Figure 4.

IFN-γ- and TNF-α-pretreated HUVECs are vulnerable to CART123 in vitro. (A) Expression of CD123 on HUVECs after treatment with IFN-γ, TNF-α and neutralizing antibodies at different concentrations for 24 hrs (mean±SEM, n=3). Data show one representative experiment. (B) Expression of CD123 on HUVECs after treatment with IFN-γ, TNF-α, and IL-4 at different concentrations for 24 hrs (mean ±SEM, n=3). Data show one representative experiment. (C-D) In in vitro co-culture model, HUVECs were incubated with effector cells (CART123 or NT), target cells (MOLM-13), IFN-γ and TNF-α neutralizing antibodies, IL-4 (C) and tocilizumab (D) for 36 hrs and detected the CD123 expression by flow cytometry (mean±SEM, n=3). Data show one representative experiment. (E) Cytotoxicity of CART123 or NT at 6 hrs when co-cultured with HUVECs pretreated with cytokines (IFN-γ, TNF-α, and IL-4) for 24 hrs and treated with neutralizing antibodies at E:T of 5:1 was detected using 7-AAD and Annexin V (mean±SEM, n=3). Data show one representative experiment. (F) HUVECs (red) and CART123 or NT (green) were co-cultured for 24 hrs and recorded by Nikon Ti-E Inverted Live Cell Imaging System Manuals at 5% CO₂ and 37°C. The number of detached cells after co-culture for 24 hrs is shown (mean±SEM, n=2). (G) Cytotoxicity at 14 hrs of CART123 or NT when co-cultured with AML cell lines KG-1a and MOLM-13 treated with neutralizing antibodies at 5:1 E:T was detected using 7-AAD and Annexin V (mean±SEM, n=3). Data show one representative experiment. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Abbreviations: IFN, interferon; TNF, tumor necrosis factor, HUVECs, human umbilical vein endothelial cells; CAR, chimeric antigen receptor; NT, non-transduced T; 7-AAD, 7-Aminoactinomycin D; AML, acute myeloid leukemia; E:T, the effector to target cell ratio.