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. 2021 Oct 13;8:745286. doi: 10.3389/fmolb.2021.745286

FIGURE 3.

FIGURE 3

CAR19-T2A-aPD-1-PEBL construct effectively removes PD-1 from the T cells surface and releases PD-L1-mediated suppression. (A) Jurkat PD-1 cells were transduced by the different amounts of the CAR19-T2A-aPD-1-PEBL lentiviral supernatant and analyzed for PD-1 and CAR19 expression by flow cytometry. (B) Schematic representation of the Jurkat-Lucia™ NFAT PD-1 CAR19 reporter system. (C) Surface expression analysis of the PD-L1 on Nalm-6 and Nalm-6 PD-L1 cells. (D) CAR19 T cells activation luciferase assay. PD-1 positive and negative Jurkat-Lucia™ NFAT cells were modified by CAR19 or CAR19-T2A-aPD-1-PEBL and then co-incubated with Nalm-6 and Nalm-6 PD-L1. 48 h after co-incubation started, supernatant was collected and analyzed for luciferase activity. (E) Inhibitory effect of purified 102c3 VHH antibody on PD-L1 mediated suppression of Jurkat PD-1 CAR19 cells. Reporter Jurkat PD-1 CAR19 cells were cocultured with the Nalm-6 PD-L1 cells in presence of different concentrations of the 102c3 VHH. All data represent mean ± SD. The p values were determined by multiple t test. Statistical significance: wf****p < 0.000001.