Figure 3.

2G B7-H3-CAR T Cell Expansion, Basal Apoptosis, Cytokine Secretion, and Repeat Killing Capacity

2G B7-H3-CAR T cells were evaluated for in vitro expansion and effector function. (A) Expansion of NT and CAR T cells (N = 10). (B) Basal apoptosis of NT and CAR 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, CD8α/Δ versus functional CARs; red asterisks, CD8α/41BB-CAR or CD28/41BB-CAR versus CD8α/Δ-CAR in media alone or coculture with LM7KO). (E and F) Repeat impedance-based cytotoxicity assay (xCELLigence) using LM7 cells as targets and CAR T cells as effectors (N = 5 in triplicate). (E and F) First (E) and final (F) stimulation (black asterisks, CD8α/Δ-CAR versus functional CARs; blue asterisks or ns, CD28/41BB-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, not significant.