PD-1 inhibits antiviral immunity at the effector phase in the liver

Abstract

Unlike naive T cells, effector T cells can be activated by either T cell receptor signal or costimulatory signal alone and therefore the absence of costimulatory molecules on tissue cells cannot explain the tolerance mechanism at the effector phase. Here we report that PD-L1, the ligand for the immunoinhibitory receptor PD-1, was expressed on vascular endothelium in peripheral tissues. Liver nonparenchymal cells including sinusoidal endothelial cells and Kupffer cells constitutively expressed PD-L1 and inhibited proliferation and cell division of activated T cells expressing PD-1. The absence of PD-1 induced proliferation of effector T cells in the adenovirus-infected liver and resulted in rapid clearance of the virus. These results indicate that PD-1 plays an important role in T cell tolerance at the effector phase and the blockade of the PD-1 pathway can augment antiviral immunity.

Figure 1.

PD-L1 expression on vascular endothelium. (a) ECs were freshly isolated from murine heart and stained with anti-PD-L1 or anti-PD-L2 (open curves). The filled curves represent control IgG. (b-g) Biotinylated F(ab′)₂ of anti-PD-L1 (b-g1) or control IgG (b-g2) was intravenously injected into mice and organs were harvested 1 h later. Sections were stained with streptavidin-FITC (green) and counterstained with phalloidin (red). (b) Eye, (c) submandibular gland, (d) lung, (e) heart, (f) liver, (g) kidney. Ch, choroid; CV, central vein; Gl, glomerulus; Re, retina. The arrows represent vascular endothelial cells. Original magnification, ×40.

Figure 1.

PD-L1 expression on vascular endothelium. (a) ECs were freshly isolated from murine heart and stained with anti-PD-L1 or anti-PD-L2 (open curves). The filled curves represent control IgG. (b-g) Biotinylated F(ab′)₂ of anti-PD-L1 (b-g1) or control IgG (b-g2) was intravenously injected into mice and organs were harvested 1 h later. Sections were stained with streptavidin-FITC (green) and counterstained with phalloidin (red). (b) Eye, (c) submandibular gland, (d) lung, (e) heart, (f) liver, (g) kidney. Ch, choroid; CV, central vein; Gl, glomerulus; Re, retina. The arrows represent vascular endothelial cells. Original magnification, ×40.

Figure 2.

PD-L1 expression on liver nonparenchymal cells. (a) Cryosections of murine liver were stained with anti-ICAM-1 (top, green) or anti-PD-L1 (bottom, green). Nuclei were counterstained with propidium iodide (red). CV, central vein. Original magnification, ×40. (b) Surface phenotype of Kupffer cells and LSECs. LNPCs were isolated from murine livers and stained with anti-CD54 (ICAM-1)-FITC and anti-CD11b-APC, in combination with biotinylated mAb for ICAM-1, PD-L1, B7–1, or B7–2, followed by streptavidin-PE. Kupffer cells and LSECs were gated as CD54⁺CD11b^high and CD54⁺CD11b^low cells, respectively.

Figure 2.

PD-L1 expression on liver nonparenchymal cells. (a) Cryosections of murine liver were stained with anti-ICAM-1 (top, green) or anti-PD-L1 (bottom, green). Nuclei were counterstained with propidium iodide (red). CV, central vein. Original magnification, ×40. (b) Surface phenotype of Kupffer cells and LSECs. LNPCs were isolated from murine livers and stained with anti-CD54 (ICAM-1)-FITC and anti-CD11b-APC, in combination with biotinylated mAb for ICAM-1, PD-L1, B7–1, or B7–2, followed by streptavidin-PE. Kupffer cells and LSECs were gated as CD54⁺CD11b^high and CD54⁺CD11b^low cells, respectively.

Figure 3.

Phenotypic analysis of activated T cells. In vitro–activated CD4⁺ or CD8⁺ T cells from PD-1^−/− or WT mice were stained with a panel of mAbs (thick lines) and control IgG (thin lines).

Figure 4.

PD-L1 effect of LNPCs on T cell proliferation and cytokine production. (a and d) Naive T cells from PD-1^−/− mice or WT mice were stimulated with or without plate-bound anti-CD3 for 48 h. T cell proliferation was measured by BrdU incorporation (a). The culture supernatants were harvested and IFN-γ production was measured by ELISA (d). (b and e) In vitro–activated PD-1^−/− or WT T cells were cocultured for 48 h with or without mitomycin C–treated LNPCs from WT mice in the presence or absence of anti-PD-L1 (control rat IgG) or CTLA4-Ig (control human IgG). T cell proliferation was measured by BrdU incorporation (b). The culture supernatants were harvested and IFN-γ production was measured by ELISA (e). (c) In vitro–activated PD-1^−/− or WT T cells were labeled with CFSE and cocultured with LNPCs. After 48 h of incubation, T cell division was examined by FACS^® analysis of CFSE intensity.

Figure 5.

The effect of Kupffer cells and LSECs on proliferation of activated T cells. In vitro–activated PD-1^−/− or WT T cells (3 × 10⁴ cells/well) were cocultured for 48 h with or without mitomycin C–treated LNPCs (before separation, 2 × 10⁵ cells/well, consisting of 20% Kupffer cells and 80% LSECs), purified KCs alone (4 × 10⁴ cells/well), purified LSECs alone (1.6 × 10⁵ cells/well), or mixture of purified Kupffer cells + LSECs (4 × 10⁴ cells: 1.6 × 10⁵ cells/well). T cell proliferation was measured by BrdU incorporation.

Figure 6.

BrdU⁺ cells increased in the adnovirus-infected liver of PD-1^−/− mice. PD-1^−/− or WT mice (3 mice/group) were intravenously injected with Ad-lacZ. At day 0 (a) or day 7 (b) of infection, mice were intraperitoneally injected with BrdU 1 h before sacrifice. Splenocytes and intrahepatic lymphocytes were doubly stained for BrdU and CD19, CD3, CD4, or CD8. (c) The bars represent percentages of BrdU⁺ cells in the CD19, CD3, CD4, or CD8⁺ population at day 7 after infection. The results are representative of two separate experiments.

Figure 7.

Rapid clearance of adenovirus in the liver of PD-1^−/− mice. PD-1^−/− or WT mice were intravenously injected with Ad-lacZ. (a–d) At day 0 or day 7 of infection, WT (top) or PD-1^−/− mice (bottom) were intraperitoneally injected with BrdU 1 h before killing. Liver sections were stained with anti-BrdU (red). Original magnification, ×20. (e and f) At day 7, liver sections of PD-1^−/− mice were doubly stained with anti-BrdU (red), in combination with anti-CD4 (blue, e) or anti-CD8 (blue, f). Original magnification, ×40. (g–r) At day 7 or 30, liver sections of WT mice (top) or PD-1^−/− mice (bottom) were analyzed by H&E staining (g–j, ×20; k–n, ×40) and X-Gal staining (×40). The arrows represent apoptotic hepatocytes.

Figure 7.

Rapid clearance of adenovirus in the liver of PD-1^−/− mice. PD-1^−/− or WT mice were intravenously injected with Ad-lacZ. (a–d) At day 0 or day 7 of infection, WT (top) or PD-1^−/− mice (bottom) were intraperitoneally injected with BrdU 1 h before killing. Liver sections were stained with anti-BrdU (red). Original magnification, ×20. (e and f) At day 7, liver sections of PD-1^−/− mice were doubly stained with anti-BrdU (red), in combination with anti-CD4 (blue, e) or anti-CD8 (blue, f). Original magnification, ×40. (g–r) At day 7 or 30, liver sections of WT mice (top) or PD-1^−/− mice (bottom) were analyzed by H&E staining (g–j, ×20; k–n, ×40) and X-Gal staining (×40). The arrows represent apoptotic hepatocytes.