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Updated: Jul 14, 2026
Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
Published on: September 28, 2018
Specific and high-affinity binding of tetramerized PD-L1 extracellular domain to PD-1-expressing cells: possible
Seigo Terawaki1, Yoshimasa Tanaka, Tomokazu Nagakura
1Department of Immunology and Genomic Medicine, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-Ku, Kyoto 606-8501, Japan.
Abstract:
The negative co-stimulatory receptor, programmed cell death 1 (PD-1), is induced on activated T cells and delivers inhibitory signals upon engagement with its ligands PD-L1 and PD-L2, which are expressed on various somatic cells and certain cancers. Accumulating evidence suggests that interfering with the PD-1-PD-L1 interaction may result in the restoration of defective T cell functions in cancer and chronic viral infection. Herein, we established procedures to produce large amounts of renatured recombinant extracellular domain proteins of mouse PD-1 (mPD-1) and PD-L1. While monomeric mPD-1 and mouse PD-L1 (mPD-L1) only marginally interacted with the cells expressing their counterpart proteins, their tetramerization markedly enhanced the affinity with the K(d) of mPD-L1 tetramer being nearly 100-fold lower than that of the corresponding monomer. The affinity of mPD-L1 tetramer was even higher than a high-affinity anti-PD-1 mAb, and it efficiently inhibited the binding of mPD-L1/Fc-chimeric protein to mPD-1(+) cells. Functionally, mPD-L1 tetramer significantly enhanced the proliferative responses as well as the cytotoxic activity of T cells against specific target cells in vitro. The results suggest that oligomeric PD-L1 extracellular domains may provide a potential means to restore T cell functions in cancer and viral infection in humans.
Insights
Tetramerized programmed cell death 1 ligand 1 (PD-L1) proteins significantly enhance T cell proliferation and cytotoxic activity. This finding suggests oligomeric PD-L1 may restore T cell functions in cancer and viral infections.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Programmed cell death 1 (PD-1) is a negative co-stimulatory receptor on T cells.
- PD-1 engagement with its ligands (PD-L1, PD-L2) inhibits T cell function.
- Interfering with PD-1/PD-L1 interaction can restore T cell function in cancer and chronic viral infections.
Purpose of the Study:
- To produce large amounts of renatured recombinant extracellular domain proteins of mouse PD-1 (mPD-1) and PD-L1.
- To investigate the effect of tetramerization on the binding affinity and function of PD-L1.
Main Methods:
- Production of renatured recombinant mouse PD-1 and PD-L1 extracellular domains.
- Assessment of monomeric and tetrameric mPD-1 and mPD-L1 binding affinity to cells.
- Evaluation of the inhibitory effect of mPD-L1 tetramer on PD-1/PD-L1 binding.
- In vitro assessment of T cell proliferation and cytotoxic activity.
Main Results:
- Tetramerization of mPD-1 and mPD-L1 markedly enhanced their binding affinity.
- The mPD-L1 tetramer exhibited a nearly 100-fold lower K(d) compared to its monomer.
- mPD-L1 tetramer showed higher affinity than a high-affinity anti-PD-1 mAb and inhibited PD-1/PD-L1 binding.
- mPD-L1 tetramer significantly enhanced T cell proliferation and cytotoxic activity in vitro.
Conclusions:
- Oligomeric PD-L1 extracellular domains demonstrate significantly enhanced binding affinity and functional activity.
- Tetramerized PD-L1 holds potential for restoring T cell functions in conditions like cancer and viral infections.
