Figure 6.

The TCR-CD3 complex regulates TRuC surface expression

(A) Surface organization and schematic representation of the FVIII TRuC construct. The V_L and V_H regions of the FVIII-specific scFv, linker, extracellular, transmembrane, and intracellular signaling regions of murine CD3ε domains are indicated. (B) Representative density and histogram plot of TRuC-transduced Tregs (indicated by mScarlet reporter protein) to show binding of 1 IU/mL FVIIIFc and detection with α-human IgG Fc conjugated to AF647. (C) Comparison of surface scFv expression between CAR and TRuC Tregs at comparable transduction levels (indicated by mScarlet MFI) by α-human F(ab′)2 binding and detection with α-goat AF647. (D) Surface expression of FVIII TRuC is dependent on incorporation into the TCR-CD3 complex. Single or co-transduction of FVIII CAR or TRuC and TCR-α/β into the TCR-α/β-deficient murine T cell line 5KCα⁻/β⁻. Incubation with 1 IU FVIIIFc and detection of frequencies of mScarlet⁺ cells that bind FVIIIFc by αFc conjugated to AF647 indicates dependence of TRuC but not CAR surface expression and CD69 upregulation on co-transduction with TCR-α/β. Data points are averages ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.001 by unpaired t test for (C).