Effects Of CD4~+T-cell Exosomes On The Antibody Production In HBsAg Vaccine Vaccinated Mice

Objective:To observe the effects of CD4⁺T-cell exosomes on mice vaccinated with Hepatitis b surface antigen?HBsAg?vaccine,investigate its roles in B cell response in vitro,and reveal its possible mechanism.Methods:?1?100?L 20?g/mL HBsAg vaccine was intramuscular injected into left hindthigh muscle of 6-8w female Babl/c mice.After 14 days,same dose of HBsAg vaccine was used to boost the immune response.Spleen CD4⁺T cells from HBsAg vaccine vaccinated mice and wild type mice were isolated by magnetic cells sorting and activated with 2?g/m L anti-CD3 and CD28 functional mAbs for 24h in vitro,exosomes in CD4⁺T cells cultural supernatant were extracted by differential centrifugation and ultrafiltration membrane technology followed by precipitation,termed as HB-T-Exo and WT-T-Exo,respectively.The morphology and size distribution of exosomes were observed by electron microscopy and nanoparticle tracking analyzer,exosomes surface molecules were analyzed by flow cytometry and its characteristic protein expression were detected by western blot.?2?100?L HBsAg vaccine?containing 2?g HBs Ag?was intramuscular injected into mouse thigh muscle at day0 and day14,at the same time,50?g HB-T-Exo was intravenous injected into the tail vein,WT-T-Exo was exosomes control,PBS injection was used as a blank control.Serum was collected from tail blood at day16,23,30,40 and 50.The levels of Hepatitis B surface antibody?HBs Ab?and its subtypes were detected by ELISA.Mice were sacrificed at day50,the proportion of spleen Th1 cells,Th2 cells,B cells,plasma cells and bone marrow plasma cells were analyzed by flow cytometry.?3?30?g PKH26-labeled CD4⁺T-cell exosomes were co-cultured with mouse spleen lymphocytes for 4h,exosomes binding T cells and B cells were analyzed by flow cytometry.After co-cultured of B cells with PKH67-labeled CD4⁺T-cell exosomes for 4h,laser confocal microscopy was performed.?4?CD4⁺T-cell exosomes were extracted from ovalbumin?OVA?immunized mice and wild type mice,termed as OVA-T-Exo and WT-T-Exo,respectively.B cells were co-cultured with corresponding exosomes,the expression of CD86,CD80,CD40 and MHC?were analyzed by flow cytometry.CFSE staining was used to analyze the proliferation of B cells,ELISA was used to detect the production of antibodies in the co-culture supernatant.?5?Mouse T-cell lymphoma EL-4 were used to establish CD40L knock down cell line by CD40L ShRNA plasmid,and CD40L knock down EL-4 exosomes were prepared.Then observe the role of CD40L molecules in T-cell exosomes mediated B cell responses in vitro.Results:?1?CD4⁺T-cell exosomes showed a circular or elliptical disk-like structure by electron microscopy,with a diameter of 50-150nm.Flow analysis showed that it expressed CD4,CD25 and ICOS surface molecules.Western blot showed it expressed CD63,CD9 and Hsp70 protein,moreover,it also express CD40L molecules.?2?After treated with different sources of CD4⁺T-cell exosomes,compare to PBS and WT-T-Exo treated group,the level of HBsAb in serum of HBsAg vaccine vaccinated mice treated with HB-T-Exo was significantly increase,which was higher than that treated with WT-T-Exo and PBS at day50?P<0.05?.However,the serum HBsAb level in WT-T-Exo treated mice was not significantly different from PBS control group,suggesting that the biological function of CD4⁺T-cell exosomes was antigen-specific.Through the detection of HBsAb antibody subtypes,we found that HB-T-Exo treatment mainly enhanced the production of HBsAb IgG2a,but had no effect on the production of HBsAb IgG1.Therefore,the promotion of humoral immune response mediated by CD4⁺T-cell exosomes mainly enhance the production of Th1 antibodies.In PBS control group,the proportion of bone marrow plasma cells was 3.143±0.9765%,and HB-T-Exo and WT-T-Exo treated groups were much higher?17.83±2.226%,17.07±3.404%,respectively?,which suggested that CD4⁺T-cell exosomes can promote the generation of plasma cells in HBsAg vaccine vaccinated mice.In addition,in PBS treated group,the proportion of Th1 cells was 2.50±0.33%,while HB-T-Exo and WT-T-Exo treated group was 4.38±0.32%and 3.62±0.36%,respectively,wihich were much higher than PBS treated group.However,CD4⁺T-cell exosomes had no effect on the proportion of spleen Th2 cells,B cells and plasma cells.?3?By flow analysis of the exosomes binding efficiency of spleen T cells and B cells,it showed that CD4⁺T-cell exosomes mainly bind to B cells,the mean fluorescence intensity?MFI?of PKH26 on B cells was almost 3.5 times than T cells?P<0.01?.Confocal microscopy analysis further confirmed that CD4⁺T-cell exosomes can bind and be internalized by B cells.?4?In vitro experiments demonstrated that exosomes derived from activated CD4⁺T cells can promote the expression of CD86 and MHC?molecules on B cells isolated from OVA-immunized mice in a dose-dependent manner.In addition,CD4⁺T-cell exosomes can also promote B cells proliferation and antibody production.Interestingly,compare to WT-T-Exo,OVA-T-Exo showed a stronger effect on promoting B cell activation,proliferation and antibody production.?5?We successfully established the CD40L knock down EL-4 cell line,and prepared CD40L knock down EL-4 exosomes,which termed as EL-4^CD40L ShRNAD40L ShRNA Exo,the control exosomes was termed as EL-4^{NC ShRNA} Exo.The MFI of CD86 and MHC?molecules on EL-4^{NC Sh RNA} Exo treated B cells were 1124±158.9 and 6565±623.9,respectively,they were significantly decrease on EL-4^CD40L ShRNAD40L ShRNA Exo treated B cells?430.5±53.18,3300±468,respectively??P<0.05?.Moreover,compare to EL-4^{NC Sh RNA} Exo treated B cells,the proliferation of EL-4^CD40L ShRNAD40L ShRNA Exo treated B cells were decreased,and the total IgG level in culture supernatant was also significantly decrease?P<0.01?.Conclusion:?1?We successfully prepared CD4⁺T-cell exosomes,which can increase the proportion of mouse bone marrow plasma cells.Antigen-specific CD4⁺T-cell exosomes can enhance the HBsAb production in HBsAg vaccine vaccinated mice in vivo,which mainly promote the production of Th1 antibodies.?2?CD4⁺T-cell exosomes mainly bind to B cells in vitro,thus promote B cells activation,proliferation and antibody production,which partly by CD40L.