Figure 2.

ITAM1⁻ mutation on CD3ζ increases CAR Treg persistence in vivo but does not improve suppressive function

(A) BDD-FVIII or FVIIIFc stimulation of WT, ITAM1⁻, and ITAM3⁻ but not ITAM1⁻ITAM3⁻ FVIII-transduced CAR Tconvs leads to cell death of activated cells, whereas stimulation with FIXFc does not affect viability in vitro. Viability is normalized to 100% using unstimulated controls. (B) BDD-FVIII or FVIIIFc stimulation of WT, ITAM1⁻, or ITAM3⁻ FVIII CAR Tregs does not affect viability in vitro. Viability is normalized to 100% using unstimulated control Tregs. (C) Proliferation fit statistics indicating division index of CTV-labeled, adoptively transferred WT, ITAM1⁻, or ITAM3⁻ FVIII CAR Tregs on day 3. A representative proliferation histogram for ITAM1⁻ FVIII CAR Tregs is indicated. (D) Flow cytometric detection of CTV-labeled, adoptively transferred (1 × 10⁶ cells/mouse) WT, ITAM1⁻, or ITAM3⁻ FVIII CAR Tregs from splenocytes of recipient animals on days 3 and 8. Recipient mice were intravenously injected with 1.5 IU of BDD-FVIII 1 day following adoptive transfer (n = 4). Frequencies of adoptively transferred FVIII CAR Tregs in splenic CD4⁺ T cells are indicated. (E) Timeline for investigating in vivo prevention of inhibitor formation by CAR Treg cellular therapy. 1 × 10⁶ freshly isolated tTregs, 5 × 10⁵ WT, ITAM1⁻, ITAM3⁻ FVIII CAR Tregs, or WT FVIII CAR Tregs expanded in the presence of rapamycin 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 quantified by (F) Bethesda assay and (G) α-FVIII IgG1 ELISA. 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 Tukey’s multiple comparisons (A) or Dunnett’s comparisons (F and G). Multiple unpaired t tests (D).