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. 2020 Sep;8(17):1048. doi: 10.21037/atm-20-4363

Figure 2.

Figure 2

Preparation of Anti-CD19 CAR T Cells. The CAR19 gene was composed with an anti-CD19 scFv (FMC63), an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB-derived costimulatory domain, and a CD3ζ intracellular domain. The 1904B gene was composed with an anti-CD19 scFv (FMC63), a CD8 hinge and transmembrane domain, a 4-1BB-derived costimulatory domain, and a CD3ζ intracellular domain. Both CAR19 and 1904B were combined with a truncated EGFR protein, which was linked by T2A sequence at the C-terminus of CAR. CAR-T cells were generated as previously described. In brief, CD3+ T cells were isolated from the peripheral blood mononuclear cells by CliniMACS CD3 (Miltenyi Biotec). T cells were activated with CD3/CD28 microbeads (Gibco), and cultured in TexMACS (Miltenyi Biotec) with 200 IU/mL of IL-2 (Miltenyi Biotec). Lentivirus-mediated CAR transduction was performed on day 2 post-activation. The composition and purity of the CAR-T cells were assessed by flow cytometry. The CAR-T cells were harvested on day 14. CAR, chimeric antigen receptor; IL-2, interleukin-2.