MyD88 Signaling in B Cells Regulates the Production of Th1-dependent Antibodies to AAV

Abstract

The administration of recombinant adeno-associated viral vectors (rAAV) for gene transfer induces strong humoral responses through mechanisms that remain incompletely characterized. To investigate the links between innate and adaptive immune responses to the vector, rAAVs were injected intravenously into mice deficient in cell-intrinsic components of innate responses (Toll-like receptors (TLRs), type-1 interferon (IFN) or inflammasome signaling molecules) and AAV-specific antibodies were measured. Of all molecules tested, only MyD88 was critically needed to mount immunoglobulin G (IgG) responses since MyD88−/− mice failed to develop high levels of AAV-specific IgG2 and IgG3, regardless of capsid serotype injected. None of the TLRs tested was essential here, but TLR9 ensured a Th1-biased antibody responses. Indeed, capsid-specific Th1 cells were induced upon injection of rAAV1, as directly confirmed with an epitope-tagged capsid, and the priming and development of these Th1 cells required T cell-extrinsic MyD88. Cell transfer experiments showed that autonomous MyD88 signaling in B cells, but not T cells, was sufficient to produce Th1-dependent IgGs. Therefore, rAAV triggers innate responses, at least via B cells, controlling the development of capsid-specific Th1-driven antibodies. MyD88 emerges as a critical and pivotal regulator of both T- and B-cell adaptive immunity against AAV.

Unique Roles of TLR9- and MyD88-Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-Mediated Gene Transfer

“…As opposed to the initial findings, contradictory data in the literature suggest an incomplete role for TLR9 signaling or a function for MyD88 but not TLR9 in shaping antibody responses [18,20,22]. As a whole, our data agree with the latter conclusions.…”

“…Others have previously reported that B cell-intrinsic MyD88 signaling may be required for the induction of IFN-γ-producing CD4 + T cells and subsequent formation of Th1-associated IgG2c antibody responses [22,28,29]. To verify this, we adoptively transferred WT B cells into MyD88 −/− mice 1 day prior to vector administration.…”

“…Human cells have been shown to sense AAV capsid through TLR2, a receptor recognizing various microbial protein and glycolipid structures, though no correlation has yet been made to adaptive immunity [21]. Finally, B cell-intrinsic MyD88, a downstream mediator of TLR2 and TLR9 signaling, has been suggested to be critical in the formation of Th1-dependent antibodies to AAV [22]. …”

Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung

“…However, it has been reported that in murine and human plasmacytoid dendritic cells in vitro, the AAV vector interacts with the innate immune system through the Toll-like receptor (TLR)9/MyD88 and type I interferon cascade [38,39]. AAV vectors also trigger nuclear factor kB-dependent production of cytokine and chemokine release in mouse liver, and MyD88 signaling in B cells also seems to control the production of capsid-specific T helper 1 antibody responses [40,41].…”

Complexity of immune responses to AAV transgene products – Example of factor IX

“…However, the effect on antibody formation overall is modest, as antibodies form even in TLR9 knockout mice [37]. IgG2a formation (the Th1-dependent murine equivalent of human IgG1) is dependent on intrinsic MyD88 in B cells [37, 39]. Antibodies that form against FIX upon AAV gene transfer in mouse and dog models, however, primarily reflect class-switch to Th2-dependent immunoglobulin (IgG1 in the mouse, equivalent to human IgG4).…”