Fig. 3
MUC21 attenuates T cell activation by hindering their antigen recognition. (A-C) CD8+T cells isolated from human PBMCs were co-cultured with 293FT cells that were transfected with mock (control) or MUC21 expressing plasmids for five days. The co-culture was performed in the presence of plate-coated anti-CD3 antibody (3 µg/ml). (A) Representative FACS plots (left panel) and a summary graph (right panel) showing the percentages of CD69+ or CD25+ CD8+T cells. Each dot represents an individual human sample. (B) Representative FACS plots showing proliferating CTVlow CD8+T cells (left panel) and a summary graph showing the division index of CTVlow CD8+T cells (right panel). (C) ELISA of IFN-γ secretion by CD8+T cells. (D-G) CD8+T cells were isolated from human PBMCs and co-cultured with p815 cells expressing membrane bound anti-CD3 scFv and Dox inducible MUC21 (p815-OKT3-tet-MUC21) at various E:T ratios for 3 days, in the presence or absence of Dox (1 µg/ml). (D) Schematic illustration of the p815-OKT3-tet-MUC21 artificial APC assay. (E) Representative FACS plots showing the expression of MUC21 in p815-OKT3-tet-MUC21 cells upon Dox (1 µg/ml) treatment. (F) Representative FACS plots showing proliferating CTVlow CD8+T cells (above panel) and a summary graph showing the division index of CTVlow CD8+T cells (below panel). (G) ELISA analysis of IFN-γ secretion by CD8+T cells. (H-I) 1G4 TCR-engineered CD8+T (1G4 TCR-CD8+T) cells were co-cultured with Raji cells expressing A*02:01/NY157–165 single-chain trimers, PD-L1 and Dox-inducible MUC21 (Raji-A2-ESO-1-PD-L1-MUC21) for three days, in the presence of the indicated antibodies (10 µg/ml) or Dox (1 µg/ml). (H) Schematic illustration of 1G4 TCR-engineered CD8+T cell-mediated Raji-A2-ESO-1-PD-L1-MUC21 cell killing. (I) Percentages of antigen-specific killing of Raji-A2-ESO-1-PD-L1-MUC21 cells by 1G4 TCR-CD8+T cells at an E:T ratio of 1:10 in the presence of the indicated antibodies with or without Dox. Data were compiled from three independent experiments with two replications. Statistical significance was determined by two-tailed unpaired t-test in (A), (B), (C), (F) and (G) or one-way ANOVA with Holm-Sidak multiple comparisons in (I). *P < 0.05, **P < 0.01, ***P < 0.001; and ns, not significant