Fig. 4. Assessing functionality of the TGF-β CAR.

Data presented are reprinted with permission from a previously published study that employed the protocols mentioned in this article⁶. a, An NFAT reporter Jurkat T-cell line demonstrates that TGF-β stimulates NFAT signaling in TGF-β CAR-expressing Jurkat-T cells. b, Similarly, an NFκB reporter Jurkat T-cell line demonstrates that TGF-β stimulates NFκB signaling in TGF-β CAR-expressing Jurkat-T cells. c, Exposure of TGF-β CAR-expressing primary human CD4⁺ or CD8⁺ T cells to TGF-β triggers CD69 upregulation. Data in a-c are from experiments described in steps 43–48. d, Primary human CAR-T cell lines were assessed for cytokine production in the presence or absence of TGF-β as described in steps 49–51 (using a cytometric bead array). e, TGF-β CAR-expressing NFAT reporter Jurkat T cells were incubated with or without cognate ligand at varying cell densities as described in steps 58–65. Flow cytometry analysis showed the presence of a density-independent regime at low cell densities, a density-dependent regime at intermediate cell densities, and a density-independent regime at high cell densities where the signal output is likely saturated. The presence of a density-independent regime at low cells densities is consistent with the hypothesis that the TGF-β CAR can be activated by soluble TGF-β ligand independent of cell-to-cell interfaces. f, Microscopy of a TGF-β CAR-expressing Jurkat-T cell loaded with the Ca²⁺ flux indicator Fluo-4 AM was performed as described in steps 66–74. TGF-β ligand was added at time 0, and an activation event in the absence of cell-cell contact was observed. Scale bar represents 10 μm. In all graphs, data points from n = 3 samples are shown with means ± 1 standard deviation. Two-tailed Student’s t-tests with unequal variances were used to generate the p-values presented in a-d.