OPEN ORIGINAL ARTICLE Role of antigen-specific regulatory CD4þCD25þ T cells

Neonatal AAV8-mediated Factor IX (F.IX) gene delivery was applied as a model for exploring mechanisms of tolerance induction during immune ontogeny. Intraperitoneal delivery of AAV8 / Factor IX (hF.IX) during weeks 1–4 of life, over a 20-fold dose range, directed stable hF.IX expression, correction of coagulopathy in F.IX-null hemophilia B mice, and induction of tolerance to hF.IX; however, only primary injection at 1–2 days of life enabled increasing AAV8-mediated hF.IX expression after re-administration, due to the absence of anti-viral capsid antibodies. Adoptive splenocyte transfer from tolerized mice demonstrated induction of CD4þCD25þ T regulatory (Treg) populations that specifically suppressed anti-hF.IX antibody responses, but not responses to third party antigen. Induction of hF.IX antibodies was only observed in tolerized mice after in vivo CD4þCD25þ cell depletion and hF.IX challenge. Thus, primary injection of AAV during a critical period in the first week of life does not elicit antiviral responses, enabling re-administration of AAV and augmentation of hF.IX levels. Expansion of hF.IX-specific CD4þCD25þ Tregs has a major role in tolerance induction early in immune ontogeny. Neonatal gene transfer provides a useful approach for defining the ontogeny of immune responses and may suggest approaches for inducing tolerance in the context of genetic therapies