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Research Article: Hypoxic conditioning of neuroblastoma cells promotes CD8 + T-cell dysfunction through the HIF-1 ? /PD-L1 axis

Date Published: 2026-10-14

Abstract:
To investigate whether hypoxic conditioning of neuroblastoma (NB) cells induces CD8 + T cell dysfunction/exhaustion-like phenotype via the HIF-1 ? /PD-L1 axis and whether targeting this axis reverses immune suppression. Human NB cell lines SK-N-SH, SK-N-BE(2), and SK-N-AS were exposed to 1% O? hypoxia. HIF-1 ? and PD-L1 expression were assessed by Western blot, qRT-PCR, and flow cytometry. ChIP-qPCR and dual-luciferase assays were used to verify HIF-1 ? binding and transcriptional activation of the PD-L1 promoter. SK-N-SH cells pre-conditioned under normoxia or hypoxia were co-cultured with primary human CD8 + T cells. CD8 + T cell dysfunction/exhaustion-like phenotype (PD-1 + TIM-3 + ), apoptosis, IFN- ? , Granzyme B, CD107a, and specific cytotoxicity were evaluated. HIF-1 ? siRNA, YC-1, and PD-L1 neutralizing antibody were applied. The direct effect of YC-1 on T cells was also assessed. Hypoxia increased HIF-1 ? and PD-L1 expression in a time-dependent manner and also upregulated PD-L1 in additional NB cell lines. ChIP-qPCR and dual-luciferase assays confirmed direct HIF-1 ? binding to the HRE (5’-AGGTGC-3’) and transcriptional activation. PD-L1 elevation declined after reoxygenation. Hypoxia-preconditioned NB cells increased PD-1 + TIM-3 + CD8 + T cells (5.49%–32.24%) and apoptosis (10.61%–26.89%), while reducing IFN- ? + , Granzyme B + , CD107a + cells and specific cytotoxicity (79.94%–38.40%). HIF-1 ? knockdown or PD-L1 blockade alleviated these effects. YC-1 at 25 ?M did not significantly affect T cell viability or IFN- ? secretion. Hypoxic conditioning of NB cells stabilizes HIF-1 ? , enhances its binding to the PD-L1 promoter HRE, upregulates PD-L1, and induces CD8 + T cell dysfunction/exhaustion-like phenotype and impaired antitumor function. Targeting HIF-1 ? or PD-L1 partially restores T cell function in this co-culture model. The hypoxia-driven HIF-1 ? /PD-L1 axis contributes to immune suppression in NB and may be a potential therapeutic target.

Introduction:
Neuroblastoma (NB) is the most common extracranial solid tumor in children. It comes from nerve crest cells during sympathetic nervous system development and often grows in the adrenal gland or sympathetic ganglia. NB causes nearly 15% of childhood cancer deaths ( 1 – 3 ). Even with intensive treatments like surgery, chemotherapy, bone marrow ablation with stem cell support, and radiotherapy, the outcome for high-risk NB patients remains poor ( 4 , 5 ). Immune checkpoint inhibitors (such as anti-PD-1/PD-L1…

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