Fig. 5.

Tn-peptide binding and activity of CAR-transduced T cells. (A) Splenic T cells from C57/BL6 mice were mock transduced or transduced with 237-CAR (the wild-type), WE-CAR, LGQ-CAR, TNGK-CAR, or 5E5-CAR. Transduced T cells were stained with various concentrations of Tn-OTS8 or Tn-MUC1 biotinylated monomeric peptides, followed by streptavidin-PE to assess binding by flow cytometry. Histograms of cells stained with streptavidin-PE only are shown in gray. Similar results were obtained in three independent experiments. (B) To assess activation, mock (shown in gray), 237-CAR, WE-CAR, LGQ-CAR, TNGK-CAR, or 5E5-CAR transduced T cells were added to plates containing various concentrations of immobilized Tn-OTS8 or Tn-MUC1 peptide and incubated at 37 °C, 5% CO₂. After 24 h, IFN-γ released in the culture supernatants was measured by ELISA. Activation of transduced cells was measured in duplicate. The 237-, TNGK-, and 5E5-CARs were assessed in three independent experiments. WE- and LGQ-CARs were assessed in one experiment. Error bars indicate SEM.