NOD1-TLR3Ligands Stimulated Mouse LSECs Trigger HBV Specific T Cell Proliferation

ObjectiveLiver sinusoidal endothelial cells (LSECs) are organ resident antigen-presentingcell in liver capable of antigen presentation and subsequent tolerization of differentkinds of T cells. Here we investigated the the effect of LSECs in anti-HBV acquiredimmune response, and elucidated anti-HBV specific T cell immunity and mechanismtriggered by NOD1-TLR3receptor ligands stimulated LSECs. To provide atheoretical basis for the new strategy of anti-HBV immune.Methods1. LSECs were isolated using Liberase Blendzymes perfusing and Anti-CD146MicroBeads from C57BL/6mouse.2. HBV specific T cell mice model was established by hydrodynamic injection ofPSM2into the tail veins of C57BL/6mice. HBV specific T cells were isolatedusing Anti-CD90.2MicroBeads from HBV specific T cell mice.3. CFSE staining was used to detect the HBV-specific T cell proliferation induced byNOD1-TLR3stimulated LSECs.4. LSECs were stimulated with the NOD1-TLR3agonists respectively orNOD1/TLR3agonists costimulatory for3hours, the LSECs were collected andTotal RNA was isolated. The mRNA of IFN-β, IFN-γ, IRF3, ISG15, TNF-α, IL-6, TGF-β, IL-10, CCL2, CCL5, CXCL1, CXCL2, CXCL9, CXCL10and β-actin byreal time RT-PCR. the LSECs and supernant were collected for20hours, LSECssurface molecules (MHC-Ⅱ, CD80, CD86, PD-L1, CD106, CD54and CD80)were detected by flow cytometry. The cytokines (IFNα/β/γ, TNF-α, IL-6) in thecells supernatant were detected by quantitative ELISA.Results1. NOD1and TLR3ligands Stimulated LSECs trigger HBV-specific T cellproliferation.2. After costimulated by NOD1and TLR3ligands, LSECs are induced toup-regulate the chemokines（CCL2, CCL5, CXCL1, CXCL2, CXCL9,CXCL10）mRNA expression.3. After costimulated by NOD1and TLR3ligands, It is found that up-regulation ofAdhesion molecule(CD54, CD106) in the LSECs detected by flow cytometry.4. After costimulated by NOD1and TLR3ligands, LSECs are induced toup-regulate the cytokine IL-6and TNF-α mRNA expression.Conclusion1. HBV（HBsAg/HBeAg）can not induce HBV-specific T cell proliferation byLSECs.2. NOD1and TLR3can Mediate LSECs induce HBV-specific T cell proliferation.The mechanism can be conclused as follows: Fistly, after costimulated by NOD1and TLR3ligands, LSECs induced secretion of various chemokines to regulate Tcell recruitment and activation by immune synapse formation.In Addition theAdhesion molecule(CD54, CD106) on the LSECs up-regulated, they can inducethe T cells and LSECs adhesion,Then provied the proliferation signals for T cells.Furthermore the up-regulate of IL-6and TNF-α may enhance some moleculesindeced lymphocyte proliferative effect.