Development and Validation of a Multiplex Flow Cytometry‐Based PBMC Assay for Immunomodulator Screening

Abstract

The identification of novel compounds capable of stimulating or suppressing immune responses is critical for advancing immunotherapy and improving our understanding of immune regulation. However, traditional screening approaches are often limited by low throughput, poor physiological relevance, and insufficient resolution to detect subtle immunomodulatory effects. In this study, we introduce a validated and robust multiplexed flow cytometry‐based functional screening assay that enables efficient and precise identification of immunomodulatory compounds, while addressing the key limitations of conventional methods. By using human peripheral blood mononuclear cells (PBMCs) and defined stimulation conditions, the assay mimics complex immune interactions in a physiologically relevant context. It simultaneously monitors T‐ and B‐cell responses across multiple functional readouts, including T‐cell proliferation (carboxyfluorescein succinimidyl ester [CFSE]), activation (CD25, CD71), and B‐cell costimulatory marker expression (CD86), enabling an in‐depth assessment of immune modulation within heterogenous cell populations. The incorporation of robust statistical validation using the Z‐factor further supports assay reliability and reproducibility. In a pilot screen with 295 immunology‐related compounds, the assay demonstrated its ability to identify known immunomodulators with distinct functional profiles. This multiplex functional screening approach bridges the gap between mechanistic immunology and drug discovery by enabling systematic identification and characterization of immunotherapeutic candidates, while providing new insights into the complex dynamics of immune regulation.