Functional Flow Cytometry to Predict PD-L1 Conformational Changes

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.