33. Induction of Antigen-Specific T Cell Anergy and Deletion by Hepatic AAV Gene Transfer

Abstract

Gene therapy for genetic disease may be limited by immune responses because of a lack of tolerance to the therapeutic gene product. Therefore, it is desirable that expression of a transgene product representing a neo-antigen induces immune tolerance. In published experiments, we have shown that adeno-associated virus (AAV)-mediated, hepatic in vivo gene transfer of a coagulation factor IX (F.IX) transgene can induce tolerance to the transgene product in adult immunocompetent mice (JCI 111:1347). Tolerized animals failed to form anti-F.IX even after challenge with F.IX in adjuvant, and F.IX-specific lymphocyte proliferation was substantially reduced. However, direct evidence for induction of T cell tolerance was elusive because of the low number of antigen-specific T cells and inability to physically identify these cells. Here, we demonstrate induction of antigen-specific CD4+ T cell tolerance in ovalbumin-specific T cell receptor (TCR) transgenic mice by hepatic AAV-mediated gene transfer of ovalbumin (ova), a secreted protein. Transduced BALB/c mice and BALB/c mice transgenic for MHC class II-restricted ova-specific DO11.10 TCR maintained stable, vector dose-dependent systemic ova levels without evidence for immune responses. Infusion of 3×1012 vg of AAV2-EF1α-ova into the portal circulation of DO11.10 transgenics resulted in levels of ~50 ng/ml plasma at day 10 and 100–200 ng/ml at days 30 and 60 after gene transfer. Lymph node (LN) cells and splenocytes were hypo-responsive to ova as early as day 10 after gene transfer, as demonstrated by reduced IL-2 cytokine release compared to controls. Dual color flow cytometry showed that numbers of TCR+CD4+ cells were reduced in secondary lymphoid organs and in the thymus by 1–2 months after vector administration (up to 2.5-fold). At 2 months, numbers of CD4+ cell lacking DO11.10 TCR expression were increased, consistent with a central deletion mechanism. The remaining TCR+CD4+ cell population was anergic to ova antigen in vitro. Splenocytes and LN cells showed markedly reduced IL-2 secretion and proliferation after stimulation with ova. Proliferation could largely be restored by addition of murine IL-2 to conditioned media, indicating T cell anergy. At day 60, percentage of CD25+ cells among TCR+CD4+ cells was increased in all examined lymphoid organs. CD4+CD25+ splenocytes isolated from hepatic AAV-ova transduced DO11.10 mice failed to express IL-2 in vitro after stimulation with ova, and suppressed IL-2 expression of CD4+CD25- splenocytes from naive DO11.10 mice in a cell dose-dependent manner, supporting the hypothesis that hepatic gene transfer promotes regulatory CD4+ T cell responses. These data provide direct evidence that transgene expression following in vivo viral gene transfer can induce CD4+ T cell tolerance to the transgene product, involving anergy and deletion mechanisms. Increase in regulatory T cell proportion likely enhances T cell anergy among the remaining ova-specific CD4+ T cell population. Induction of T cell tolerance correlated with absence of B cell responses, as AAV-ova transduced mice failed to form anti-ova IgG after subcutaneous injection of ova in complete Freund's adjuvant. This immunization protocol caused IgG1 anti-ova formation in naive control DO11.10 transgenics.