Tumor relapses after chemoimmunotherapy correlate with acquisition of the tolerized phenotype and loss of function in tumor-specific CD4⁺ T cells. The timeline of the procedures is outlined. Mice with established systemic A20HA tumors were not treated or treated as indicated. (A) The Kaplan-Meier plot depicts overall survival. The number of mice in each group is shown. (B) Retrospective correlation analysis of CD4⁺ T-cell frequency in peripheral blood and mouse survival. The frequencies of the transferred CD4⁺ T cells in peripheral blood were periodically monitored by FACS after mice received the combination treatment of Cy and CD4⁺ T-cell transfer. (C) Phenotype of the transferred CD4⁺ T cells in cured or relapsed mice. Spleen cells from cured mice or sick (relapsed) mice were analyzed as described in Figure 1C. (D) Costaining of PD-1 and IFNγ. Purified CD4⁺ T cells were stimulated with HA peptide-pulsed BALB/c splenocytes for 4 hours in the presence of GolgiStop. Plots shown are gated on divided donor CD4⁺ T cells. (E) IFNγ production by endogenous CD8⁺ T cells. Purified CD8⁺ T cells were assayed for IFNγ expression after 4 hours stimulation with PMA/ionomycin. CD8⁺ T cells from a naive mouse were used as control. Numbers represent the percentages of cells in the given quadrant. Results shown are representative of 3 independent experiments with similar results.