Laponite^? Nanosheets Increase Dendritic Cell Homing And The Efficacy Of Anti-viral CD8⁺ T Cell Immune Responses

Background and ObjectiveHepatitis B Virus（HBV）infection and the caused hepatitis,cirrhosis,and hepatocellular carcinoma（HCC）are major health problems.Immunotherapy is one of the most promising therapies available to overcome chronic HBV infection,breaking the immune tolerance caused by HBV and rebuilding the patient’s antiviral immunity.Dendritic cells（DCs）are the most powerful antigen-presenting cells known and are the initiators of antiviral immune responses.There have been domestic and international studies using DC vaccine loaded with HBV antigens for the treatment of chronic HBV,and some therapeutic effects have been initially observed,but they still do not meet expectations.Our previous study found that the extremely low in vivo homing ability of DCs（only 3-5%）was the key to their dysfunction.This study focuses on the development nanoadjuvants for DC vaccine,and systematically investigates the role and molecular mechanism of Laponite（?）（Lap）,a synthetic nano-sheeted magnesium-lithium silicate mineral,in enhancing the homing ability of DCs and the activation of anti-HBV viral CD8⁺T cells.MethodsThe particle size,morphology,and surface charge properties of the prepared Lap nanosheets were characterized by atomic force microscopy（AFM）,transmission electron microscopy（TEM）,and particle size analyzer;the subcellular organelle localization of Lap in DCs was visualized by confocal imaging;the immune synapse（IS）formation between Lap-treated DCs and CD8⁺T cells was examined under the live cell imaging system.Finally,the in vivo homing of DCs and the clearance of HBV were analyzed by the small animal live imaging system（IVIS）.ResultsThe prepared Lap was disc-shaped with a diameter of about 27.98 nm and a thickness of about 0.803 nm.the overall Lap dispersion was negatively charged with a potential of-38.50±3.01 m V.Lap had no significant effect on the cell viability of DCs in the concentration range of 25-100μg/m L.Lap was internalized by DCs in the lysosome via the giant cell drinking pathway,causing an increase in lysosomal membrane lipid peroxidation（LPO）and reactive oxygen species（ROS）levels.The increase in LMP triggered Ca²⁺efflux from lysosomal stores and promoted cytoskeletal rearrangement and chemokine receptors CCR5,CCR7,and CXCR4 through the"TFEB-Rab7b"signaling axis,which greatly enhanced the lymphoid tissue homing ability of DCs（PBS VS.Lap:7.9±4.9%VS.37.6±5.7%）.Further,Lap stimulated DCs to activate higher levels of antiviral Cytotoxic T Lymphocytes（CTLs）in liver-draining lymph nodes compared to the conventional cytokine cocktail adjuvant Cytokine-Cocktail（C-C）（C-C VS.Lap:0.46±0.07%VS.0.68±0.1%）,increased expression of CD69,CD44,CD25 and CD107a on the surface of CD8⁺T cells and synthesis of TNF-ɑand IFN-γ.Finally,the modified cytokine cocktail Lap@C-C was prepared by replacing the PGE₂ in C-C with Lap,and then injected the optimized DC vaccines into HBV long-term expression mouse model,with the results showing that Lap@C-C stimulated DCs had higher HBV clearance rate of 99.9%。ConclusionAs an easy-to-prepare and highly biocompatible disc-shaped inorganic nanosheet material,Laponite（?）can be internalized by DCs in large quantities and cause an increase in lysosomal oxidative stress levels in DCs,promoting cytoskeletal rearrangement,enhanced lymphoid tissue homing ability,and activation of antiviral CTLs by inducing the release of lysosomal Ca²⁺.In conclusion,this study reports the great potential of Laponite（?）in immunomodulation,which provides a reference and research basis for developing novel nano-vaccine adjuvants.