Polyfunctional CD4⁺ T cells acquire a PD1-dominant dysfunctional phenotype in a tumor environment. Following the same experimental procedures depicted in Figure 1, tumor-bearing mice were treated with Cy followed by adoptive transfer of HA-specific CD4⁺ T cells. Seven days after T-cell transfer, spleen cells were collected for analysis. (A) Ex vivo staining for surface (s) and intracellular (intra) expression of CD40L in the transferred CD4⁺ T cells. Numbers represent the percentages of cells in the given quadrant. (B) CD40L surface expression after antigenic stimulation and its correlation with IFNγ. Purified CD4⁺ T cells were stimulated with peptide/APC for 4 hours, then stained for surface CD40L and intracellular IFNγ. Plot showing the costaining of CD40L and IFNγ is gated on the divided cells. Data shown are representative of 3 indepen-dent experiments with similar results. (C) The CFSE^lowCD40L^high cells were collected by FACS-sorting. The phenotype of these cells was documented (top panel). The sorted cells were reinfused to mice bearing 10-day-old A20HA tumors. Two weeks after T-cell transfer, the transferred CD4⁺ T cells were recovered from the spleens and assayed for the expression of the indicated markers (bottom panel). Results shown are representative of 2 independent experiments with similar results.