Figure 4. Cytokine release and cytotoxicity assay. Cytokine release in target cells in response to effector non-transduced T cells, con-CAR-T cells, EGFR-CAR-1 T cells, and EGFR-CAR-2 T cells. Effector cells were co-cultured with target cells (HS578T, MDA-MB-468, MDA-MB-231, and MCF-7) at an E:T ratio of 10:1 for 24h. (A) IFN-γ, (B) IL-4, and (C) IL-2 levels were assayed in the co-culture supernatants. Cytotoxicity was measured in each group using a standard LDH release assay. Effector cells were co-cultured with (D) HS578T, (E) MDA-MB-468, (F) MDA-MB-231, and (G) MCF-7 target cells at E:T ratios of 5:1, 10:1, or 20:1 for 24h.