Figure 2.

Expression of anti-CD30 CARs. (A) Three CARs were compared. All three CARs had the 5F11 scFv. 5F11-28Z had hinge, transmembrane, and costimulatory domains from CD28 and a CD3ζ T cell activation domain. 5F11-CD828Z had hinge and transmembrane domains from the CD8α molecule, a CD28 costimulatory domain, and a CD3ζ T cell activation domain. 5F11-CD8BBZ was identical to 5F11-CD828Z, except a 4-1BB domain was included instead of a CD28 domain. (B) Human PBMCs were stimulated to proliferate with anti-CD3 in IL-2-containing medium. One day after culture initiation, the cells were transduced with vectors encoding the indicated CARs, or the cells were left untransduced. Six days after transduction, the cells were stained with antibodies against CD3, CD4, and CD8. Cells were also stained with Protein L to detect CARs. Plots are gated on live CD3⁺ lymphocytes and then on either CD4⁺ or CD8⁺ cells. The mean + SEM percentages of (C) CD4⁺ T cells and (D) CD8⁺ T cells that expressed the indicated CARs are shown. There was no statistically significant difference between any pair of CARs for either CD4⁺ or CD8⁺ T cells. The mean + SEM MFI of CAR expression is shown for (E) CD4⁺CAR⁺ T cells and (F) CD8⁺CAR⁺ T cells. p-values are shown on the graphs with brackets connecting the groups being compared by each p-value. For (C–F), statistical analysis was by Wilcoxon matched-pairs signed rank test; n = 9. For (C–F), correction for multiple comparison was performed by the Bonferroni method, so p < 0.017 was considered statistically significant. CAR, chimeric antigen receptor; MFI, median fluorescence intensity; scFv, single-chain variable fragment; SEM, standard error of the mean.