Remarkably, whereas CTLA-4/Ig alone was inefficient, rAAV2/1-PD-L1 co-administered with CTLA-4/Ig at day 0 significantly improved transgene persistence, as measured by quantification of Ova DNA by qPCR (Figure 3C, left panel) . Accordingly, mRNA analysis by qRT-PCR at day 80 revealed sustained transcription of the transgene in muscle (Figure 3C, right panel) . Of note, rAAV2/1-PD-L2 appeared equally as effective as rAAV2/1-PD-L1 to provide this effect when combined with CTLA-4/Ig. Importantly, this protective effect occurred in a setting where neither CTLA-4/Ig alone nor the tested combination therapies could block immune response at days 40 and 80 (Figures 3A,B) . To further attest the presence of functional anti-Ova cytotoxic T cells at day 80 (a time point when pentamer staining is not sensitive enough to detect anti-Ova T cells, not shown), we analyzed by ELISpot the capacity of CD8 + T cells to secrete IFNγ when stimulated with the Ova-derived immunodominant SIINFEKL peptide. This assay revealed the presence of low numbers of functional anti-Ova CD8 + T cells in the three groups of animals that had received CTLA-4/Ig with or without rAAV-PD-L1 or rAAV-PD-L2, whereas the assay was negative for mice that did not receive rAAV-Ova and strongly positive for those that received rAAV-Ova in the absence of any immunoregulatory adjuvant therapy ( Figure 4A) . To definitively demonstrate that these IFNγ-secreting T cells were functional in vivo, we performed adoptive transfer experiments to non-lethally irradiated syngenic recipients that were inoculated with Ova-bearing tumor cells. Whereas tumors rapidly developed in recipients that had received T cells from control mice that only received PBS, recipient mice receiving lymphocytes from rAAV2/1-Ova injected donors rapidly developed an anti-tumor immune response that resulted in complete tumor rejection (Figure 4B ). In agreement with the lower numbers of anti-Ova CD8 + T cells detected by ELISpot, recipient mice that had received immune cells from donors treated with CTLA-4/Ig or the combined CTLA-4/Ig plus rAAV-PD-L1 or rAAV-PD-L2 therapy reacted with a slight delay but were also was also capable to efficiently control tumor growth ( Figure 4B) . Together, these results indicate that the cytotoxic activity of transgene product specific T cells that have escaped costimulation blockade can be functionally blocked by AAV-mediated gene transfer of PD-1 ligands in muscle tissues while they remain capable to reject tumor cells.