MBTP 34: Immunophenotyping of CAR T cell persistence and differentiation in blood samples using flow cytometry

Miltenyi Biotec-tested panel 34 (MBTP 34)

This application protocol describes the characterization of CAR T cell persistence and differentiation using flow cytometry. For this, CAR T cells were spiked into whole blood. The data was analyzed using the automated data acquisition and analysis tool Express Mode “CAR_T_Cell_Persistence_h_01”. 


Gating strategy showing the analysis of persistence and differentiation of CAR T cells. Samples were initially gated on lymphocytes based on SSC-A/FSC-A gating (A). Cells were further gated on live cells using 7-AAD-negative gating (B). T cells were gated as CD3+ (C). CAR T cells can be selected using CAR Detection Reagent-based gating (D). Subsets of CAR T cells were further analyzed based on their CD4 and CD8 expression (E). The differentiation state was determined based on expression of CD45RO, CD62L, and CD45RA by CD4+ CAR T cells (F, G) and CD8+ CAR T cells (H, I). N = naive, CM = central memory, E = effector; EM = effector memory. Same differentiation gating strategy can be applied to CAR+ T cells to monitor their differentiation state (data not shown).


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