Suppression of NK cell-mediated immunosurveillance by IL-35 drives tumor progression in EGFR-mutant non-small cell lung cancer

Na Li¹Yinsong Zhang²Xiaojie Zhang³Haizhi Wang⁴Fang Yang⁵Fengju Zhou⁵Daqing Wang⁵Yan Zhang^6*

• ¹Department of Oncology, Hebei Medical University, Hebei Province, China Department of Oncology, Hengshui People's Hospital, Hebei Province, China, Harrison International Peace Hospital, Hengshui, China
• ²Department of Neurosurgery, Hengshui People's Hospital, Hebei Province, China, Harrison International Peace Hospital, Hengshui, China
• ³Department of Interventional Therapy, Hengshui People's Hospital, Hebei Province, China, Harrison International Peace Hospital, Hengshui, China
• ⁴Department of Laboratory Medicine, Hengshui People's Hospital, Hebei Province, China, Harrison International Peace Hospital, Hengshui, China
• ⁵Department of Oncology, Hengshui People's Hospital, Hebei Province, China, Harrison International Peace Hospital, Hengshui, China
• ⁶Department of Oncology, Shijiazhuang People's Hospital, Affiliated to Hebei Medical University, Hebei Province, China, Harrison International Peace Hospital, Hengshui, China

Background: Interleukin-35 (IL-35), a member of the IL-12 family composed of EBI3 and p35 subunits, is implicated in tumor immune evasion. This study investigates its expression and immunosuppressive role in EGFR-mutant non-small cell lung cancer (NSCLC), focusing on natural killer (NK) cells. Methods: IL-35 expression was assessed in 82 NSCLC tissues (47 EGFR-mutant, 35 wild-type) via immunohistochemistry. ELISA, Western blot, and PCR validated findings in fresh tissues. NK-cell infiltration was quantified as NKp46⁺ cells among CD45⁺ cells. The functional impact of IL-35 on healthy donor-derived NK cells was evaluated using proliferation, flow cytometry, cytokine ELISA, and cytotoxicity assays. In vivo studies utilized H1975 and PC-9 xenograft models. Results: IL-35 was significantly overexpressed in EGFR-mutant NSCLC tissues, correlating with larger tumor size and EGFR mutation status. High IL-35 expression was associated with poorer patient prognosis. A significant inverse correlation was found between IL-35 expression and NKp46⁺ cell density. In vitro, IL-35 suppressed NK cell function, reducing the proportion of CD3⁻CD56⁺ cells, NKG2D expression, and secretion of IFN-γ, perforin, and granzyme B. Conditioned medium from IL-35-treated NK cells enhanced the proliferation, invasion, and migration of NSCLC cells. In vivo, IL-35 promoted tumor growth in xenograft models, while IL-35 neutralization suppressed it and increased NK cell infiltration. Conclusion: IL-35 is upregulated in EGFR-mutant NSCLC and contributes to immune suppression by directly impairing NK cell activity. Targeting the IL-35 pathway represents a promising therapeutic strategy to restore NK cell function and anti-tumor immunity in this setting.