T lymphocytes from B-CLL patients expressing the κ-specific CAR kill primary κ⁺ B-CLL cells and expand when CD28 endodomain is present in the CAR. CD3⁺ T lymphocytes were isolated from patients with κ⁺ B-CLL. After activation with CD3/CD28 antibodies, T lymphocytes were transduced either with CAR46ζ or CAR46/28ζ or control GFP vector. After transduction, T lymphocytes were stained either with CD4-PE or CD8-PE antibodies and with Fc-γCy5 antibody to detect the expression of the CAR. Panel A illustrates the profile for Fc-γCy5 antibody in control T lymphocytes (top panels) and CAR46/28ζ⁺ T lymphocytes (bottom panels). Both CD4 and CD8 cells were transduced. Control, CAR46ζ⁺ and CAR46/28ζ⁺ T lymphocytes were stimulated weekly with autologous B-CLL cells (ratio, 1:1) without exogenous cytokines. Panel B illustrates that only CAR46/28ζ⁺ T lymphocytes expanded (for at least 3 weeks) after antigenic stimulation. Panel C shows that CAR46/28ζ⁺ T lymphocytes (▪) released significantly more IFN-γ (P = .01) and IL-2 (P = .02) than CAR46ζ T cells (▦) or control T cells (□) after stimulation with autologous B-CLL cells. Data represent mean ± SD of 4 different donors. The specificity of expanded T lymphocytes was then evaluated using a CFSE-based cytotoxicity assay (E/T ratio, 20:1). Panel D illustrates that CAR46/28ζ T lymphocytes (▪) killed autologous and allogeneic κ⁺ B-CLL cells, but not allogeneic κ⁺ B-CLL cells. In contrast, no significant killing was observed for control transduced T lymphocytes expanded in the presence of exogenous IL-2 (□). Data represent mean ± SD of 3 different donors.