Neutrophils act as a “double-edged sword” in the tumor microenvironment by either supporting or suppressing tumor progression. Thus, eliciting a neutrophil antitumor response remains challenging. Here, we showed that tumor cell–derived microparticles induced by methotrexate (MTX-MP) acts as an immunotherapeutic agent to activate neutrophils, increasing the tumor-killing effect of the cells and augmenting T-cell antitumor responses. We found that lactate induced tumor-associated neutrophils to elevate expression of programmed cell death protein 1 (PD-1) and that PD-1+ neutrophils had the properties of N2 neutrophils and suppressed T-cell activation through PD-1/programmed death-ligand 1 (PD-L1) signaling. By performing ex vivo experiments, we found that MTX-MPs–activated neutrophils had reduced surface expression of PD-1 as a result of PD-1 internalization and degradation in the lysosomes, leading to the cells showing a decreased capacity to suppress T-cell responses. In addition, we also found that MTX-MP–activated neutrophils released neutrophil elastase which could kill tumor cells and disrupt tumor stroma, leading to increased T-cell infiltration. Furthermore, using a combination of anti–PD-L1 and MTX-MPs, we observed that long-term survival increased in a mouse model of lung cancer. Collectively, these findings highlight the potential use of a combination of anti–PD-L1 and MTX-MPs to enhance the therapeutic effect of anti–PD-L1 alone.

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