Pe conjugated anti cd27

To measure CAR expression, cells were first suspended in 100 μL of cold buffer (PBS containing 0.5% BSA and 2 mM EDTA) supplemented with 2 μg of goat IgG (Jackson Immunoresearch, West Grove, PA, USA) and incubated on ice. CD19-FLAG CAR-T cells were stained with 2 μL of PE- or Alexa Fluor 488-conjugated anti-FLAG (Biolegend), whereas BCMA CAR-T cells were first stained with 0.4 μg of BCMA-huFc protein (Acro Biosystems, Newark, DE, USA) and then stained with 1 μL of PE- or Alexa Fluor 488-conjugated goat anti-human IgG (Jackson Immunoresearch). Cells were co-stained with either FITC-conjugated anti-CD4 and APC-conjugated anti-CD8; PE-conjugated anti-CD27 and APC-conjugated CD45RO; or APC-conjugated anti-PD-1 (all from Biolegend). Dead cells were identified with 7-aminoactinomycin D (7-AAD, BioLegend). The cells were rinsed with 3 mL of buffer, then suspended in buffer and acquired on a FACSCalibur (BD Biosciences, San Jose, CA, USA). Gating strategies are shown in Supplemental Figure S1.