Figure 5.

AAV-CAR Tregs suppress capsid-specific immune response in vivo

(A) Expression of hAAT protein in the serum of animals injected i.m. with rh32.33-AAT followed by AAV-CAR Tregs (red), polyclonal Tregs (orange), or PBS (blue) as measured by ELISA (n = 6 for AAV-CAR Treg and PBS-treated groups, n = 5 for polyclonal Treg-treated group). (B) Anti-rh32.33 antibodies in the serum measured by ELISA. Anti-capsid antibodies increased in all groups over time (n = 6 for AAV-CAR Treg group, n = 7 for PBS group, n = 5 for polyclonal Treg-treated group). (C) Serum level of free active TGF-β1 measured by CBA assay over time. A significant increase in free active TGF-β1 was observed in AAV-CAR Treg group (red) at week 11 post injection compared with the PBS control group (blue). (D) Serum levels of IL-10 measured by CBA assay over time. Two-way ANOVA repeated measure with Tukey's multiple comparisons was used (n = 6 for AAV-CAR Treg and PBS-treated groups, n = 5 for polyclonal Treg treated group for C and D). (E) Quantification of number nuclei in right limb muscles of animals 2 weeks after AAV injection. Each dot represents one animal, which is the average of 10 images per animal (HPF, high-power field). One-way ANOVA, n = 3. Error bars are mean ± SEM; ∗p ≤ 0.05, ∗∗p ≤ 0.01. (F) Representative images of rh32.33-AAT muscles 2 weeks post injection stained for H&E for cellular infiltration. (Upper) scale bar, 527 μm; (lower) scale bar, 131 μm. (G) Representative images of rh32.22-AAT-injected muscles stained by immunohistochemistry for AAT protein (brown), 26 weeks after AAV injection. (Upper) scale bar, 527 μm; (lower) scale bar, 131 μm. ∗AAV-CAR Tregs compared with PBS; #AAV-CAR Tregs compared with polyclonal Tregs; ˆpolyclonal Tregs compared with PBS.