The Capacity Of Antigen Presentation Of BMDCs Enhanced By HBc-VLPs

Objective: Dendritic cells (DC) are the strongest professional antigen presenting cells currently found that control the quality, magnitude and memory of the immune response. With the establishment of DC culture technology in vitro, it have be shown that the immature DC cultured in vitro can efficiently uptake virus-like particles (VLPs) and induce not only humoral immunity but also cell-mediated immune response by cross-antigen presenting. During this process, in addition to providing specific antigen to DCs as antigen expression vector, VLPs provide stimulate mature of the cells and induce the expression of MHC I and class II molecules and some adhesion molecules also. Thus the construction of DC vaccines by loading VLPs set up a new way to develop vaccines for cancer and intracellular pathogen infection.HBV core antigen (Hepatitis B core, HBc), as the important structural proteins of hepatitis B virus with stronger immunogenicity, can be expressed efficiently in bacteria, yeast and mammalian cells and can auto-assemble into VLPs even exogenous peptides are inserted. Thus loading DCs with HBC-VLPs may be a better way DC vaccine research.With the human genome project implementation and development, life science research has progressed into the post-genome era. The new disciplines-Proteomics was born, which could be used to study the dynamic of all proteins of cells to get a deeper understanding of the laws of life activities.Based on the reasons above, in this study, a DC vaccine model was set up by loading BMDC with HBC-VLP obtained from prokaryotic expressing system and the proteins extracted from BMDC or HBc-VLPs-pulsed-BMDC were separated by typical two-dimensional electrophoresis (2DE) to find the proteins changed in related to the activation and antigen presentation of DC. Several proteins expressed differently in BMDC and HBc-VLPs-pulsed-BMDC were separated and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS), which provide new clues and ideas to understanding the mechanisms of cell activation and immune response inducing of DC vaccines.Methods:⑴HBC-VLPs preparation: E.coli BL21 transformed with pET28a-HBc were cultured in 2YT medium and induced by IPTG; the expressed HBC proteins in form of inclusion bodies were extracted and purified by Ni-NTA affinity chromatography.⑵Identification of HBc-VLPs: HBc protein was identified by SDS-PAGE and its immunogenicity was tested by Western blot; the formation of HBc-VLPs was observed by transmission electron microscopy.⑶Cultivation of mouse bone marrow-derived dendritic cells (BMDC) in vitro: BMDC were cultured in R10 medium and induced by rmIL-4 and rmGM-CSF.⑷The uptaking of HBc-VLPs by BMDC was observed by laser scanning confocal microscopy at different time points.⑸Flow cytometry analysis was used to observe the surface molecular markers on BMDC that had been stained with FITC labeled anti-mouse CD11c antibody and FITC labeled anti-mouse CD86, CD80 and MHC-Ⅱ.⑹T he levels of IFN-γand IL-12 mRNA was determined by RT-PCR in three groups (untreated-BMDC, LPS-BMDC and HBc-VLPs-BMDC), usingβ-actin as the reference.⑺①The typical two-dimensional electrophoresis (2DE) was used to separate proteins from BMDC and HBc-VLPs-pulsed-BMDC:Ⅰprotein sample preparation: cell proteins were extracted and treated with 2D-clean-up kit according to the instruction of the manufacturer and the concentrations of protein were determined by BCA method;Ⅱ2DE: isoelectric focusing:150μg protein sample was loaded to IPG and isoelectic focusing was performed by hydration at 30 v for 1 h, demineralization at 200 v for 1 h and then gradient boosting to 8000 v for 6-8h, total 50000 vh; SDS-PAGE: the plastic strips were balanced and transferred to SDS-PAGE gel and then vertical electrophoresis were carried out at 20 mA/gel for 5 h-6 h;ⅢSilver nitrate staining: after the fixing, sensitizing and rinsing, the gels were stained with silver nitrate; the stained gels were rinsed, colorized and then terminating the colorization by rinsing, and preserved in 1% acetic acid.②analysis and selection of the proteins: digital images were gotten by gel imaging system ChemiIamgerTM5500. IamgerMasterTM 5.0 software were used to analysis the images and the points that showed more than three times of differences were selected.③The dates of selected proteins got by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry MS were put in NCBInr UniProt database and searched for the matched peptides to make sure which peptides they might be.⑻According to the results MS identification,Anexin 2 protein confirmed by Western Blot.⑼in vivo target killing experiment:①immunization of C57BL / 6 mice: BMDC were incubated with HBc-VLPs 24 h, and treated with LPS to induced maturation of the cells, and then HBc-VLPs pulsed BMDC (about 3×106) were injected subcutaneously in mice;②Spleen cells that were obtained from the normal C57BL / 6 mice, and labeled with 5 nm of 5(6)-carboxyl fluorescein succinimidyl-ester (CFSE) for HBc peptide treated and 0.5 nm for untreated cells, were inoculated intravenously to the immunized mice in a rate of 1:1 of the labeled cells 7 days after the immunization. Flow cytometry analysis was carried out and the CTL activities were determined according to the following formula: the CTL activity= (1 - CFSEhigh / CFSElow)×100.⑽Statistics: All data were analyzed by Student's t-test.Results:⑴The HBc-VLPs were formed from proteins produced in E.coli and purified by Ni2+ columm affinity chromophotography; As confirmed by SDS-PAGE, Western blotting and electron microscopy, the recombinant HBcAg maintained its exact molecular weigh, immunogenic properties and the abilities to correctly fold and auto-assemble into virus-like particles, the particle size of VLP was about 22 nm.⑵The successful culture of mice BMDC was verified by using optical microscopy and flow cytometry. BMDC surface marker expression of CD11c+ rate was more than 75%.⑶By using Laser scanning confocal microscopy, we observed that Mice BMDC have strong phagocytic capacity and a large number HBc-VLPs could be quickly uptoken by BMDC in vitro.⑷Flow cytometry analysis was used to detect whether HBc-VLPs can induce antigen-presenting ability of BMDC or not. The positive rate of surface marker CD86 in the three groups (untreated-BMDC, HBc-VLPs-BMDC, LPS-BMDC) were 17-20%, 42-45%, 46-51% respectively; those of CD80 were 16-17%, 47-48.5%, 56-58% respectively; and MHC-II,40-42.3%, 63.5-68%, 78-81%. The results RT-PCR confirmed that mRNA levels of IL-12 and IFN-γin BMDC-HBc-VLPs were significantly higher than those of untreated-BMDC group.⑸2DE was used to detect differential expression of the total cells protein before and after BMDC loaded by HBc-VLPs. Through ImageMaster TM 5500 and the gel analysis, we got 500-600 protein points on each gels, the match rate between the gels was more than 70%. Eight protein points were shown to have differences of more than 3 times between HBc-VLPs-BMDC group and the PBS-BMDC group. Six of the highly expressed protein spots were identified by MALDI-TOF mass spectrometry. Four protein spots were known by searching in the UniProt database and NCBInr peptide mass: which were Isocitrate dehydrogenase (NAD+) alpha, isoform CRA_e; growth factor receptor bound protein 2, and Annexin A2. Annexin A2 expression was found down-regulated and that was subsequently confirmed by western blot.⑹HBc-VLPs-BMDC could induce specific CTL activity which was detected by target killing test in vivo: histogram of flow cytometry showed that the percentage of target cells (M2) were 46-49%, 47-51%, 17-19% in the untreated group, PBS-treated group, HBc-VLPs-BMDC group, respectively. The M2 was inversely proportional to the number of cells killed that indicated that HBc-VLPs-load-BMDC could induce high level of specific CTL activity.Conclusion:⑴The highly purified HBc-VLPs were successfully obtained from the inclusion of HBc encoding plasmid transformed E.coli.⑵BMDC were cultured successfully in vitro that was confirmed by morphology observation and specific maker expression analysis on the cells.⑶Laser scanning confocal microscopy observation and flow cytometry analysis showed that mice BMDC could uptake effectively HBc-VLPs in vitro; BMDC went a further mature process after the uptaking of HBC-VLPs that was indicated by the expression of cell surface markers CD86, CD80, increased expression of MHC-Ⅱ, In vivo killing experiments showed that HBc-VLPs-load-BMDC in mice could induce antigen-specific T cell responses and increased CTL activity.⑸2DE experiments found that there were differences in the expression of the protein between HBc-VLPs-BMDC group and the untreated-BMDC group.⑹Four proteins were identified by Protein spectrum analysis and database searching: Isocitrate dehydrogenase (NAD+) alpha, isoform CRA_e, similar to Epiplakin, growth factor receptor bound protein 2, and annexin A2. The last two proteins have been reported to playe an important role in cell differentiation, expansion and the process of apoptosis.⑺Protein immunoblotting experiment confirmed that compared to PBS-BMDC group, the expression of Annexin-A2 in HBc-VLPs-BMDC was decreased, suggesting that the reduction of inhibitory factors of DC might be responsible to enhanced ability of DC to activation of T-cell.