Figure 7.

Simultaneous Checkpoint Blockade and Treg Depletion Breaks Liver-Induced Anti-transgene Tolerance

(A and B) Five-week-old C57BL/6J mice were intramuscularly injected (TA) with 1 × 10¹⁰ vg/mouse of AAV1-SPc5.12-hSGCA-SIINFEKL vector and intravenously injected with PBS (Muscle group) or 1 × 10¹¹ vg/mouse of AAV9-hAAT-hSGCA-SIINFEKL (Muscle-Liver group). A group of mice treated with the combination of vectors received a simultaneous injection of NAD and anti-CD25 at day 3 and anti-CD25 alone at days 6 and 7 (Muscle-Liver-Treg groups). Finally, another group of mice received the same treatment combined with the injection of anti-PD-1, anti-PD-L1, and anti-LAG3 antibodies every 3 days from days 3 to 12 (Muscle-Liver-Treg+Ab group). Two weeks after treatment, mice were sacrificed, and tissues were collected. (C) IFN-γ-ELISPOT performed on splenocytes stimulated with SIINFEKL peptide. (D) Liver intraparenchymal CD8⁺ cells gated on CD45⁺ cells. (E) Liver intraparenchymal CD8⁺CD44⁺Dextramer⁺ cells gated on CD8⁺ cells. (F) Vector genome copy number (VGCN) per diploid genome measured in TA. (G) Immunostaining with anti-hSGCA (green), CD8 (red), and DAPI (blue) in TA. White arrow indicates CD8 cell. Data were expressed as mean ± SD. Statistical analyses were performed by ANOVA except for (B), where a Kruskal-Wallis test was used (*p < 0.05; n = 4 per group).