Figure 5.
NKG2D upregulation in CAR-iNKT cells accounts for their ability to outperform CAR T cells in vitro. (A) Flow cytometric analysis of NKG2D expression on bispecific CD19-CD133 CAR T cells and CAR-iNKT cells before and after overnight coculture with SEM cells. (B) NKG2D expression as measured by per cent of cells and mean fluorescence intensity (MFI) in SEM and RS4;11 KMT2Ar cells. ∗∗P < .01, ∗∗∗∗P < .0001, by 1-way ANOVA. (C) NKG2D ligand expression in the indicated KMT2Ar leukemia cell lines as assessed by staining with biotinylated NKG2D-Fc protein followed by fluorescent streptavidin. (D-E) Cytotoxicity of bispecific CAR T cells and CAR-iNKT cells that had been precultured with SEM cells against SEM cells in the presence of different concentrations (D), or 5 mg of NKG2D mAb or immunoglobulin (Ig) isotype control (E). (F) Avidity measurement of bispecific CAR-iNKT cells vs CAR T cells against SEM cells in the presence of NKG2D mAb or Ig isotype control. Data are presented as the mean ± SEM. NS, not significant; ∗P < .05, by 1-way ANOVA. (G) Left: schematic of experiment. Right: NKG2D expression (% and MFI) in untransduced T cells/iNKT cells and CAR T cells/CAR-iNKT cells before coculture with leukemia cells. (H) Left: schematic of experiment. Right: NKG2D expression (% and MFI) in untransduced T cells/iNKT cells and CAR T cells/CAR-iNKT cells after coculture with the different SEM cell lines as shown. (I) Left: schematic of experiment. Middle: cytotoxicity of bispecific CAR T cells and CAR-iNKT cells that had been precultured with CD19+CD133+ SEM cells against CD19–CD133– SEM cells in the presence of NKG2D mAb or Ig isotype control. Right: cytotoxicity of bispecific CAR T cells and CAR-iNKT cells that had been precultured with CD19–CD133– SEM cells against CD19–CD133– SEM cells in the presence of NKG2D mAb or Ig isotype control. (A-I) Data are from 2 independent experiments and 2 different iNKT cell donors.

NKG2D upregulation in CAR-iNKT cells accounts for their ability to outperform CAR T cells in vitro. (A) Flow cytometric analysis of NKG2D expression on bispecific CD19-CD133 CAR T cells and CAR-iNKT cells before and after overnight coculture with SEM cells. (B) NKG2D expression as measured by per cent of cells and mean fluorescence intensity (MFI) in SEM and RS4;11 KMT2Ar cells. ∗∗P < .01, ∗∗∗∗P < .0001, by 1-way ANOVA. (C) NKG2D ligand expression in the indicated KMT2Ar leukemia cell lines as assessed by staining with biotinylated NKG2D-Fc protein followed by fluorescent streptavidin. (D-E) Cytotoxicity of bispecific CAR T cells and CAR-iNKT cells that had been precultured with SEM cells against SEM cells in the presence of different concentrations (D), or 5 mg of NKG2D mAb or immunoglobulin (Ig) isotype control (E). (F) Avidity measurement of bispecific CAR-iNKT cells vs CAR T cells against SEM cells in the presence of NKG2D mAb or Ig isotype control. Data are presented as the mean ± SEM. NS, not significant; ∗P < .05, by 1-way ANOVA. (G) Left: schematic of experiment. Right: NKG2D expression (% and MFI) in untransduced T cells/iNKT cells and CAR T cells/CAR-iNKT cells before coculture with leukemia cells. (H) Left: schematic of experiment. Right: NKG2D expression (% and MFI) in untransduced T cells/iNKT cells and CAR T cells/CAR-iNKT cells after coculture with the different SEM cell lines as shown. (I) Left: schematic of experiment. Middle: cytotoxicity of bispecific CAR T cells and CAR-iNKT cells that had been precultured with CD19+CD133+ SEM cells against CD19–CD133– SEM cells in the presence of NKG2D mAb or Ig isotype control. Right: cytotoxicity of bispecific CAR T cells and CAR-iNKT cells that had been precultured with CD19–CD133– SEM cells against CD19–CD133– SEM cells in the presence of NKG2D mAb or Ig isotype control. (A-I) Data are from 2 independent experiments and 2 different iNKT cell donors.

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