Related Experiment Video
Updated: Feb 16, 2026
Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
Published on: September 28, 2018
An optimized protocol for the in vitro generation and functional analysis of human PD1/PD-L1 signal
Mostafa Khedri1, Khalil Abnous2, Houshang Rafatpanah3
1a Department of Immunology, Faculty of Medicine , Mashhad University of Medical Sciences , Mashhad , Iran.
Abstract:
Programmed cell death-1 (PD1) is an inhibitory receptor expressed on the activated T and B cells. Binding of PD1 to its ligands, PD-L1 and PD-L2 has led to deliver an inhibitory signal into the activated T cells. Recently, blocking PD1/PD-L1 pathway has emerged as a new treatment paradigm across a broad spectrum of malignancies. Remarkable clinical responses of monoclonal antibodies specific for PD-1 or its ligands in patients with many different types of cancer, attracted several pharmaceutical companies and researchers to investigate the agents that block PD1/PD-L1 signal. The safety and efficacy of the agents are needed to examine in the preclinical studies. In this study, we optimized a facile and cost-effective protocol for in vitro generation and functional analysis of human PD1/PD-L1 pathway. Activation of CD8 + CD279 + T cell was performed by anti-CD3 and D28 antibodies and the recombinant PD-L1 was used for inactivation of T cells through PD1/PD-L1 pathway. In this protocol, T-cell cytokine production (IL-2 and IFN-γ) and proliferation assay confirmed that a measurable PD1/PD-L1 signal was generated. We expected that in vitro PD1/PD-L1 signal that has been optimized in this study will serve as a valuable protocol for preclinical studies involving PD1/PD-L1 pathway.
Insights
We developed a cost-effective method to study the Programmed Cell Death-1 (PD1) and its ligand PD-L1 pathway in vitro. This optimized protocol aids in preclinical research for cancer immunotherapies targeting the PD1/PD-L1 signal.
Area of Science:
- Immunology
- Cancer Biology
- Drug Discovery
Background:
- Programmed Cell Death-1 (PD1) is an inhibitory receptor on T and B cells.
- PD1/PD-L1 pathway binding delivers inhibitory signals, impacting T cell activity.
- Blocking the PD1/PD-L1 pathway is a promising cancer treatment strategy.
Purpose of the Study:
- To optimize a facile and cost-effective protocol for in vitro generation and functional analysis of the human PD1/PD-L1 pathway.
- To establish a reliable method for preclinical assessment of agents targeting the PD1/PD-L1 pathway.
Main Methods:
- Activation of CD8+CD279+ T cells using anti-CD3 and anti-CD28 antibodies.
- Utilized recombinant PD-L1 to induce T cell inactivation via the PD1/PD-L1 pathway.
- Assessed T-cell cytokine production (IL-2, IFN-γ) and proliferation to confirm pathway engagement.
Main Results:
- Successfully generated a measurable PD1/PD-L1 inhibitory signal in vitro.
- Confirmed pathway functionality through observed changes in T-cell cytokine production and proliferation.
- Demonstrated the protocol's ability to functionally analyze the PD1/PD-L1 pathway.
Conclusions:
- The optimized in vitro protocol provides a valuable tool for preclinical studies of the PD1/PD-L1 pathway.
- This method facilitates the investigation of novel immunotherapies targeting cancer malignancies.
- The cost-effective nature of the protocol enhances its accessibility for research.
