Figure 4.

Functional evaluation of CARs from novel binders on HBsAg-expressing target cells. (A) CAR-transduced T cells were co-cultured at indicated effector-to-target (E:T) ratios on HepG2, HepG2-SML, or HepG2-2.15 cells. IFNγ secretion was measured via ELISA after 72h. Data points represent mean values ± SD from triplicates. (B) CAR-transduced T cells were pre-incubated with CellTrace Violet (CTV) and cultured at an E:T ratio of 2:1 on indicated target cells. Proliferation as a measure of CTV decrease was observed by flow cytometry after 72h. Histograms are representatives of duplicates. (C) Intracellular cytokine staining of CAR-transduced T cells co-cultured at an E:T ratio of 2:1 for 48h on indicated target cells (2 µg/ml Brefeldin A added after 24h). Statistical differences were calculated using unpaired student t-test; *=p≤0.05, **=p≤0.01, ***=p≤0.001. (D) Polyfunctionality of CAR-transduced T cells derived from intracellular cytokine staining described in (C), indicating single (SP), double (DP), triple (TP), or quadruple positive (QP) populations of CD4⁺ or CD8⁺ CAR-transduced T cells incubated on HepG2-SML cells. (E) xCELLigence cytotoxicity assay of the different CAR-transduced T cells co-cultured at various E:T ratios for 96h with the indicated target cells. Data points represent mean values from triplicate analyses.