(A) The mechanism of DC activation by rAAV. Vector genome sensing by TLR9 in pDCs’ endosomes triggers the activation of the TLR9/MyD88 signaling pathway that culminates in pDCs producing type I IFNs that directly signal to immature cDCs. This signaling event is required for effective priming and leads to activation and licensing of immature cDCs to mature cDCs. Licensing of cDCs enhances their ability to activate T cells. Activated cDCs also interact with CD4⁺ T cells, which may additionally contribute to licensing the cDCs to activate rAAV capsid–specific CD8⁺ T cells. Activated CD4⁺ Th cells also promote antibody formation against the rAAV capsid. (B) Depiction of rAAV-specific antigen presentation by APCs. Upon entry of AAV virions into cells by endocytosis, rAAV capsids can either be degraded in lysosomes or escape into the cytoplasm. Transcription and translation of the vector genome in the nucleus generate transgene proteins that, along with viral capsids, can be ubiquitylated and degraded in the proteasome into small peptides. These peptides are transported into the Golgi/endoplasmic reticulum by transporter associated with antigen presentation (TAP), loaded onto the MHC class I molecule, and presented on the surface of the target cell. This causes the cell to be recognized by a CD8⁺ T cell and, finally, eliminated by a capsid-specific CTL response. Capsid peptides are also loaded onto MHC class II molecules for presentation to CD4⁺ T cells for subsequent B cell activation and antibody production.