Figure 5.

IL-10-overexpressing FVIII CAR Tregs are not suppressive

(A) Schematic showing incorporation of the murine IL-10 sequence into WT, Y170F, or AYAA FVIII CAR constructs, separated by a P2A self-cleaving peptide sequence. (B) Effect of constitutive IL-10 expression on production of cytokines: IL-10, IL-4, IL-17, IFN-γ, IL-2, and TNF-α. Cell-sorted WT, WT-IL-10, CD28 AYAA-IL-10, or CD28 Y170F-IL-10 FVIII CAR Tregs were either left unstimulated (Ctrl) or stimulated with BDD-FVIII or FIX. Cell supernatants were estimated for cytokine production at 48 h in vitro. (C) CD28 AYAA-IL-10 or CD28 Y170F-IL-10 mutations do not adversely affect proliferation of activated FVIII CAR Tregs. In vitro proliferation of CTV-labeled WT-IL-10, CD28 AYAA-IL-10, or CD28 Y170F-IL-10 FVIII CAR Tregs, 72 h post-stimulation with low-dose (0.1 IU/mL) or high-dose (5 IU/mL) BDD-FVIII or no stimulation (Ctrl). Representative histogram plots to compare proliferation and graphical representation of division indices are shown. (D) 5 × 10⁵ WT-IL-10, CD28 AYAA-IL-10, or CD28 Y170F-IL-10 FVIII CAR Tregs were adoptively transferred into BALB/c F8e16^−/− recipient mice (n = 6−10/group). Mice received 4 weekly i.v. injections of 1.5 IU BDD-FVIII before functional inhibitors were assayed by the Bethesda assay. Control mice received only BDD-FVIII injections without cell transfer. (E) αFVIII IgG1 ELISA for the same. Data points are averages ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.001 by 2-way ANOVA with Sidak’s comparisons for (B), 1-way ANOVA test with Tukey’s multiple comparisons for (C), and 1-way ANOVA test with Dunnett’s comparison for (D) and (E).