419. Generation of Transgene Product-Specific Regulatory CD4+CD25+ T Cells by Hepatic AAV Gene Transfer
Authors: O. Cao, E. Dobrzynski, B.L. Mingle, L. Wang, R.W. Herzog
Journal: Molecular Therapy (2005)

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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.