PD-1 blockade after chemoimmunotherapy markedly reduces the occurrence of relapse. (A) PD-1 blockade in combination with Cy and adoptive transfer of tumor-specific CD4⁺ T cells. Following the timeline of the procedures outlined, mice with localized large A20HA tumors were treated with Cy followed by HA-specific CD4⁺ T-cell transfer. Some mice were injected with a mixture of αPD-1 and αPD-L1 blocking antibodies (100 μg of each) for 5 consecutive days with the first injection given right after T-cell transfer. Tumor growth kinetics in mice was monitored. The results for mice receiving Cy plus CD4⁺ T-cell transfer without and with PD-1 blockade are depicted in the left graph and the right graph, respectively. Asterisks in the left graph mark 2 mice whose tumors initially regressed and became impalpable, but later regrew on the original location. (B) Tumor growth curves in control mice receiving Cy and PD-1 blockade. A group of tumor-bearing mice were treated with the combination of Cy and PD-1 blockade following the same administration schedule depicted in panel A schema. (C) PD-1 blockade in combination with Cy and adoptive transfer of vaccine-primed polyclonal CD4⁺ T cells. Following the procedures depicted in the schema, tumor-reactive CD4⁺ T cells were obtained from the DLNs of tumor-free mice that had been vaccinated with irradiated GMCSF-producing A20 tumors (GVAX). Tumor-reactivity of these CD4⁺ T cells were evaluated by incubating aliquot of vaccine-primed CD4⁺ T cells (GVAX-CD4) with irradiated A20 tumor cells overnight before conducting IFNγ ICS. Naive CD4⁺ T cells were cultured the same way and used as control. Dot plots shown represent the ICS results. Numbers represent the percentages of the gated cells. Purified vaccine-primed CD4⁺ T cells were expanded in vitro, and then adoptively transferred to Cy-treated tumor-bearing mice, with or without subsequent PD-1 blockade as described in panel A. Tumor growth curves in mice of each group are shown in panel D.