Figure 6.

Immune-Checkpoint Blockade Induces Liver Intra-parenchymal Tregs

(A) 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 1 × 10¹¹ vg/mouse of AAV9-hAAT-hSGCA-SIINFEKL (Muscle-Liver group). A group of mice treated with the same combination of vectors was treated every 3 days from day 2 to day 14 with the combination anti-PD-1, anti-PD-L1, and anti-Lag3 antibodies (Muscle-Liver+Ab group). Another group of mice received only the AAV1 vector intramuscularly (Muscle group). PBS-injected mice were used as controls (Control group). Two weeks after treatment, mice were sacrificed, and tissues were collected. (B) IFN-γ-ELISPOT performed on splenocytes stimulated with the SIINFEKL peptide. (C) Immunostaining with anti-hSGCA (green), CD8 (red), and DAPI (blue) in TA muscle (scale bar: 50 μm). White arrow indicates CD8 cell. (D) Immunostaining with anti-hSGCA (green), CD8 (red), and DAPI (blue) performed on liver (scale bar: 50 μm). White arrow indicates CD8 cell. (E) Immunostaining with anti-CD3 (green), anti-FoxP3 (red), and DAPI (blue) (scale bar: 25 μm). White arrow indicates FoxP3 cell. (F and G) Flow cytometry dot plots representing the liver non-parenchymal CD4⁺FoxP3⁺ population gated on CD4⁺ cells (F). The histogram shows the quantification of the dot plots (G). 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 versus all groups; n = 4 per group).