Transgene expression in muscle was found to be high early after transduction but rapidly declined over time, suggesting the occurrence of an immune response against transduced cells ( Figure 1A) . To investigate this point, we quantified the cellular and humoral immune responses directed against the xenogenic Ova transgene product by flow cytometry and ELISA. Staining with MHC-I pentamers presenting the immunodominant Ova-derived SIINFEKL peptide, designated hereafter as H-2K b /Ova pentamers, allowed to monitor the expansion of CD8 + lymphocytes in spleen and draining lymph nodes of rAAV-Ova challenged animals. This analysis revealed that both rAAV2/1-Ova and rAAV2/8-Ova injected in the gastrocnemius muscles induced a robust expansion of anti-Ova CD8 + lymphocytes (Figures 1B,C) . Interestingly, the kinetics and intensity of T cell priming was found to be different when comparing the cells harvested from the draining lymph nodes and from the spleen. In the lymph nodes draining the injected muscles, anti-Ova CD8 + T cells expanded early after AAV challenge to reach around 2% of the CD8 + T cell compartment at day 7 post-injection ( Figure 1C , left panel). In contrast, when analyzing splenocytes, anti-Ova CD8 + T cells were found to reach up to 10% of the total CD8 + subset but only at day 14 after i.m. AAV injection ( Figure 1C , right panel). These results are consistent with a model in which the local cellular immune response measured in the draining lymph nodes would be induced by an rAAV leakage from the injected muscle and the consequent transduction of non-muscle cells such as dendritic cells, as shown for AAV5 (Xin et al., 2006) or AAV1 (Lu and Song, 2009 ) for instance. The systemic response detected in the spleen would rather reflect systemic immunization directed against the circulating sOva protein after it has been produced by transduced muscle cells. This interpretation is supported by the kinetics of sOva apparition and antibodies production ( Figure 1D) . Indeed, in the serum of AAVtreated mice, sOva reached a maximum at day 7 post-injection and rapidly declined thereafter ( Figure 1D , green curves). Consistently, anti-Ova IgG appeared between day 7 and 12 after AAV i.m. injection, correlating with the disappearance of sOva in the serum after day 7 ( Figure 1D , blue curves). The latter probably reflects the in vivo clearance of sOva following the formation of antigenantibody immune complexes, although we cannot completely rule out that anti-Ova IgG antibodies may mask Ova epitopes in the immunoassay. Together, these data reveal that i.m. injection of www.frontiersin.org rAAV encoding sOva is strongly immunogenic and provides a mouse model that is suited to stringently evaluate the efficacy of tolerance induction protocols.