AAV8 Induces Tolerance in Murine Muscle as a Result of Poor APC Transduction, T Cell Exhaustion, and Minimal MHCI Upregulation on Target Cells

Abstract

Following gene transfer of adeno-associated virus 2/8 (AAV2/8) to the muscle, C57BL/6 mice show long-term expression of a nuclear-targeted LacZ (nLacZ) transgene with minimal immune activation. Here, we show that pre-exposure to AAV2/8 can also induce tolerance to the more immunogenic AAV2/rh32.33 vector, preventing otherwise robust T-cell activation and allowing stable transgene expression. Depletion and adoptive transfer studies showed that a suppressive factor was not sufficient to account for AAV2/8-induced tolerance, whereas further characterization of the T-cell population showed upregulation of the exhaustion markers PD1, 2B4, and LAG3. Furthermore, systemic administration of Toll-like receptor (TLR) ligands at the time of AAV2/rh32.33-administration broke AAV2/8-induced tolerance, restoring T-cell activation and β-gal clearance. As such, AAV2/8 transduction appears to lack the inflammatory signals necessary to prime a functional cytotoxic T-cell response. Inadequate T-cell priming could be explained upstream by AAV2/8's poor transduction and activation of antigen-presenting cells (APCs). Immunohistochemical analysis indicates that AAV2/8 transduction also fails to upregulate major histocompatibility complex class I (MHCI) expression on the surface of myocytes, rendering transduced cells poor targets for T-cell-mediated destruction. Overall, AAV2/8-induced tolerance in the muscle is multifactorial, spanning from poor APC transduction and activation to the subsequent priming of functionally exhausted T-cells, while simultaneously avoiding upregulation of MHCI on potential targets.

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