Quantification of PD-1/PD-L1 Interaction between Membranes from PBMCs and Melanoma Samples Using Cell Membrane Microarray and Time-Resolved Förster Resonance Energy Transfer

Abstract

Melanoma is a carcinoma known to evade the host immune defenses via the downregulation of the immune response. One of the molecules involved in this mechanism is programmed cell death ligand 1 (PD-L1), which interacts with its receptor, programmed cell death protein 1 (PD-1), expressed on T cells, leading to a reduction in cytokine release and cytotoxic activity, as well as a halt in T-cell proliferation. The approved therapeutic monoclonal antibodies, such as pembrolizumab, target the PD-1/PD-L1 interaction and are revolutionizing cancer treatments. We developed an assay that provides a quantitative readout of PD-1/PD-L1 interactive states between cell membranes of human immune cells (peripheral blood mononuclear cells, PBMCs) and PD-L1-expressing samples. For this purpose, cell membrane microarrays (CMMAs) were developed from membranes isolated from a HT144 cell line and melanoma samples, and PD-L1 expression was quantified using immunofluorescence (IF). CMMAs were incubated with cell membranes of PBMCs expressing PD-1, and the interaction with PD-L1 was quantified by time-resolved Förster resonance energy transfer, in the presence and absence of pembrolizumab as a blocking drug. The developed assay was able to quantify the PD-1/PD-L1 interaction, and this engagement was disrupted in the presence of the blocking antibody. This demonstrates the potential of the method to analyze monoclonal antibody drugs, as well as the functional states of immune checkpoint regulators. Furthermore, our findings provide evidence to support the future implementation of this methodology for both drug discovery and immune system monitoring in cancer, transplantation, and inflammatory and autoimmune diseases.

Elevation of Tear MMP-9 Concentration as a Biomarker of Inflammation in Ocular Pathology by Antibody Microarray Immunodetection Assays

“…Cell membranes, whole cells, antibodies, enzymes, nucleic acids, and other proteins can be immobilized on diverse surfaces using microarray technology without losing their functional structure. As a result, they are used in immunochemistry, autoradiography, radioligand and binding investigations, mitochondrial toxicity assays, as well as other techniques such as colorimetry and mass spectrometry [ 33 , 34 , 35 , 36 , 37 ]. Microarrays allow the reduction of the number of samples, medications, chemicals, and residues.…”

Study of Tissue-Specific Reactive Oxygen Species Formation by Cell Membrane Microarrays for the Characterization of Bioactive Compounds

“…This study has described the application of cell membrane microarrays to determine the specific actions of antimalarial drugs on human tissues by monitoring the production of superoxide, one of the main ROS, as indicator of celular stress. For this purpose, firstly, the experimental protocol was validated using CMMA from a set of rat brain areas, whose stability and functionality has already been confirmed by different methods, together with specific inhibitors of the oxidative phosphorylation [53][54][55][56][57].…”

Microarrays, Enzymatic Assays, and MALDI-MS for Determining Specific Alterations to Mitochondrial Electron Transport Chain Activity, ROS Formation, and Lipid Composition in a Monkey Model of Parkinson’s Disease

“…The tubes were finally decanted, and the pellets were frozen at −80 °C, except for one aliquot, which was used to determine the protein concentration. The protein concentration was determined using the Bradford method [ 63 , 64 ], and adjusted to the final concentration.…”