Functional Flow Cytometry to Predict PD‐L1 Conformational Changes

Abstract

AbstractThe 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 cytometryBasic Protocol 2: Protocol preparation, sample acquisition, and gating strategy for flow cytometric screening of PD‐L1+ myeloid‐derived suppressor cells in patients with lung cancerSupport Protocol 1: Bioinformatic tools for the analysis of flow cytometric data