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Surface PD-1 expression in T cells is suppressed by HNRNPK through an exonic splicing silencer on exon 3

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Abstract

Objective

Immunotherapy targeting programmed cell death 1 (PDCD1 or PD-1) and its ligands has shown remarkable promise and the regulation mechanism of PD-1 expression has received arising attention in recent years. PDCD1 exon 3 encodes the transmembrane domain and the deletion of exon 3 produces a soluble protein isoform of PD-1 (sPD-1), which can enhance immune response by competing with full-length PD-1 protein (flPD-1 or surface PD-1) on T cell surface. However, the mechanism of PDCD1 exon 3 skipping is unclear.

Methods

The online SpliceAid program and minigene expression system were used to analyze potential splicing factors involved in the splicing event of PDCD1 exon 3. The potential binding motifs of heterogeneous nuclear ribonucleoprotein K (HNRNPK) on exon 3 predicted by SpliceAid were mutated by site-directed mutagenesis technology, which were further verified by pulldown assay. Antisense oligonucleotides (ASOs) targeting the exonic splicing silencer (ESS) on PDCD1 exon 3 were synthesized and screened to suppress the skipping of exon 3. The alternative splicing of PDCD1 exon 3 was analyzed by semiquantitative reverse transcription PCR. Western blot and flow cytometry were performed to detect the surface PD-1 expression in T cells.

Results

HNRNPK was screened as a key splicing factor that promoted PDCD1 exon 3 skipping, causing a decrease in flPD-1 expression on T cell membrane and an increase in sPD-1 expression. Mechanically, a key ESS has been identified on exon 3 and can be bound by HNRNPK protein to promote exon 3 skipping. Blocking the interaction between ESS and HNRNPK with an ASO significantly reduced exon 3 skipping. Importantly, HNRNPK can promote exon 3 skipping of mouse Pdcd1 gene as well.

Conclusions

Our study revealed a novel evolutionarily conserved regulatory mechanism of PD-1 expression. The splicing factor HNRNPK markedly promoted PDCD1 exon 3 skipping by binding to the ESS on PDCD1 exon 3, resulting in decreased expression of flPD-1 and increased expression of sPD-1 in T cells.

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Data availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

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Funding

This research was funded by the National Natural Science Foundation of China, grant number 81970933, the Fundamental Research Funds for the Central Universities, grant number, 2042023kfyq02 and the International Scientific Collaboration Project of Hubei Province, grant number 2022EHB036.

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Author notes

  1. Jiayun Wang and Lingyan Yan have contributed equally to this study and share first authorship.

Authors and Affiliations

  1. State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University, Wuhan, 430079, China

    Jiayun Wang, Lingyan Yan, Xu Wang, Rong Jia & Jihua Guo

  2. RNA Institute, Wuhan University, Wuhan, 430072, China

    Rong Jia

  3. Department of Endodontics, School & Hospital of Stomatology, Wuhan University, Wuhan, 430079, China

    Jihua Guo

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  1. Jiayun Wang
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  2. Lingyan Yan
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  3. Xu Wang
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  4. Rong Jia
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Contributions

Conceptualization, J.G. and R.J.; methodology, J.W. and L.Y.; validation, J.W. and L.Y.; formal analysis, J.W.; investigation, J.W. and L.Y.; resources, J.G. and R.J.; data curation, J.W. and L.Y.; writing—original draft preparation, J.W. and L.Y.; writing—review and editing, J.G. and R.J.; visualization, J.W.; supervision, J.G. and R.J.; project administration, J.G. and R.J.; funding acquisition, J.G. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Jihua Guo.

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Wang, J., Yan, L., Wang, X. et al. Surface PD-1 expression in T cells is suppressed by HNRNPK through an exonic splicing silencer on exon 3. Inflamm. Res. (2024). https://doi.org/10.1007/s00011-024-01887-4

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  • DOI: https://doi.org/10.1007/s00011-024-01887-4

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