Immunophenotyping of CAR T cell differentiation using flow cytometry

Gating strategy showing the analysis of CAR T cell differentiation. Lymphocytes were identified by a SSC^low gate which also excluded debris (A). Dead cells were excluded by gating on 7-AAD⁻ cells (B). CD3⁺ cells were identified (C) and discriminated in CAR⁺ and CAR⁻ cells (D). Subsets of CAR⁻ T cells were further analyzed based on CD4 and CD8 expression (E). The differentiation state of CD4⁺CAR⁻ and CD8⁺CAR⁻ T cells was determined based on expression of CD45RO and CD62L. CD8⁺CAR⁻ cells are shown here as an example (F). e = effector, n = naive, cm = central memory, em = effector memory. CD8⁺CAR⁻ cells were further analyzed for CD45RA and CD45RO expression (G). The same differentiation gating strategy can be applied to CAR⁺ T cells to monitor their differentiation state (data not shown).