Figure 1.

FVIII CAR Tregs bind and respond to FVIII stimulation in vitro

(A) Schematic of the FVIII CAR construct. The variable light (V_L) and heavy (V_H) regions of the FVIII-specific scFv, hinge, transmembrane, and intracellular signaling regions of murine CD28 and CD3ζ signaling domains are indicated. ITAM tyrosine motifs in the CD3ζ chain are indicated (Y), as well as tyrosine to phenylalanine point mutations (Y-F) in ITAM1 and ITAM3. (B) Schematic of Treg isolation, anti (α)-CD3/28 microbead activation, and retroviral vector transduction of FVIII CAR and FVIII TRuC Tregs. Representative plots showing pre-and post-sort purity analysis at the Treg isolation step and after the transduction step. (C) Representative density plots of FVIII CAR Tregs (indicated by mScarlet fluorescent reporter protein) to show surface expression of the Myc tag, detected with α-Myc PE, surface binding of 1 IU/mL FVIIIFc detected with α-human Fc conjugated to Alexa Fluor (AF) 647. (D) In vitro upregulation of Treg-associated activation markers CD69, LAP, GITR, FoxP3, CD49b, Ki67, and CD28 by BDD-FVIII- or FVIIIFc-stimulated murine CAR Tregs at 48 h. Controls include unstimulated cells, cells stimulated with an irrelevant protein, FIXFc, or α-Fc antibody. (E) In vitro proliferation of CTV-labeled FVIII CAR Tregs, 72 h post-stimulation with BDD-FVIII, FVIIIFc, FIXFc, or no stimulation. Proliferation assay was carried out in the absence of exogenous IL-2. Data points are averages ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by 1-way ANOVA test with Dunnett’s multiple comparisons test relative to control (Ctrl; unstimulated) for (D).