Figure 2.

Functional assay of Lp-2-CAR-T cells in vitro

(A) PDPN-positive glioma cells (LN229/hPDPN) are lysed significantly by Lp2-CAR–T compared with peripheral blood mononuclear cells (PBMCs) and mock vector-transduced T cells (mock CAR-T) in an effector:target (E:T) ratio-dependent manner; however, specific lysis was not observed in PDPN-deficient glioma cells. Mock versus Lp2-CAR-T, p < 0.05 for ET ratio of 12 or more. Means and standard deviation (SD) of 3 wells of the same experiment are shown. (B) Interferon (IFN)-γ release (enzyme-linked immunosorbent assay [ELISA]). The co-culture of LN229/hPDPN or TGS01 with Lp2-CAR-T produced approximately 90–100 pg/mL IFN-γ. The IFN-γ levels were significantly higher than those from mock CAR-T cells (p < 0.05). LN229 and MeT-5A cells produced only a similar amount of IFN-γ when co-cultured with both Lp2-CAR-T and mock CAR-T cells. Means and SD are shown. (C) PDPN-expressing glioma cells (LN229/hPDPN and LN319) showed significantly higher cytolysis by Lp2-CAR-T when compared with PBMCs added at the same E:T ratio (6); ∗p < 0.05, for each hour starting at 4 h after addition of CAR-T cells. However, LN229 cells (which do not express PDPN) did not show any specific cytolysis by Lp2-CAR-T when compared with PBMCs. Lp2-CAR-T cells also showed significant cytolysis in patient-derived glioma stem cells, GIC0222 (∗p < 0.05 after 5 h) and TGS01 (∗p < 0.05 after 4 h). Both stem cells express PDPN. The means and standard error of the mean (SEM) of three wells of the same experiment are shown.