FIGURE 1.

Design of bicistronic expression vector for CARs and catalase expression. (A) Schematic diagram depicting the two sets of CARs used. (B) PBMCs from healthy donors were cultured for a 4 d and transduced using either bicistronic retroviral expression vectors for CARs and catalase or retroviral expression vector for CARs alone. Expression of CARs on transduced T cells was assessed by staining with PE conjugated F(ab′)₂ anti-human IgG that binds to the extracellular Fc region of the CAR and allophycocyanin-conjugated anti-CD3. PE-conjugated isotype Abs were used to confirm lack of nonspecific binding. CAR cells were gated on lymphocyte population in forward scatter and side scatter prior to gating CAR⁺ cells. (C) Protein lysates from MACS-sorted transduced T cells were analyzed by Western blot. Relative protein expression was determined by ImageJ analysis of the intensity of the bands from the Western blot. (D) MACS-sorted lysates were used to measure catalase activity. (E) Luminescence from 10⁵ transduced or nontransduced cells was measured after adding L-012 and H₂O₂. (F) Transduced T cells were permeabilized and rabbit polyclonal anti-human catalase Ab was used to stain for intracellular catalase. FITC-conjugated anti-rabbit IgG Ab was used to analyze the samples with flow cytometry. Data are presented as means ± SD. ***p < 0.005 by Student t test using GraphPad Prism 5.