Figure 2.

CD28 and 4-1BB signaling domains endow CAR-T cells with comparable function in vitro

(A) OVCAR-3 (left) and MESOV (right) target cells were co-cultured with T cells (no CAR), TAG-72.CD28 CAR, or TAG-72.4-1BB CAR at an E:T ratio of 5:1, and the response was monitored in real time using xCELLigence where a decrease in the normalized cell index is indicative of target cell death relative to target cells alone (black). (B and C) Additionally, OVCAR-3 (B) or MESOV (C) target cells were co-cultured with effector cells at E:T ratios of 1:1 and 1:5. Cytotoxic functions for all E:T ratios were characterized at 5, 10, 15, and 20 h of co-incubation and are presented here as % cytotoxicity. Biological and intra-assay triplicates were used and are denoted as average ± SD. (D) CD4⁺ and CD8⁺ CAR-T cells elicit a cytotoxic response to antigen exposure. CD4⁺ (left), CD8⁺ (middle), or a controlled CD4⁺/CD8⁺ ratio (right) of TAG-72.4-1BB CAR-T (Δ) cells were co-cultured with OVCAR-3 cells at an E:T ratio of 1:1. CD4⁺ or CD8⁺ T cells (no CAR) were cultured in parallel (blue). A reduction in normalized cell index relative to target cells alone (▪) demonstrates effector cell-mediated cytotoxicity. Numerical values embedded within each graph represent average percent cytotoxicity ± SD (n = 5) at 20 h relative to the target cells alone control. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.