78. Distinct Roles of Dendritic Cell Subsets in Innate Immune Sensing and Cross-Presentation of AAV Capsid In Vivo

Immune responses to AAV vectors currently represent one of the greatest hurdles facing successful gene therapy. Adeno-associated virus (AAV) vectors have been investigated in clinical trials for treatment of hemophilia B. However, in these trials, it was shown that transduced hepatocytes are eliminated by capsid specific CD8+ T cells, resulting in a loss of transgene expression and therapeutic efficacy. AAV capsid proteins are not endogenously expressed and therefore must be loaded on MHC class I via cross-presentation, a poorly understood mechanism relatively unique to conventional dendritic cells (cDCs). Previous work in our lab has shown that selective depletion of cDCs using CD11c-DTR mice ablates the anti-capsid CD8+ T cell response, and that innate sensing of the AAV genome is mediated through TLR9-sensing of the DNA genome and downstream signaling through its adaptor protein, MyD88. A second subset of dendritic cells, called plasmacytoid DCs (pDCs), highly express TLR9 and secrete large amounts of type I interferon following activation from a viral stimulus. Herein, we investigated the respective roles of cDCs and pDCs in the cross-presentation of capsid antigen. To measure capsid-specific T cell responses, we have created a modified AAV2 capsid that contains the peptide sequence SIINFEKL (AAV2-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 H2-Kb-SIINFEKL tetramer. We first confirmed the requirement of cDCs in capsid cross-presentation by measuring the proliferation of adoptively transferred T cells from TCR transgenic OT-1 mice, which produce CD8+ restricted, OVA-specific T cells, into cDC-depleted CD11c-DTR mice (n=4) that received AAV2-SIINFEKL. After 7 days, OT-I T cell proliferation was ~3% in the absence of cDCs compared to ~27% in the presence of cDCs. Strikingly, we found that the specific depletion of pDCs in BDCA2-DTR mice (n=4) that received AAV2-SIINFEKL also resulted in suppressed capsid CD8+ T cell formation, suggesting discrete yet essential roles for both pDCs and cDCs in the initiation of a cytotoxic T lymphocyte (CTL) response directed against AAV capsid antigen. To dissect the specific roles of cDCs and pDCs, we first investigated the requirement for MyD88 in cDCs by generating a transgenic mouse expressing Cre-recombinase under the control of the CD11c promoter with the MyD88 gene flanked by LoxP sites, specifically ablating MyD88 expression in cDCs (DC-MyD88−/− mice). We observed no significant difference in tetramer positive CD8+ T cells between DC-MyD88−/− mice (n=4) and WT mice, indicating that cDC-intrinsic MyD88 signaling is not required for the formation of anti-capsid CTL responses. Importantly, pDCs retained MyD88 expression in this model. To precisely define the role of TLR9 in these DC subsets, we adoptively transferred either WT pDCs (n=9) or WT cDCs (n=9) into TLR9−/− mice followed by injection of AAV2-SIINFEKL. Adoptive transfer of WT pDCs but not of WT cDCs restored induction of tetramer positive CD8+ T cells in the TLR9−/− mice. Therefore, we propose a 2-step model for anti-capsid CD8+ T cell responses to AAV vectors in which innate recognition of the genome occurs in pDCs via the TLR9-MyD88 pathway, promoting the cross-presentation of capsid antigen by cDCs. These results establish both DC subsets as critically important in the initiation of anti-capsid CD8+ T cell responses to AAV vectors and unveil potential targets to mitigate the deleterious effects of the immune response to AAV gene therapy.