Figure 7.

TRuC Tregs exhibit controlled signaling in vitro

(A) Upregulation of Treg-associated activation markers CD69, Ki67, CD28, CTLA-4, and FoxP3 by BDD-FVIII-stimulated murine TRuC Tregs at 48 h in vitro. Controls include unstimulated cells or stimulation with an irrelevant protein, FIX. (B) Comparison of pAKT (S473), pERK, or pS6 at indicated times following stimulation with low-dose (0.1 IU/mL) or high-dose (5 IU/mL) BDD-FVIII or TCR triggering with α-CD3/28 microbeads by flow cytometry for WT CAR (solid line) or TRuC (dotted line) Tregs. (C) Comparison of western blot analysis for pERK and pS6 at indicated times following stimulation with high-dose (5 IU/mL, blue line) or low-dose (0.1 IU/mL, pink line) BDD-FVIII. Densitometric analysis for pERK and pS6 for WT CAR Treg (solid line) or TRuC Treg (dotted line) is indicated. (D) Detection of IL-2, IL-10, IL-4, IL-17, and IFN-γ cytokines from BDD-FVIII-stimulated TRuC and CAR Treg cell supernatants at 48 h in vitro. (E) Normalized in vitro suppression of CTV-labeled FVIII TRuC Tconv proliferation when co-cultured with FVIII TRuC Tregs at the indicated Treg:Tconv ratios. Cells were stimulated with high-dose (5 IU/mL) or low-dose (0.1 IU/mL) BDD-FVIII or left unstimulated for 72 h in vitro. Percentage suppression calculated as ([mean proliferation Tconv − mean proliferation Treg + Tconv]/[mean proliferation Tconv]) × 100%. Data points are averages ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.001 by 1-way ANOVA with Dunnett’s comparisons for (A), multiple unpaired t tests for (B), 2-way ANOVA test with Tukey’s multiple comparison for (C) and (D), and 2-way ANOVA with Sidak’s multiple comparisons for (E).