Bead-Based Multiplex Flow Cytometry Assay for Functional Profiling of CAR T Cells

Profiling CAR T cells presents challenges due to their heterogeneity, complex immune responses, and limited sample availability. Multiplex assays enable functional assessment by allowing the simultaneous detection of a broad range of effector molecules, including cytokines, chemokines, and cytotoxic mediators. Here, we describe a bead-based multiplex assay compatible with flow cytometry for the analysis of effector molecule secretion by human chimeric antigen receptor (CAR)-T cells. CAR T cell supernatants are incubated with a mixture of capture beads comprising multiple fluorescence-coded populations coupled to analyte-specific antibodies. These beads bind soluble targets in the sample, forming bead-analyte complexes that are subsequently detected using labeled detection antibodies. The assay includes a panel of immune mediators such as interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Granzyme B. The use of predefined standards allows quantitative measurement of multiple analytes from a single sample. Data acquisition and analysis can be performed using automated gating approaches provided by instrument-associated or web-based software tools, facilitating data processing. In conclusion, the presented multiplex assay enables multiparametric analysis of CAR T cell functionality and can be applied in contexts such as research, quality control testing, mechanistic studies, and functional characterization.