Figure 4.

Comparison of 2G-, 3G-, and 41BBL-CAR T Cell Effector Function In Vitro

(A) Schematic representation of the B7-H3-CAR with a CD8α/CD28 backbone combined with a 41BB endodomain (3G) or surface 41BB ligand (41BBL). (B) Representative flow plots of NT and transduced T cells. (C and D) IFNγ (C) and IL-2 (D) production after coculture with B7-H3-positive (LM7, A549, U373) or B7-H3-negative (LM7KO) tumor cells, or media alone. Media were collected after 24 h and cytokines were determined by ELISA (N = 4 in duplicate; blue asterisks, LM7KO versus LM7 for functional CARs; black asterisks and ns, CD8α/Δ-CAR versus functional CARs). (E and F) Repeat impedance-based cytotoxicity assay (xCELLigence) using LM7 cells as targets and CAR T cells as effectors (N = 4 in triplicate). First (E) and final (F) stimulation (black asterisks and ns, CD8α/Δ-CAR versus functional CARs; blue asterisks, 41BBL-CAR versus other functional CARs). One-way ANOVA was used for all analyses except for blue asterisks in (C) and (D) (two-way ANOVA). Data, mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ns, non-significant.