Tumor cell-derived microparticles induced by methotrexate augment T-cell antitumor responses by down-regulating expression of PD-1 in neutrophils.

Neutrophils act as a "double-edged sword" in the tumor microenvironment (TME) 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 (TANs) to elevate expression of PD-1 and that PD-1+ neutrophils had the properties of N2 neutrophils and suppressed T-cell activation through PD-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.