TGF-β1 drives neutrophil extracellular traps formation to promote CD8 ⁺ T cell exhaustion via the ERK-c-Fos-JunB axis, mediating gastric cancer immunotherapy resistance

Background Resistance to anti-programmed cell death protein-1 (PD-1) treatment in gastric cancer (GC) is closely associated with an immunosuppressive tumor microenvironment. However, the role of neutrophils in resistance to anti-PD-1 therapy remains unclear. Methods Single-cell RNA sequencing was performed on tumor samples from patients with advanced GC receiving anti-PD-1 therapy to identify neutrophil subsets associated with neutrophil extracellular traps (NETs). Multilevel experimental validation was conducted using multiomics analysis, flow cytometry, multiplex immunofluorescence, and in vitro co-culture. Therapeutic strategies targeting NETs and CD8 + T-cell exhaustion were evaluated in a mouse model of YTN16 tumors. Results We identified a NETs-associated neutrophil subset enriched in patients with GC resistant to anti-PD-1 treatment. This subset was marked by CD177, and it exhibited a high potential for NETs release. Peripheral blood NETs levels and CD177 + neutrophil ratios in patients with GC act as markers for evaluating the efficacy of PD-1 inhibitors. Furthermore, transforming growth factor-β1 (TGF-β1), which was highly expressed in GC and spatially colocalized with CD177 + neutrophils, might induce neutrophils to release NETs via the Smad3-NFE2 axis. NETs promoted CD8 + T cell exhaustion by activating the MEK/ERK-c-Fos/JunB axis, as evidenced by increased PD-1/TIM3 expression and reduced interferon-gamma/tumor necrosis factor-alpha secretion. In vivo experiments confirmed that targeted inhibition of NETs formation using DNase I or TGF-β1 inhibitors significantly suppressed tumor growth and CD8 + T cell exhaustion. Notably, the MEK inhibitor trametinib reversed the immunosuppressive microenvironment associated with CD8 + T cell exhaustion and synergistically enhanced the antitumor efficacy with anti-PD-1 therapy. Conclusions TGF-β1 drives CD177 + neutrophils to release NETs, which induce CD8 + T cell exhaustion via the ERK-c-Fos-JunB pathway, thereby mediating resistance to anti-PD-1 treatment in GC. Furthermore, targeting NETs formation and combining trametinib with PD-1 inhibitors can significantly reverse CD8 + T cell exhaustion, exert synergistic antitumor effects, and offer a potential therapeutic strategy for overcoming resistance to anti-PD-1 therapy in GC.