Tolerance Induction to Cytoplasmic \(\beta\)-Galactosidase by Hepatic AAV Gene Transfer — Implications for Antigen Presentation and Immunotoxicity

Background: Hepatic gene transfer, in particular using adeno-associated viral (AAV) vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein. Major Findings: AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic b-galactosidase (b-gal) was performed in immune competent mice, followed by a secondary b-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in ,2% of hepatocytes almost completely protected from inflammatory T cell responses against b-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, ,10% of hepatocytes continued to express b-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8 T cell responses to b-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer. Conclusions: These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity. Citation: Martino AT, Nayak S, Hoffman BE, Cooper M, Liao G, et al. (2009) Tolerance Induction to Cytoplasmic b-Galactosidase by Hepatic AAV Gene Transfer — Implications for Antigen Presentation and Immunotoxicity. PLoS ONE 4(8): e6376. doi:10.1371/journal.pone.0006376 Editor: William Giannobile, University of Michigan, United States of America Received May 20, 2009; Accepted June 29, 2009; Published August 4, 2009 Copyright: 2009 Martino et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by NIH grant P01 HL078810 (Projects 3 and 4) to RW Herzog and C Terhorst. This source of funding was used to pay for all reagent, supply, and animal costs. The same grant also provided salary support for BE Hoffman, M. Cooper, G. Liao, C. Terhorst, and RW Herzog. In addition, salary support for S. Nayak and AT Martino was provided by NIH grants T32DK074367 and 2R01 AI/HL51390, respectively. BE Hoffman was additionally supported by a postdoctoral fellowship from the American Heart Association. DM Markusic and BJ Byrne did not receive funding for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no corporate support for this study. Competing Interests: RW Herzog has been receiving royalty payments from Genzyme Corp. for AAV-F.IX technology license. However, no AAV-F.IX gene transfer was performed as part of this study, and no funding of research has been received from Genzyme. No patents are associated with this study. * E-mail: [email protected] . These authors contributed equally to this work.