TLR9 and Dendritic Cells Are Required for CD8⁺ T Cell Responses to the AAV Capsid

CD8⁺ T cell responses to the adeno-associated virus (AAV) capsid have posed a significant barrier to transduction in clinical trials of AAV-mediated gene therapy for hemophilia B, as reactivation of a memory CTL response to the capsid is capable of eliminating transduced hepatocytes in the absence of immunosuppression. Recently, it has been suggested that innate immune responses induced by the toll-like receptor (TLR) pathway can influence the development of adaptive immune responses to AAV-mediated gene transfer. In particular, reports have implicated TLR2 (AAV capsid), TLR9 (AAV genome), and MyD88 (downstream signaling adaptor of both these TLRs). Herein, we have used a modified AAV2 with an insertion of the immunodominant MHC class I epitope of ovalbumin into the capsid (AAV2-SIINFEKL) to study the mechanism of CD8⁺ T cell responses to the AAV capsid. Using an H2-K^b-SIINFEKL tetramer reagent, we determined that anti-capsid CD8⁺ T cell responses depended on the TLR9-MyD88 pathway. While the frequency of circulating capsid-specific CD8+ T cells peaked around 7-10 days post-injection and subsided after about 21 days in wild type (WT) mice, tetramer-positive cells were not detected in TLR9^-/- or MyD88^-/- mice. The kinetics and magnitude of the response was unaltered in TLR2^-/- mice. Mice deficient in STING, a downstream adaptor of multiple cytoplasmic DNA sensing pathways, also developed comparable capsid-specific CD8⁺ T cell frequencies to WT mice, suggesting that this is not a general effect of pattern recognition of DNA. Interestingly, the frequency of capsid-specific CD8⁺ T cells was not reduced in AP3^-/- mice, which are deficient in type I IFN signaling downstream of TLR9. Adoptively transferred OVA-specific OT-1 T cells proliferated in WT but not TLR9^-/- mice that received AAV2-SIINFEKL, confirming the importance of TLR9. The effect was antigen-specific, as OT-1 cells in WT mice that received AAV2 lacking SIINFEKL showed minimal proliferation comparable to TLR9^-/- mice. In addition to pattern-recognition receptors, we also assessed the role of antigen-presenting cells in the CD8⁺ T cell response to capsid. The formation of capsid-specific CD8⁺ T cells was unaltered in mice that received gadolinium chloride to inactivate macrophages, or in B cell-deficient μMT mice. Depletion of B cells in WT mice prior to vector administration also failed to affect the anti-capsid CD8⁺ T cell response. However, transient depletion of dendritic cells (DCs) in CD11c-DTR mice resulted in a delayed development of capsid-specific CD8⁺ T cells. Seven days post-injection, DC-depleted mice had a significantly reduced frequency of tetramer-positive CD8⁺ T cells which recovered to normal by 10 days, likely due to the repopulation of DCs before the input capsid was completely cleared. Overall, our results show that TLR9 signaling, most likely in DCs, is required for the formation of de novo anti-capsid CD8⁺ T cell responses.