The Expression Of The Programmed Cell Death 1 Ligands In Woodchuck Hepatitis Virus Infection And Their Involvement In Regulation Of Specific T Cell Function In Woodchucks

Objective1. To learn the sequence of the woodchuck PD-L1 and PD-L2, produce the extracellular region of the moleculars by prokaryotic expression, and generate the polyclonal antibodies.2. To study the induction of wPD-L1 expression in the PWH and PBMC subsets by TLR ligands and IFNs. Evaluate the relationship between the PD-1/PD-Ls immune-suppression pathway and the progress of WHV infection in woodchuck model.3. To learn whether blockade of PD-1/PD-L1 pathway could restore T cell response and clear the virus in chronic WHV infected woodchuck model, and provide a new idea to develop effective therapeutic methods of HBV.Methods1. Clone the CDS region of PD-L1 and PD-L2 sequence from the chronically WHV infected woodchuck liver RNA. Sequence and analyze the homology with other mammalian species at nucleotide level and amino acid level. 2. Separate the PWH and PBMCs from healthy woodchuck, stimulate with TLR ligands and IFNs. Detect the expression of PD-L1 at RNA and protein level by Real time PCR and FACS staining. Analyze the different reaction of PBMC subsets after the TLR ligand or IFNs stimulaion.3. Separate total RNA from the liver tissue of the woodchucks at different infection stages (healthy control, acute infection, chronic infection), and detect the PD-L1 expression at RNA level by Real time PCR.4. Separate PBMCs from the woodchucks at different time point of WHV infection (healthy control, early stage and late stage of acute infection, chronic infection), and detect the PD-1 and PD-L1 expression of different cell subset (CD4+and CD4-cell populations) by FACS.5. Prokaryotic expression of the extracellular region of wPD-L1/-L2, immunize rabbit with purified proteins to obtain anti-wPD-L1/-L2 antiserums, and purify the polyclonal antibodies. Detect the antibody titers by ELISA. Construct the mammalian expression vectors expressing full CDS of wPD-L1 and extracellular and transmembrane region of wPD-L2. Transfect BHK cells with the plasmids and detect the avidity and specify of the antibodies by western blot and immunoflourences.6. Separate PBMCs from the woodchucks at different time point of WHV infection (healthy control, early stage and late stage of acute infection, chronic infection), block the PD-1/PD-Ls pathway by incubation with wPD-L1/-L2 polyclonal antibodies, and detect the proliferation or function of antigen-specifc T cells by [H]3-Adenin or FACS staining, respectively.Results1. The CDS of wPD-L1 and-L2 were obtained and subjected to sequence analysis. The CDS region of wPD-L1 contains 864 base pairs, and had a homology of 84.9%,82.1%, 75.3%,80.9% at nucleotide level, or 75.9%,71.9%,66.2%,70.4% at amino acid level to the counterparts of human, pig, mouse and cattle, respectively; wPD-L2 contains 822 base pairs, and had a homology of 83.8%,83.2%,80.3%,78.0%at nucleotide level, or 72.9%, 72.5%,68.8%,70.9%at amino acid level to the counterparts of human, monkey, pig, and mouse, respectively. The homology between wPD-L1 and-L2 was 31.4% for the complete coding region2. WPD-L1 expression in PWH and PBMCs was low and could be stimulated by TLR ligands and IFNs at RNA and both protein level. It could be shown that the ligands of TLR1/2,3,4, and 7, IFN-αand-γcould significantly up-regulate the wPD-L1 expression in primary woodchuck hepatocytes (PWH). TLR4 and 7 have a strongest upregulation to PBMCs, while TLR1/2,3, IFN-αand-γhave lower function, and the other ligands. Stimulation with TLR2/6,5 and 9 ligands did not generate consistent results with PBMCs from different woodchucks. CD4-cells have a stronger upregulation to the stimulation than CD4+cell subsets.3. WPD-L1 in liver tissues was significantly higher than that measured in cultured primary hepatocytes, and was only slightly elevated in woodchucks with acute and chronic WHV infection. In the HDV and HBV superinfection, the wPD-L1 was significantly upregulated in the liver tissue.4. WPD-1 and wPD-L1 expression on PBMCs was significantly up-regulated during acute and chronic WHV infection. During the acute infection, wPD-1 show a high expression at the early stage while wPD-L1 at late stage.5. The prokaryotic expression vectors, pET28a-wLl and pET30a-wL2, contained the coding sequences nucleotides for the extracellular region in the putative mature wPD-L1 and-L2 proteins. The recombinant proteins His-wPD-L1 and-L2 were expressed in the E. coli strain BL21(DE3)plyS by IPTG induction and purified with HisTrap Kit. Rabbit antiserums to wPD-L1 and-L2 were generated by subcutaneous immunization with purified proteins. The antiserums were detected by ELISA and had titers over 1:1,000,000. The polyclonal antibody IgG were then purified by SPA columns. 6. The rabbit antibodies were identified by transient infection with the eukaryotic expression wPD-L1/-L2 plasmids, the titer of Western blot were around 1:2000, and the titer of IF is around 1:100 or 1:200 for anti-wPD-L1 or anti-wPD-L2, respectively.7. In vitro blockade with antibodies to wPD-L1 enhanced lymphoproliferation and CD 107a degranulation in some acutly or chronically WHV-infected woodchucks, while blockade with anti-wPD-L2 could only enhance lymphoproliferation in few animals and has no effect on the T cell function.Conclusions1. Successfully obtained the full CDS of woodchuck PD-L1 and-L2.2. TLR ligands and IFNs could upregulate the expression of PD-L1 on PWH and PBMC, and PD-1 and PD-L1 were significantly upregulated on the PBMC during acute and chronic infection. These results show some relationship between the innate and adaptive immune response during hepatitis virus infection.3. Hepatocytes were not the main source of liver PD-L1. PD-L1 expression during the infection is only slightly changed, and may be because of the immune tolerant environment in the liver.4. Successfully prepared anti-wPD-L1/PD-L2 polyclonal antibodies. In vitro blockade with antibodies to wPD-L1 and-L2 enhanced lymphoproliferation and CD 107a degranulation in some WHV-infected woodchucks.5. Not every chronic woodchuck was responsive to the blockade, which indicates that the wPD-1/PD-L1 system may be not the only cause for low T cell responses in chronic WHV infection. Thus, a combined approach would be preferred to restore T cell functions in chronically infected individuals.