Expansion of Antigen-Specific CAR-T Cells

CAR-T cells were labeled with FITC-conjugated anti-human NGFR mAb (clone ME20.4) and isolated using anti-FITC microbeads (Miltenyi Biotec). Isolated CAR-library T cells were stimulated with the K562-based APCs established as above at an E/T ratio of 20:1^{35 (link)–37 (link)}. To expand the A2/NY-ESO-1₁₅₇-specific CAR-library T cells, A2-APCs were pulsed with 10 μg/mL NY-ESO-1₁₅₇ peptide, irradiated, and the floating peptide was removed to stimulate T cells. To expand the CD19-specific CAR-library T cells, CD19-APCs were utilized without loading any peptides. These CAR-library T cells were cultured in RPMI1640 supplemented with 50 μg/mL gentamicin and 10% human AB serum in the presence of 10 IU/mL human IL-2 and 10 ng/mL human IL-15 (PeproTech). Following three stimulations, antigen-specific CAR-library T cells were used for analyses. In some experiments, newly identified CD19 CAR-T cells as well as the original CD19 CAR-T cells were stimulated with Raji cells (E/T 1:1) or CD19-APCs (E/T 10:1). After stimulation, the proliferation activity of the CD19 CAR-T cells was assessed by calculation of the fold increase.