Figure 4.

Targeted mutations in the CAR CD28 signaling domain do not confer suppressive ability

(A) Schematic representation of FVIII CAR constructs with targeted Y170F or AYAA mutations in the CD28 PI3K- and LCK-binding sites, respectively. (B) Y170F- or AYAA-targeted mutations in the CD28 signaling chain of the FVIII CAR construct do not adversely affect upregulation of CD69, CD28, Ki67, or CTLA-4 in transduced Tregs. MFIs for WT FVIII CAR Tregs compared to Y170F or AYAA variants at 48 h in vitro (WT, top panel; CD28-Y170F, middle panel; CD28-AYAA, bottom panel). Cells are either left unstimulated (Ctrl), stimulated with 5 IU/mL BDD-FVIII, or stimulated with an irrelevant protein, FIX, as indicated. (C) Y170F- or AYAA-targeted mutations in the CD28 signaling chain of the FVIII CAR construct downregulate the production of multiple cytokines, including IL-2, IL-10, IL-4, IL-17, and IFN-γ. WT, Y170F, or AYAA variants of FVIII CAR Tregs were stimulated with 5 IU/mL BDD-FVIII or left unstimulated, and cytokines were assayed after 48 h in vitro. (D) 5 × 10⁵ WT-, Y170F-, or AYAA-mutated FVIII CAR Tregs were adoptively transferred into BALB/c F8e16^−/− recipient mice (n = 4−10/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. Data points are averages ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by 1-way ANOVA test with Dunnett’s multiple comparisons for (B) and (D); 2-way ANOVA with Dunnett’s multiple comparisons for (C).