We further analyzed the presence of Ova DNA and mRNA in muscles at day 40 in the different groups of treated mice by qPCR and qRT-PCR, respectively. This further confirmed that suppression of immune priming by CTLA-4/Ig promotes the persistence of transduced cells in vivo as measured by the detection of significantly higher levels of Ova DNA and mRNA in the muscles of treated animals ( Figure 2D ). By contrast, injection Frontiers in Microbiology | Microbial Immunology Figure 1 with 1 × 10 11 vg rAAV2.1-Ova in the gastrocnemius muscles at day 0. Splenic CD8 + T cells were analyzed at day 14 by flow cytometry for expression of CD44 and PD-1. (B) rAAV2/1-PD-L1 and rAAV2/1-PD-L2 vectors were designed, produced, and tested for their capacity to transduce HEK-293 cells in vitro. For this, cells were analyzed by flow cytometry 3-5 days after their transduction with 1/10th dilution of the concentrated virus stocks. Controls correspond to unmanipulated HEK-293 parental cells stained with the same antibodies (green histograms). (C) The immunosuppressive potential of rAAV2/1-PD-L1 and CTLA-4/Ig were evaluated in vivo. Mice were injected with 10 11 vg rAAV2/1-Ova in the gastrocnemius muscles and received or not a co-injection of 10 11 vg rAAV2.1-PD-L1, or 200 μg of CTLA-4/Ig injected contemporaneously by the i.p. route. Blood samples were then collected 14 days later to analyze the percentage of anti-Ova CD8 + T cells, the level of anti-Ova IgG and the presence of sOva in the serum. (D) Gastrocnemius muscles were then collected at day 40, and Ova DNA and mRNA were quantified by qPCR and qRT-PCR. of rAAV2/1-PD-L1 did not significantly improve transgene persistence in these settings, in line with the prominent systemic immune response detected at day 14 ( Figure 2D ).