The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice
Jiangao Zhu, … , Xiaopei Huang, Yiping Yang
Jiangao Zhu, … , Xiaopei Huang, Yiping Yang
Published July 1, 2009
Citation Information: J Clin Invest. 2009;119(8):2388-2398. https://doi.org/10.1172/JCI37607.
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Research Article Genetics

The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice

Abstract

Recombinant adeno-associated viruses (AAVs) have been used widely for in vivo gene therapy. However, adaptive immune responses to AAV have posed a significant hurdle in clinical application of AAV vectors. Recent advances have suggested a crucial role for innate immunity in shaping adaptive immune responses. How AAV activates innate immunity, and thereby promotes AAV-targeted adaptive immune responses, remains unknown. Here we show that AAV activates mouse plasmacytoid DCs (pDCs) via TLR9 to produce type I IFNs. In vivo, the TLR9-MyD88 pathway was crucial to the activation of CD8+ T cell responses to both the transgene product and the AAV capsid, leading to loss of transgene expression and the generation of transgene product–specific and AAV-neutralizing antibodies. We further demonstrate that TLR9-dependent activation of adaptive immunity targeting AAV was mediated by type I IFNs and that human pDCs could be activated in vitro to induce type I IFN production via TLR9. These results reveal an essential role for the TLR9-MyD88–type I IFN pathway in induction of adaptive immune responses to AAV and suggest that strategies that interfere with this pathway may improve the outcome of AAV-mediated gene therapy in humans.

Authors

Jiangao Zhu, Xiaopei Huang, Yiping Yang

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Figure 6

Lack of TLR9-MyD88 signaling diminishes CD8+ T cell responses to the AAV capsid and the transgene product and prolongs the transgene expression.

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Lack of TLR9-MyD88 signaling diminishes CD8+ T cell responses to the AAV...
AAV2-HA (1 × 1011 vg) was injected intramuscularly into WT, Tlr9–/–, or Myd88–/– BALB/c mice. (A) After 12, 26, and 60 days, the infected muscles were harvested and analyzed for HA expression by immunohistochemistry. Original magnification, ×100. (B) CD5+ T cells purified from splenocytes at day 26 after infection, along with uninfected WT splenocytes (control), were restimulated with AAV2-HA at 0, 50, 500, or 5,000 vg/cell. Proliferation of AAV-specific T cells was analyzed by 3H-thymidine incorporation. Data reflect the mean ± SD of the stimulation index, which was calculated by dividing 3H counts in cpm in the presence of viral stimulation by those in the absence of stimulation, as a function of different virus doses. (C–F) At days 12 and 26 after infection, splenocytes were harvested and stimulated with either AAV2 capsid epitope peptide (C and D) or HA epitope peptide (E and F) for 5 hours and assayed for intracellular IFN-γ secretion by CD8+ T cells. (C and E) The FACS plots show percentages of IFN-γ–producing CD8+ T cells among total CD8+ T cells. (D and F) The mean percentages ± SD of IFN-γ–producing CD8+ T cells among total CD8+ T cells are also shown. Representative results of 3 independent experiments are shown.