419. Generation of Transgene Product-Specific Regulatory CD4+CD25+ T Cells by Hepatic AAV Gene Transfer

Abstract

Top of pageAbstract Gene therapy for genetic disease may be limited by immune responses to the therapeutic gene product. Previously, we found induction of antigen-specific immune tolerance to secreted transgene products by hepatic AAV gene transfer. Immune tolerance to coagulation factor IX (F.IX) was associated with activation of regulatory CD4+ T cells (Tregs) capable of suppressing antibody formation to F.IX (JCI 111:1347). Experiments in a T cell receptor (TCR) transgenic model provided evidence for clonal deletion and T cell anergy of ovalbumin (ova)-specific CD4+ T cells after gene transfer (Blood 104:969). Here, we sought to identify the type of Tregs activated by hepatic gene transfer. First, we injected AAV vector expressing hF.IX from a hepatocyte-specific promoter into portal veins of C57BL/6 mice. At 6 weeks, splenocytes were adoptively transferred to na|[iuml]|ve C57BL/6 mice. After 24 hrs, hF.IX protein in adjuvant was injected subcutaneously. MACS purified CD4+CD25+ cells from tolerized mice, but not CD4- or CD4+CD25- cells suppressed anti-hF.IX IgG formation. Compared to controls, antibody titers were 3-fold lower, with statistical significance of p|[le]|0.02 (n=7|[ndash]|8/group). Small numbers of CD4+CD25+ cells (1x106/animal) were as potent in suppressing antibodies as total CD4+ cells (1x107/animal). CD25 is transiently expressed by activated T cells and constitutively expressed by |[ldquo]|naturally occurring|[rdquo]| Tregs. To further define CD4+CD25+ Tregs activated by AAV gene transfer, we switched to a better defined model system, mice transgenic for DO11.10 TCR. This TCR (encoded by rearranged V|[aacute]|13 and V|[beta]|8.2 genes) is specific for a ova peptide, amino acids 323|[ndash]|339, presented by the MHC class II molecule I-Ad to CD4+ T cells. DO11.10 TCR+CD4+ cells can be identified by flow cytometry using dual antibody stain (anti-CD4 and KJ1-26 anti-DO11.10 TCR). Due to lack of endogenous TCRs and therefore lack of stimulation with self-antigens, BALB/c mice transgenic for DO11.10 TCR and Rag-2 -/- are devoid of Tregs, as evident from absence of CD4+CD25+ cells and of expression of FoxP3 transcription factor. Hepatic AAV-ova gene transfer induced a population of CD4+CD25+ cells in all lymphoid organs analyzed (thymus, lymph nodes, spleen, 1|[ndash]|3.5% of CD4+ cells, 3- to 18-fold increase compared to untreated animals by day 60 after gene transfer). Moreover, these cells were anergic in vitro to ova antigen, but efficiently suppressed ova-specific CD4+CD25- cells in a standard assay for Tregs function in a cell dose-dependent manner. Furthermore, quantitative RT-PCR showed a 190-fold increase in FoxP3 transcript levels in the CD4+CD25+ cell populated induced by transgene expression compared to CD4+CD25- cells. Taken together, our study demonstrates that hepatic AAV-mediated gene transfer generated CD4+CD25+ Tregs comparable to naturally occurring Tregs and capable of suppressing lymphocyte responses to the transgene product. In vitro cytokine release assays for F.IX and ova-tolerized mice failed to show increases in IL-10 (expressed by Tr1 Tregs) or TGF- |[beta]|(expressed by Th3 Tregs), indicating that the mechanism of tolerance induction by hepatic gene transfer is distinct from other approaches such as cytokine- or mucosal induced tolerance.