Indeed, previous studies (9, 13) have revealed that IL-27 delivered systemically can inhibit the development of experimental autoimmune encephalomyelitis (EAE) in mice, an experimental model of MS. However, systemic injection of IL-27 is costly, and it is also difficult to maintain an effective concentration in the circulation. In this context, gene therapy could serve as an effective alternative approach. For instance, IL-30 gene therapy has been shown to efficiently inhibit autoimmune inflammation in the central nervous system (CNS) and eye (16), and lentiviral IL-27 gene delivery to the CNS inhibits neuroinflammation (17). Adeno-associated viral vectors (AAVs) are highly efficient delivery agents for gene therapy (18). AAV vectors can efficiently transfer genes of interest to a broad range of mammalian cell types leading to high levels of stable and long-term expression after a single application (19). AAV vectors are also known to have low immunogenicity and have been used in human clinical trials (2022). In this study, we have evaluated the therapeutic efficacy of AAV-delivered IL-27 (AAV-IL-27) and IL-30 (AAV-IL-30) in T cell-mediated autoimmune encephalomyelitis, where the inflammation in the CNS is considered to be mediated mainly by Th17/Th1 responses and T cells producing GM-CSF (23, 24). We found that one single administration of AAV-IL-27, but not AAV-IL-30 completely prevented EAE development. Experiments involving IL-10-deficient mice and PD-1 blockade revealed that AAV-IL-27-induced IL-10 and PD-L1 expression were not required for the inhibition of EAE development. However, neither AAV-IL-27 nor AAV-IL-30 treatment inhibited EAE development and Th17 responses when given at disease onset. We found that mice with established EAE had significant expansion of CD11b+Gr-1+ myeloid cells, and AAV-IL-27 treatment further expanded these cells and induced their expression of multiple cytokines including Th17-promoting cytokines such as IL-6 and IL-23. Adoptive transfer of AAV-IL-27-expanded CD11b+Gr-1+ cells enhanced EAE development. Thus, systemic delivery of IL-27 can efficiently prevent EAE development and the priming of Th17 responses. However, the therapeutic potential of IL-27 is limited by its failure in inhibiting ongoing EAE, and shutting down established Th17 responses, presumably due to the expansion of CD11b+Gr-1+ myeloid cells.