A human CXCL13-induced actin polymerization assay measured by fluorescence plate reader

Area of Science:

• Immunology
• Cell Biology
• Drug Discovery

Background:

• The chemokine receptor CXCR5 is crucial for B cell and T-helper cell function.
• CXCR5 and its ligand CXCL13 are implicated in ectopic germinal center formation in autoimmune diseases like rheumatoid arthritis, multiple sclerosis, and Sjogren's syndrome.
• Targeting CXCL13-induced chemotaxis presents a therapeutic strategy for these immune disorders.

Purpose of the Study:

• To develop a high-throughput, cost-effective actin polymerization assay for screening CXCR5-targeting therapeutics.
• To establish a surrogate readout for chemokine receptor activation that is more proximal than chemotaxis.
• To validate the assay's utility in drug discovery pipelines.

Main Methods:

• Development and optimization of a 96-well actin polymerization assay using standard fluorescence microplate readers.
• Optimization of cell density, actin staining reagents, staining buffer, and cell culture conditions.
• Pharmacological validation of the assay using human CXCR5 and its ligand CXCL13.

Main Results:

• A robust 96-well actin polymerization assay capable of processing 1,000–1,500 samples daily was established.
• The assay demonstrated expected pharmacology for human CXCR5.
• The assay proved suitable for primary screening of neutralizing scFv and secondary screening of small compound libraries.

Conclusions:

• The developed 96-well actin polymerization assay is a valuable tool for high-throughput screening of CXCR5-targeting agents.
• This assay facilitates the discovery of novel therapeutics for immune diseases driven by CXCR5/CXCL13 signaling.
• The assay offers a practical and efficient alternative to conventional methods for measuring actin polymerization.