519. Unique Role of the TLR9-MyD88 Signaling Pathway in Dendritic Cells in AAV Capsid-Specific CD8+ T Cell Activation

Abstract

Clinical trials of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B have revealed a deleterious role for memory CD8+ T cell responses to the AAV capsid, which can eliminate transduced hepatocytes and transgene expression. However, the mechanisms underlying this response remain unclear, and have been difficult to study because the functional anti-capsid CTL response is not observed in murine models. To better identify capsid-specific T cells, we have created a modified AAV2 vector with a series of substitution mutations that contains the peptide sequence SIINFEKL, which is the CD8+ immunodominant epitope of the model antigen ovalbumin in C57BL/6 mice. With this vector, capsid-specific CD8+ T cells can be identified using an H2-Kb-SIINFEKL tetramer. We have used this vector to study the innate immune mechanisms underlying the formation of AAV capsid-specific CTLs—which pattern recognition receptors and which antigen-presenting cell types are required. Using knockout mice, we determined that sensing of the viral genome by toll-like receptor 9 (TLR9) and its downstream signaling through MyD88 are required for the formation of tetramer-positive (capsid-specific) CD8+ T cells after i.m. injection of AAV2-SIINFEKL. Similarly, OT-1 CD8+ T cells (TCR transgenic specific for SIINFEKL) adoptively transferred to wild-type (WT) mice proliferated following administration of AAV2-SIINFEKL but not with unmodified AAV2 lacking the SIINFEKL motif. In contrast, there was a lack of proliferation in TLR9−/- mice. TLR2 (which has been reported to sense the AAV capsid), AP3 (required for type I interferon signaling downstream of TLR9), and STING (a downstream signaling adaptor of several cytoplasmic sensors of DNA) were dispensable for the anti-capsid CTL response. In terms of the antigen presenting cell requirements, we found that transient depletion of dendritic cells (DCs) in CD11c-DTR mice delayed the development of tetramer-positive CD8+ T cells relative to WT mice. The response in mice that received gadolinium chloride to inactivate macrophages or in B cell-deficient μMT mice was comparable to WT mice. To test the hypothesis that TLR9-MyD88 signaling in DCs is required for capsid-specific CTL responses, we have crossed mice expressing Cre recombinase under the control of the CD11c promoter with mice in which the MyD88 gene is flanked by LoxP sites, creating DC-MyD88−/- mice which specifically lack MyD88 expression—and thus TLR9 signaling—in DCs. Moreover, we have acquired inhibitors of TLR9 and MyD88 to determine if pharmacological inhibition of these pathways is a viable strategy to prevent the formation of capsid-specific CD8+ T cells. In conclusion, TLR9-MyD88 signaling, most likely in DCs, is required for the formation of de novo anti-capsid CD8+ T cell responses.