Pemetrexed combined with dual immune checkpoint blockade enhances cytotoxic T lymphocytes against lung cancer.

Chemotherapy in combination with immune checkpoint blockade (ICB) targeting to programmed death-1 (PD-1) or its ligand PD-L1 is one of the first-line treatments for patients with advanced non-small cell lung cancer (NSCLC). However, a large proportion of patients, especially those with PD-L1 negative tumors, do not benefit from this treatment. This may be due to the existence of multiple immunosuppressive mechanisms other than PD-1/PD-L1 axis. Human leukocyte antigen-G (HLA-G) has been identified as an immune checkpoint protein (ICP) and a neo-expressed tumor-associated antigen (TAA) in a large proportion of solid tumors. In this study, we evaluated the induction of HLA-G as well as PD-L1 by sub-lethal doses of chemotherapeutics including pemetrexed in different NSCLC cell lines. Except gefitinib, most of the chemotherapeutic agents enhanced HLA-G and PD-L1 expression in a dose-dependent manner, whereas pemetrexed and carboplatin treatments showed the most consistent upregulation of PD-L1 and HLA-G in each cell line. In addition to protein levels, a novel finding of this study is that pemetrexed enhanced glycosylation of HLA-G and PD-L1. Pemetrexed potentiated the cytotoxicity of cytotoxic T lymphocytes (CTLs) to treat NSCLC. Both in vitro and in vivo experiments revealed that the CTL-mediated cytotoxicity was most pronounced when both anti-PD-L1 and anti-HLA-G ICBs were combined to pemetrexed treatment. In conclusion, anti-HLA-G could be an intervention strategy in addition to anti-PD-1/PD-L1 pathway for NSCLC. Moreover, dual targeting of PD-L1 and HLA-G combined with pemetrexed may have a better extend of CTLs-based immunotherapy.