Fig. 7

Genomically edited TGFβ CAR T-cells can escape control by induced regulatory T-cells. A Schematic representation of the working hypothesis to assess the inability of iTregs to suppress TGFβRII KO CAR T-cells: Genomic disruption of TGFβRII expression leads to functional unresponsiveness towards TGFβ secreted by iTregs ‘in situ’. This deactivates suppression of iTregs and hence, promotes better effector functions of responder T-cells such as antigen-dependent proliferation towards antigen electroporated iDCs. B Flow cytometry analysis of in vitro generated iTregs to confirm their phenotypic differentiation into CD4+ T-cells with regulatory functions. CD25 were overexpressed in both, CD4+ T-cells being solely activated by CD3 and CD28 and iTregs while the transcription factor FOXP3 was only overexpressed in the TGFβ/All trans retinoic acid /Rapamycine treated iTreg group. C/D Bar chart of normalized proliferation for WT and TGFβRII KO CAR T-cell groups, the latter genomically edited at the TGFβRII locus with 2 different gRNAs. CAR T-cell groups were either treated with exogenous TGFβ or cocultured with in vitro generated iTregs at iTreg:CART-ratios ranging from 2:1 down to 0.5:1. From this, iTregs can suppress WT CAR T-cell proliferation at any effector:responder-ratio in a dose-dependent manner. Bulk KO CAR T-cells are also prone to suppression by TGFβ secreted by iTregs, but are able to substantially resist, in particular at lower iTreg:CART-ratios (0.5:1 for CART MSLN; 0.5:1 and 1:1 for CART CLDN6) towards iDCs pulsed with MSLN (C) or CLDN6 (D). On the right, the relative changes in proliferation suppression are calculated from C/D left. P values were determined by two-way Anova using multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. In all experiments, mean ± SD of three technical replicates are given and experiments, involving T cells, are repeated for at least three donors