Figure 5.

Regulation of CAR T Cell Activity by CAR Density and Antigen Density

A GFP reporter system indicating NFAT activity was used to evaluate CAR T cell activation upon receptor cross-linking with plate-bound Protein L or anti-(CD19 scFv)-idiotype antibody. (A) The lentiviral construct incorporates six NFAT-binding elements upstream of a minimal IL-2 promoter, and it drives GFP expression upon T cell activation. (B) ALK CAR/NFAT-GFP reporter T cells were spun onto 96-well plates coated with biotinylated Protein-L concentrations as shown, and they were assessed by flow cytometry 24 hr later for surface CAR and GFP expression. Representative plots of NFAT-GFP activity in populations of T cells with low initial CAR expression (CAR⁺), medium initial CAR expression (CAR⁺⁺), and high initial CAR expression (CAR⁺⁺⁺) are indicated. (C) Geometric mean fluorescent intensity (gMFI) of NFAT-driven GFP expression was quantified in the GFP-positive population of responding cells. Insets are visual comparisons of the NFAT response to increasing antigen density by high- (+++, blue) and low- (+, white) expressing ALK CAR T cells. (D and E) CD19.4-1BB. ζ-BFP/NFAT-GFP reporter CAR T cells were spun onto 96-well plates coated with varying concentrations of anti-(CD19 scFv)-idiotype antibody, and they were assessed by flow cytometry 24 hr later for surface CAR and GFP expression. Representative plots of NFAT-GFP activity in populations of T cells with low initial CAR expression (CAR⁺), medium initial CAR expression (CAR⁺⁺), and high initial CAR expression (CAR⁺⁺⁺) are indicated (D) and quantified (E). (F) Representative FACS plots of CAR expression and gMFI are shown for ALK CARs and sorted CD19 CARs used in the NFAT-GFP reporter assays. For the ALK CAR: dark blue, high (+++) CAR; light blue, medium (++) CAR; black, low (+) CAR; gray shaded, mock. For the CD19 CAR: dark purple, high (+++) CAR; light purple, medium (++) CAR; black, low (+) CAR; gray shaded, unsorted CAR-transduced population. (G) The percentage of CD19 CAR T cells (NFAT-GFP⁺ cells) responding to anti-idiotype stimulation is shown. (H) IFN-γ and IL-2 production was assessed after co-incubation of sorted CD19 CAR T cells with varying concentrations of plate-bound anti-idiotype antibody. Differences in the gMFI of GFP, percentage of CARs responding, and cytokines produced were determined by a two-way ANOVA followed by a Tukey’s multiple comparisons test. Experiments were repeated three times with a total of two different donors.