The chA21-28z chimeric antigen receptor T cells cultured in vitro exhibit distinct populations defined by the differentiation markers CD45RO and CD62L. (A) CD3+ T cells were the predominant cell population after 2 weeks of expansion. On day 14, peripheral blood mononuclear cells (PBMCs) cultured in vitro from three different donors contained more that 95% CD3 + CD45+ T cells. (B) HER2-specific chimeric antigen receptor (CAR) expression was detected by staining T cells with F(ab′)2 antigen-binding fragment anti-mouse antibody. Both CD4+ and CD8+ cells expressed HER2-specific CAR. (C) The T lymphocytes cultured in vitro were gated on CD3+ T cells and analyzed using the differentiation markers CD45RO and CD62L. The phenotypes of the cultured T cells from representative donors at days 0 and 14 are shown in dot plots.