Horizontal bars represent mean values. (C) PD-1–deficient CD4⁺ T cells have accelerated cell divisions. CFSE-labeled, PD-1–sufficient (PD-1WT) or PD-1–deficient (PD-1KO) HA-specific CD4⁺ T cells (Thy1.1⁺) were transferred into BALB/c mice with A20-HA tumors established 10 days earlier. At 7 days after T-cell transfer, spleen cells were collected, enumerated, and subjected to FACS analysis. Cell division status is reflected by the CFSE profile gated on Thy1.1⁺ donor CD4⁺ T cells. The numbers in each histogram represent the percentage of highly divided (> 5 divisions), divided (1-5 divisions), and undivided donor cells. (D) PD-1–deficient CD4⁺ T cells fail to accumulate in mice. The bar graph shows the absolute number of donor CD4⁺ T cells recovered from spleen (total splenocyte count × percent donor CD4⁺ T cells). Data are from 2 independent experiments and shown as mean ± SD of 4 to 5 mice per group. (E) Representative expression profiles of PD-1 and Foxp3 relative to cell division of the donor CD4⁺ T cells. Numbers in Foxp3 vs CFSE dot plots represent the percentage of Foxp3⁺ cells in the highly divided donor cells (gated). The results of all samples are summarized in bar graph. Data are shown as mean ± SD of 4 mice per group. (F) Cytokine intracellular staining for recovered donor CD4⁺ T cells. Purified donor CD4⁺ T cells were stimulated with HA peptide-pulsed fresh splenocytes for 5 hours before intracellular staining for IFN-γ and TNF-α. Plots shown are gated on the divided donor CD4⁺ T cells. The numbers represent the percentage of cells in each quad in divided donor CD4⁺ T cells. Results shown are representative of 2 independent experiments with similar results.