BACKGROUNDAutoimmune hepatitis (AIH) is a chronic inflammatory liver disease with unknown etiology. Programmed death 1 (PD-1) and its ligands (PD-L1 and PD-L2), B7-H1/PD-L1 and B7-DC/PD-L2, are new CD28-B7 family members that are involved in the regulation of immune responses. Previous observation suggests that PD-1 system plays an inhibitory role in regulating peripheral blood T cells, B cells and myeloid cells, thus their abnormality may be related to autoimmune diseases. This study aimed to explore the role of PD-1/PD-L1, L2 system in the pathogenesis of AIH.

METHODSThe mice model of experimental autoimmune hepatitis (EAH) was established in C57BL/6 mice and the expression levels of PD-1 and PD-L1, L2 in the murine liver and the cytokines, including interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and interleukin (IL)-4 in the spleen were detected using reverse transcription-polymerase chain reaction (RT-PCR), and the results were compared with those of normal controls.

RESULTSThe expression levels of PD-1, PD-L1, PD-L2 mRNA were higher in EAH compared with normal controls (P < 0.05), the PD-L2/PD-1 ratio was relatively lower in EAH (EAH -0.08 +/- 0.35, normal controls 0.52 +/- 0.07, P = 0.009). In the EAH, the expression of the three cytokines were all upregulated compared with normal controls. PD-L1 had a positive correlation with the expression of IFN-gamma (r = 0.289, P < 0.05), while PD-L2 showed a positive correlation with both expressions of IL-4 (r = 0.378, P< 0.01) and IFN-gamma (r = 0.261, P < 0.05). While TNF-alpha showed no correlation with PD-L1 (r = 0.044, P = 0.736) or PD-L2 (r = 0.127, P = 0.335).

CONCLUSIONSThe expression of PD-1/PD-L1, L2 is upregulated in EAH and regulated by IFN-gamma and IL-4. PD-1 system may play an important role in the pathogenesis of AIH.

Animals ; Antigens, Surface ; genetics ; Apoptosis Regulatory Proteins ; genetics ; B7-1 Antigen ; genetics ; B7-H1 Antigen ; Hepatitis, Autoimmune ; genetics ; Interferon-gamma ; genetics ; Interleukin-4 ; genetics ; Membrane Glycoproteins ; genetics ; Mice ; Mice, Inbred C57BL ; Peptides ; genetics ; Programmed Cell Death 1 Ligand 2 Protein ; Programmed Cell Death 1 Receptor ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; genetics

OBJECTIVETo analyze the expression levels of PD-1 (program death factor-1) in peripheral T cells from patients infected with HBV, and to investigate its relationship with HBV serological markers.

METHODSA total of 65 HLA-A2+ subjects, including 31 patients with chronic hepatitis B (CHB), 9 with acute resolved hepatitis B (AHB), 15 with HBV related liver cirrhosis (LC) and 10 healthy blood donators, were enrolled. The expression of PD-1 in peripheral T cells and PD-1 ligands PD-L1 and PD-L2 in PBMCs were determined by relative quantitative real-time PCR. The serum HBV markers, HBV DNA load and liver function were also measured.

RESULTSTaken the PD-1 and PD-ligands expression in normal controls as a baseline level, the expression of PD-1 and PD-L1 from CHB patients was significantly increased, while the expression of PD-L2 was relatively low in all groups. In CHB patients, the PD-1 expression in peripheral T cells from patients with high viral load was much higher than that from those with low viral load or from normal controls. And the PD-1 expression level positively correlated with serum HBV DNA load (r=0.41, P<0.01) but not with serum ALT level.

CONCLUSIONLong-term exposure to HBV antigens in CHB patients may increase the expression of PD-1 in T cells and thus leads to the virus persistent infection.

Adult ; Antigens, CD ; genetics ; metabolism ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; B7-H1 Antigen ; DNA, Viral ; blood ; Female ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; metabolism ; virology ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Male ; Middle Aged ; Programmed Cell Death 1 Ligand 2 Protein ; Programmed Cell Death 1 Receptor ; RNA, Messenger ; genetics ; metabolism ; T-Lymphocytes ; metabolism ; Viral Load

By using semi-quantitative RT-PCR method, it was found that PD-L1 mRNA but not PD-L2 mRNA was expressed in H22 hepatoma cells and both PD-L1 and PD-L2 mRNAs were expressed in tumor tissues of tumor-bearing mice and upregulated as compared with muscle tissues in normal mice and H22 hepatoma cells. PD-L1 and PD-L2 were also expressed on the surface of the activated T cells. The soluble recombinant sPD-1 expressed from the constructed eukaryotic expression vector could enhance the lysis of tumor cells by lymphocytes stimulated specifically with antigen. The expresssion of sPD-1 by local gene therapy on the inoculation site of H22 hepatoma cells could inhibit the growth of tumor. The results of this study indicate that expression of soluble receptor of negative costimulatory molecules could reduce the inhibitory effect on T cells in tumor microenvironment and enhance the cytotoxicity of T cells on tumor cells. This possibly provides a new method of improving efficacy of tumor gene therapy.
Animals ; B7-1 Antigen ; biosynthesis ; genetics ; B7-H1 Antigen ; Carcinoma, Hepatocellular ; immunology ; pathology ; Genetic Therapy ; Liver Neoplasms ; immunology ; pathology ; Male ; Membrane Glycoproteins ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Peptides ; genetics ; metabolism ; Programmed Cell Death 1 Ligand 2 Protein ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Spleen ; cytology ; T-Lymphocytes ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Tumor Cells, Cultured