Fig. 4

Expression of MUC21 on the cell membrane suppresses the cytotoxic functions of anti-CD19 CAR-T and CAR-NK cells. (A-D) CD3⁺T cells isolated from human PBMCs were stimulated with anti-CD3/CD28 beads and transduced with anti-CD19 CAR lentivirus. CD19 CAR-T cells were co-incubated with Raji-tet-MUC21 cells at variable E:T ratios for two days in the presence or absence of Dox. (A) Schematic illustration of the killing of Raji-tet-MUC21 cells by CD19-CAR T or NK cells. (B) FACS analysis of the expression of anti-CD19 CAR on T cells using biotinylated CD19 protein. (C) The percentages of killing of Raji-tet-MUC21 cells by CD19 CAR-T cells were determined at the indicated E:T ratios. (D) Representative FACS plots showing the percentages of IFN-γ, TNF-α or Granzyme B (GrzB) expression by CD19 CAR-T cells co-incubated with Raji-tet-MUC21 cells at an E:T ratio of 1:2. (E-G) NK-92 cells were transduced with anti-CD19 CAR lentivirus. CD19 CAR-NK-92 cells were co-incubated with Raji-tet-MUC21 cells at variable E:T ratios for four hours in the presence or absence of Dox (1 µg/ml). (E) FACS analysis of the expression of anti-CD19 CAR on NK-92 cells using biotinylated CD19 protein. (F) Percentages of the specific killing of Raji-tet-MUC21 cells by CD19 CAR-NK-92 cells at the indicated E:T ratios. (G) Summary graph showing the mean fluorescence intensity (MFI) of CD107a and GrzB expression in CD19 CAR-NK-92 cells co-incubated with Raji-tet-MUC21 cells at an E:T ratio of 1:1. Data were compiled from four independent experiments with two replicates. Statistical significance was determined by a 2-way ANOVA with Holm-Sidak comparisons in (C) and (F), or two-tailed unpaired t-tests in (G). *P < 0.05, **P < 0.01, ****P < 0.0001