and C.T.) and R01 HL097088 (to R.W.H.), and Indiana Collaborative Initiative for Talent Enrichment (INCITE) funds provided by Lilly Endowment. Footnotes Supplemental Information can be found online at https://doi.org/10.1016/j.ymthe.2019.11.011. Supplemental Information Document ZM-447439 S1. by monoclonal antibody therapy prevented cross-priming. Furthermore, experiments in cell-type-restricted knockout mice showed a specific requirement for the receptor for T1 IFN (IFNaR) in cDCs. In contrast, natural killer (NK) cells are not needed, indicating a direct rather than indirect effect of T1 IFN on cDCs. In addition, co-stimulation by CD4+ T?cells via CD40-CD40L was required for cross-priming, and blockage of co-stimulation but not of T1 IFN additionally reduced antibody formation against capsid. These mechanistic insights inform the development of targeted immune interventions. make AAV vectors ideal for many different gene therapy applications. There are now two AAV-based gene therapies that have been approved by the US Food and Drug Administration (FDA) for the treatment of Lebers congenital amaurosis (LCA) and spinal muscular atrophy (SMA).1 Several AAV gene therapies for the X-linked bleeding disorders hemophilia A and B by hepatic gene transfer are in phase III clinical trials.2 Muscle-directed and CNS gene therapy for muscle mass, neuromuscular, and storage disorders are advancing, among other disease and organ targets.3 Despite these recent successes, adaptive immune responses directed against the AAV capsid remain a significant challenge and continue to limit the power of this intervention.4 CD8+ T?cell responses against capsid can cause immunotoxicities, tissue inflammation, and loss of transgene expression unless immune suppression is ZM-447439 applied.5, 6, 7, 8, 9, 10, 11 Cells transduced with AAV vectors may become targets for CD8+ T?cells because they cross-present capsid antigen by ZM-447439 major histocompatibility complex (MHC) I as a result of proteasomal degradation of viral particles following endosomal escape, capsid phosphorylation, and ubiquitination.12, 13, 14, 15, 16 Pre-existing neutralizing antibodies (NAbs) stemming from natural contamination exclude some patients from receiving a particular vector, and NAb formation following gene transfer complicates re-administration. Crucial to achieving long-term gene therapy is usually a deeper understanding of the mechanisms that lead to adaptive immune responses to AAV capsid. CD8+ T?cells or cytotoxic T lymphocytes (CTLs) are the principal effector cells that carry out antigen-specific cell-mediated killing of infected host cells. IMPA2 antibody In the naive state, CD8+ T?cells sample peptide MHC I complexes on the surface of dendritic cells (DCs) that acquired antigen. Acknowledgement of cognate peptide in the context of MHC I (transmission?1) by CD8+ T?cells initiates a cellular program that either results in the induction of tolerance or the activation and proliferative growth of antigen-specific CTLs. The decision between these two fates hinges on the activation state of the antigen-bearing DC, which,?if properly conditioned, can provide necessary costimulation (signal?2) and inflammatory cytokines (transmission 3). DC activation is usually modulated by both DC-intrinsic and/or DC-extrinsic danger signals such as pathogen acknowledgement through pattern acknowledgement receptors or inflammatory cytokines, respectively. In addition, CD40 signaling in DCs that occurs through conversation with CD40L on CD4+ T?cell helper cells represents a potent activation or licensing transmission that can permit effective CD8+ T? cell responses even in the absence of adequate danger signals.17, 18, 19 Thus, DCs represent the critical bridge that links innate sensing to adaptive responses, and the circumstances of their activation can tip the balance between tolerance and immunity.20,21 Innate immune sensing of the AAV genome by the endosomal DNA receptor TLR9 (Toll-like receptor 9) has been recognized as a critical determinant for CD8+ T?cell activation against both capsid and transgene products.22, 23, 24, 25, 26 TLR9 is highly expressed in plasmacytoid DCs (pDCs), which in response to AAV produce type 1 interferon (T1 IFN, i.e., IFN and IFN). We previously established that cross-priming of capsid-specific CD8+ T?cells requires cooperation between pDCs, which sense the AAV genome via TLR9, and conventional DCs (cDCs), which perform the actual capsid antigen presentation.25 TLR9 was dispensable in cDCs and.