OMIP-119: A 36-Color Full-Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Peripheral Blood and Ex Vivo...

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Adoptive cell therapies, including CAR-T and TCR-T cells, are increasingly used in clinical settings.
• Effective immunophenotyping of ex vivo expanded T cells is critical for optimizing these therapies.
• Existing methods may lack the depth and breadth required for comprehensive T cell analysis.

Purpose of the Study:

• To develop and validate an extended 36-color flow cytometry panel for in-depth immunophenotyping of human T cells.
• To enable detailed analysis of T cell subsets, functional markers, activation, exhaustion, and senescence.
• To support the evaluation of T cell receptor (TCR) specificity and immune checkpoint inhibitor (ICI) therapies.

Main Methods:

• Development and optimization of a 36-color full-spectrum flow cytometry panel.
• Application of the panel to both peripheral blood and ex vivo expanded human T cells.
• Inclusion of markers for T cell subpopulations, intracellular functions (granzyme B, Ki-67), activation, exhaustion, senescence, TCR specificity, and PD-1 receptor occupancy.

Main Results:

• The panel successfully characterizes CD4+ and CD8+ memory and helper T cell subsets (e.g., Tn/Tscm, Tcm, Tem, Temra, Tfh, Th1, Th2, Th17).
• It allows for intracellular assessment of cytotoxicity and proliferation markers.
• The panel effectively evaluates T cell activation kinetics, exhaustion, senescence, TCR specificity, and PD-1 receptor occupancy.

Conclusions:

• A robust and comprehensive T cell profiling panel has been established using extended 36-color flow cytometry.
• This panel is fit-for-purpose for detailed immunophenotyping relevant to adoptive T cell therapies and ICIs.
• The methodology provides critical insights for advancing immunotherapy research and clinical applications.