<scp>OMIP‐119</scp> : A 36‐Color Full‐Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Peripheral Blood and Ex Vivo Expanded Human T Cells

The increasing uptake of adoptive CAR-T and TCR-T cell therapies into clinical practice has intensified the need for robust and detailed immunophenotyping of ex vivo expanded T cells. We have developed and optimized an extended 36-color full-spectrum flow cytometry panel suitable for in-depth immunophenotyping of both peripheral blood and ex vivo expanded human T cells. Our panel allows for analysis of CD4⁺ and CD8⁺ memory subpopulations (Tn/Tscm, Tcm, Tem, and Temra), Treg subsets (CD45RA⁺ and CD39⁺), and helper T cell subsets (Tfh, Th1, Th2, and Th17). Intracellular staining facilitates the evaluation of functional markers granzyme B (cytotoxicity) and Ki-67 (proliferation). Our panel allows detailed investigation of T cell activation using markers carrying a range of expression kinetics. Exhaustion and senescence features, known to correlate with resistance to immunotherapies, can also be thoroughly evaluated. TCR staining with pHLA-dextramer or anti-TCR antibody is also included to allow evaluation of TCR specificity and/or transduction efficiency. A PD-1 receptor occupancy component enables quantification of T cells bound to antibodies from immune checkpoint inhibitor (ICI) therapies. Overall, we have generated a fully optimized and fit-for-purpose extended T cell profiling panel with particular relevance to the field of immunotherapies including ICIs and adoptive T cell therapies.