The expression of PD-1 on immune cells (CD4, CD8, NK and macrophages) from different organs was assessed by flow cytometry (FACsAria Cytometer; Becton Dickinson, Germany). Briefly, cell suspensions of liver, spleen, blood and thymus tissues were washed and resuspended in PBS. Cells were then incubated for 30 min at room-temperature in the dark using primary antibodies (PE-PD-1, FITC-CD4, FITC-CD8, FITC-NK1.1 and FITC-CD68. eBioscience). To analyze the source of IFN-c in the liver, PD-1-deficient and WT mice were treated with MHV-3 (10 PFU). After 72 h, liver tissues were isolated and mechanically homogenized, lymphocytes were collected thereafter. Cells were then treated with Brefeldin A solution (BFA) for 4 h, and FITC-NK1.1, FITC-CD3 or PE-IFN-c mAbs (eBioscience) were added and the solution incubated for an additional 1 h. For each analysis, 10000 cells were evaluated. Flow cytometric data were analyzed with CellQuest Pro Software.