Functional Flow Cytometry to Predict PD-L1 Conformational Changes
Roser Salvia1, Laura G Rico1, Michael D Ward2
1Functional Cytomics Lab, Germans Trias i Pujol Research Institute (IGTP), Universitat Autònoma de Barcelona, Badalona, Barcelona, Spain.
Abstract:
The programmed cell death protein 1/programmed cell death protein ligand 1 (PD-1/PD-L1) axis is one of the most widely recognized targets for cancer immunotherapy. Importantly, PD-L1 conformational changes can hinder target binding when living cells are used. Antibody affinity, equilibrium binding, association and dissociation rates, and other affinity-related constants are fundamental to ensure target saturation. Here, PD-L1 changes in conformation and their potential impact on PD-L1 function and mutation are explored. Specifically, we present detailed flow cytometry procedures to analyze PD-L1 reactivity in myeloid-derived suppressor cells (MDSCs). This approach can also be used to study the contribution of protein conformational changes in living cells. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for PD-L1+ myeloid-derived suppressor cells detection by flow cytometry Basic Protocol 2: Protocol preparation, sample acquisition, and gating strategy for flow cytometric screening of PD-L1+ myeloid-derived suppressor cells in patients with lung cancer Support Protocol 1: Bioinformatic tools for the analysis of flow cytometric data.
Insights
Programmed cell death protein 1 ligand 1 (PD-L1) conformational changes affect cancer immunotherapy. This study details flow cytometry methods to analyze PD-L1 reactivity in myeloid-derived suppressor cells (MDSCs), crucial for understanding target binding.
Area of Science:
- Immunology
- Molecular Biology
- Oncology
Background:
- The programmed cell death protein 1/programmed cell death protein ligand 1 (PD-1/PD-L1) axis is a key target in cancer immunotherapy.
- Conformational changes in PD-L1 can impede antibody binding and therapeutic efficacy in living cells.
- Understanding PD-L1 dynamics is crucial for optimizing immune checkpoint inhibitor therapies.
Purpose of the Study:
- To explore the impact of PD-L1 conformational changes on its function and potential mutations.
- To present detailed flow cytometry protocols for analyzing PD-L1 reactivity in myeloid-derived suppressor cells (MDSCs).
- To provide a methodology for studying protein conformational changes in living cells.
Main Methods:
- Detailed flow cytometry procedures for sample preparation, acquisition, and gating.
- Analysis of PD-L1 reactivity in myeloid-derived suppressor cells (MDSCs).
- Utilizing bioinformatic tools for flow cytometric data analysis.
Main Results:
- Established flow cytometry protocols to assess PD-L1 conformational changes and reactivity in MDSCs.
- Demonstrated the applicability of the method for studying protein conformational dynamics in live cells.
- Provided a framework for analyzing PD-L1 expression and function in the context of lung cancer.
Conclusions:
- Flow cytometry is a viable method for analyzing PD-L1 conformational changes and reactivity in MDSCs.
- This approach can enhance the understanding of PD-L1 function in cancer immunotherapy.
- The presented protocols can be adapted for studying other cell surface proteins and their conformational dynamics.
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