Adoptive transfer of PD-1–deficient tumor-specific CD4⁺ T cells after Cy leads to durable antitumor effects. (A) Adoptive transfer of PD-1–deficient tumor-specific CD4⁺ T cells after chemotherapy. Following the depicted procedures, mice with large established subcutaneous A20HA tumors were treated with Cy, followed by adoptive transfer of PD-1–deficient HA-specific CD4⁺ T cells (PD1KOCD4). Tumor growth kinetics was monitored over time. The numbers indicate cured mice versus total mice in each group. (B) Protection from tumor rechallenge in cured mice. Cured mice were rechallenged with A20HA tumors on the flank opposite to the original tumor inoculation site. Ten naive BALB/c mice were also inoculated with tumors as controls. Graph depicts the tumor size of each mouse 20 days after tumor inoculation. (C) Persistence of transferred PD-1–deficient tumor-specific CD4⁺ T cells in cured mice. Mice protected from tumor rechallenge were killed and spleen cells were collected to examine the phenotype of the transferred CD4⁺ T cells. Representative plots show the expression profiles of the indicated markers. Numbers indicate the percentage of the gated population. (D) Requirement for endogenous CD8⁺ T cells for the curative outcome of chemoimmunotherapy. Tumor-bearing mice were injected with CD8-depleting antibody 1 day before receiving Cy-treatment and subsequent adoptive transfer of PD-1–deficient HA-specific CD4⁺ T cells. Tumor size was measured twice a week.