Figure 3.

Altered cytokine, transcription factor expression, signaling, and in vitro suppression of FVIII CAR Tregs

(A) Detection of IL-2, IL-10, IL-4, IL-17, IL-35, and IFN-γ from supernatants of sorted and BDD-FVIII-stimulated FVIII CAR Tconvs or Tregs at 48 h in vitro. Cells were cultured in the absence of exogenous IL-2. (B) Intracellular cytokine staining of cell-sorted FVIII CAR Tregs stimulated with high-dose (5 IU/mL) or low-dose (0.1 IU/mL) BDD-FVIII or left unstimulated (Ctrl) for 36 h in vitro. (C) Flow cytometric analysis for transcription factors: IRF4, T-bet, GATA3, and RORγt in cell-sorted and BDD-FVIII-stimulated FVIII CAR Tregs at 48 h in vitro. Unstimulated (Ctrl) or BDD-FVIII- or FIX-stimulated groups are indicated. (D) Normalized in vitro suppression of CTV-labeled FVIII CAR Tconv proliferation when co-cultured with FVIII CAR 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%. (E) Phospho-flow cytometry for pAKT (T308), pAKT (S473), pERK, and pS6 at indicated times following stimulation with low-dose (0.1 IU/mL) or high-dose (5 IU/mL) BDD-FVIII or 2:1 ratio of α-CD3/28 microbeads:CAR Tregs. Representative histogram plots for the same (filled in histograms represent 0 min stimulation). Data points are averages ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by multiple t tests for (A) and 1-way ANOVA with Tukey’s multiple comparisons for (B) and (C). 2-way ANOVA test with Tukey’s multiple comparisons for (D) and (E).