Hypoxic conditioning of neuroblastoma cells promotes CD8⁺ T-cell dysfunction through the HIF-1α/PD-L1 axis.
- 1Department of Pediatrics, the 960th Hospital of PLA Joint Logistics Support Force, Jinan, Shandong, China.
- 2Department of Oncology, the 960th Hospital of PLA Joint Logistics Support Force, Jinan, Shandong, China.
Abstract
To investigate whether hypoxic conditioning of neuroblastoma (NB) cells induces CD8+ T cell dysfunction/exhaustion-like phenotype via the HIF-1α/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% O₂ hypoxia. HIF-1α 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-1α 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-γ, Granzyme B, CD107a, and specific cytotoxicity were evaluated. HIF-1α 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-1α 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-1α 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-γ+, Granzyme B+, CD107a+ cells and specific cytotoxicity (79.94%-38.40%). HIF-1α knockdown or PD-L1 blockade alleviated these effects. YC-1 at 25 μM did not significantly affect T cell viability or IFN-γ secretion. Hypoxic conditioning of NB cells stabilizes HIF-1α, 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-1α or PD-L1 partially restores T cell function in this co-culture model. The hypoxia-driven HIF-1α/PD-L1 axis contributes to immune suppression in NB and may be a potential therapeutic target.
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BibTeX
@article{chao2026hypoxic,
title = {{Hypoxic conditioning of neuroblastoma cells promotes CD8⁺ T-cell dysfunction through the HIF-1α/PD-L1 axis.}},
author = {Yujin Chao and Guili Zheng},
journal = {Frontiers in pediatrics},
year = {2026},
month = oct,
doi = {10.3389/fped.2026.1923893},
url = {https://europepmc.org/article/MED/42952771},
note = {CC BY},
}
RIS
TY - JOUR
TI - Hypoxic conditioning of neuroblastoma cells promotes CD8⁺ T-cell dysfunction through the HIF-1α/PD-L1 axis.
AU - Yujin Chao
AU - Guili Zheng
PY - 2026
DA - 2026/10/14
JO - Frontiers in pediatrics
DO - 10.3389/fped.2026.1923893
UR - https://europepmc.org/article/MED/42952771
AB - To investigate whether hypoxic conditioning of neuroblastoma (NB) cells induces CD8+ T cell dysfunction/exhaustion-like phenotype via the HIF-1α/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% O₂ hypoxia. HIF-1α 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-1α 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-γ, Granzyme B, CD107a, and specific cytotoxicity were evaluated. HIF-1α 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-1α 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-1α 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-γ+, Granzyme B+, CD107a+ cells and specific cytotoxicity (79.94%-38.40%). HIF-1α knockdown or PD-L1 blockade alleviated these effects. YC-1 at 25 μM did not significantly affect T cell viability or IFN-γ secretion. Hypoxic conditioning of NB cells stabilizes HIF-1α, 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-1α or PD-L1 partially restores T cell function in this co-culture model. The hypoxia-driven HIF-1α/PD-L1 axis contributes to immune suppression in NB and may be a potential therapeutic target.
SN - 2296-2360
ER -