Advanced Cytometry Techniques for Immune-Based Therapy Analysis 2308503

Abstract

As interest in CAR-T cells and immune-based oncology therapies rises, efficient phenotyping of cell therapy products becomes essential. This involves analyzing surface markers associated with cell type characteristics. To address this need, tools for panel design and instruments for cell interrogation must be compatible. This study highlights the combined use of an advanced panel builder to simplify assay creation with quantification using the iQue® HTS Cytometer Platform. A comprehensive T cell panel was developed using the panel builder, featuring phenotype, activation, exhaustion, and memory markers, with a cell viability indicator. Two panels were created: one for iQue® 3 and a more extensive panel for iQue® 5, utilizing the enhanced laser features. Compensation particles were used to calculate spillover values using iQue Forecyt® software before applying the panel to immune cells. Human PBMCs were cultured in RPMI 1640 media with 10% serum and IL-2, with or without CD3/CD28 activation beads to activate the T cell population. Cells were collected and analyzed in a 96-well plate at 48- and 72-hours post-activation. CD3+, CD4+, and CD8+ T cell populations were gated, and their markers were characterized. The iQue® 5 enhanced capabilities allowed for more comprehensive analysis of activation, exhaustion, and memory markers from one sample. Data showed an increase in activation markers, and a transient increase in exhaustion marker expression over time. In summary, the panel design tool, and the iQue® HTS Platform streamlines the creation of flexible panels for profiling immune cells. This combined approach is suitable for the generation of HTS data in 96- or 384- well throughput. The panel can be enhanced to include cytokine profiling with the iQue Qbead® immunoassay portfolio, utilizing the ability to track cell and bead populations within the same sample. n/a Translational and Interventional Immunology (TI)