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Updated: Sep 5, 2025
Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Identification and characterization of novel CD274 (PD-L1) regulating microRNAs and their functional relevance in
Christoforos K Vaxevanis1, Michael Friedrich1, Sandy Uta Tretbar1
1Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle, Germany.
Background:
Immune checkpoint inhibitors directed against programmed cell death 1 (PDCD1/PD1) receptor and programmed cell death-ligand 1 (CD274/PD-L1) have been recently successfully implemented for the treatment of many cancers, but the response rate of tumour patients is still limited due to intrinsic and acquired resistances. However, the underlying molecular mechanisms of this limited response have still to be defined in detail. The aim of this study is to uncover processes inhibiting PDCD1/CD274 expression thereby enhancing anti-tumour immune responses. The identification and characterization of microRNAs (miRNAs) targeting the 3'-untranslated region (3'-UTR) as well as the coding sequence (CDS) of CD274 will provide the basis for a new drug development.
Methods:
Human melanoma cell lines and tissue samples were subjected to mRNA and/or protein expression analysis using qPCR, Western blot, flow cytometry, and/or immunohistochemistry. The data were correlated to clinical parameters. MiRNA trapping by RNA in vitro affinity purification (miTRAP) technology in combination with small RNA sequencing and different bioinformatics tools were employed to identify CD274-regulating miRNAs.
Results:
Screening based on miTRAP in combination with RNAseq identified a large number of novel CD274-regulating candidate miRNAs, from which eight selected miRNAs were functionally validated. Five out of eight miRNAs were able to significantly reduce CD274 surface expression indicating that these miRNAs directly bind to the 3'-UTR or CDS of the CD274 gene. The miRNA-mediated inhibition of CD274 expression was accompanied by an increased T cell recognition. Furthermore, an inverse expression of three CD274-regulating miRNAs and CD274 was demonstrated in melanoma lesions. A CD274 miRNA score was generated, which was associated with disease progression and reduced survival of melanoma patients.
Conclusions:
These data revealed a novel mechanism that miRNAs targeting the CDS of immune checkpoint genes are functional, have prognostic relevance, and also the potential for the development of novel miRNA-based therapies.
Insights
MicroRNAs targeting programmed cell death-ligand 1 (CD274/PD-L1) can enhance anti-tumour immunity by reducing CD274 expression. These findings offer potential for novel miRNA-based cancer therapies.
Area of Science:
- Immunology
- Oncology
- Molecular Biology
Background:
- Immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 improve cancer treatment but face resistance.
- Mechanisms underlying ICI resistance, particularly CD274 regulation, require detailed elucidation.
- Understanding CD274 regulation is crucial for enhancing anti-tumour immune responses.
Purpose of the Study:
- To identify microRNAs (miRNAs) that regulate programmed cell death-ligand 1 (CD274/PD-L1) expression.
- To investigate the functional impact of miRNA-mediated CD274 inhibition on anti-tumour immunity.
- To explore the therapeutic potential of miRNAs targeting immune checkpoints.
Main Methods:
- Utilized miRNA trapping by RNA in vitro affinity purification (miTRAP) and RNA sequencing to identify CD274-regulating miRNAs.
- Validated candidate miRNAs using qPCR, Western blot, flow cytometry, and immunohistochemistry in melanoma cell lines and tissues.
- Correlated miRNA and CD274 expression with clinical parameters and patient survival.
Main Results:
- Identified numerous novel miRNAs targeting CD274, with eight functionally validated.
- Five miRNAs significantly reduced CD274 surface expression by binding to its 3'-UTR or CDS.
- miRNA-mediated CD274 inhibition enhanced T cell recognition and showed prognostic relevance in melanoma patients.
Conclusions:
- Demonstrated that miRNAs targeting the coding sequence of immune checkpoint genes are functional and therapeutically relevant.
- Established a novel mechanism of miRNA-mediated regulation of CD274 expression.
- Highlighted the potential of miRNA-based therapies for overcoming ICI resistance and improving cancer treatment outcomes.
