Fig. 4

The PD-1 blocking scFv secreted by CD133 CAR-T and PD-1 s can bind bystander PD-1 + T cells and restore T cell function. A Schematic diagram of the PD-1 overexpression lentivirus with a GFP flag and a puromycin selection marker. B Flow cytometry analysis of PD-1 and GFP expression in T cells or T cells transfected with a PD-1 overexpression lentivirus and then selected with puromycin. C Flow cytometry analyses of the secreted PD-1 blocking scFv binding to normal T cells or PD-1 T cells. D Cellular immunofluorescence assay of PD-1 (GFP) and PD-1 blocking scFv (anti c-Myc-tag-647, Red) expression on normal T cells, PD-1 T cells, and PD-1 T cells incubated with the condensed supernatant from CD133 CAR-T and PD-1 s cells. E Representative flow cytometric images of granzyme B and TNFα expression in PD-1 T cells, PD-1 T cells incubated with Hep3B, and PD-1 T cells incubated with Hep3B and PD-1 blocking scFv. F, G Quantification of the frequency of granzyme B/PD-1 T cells and TNFα/PD-1 T cells by flow cytometric analysis of PD-1 + T cells, PD-1 + T cells incubated with Hep3B cells and PD-1 + T cells incubated with Hep3B cells and PD-1-blocking scFv secreted by CD133 CAR-T and PD-1 s cells. Mean ± SD, n = 3, two-tailed unpaired Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant