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. Author manuscript; available in PMC: 2020 Aug 1.
Published in final edited form as: Cell Immunol. 2017 Aug 1:S0008-8749(17)30120-X. doi: 10.1016/j.cellimm.2017.07.012

Figure 3. Combined effects of TLR9 stimulation and Treg depletion on systemic expression of and humoral immune responses after muscle-directed gene transfer.

Figure 3

(A) Timeline of experimental procedure for TLR9 stimulation and Treg depletion. The initial dose of the TLR9 agonist was combined with the vector and injected IM on day 0. Where indicated (⇒), the TLR9 agonist was injected in the same hind-leg on day 8. (B) Recovery of Tregs in peripheral blood as a function of time under various conditions. (C–F) Comparison of systemic hFIX antigen (C–D) and anti-hFIX IgG1 (E–F) levels in mice as a function of time after vector administration. (n=3–6 mice/group, *p=0.0178 AAV1-DT v. AAV1-DT-TLR9. (G) Immunofluorescent labeling of muscle tissue shows hFIX expression (red) and significant infiltration of CD8 cells (green) 2 weeks after AAV1-hFIX vector, TLR9 stimulation, and Treg depletion. (Arrows in insert point to centralized nuclei (blue) associated with new cell growth; ▲ indicate CD8+ (green) cells). (H) IFN-γ ELISpot showing a cellular immune response against hFIX in mice transduced with vector in the presence of ODN-1826 and Treg depletion, which was absent in mice treated with vector only (n=3–4/group; data are average ± SEM for epitope-stimulated minus mock-stimulated cultures).