Research Of Targeting Cytotoxity Platform Based On T Epitope And Dentritic Cell-oriented

With the development of immunology, molecular biology, and micro-electronics technology, insights regarding peptide binding with major histocompatability complex (MHC) molecules have revealed a vital role in recognition between T cell and antigen. Antigen epitope can be presented on MHC-Ⅰor II molecules by antigen presenting cells and promote an immune response. Recently, the theory that both was considered that both compound-epitopes and modified epitopes can be more effective at enhancing the function of cytotoxic lymphocytes (CTL) was proposed. Therefore, attention has focused on finding efficient epitopes that upregulate the function of dendritic cells (DC) for treating cancer and infectious diseases. It is key for the application of DC therapy in clinic to determine how to best induce CD8+CTL through MHC-I by exogenous peptides.Our research is focused on the treatment of hepatitis B virus (HBV). The HBV core protein was selected as the antigen source for presentation by DC. The HBV core protein was analysed by specialized software. Biological characteristics of HBV core protein were acquired. T epitopes were analyzed, modified and synthesized. A DC culturing platform was established and standardized. Experiments were designed to elucidate the function and mechanism of peptide-pulsed-DC.Specific Aim I: Screening of T Epitopes Objective. To predict the characteristics of efficient T epitopes restricted by HLA-A2 of the HBV core protein. Methods. B type(adw subtype) HBV core protein from Genebank was selected and predicted normal characteristics by CLC Protein Workbench3 version and SignalP3.0 software(http://www.cbs.dtu.dk/services/SignalP-2.0/)and T epitopes restricted by HLA-A2 by SYFPEITHI databank, PREDEEP databank, matrix. Results. The cross-membrane region, signal peptides, and antigenic sites of HBV core protein were identified and five T epitopes were selected for analysis. Conclusion. Combining several prediction methods can boost the prediction accuracy of T epitopes.Specific Aim II: Identifying, Modifying, and Synthesizing T EpitopesObjective. To Identify, modify, and synthesize five peptides for elucidating the mechanism of exogenous peptides entering DC by the MHC I pathway. Methods. Antigen binding experiments were performed to identify affinities, binding affinities, and MHC-peptide complex stability of five epitopes. Two peptides with the best performance were selected, modified with Th, B, and palmitoyl, then synthesized. Results. Two peptides were acquired, and nine peptides were synthesized and eleven epitopes were obtained. Conclusion. Antigen binding experiments with the Fmoc synthesis method can be used to identify and synthesize epitopes.Specific Aim III: Establishing a Platform of DC GenerationObjective. To establish a reproducible, large-scale platform to generate DC. Methods. Human peripheral blood mononuclear cells (PBMC) were labeled with CD14+ magnetic beads. CD14+ cells were isolated and cultured with IL-4 and GM-CSF in a culture bag for ten days, then harvested. The viability of DC was determined by Typanlan staining. DC phenotype was determined by FACS. The morphology of these DC was observed under a scanning electron microscope. Results. DC suspended in culture medium result in variable cell size and irregular morphology. Cytoplasmic dendrites were extended. 14% of PBMC are CD14+. Magnetic bead purification of PBMC resulted in 94.8% purity of CD14+ cells. After ten days of culture, the majority of PBMC expressed a DC phenotype with only 0.65% of cells expressing CD14. Certain cell surface molecules were expressed at ten days including HLA-I (70.43%),HLA-Dr (0.65%),CD80 (89.45%),CD86 (86.32%), and CD40 (29.61%). By electron microscopy, DC retained their specific morphology. Conclusion. CD14+ cells isolated from PBMC can be non-adherently cultured and transformed into pure DC in culture bags.Specific Aim IV: The Function and Mechanism of Peptide-Pulsed DCObjective. To compare the biological functions of DC loaded with different epitopes and select the peptide that results in the most effective CTL response. To investigate functional differences in DC pulsed with different peptides. Methods. The cytotoxity of T cells was tested by MTT assay and the amount of secretion ofγ-IFN was detected by ELISPOT. Fine structures on the DC surface loaded with different peptides were observed under atomic force microscope(AFM). The behavior of peptides in the DC at different time points were analyzed by laser scanning confocal microscope (LSCM). Results. The function of DC are upregulated when they were pulsed by peptide through MTT assay. Cell viability was significantly higher(p<0.05)when they were pulsed with T1,T1+T2,T1+Th,T1-AAA-Th,pal-KSST1,pal-KSST1AAATh,pal-KSST1Th,T1+B,T1+Th,B epitopes and especially by T1+B epitope(p<0.01). However, cell viability was significantly lower when treated with T2,Th,T1Th, and was at its lowest when treated with T2 epitope. T1 can upregulate DC function (p<0.01). T epitopes all show higher activity when they are modified by B epitope, helper T cell, and palmitoyl(p<0.01=. The results of the laser scanning confocal microscope demonstrated that small peptides can be taken up within 30 minutes by DC and transfer to branches from the cytoplasm. However, the concentration of peptide is lower in the branches than in the cell body. Peptide never was able to enter the nuclear within 12 hours in our experiment. Under TEM, peptide-pulsed DC display larger and a greater number of mitochondria in the cytoplasm. Ribosomes and endoplasmic reticulum are abundant. Conclusion. Viability of DC can change when they are treated with peptides. The reason that the epitope increased the CTL response is that exogenous peptide can induce a cellular response through the cross-presenting pathway of MHC I and promote the CTL reaction. DC function can be upregulated by epitope modified by palmitol B epitope. Both Th epitope and B epitope all have synergistic effect on T epitopes. Even if three alanine were linked between them and palmitoyl were joined to the N-terminal, the epitope did not induce an antigen-specific T cell reaction differently compared to T1 epitope. However, the way peptides entered into dendritic cell were changed. Cell-surface DC morphology changes when DC were loaded with different protein sequence under AFM. Fine structures on the surface of DC have a strong relationship with viability. Small peptides can upregulate DC function but cannot alter genetic information transmission. Small peptide still enter the cell through the endosome/lysosome pathway and are presented by MHC I on the cell surface.In our investigation, it is feasible to predict T epitopes by software, which greatly reduces conventional workload. Small peptides are taken up by DC through endocytosis. Different epitopes have different effects on DC in a CTL reaction, fine structures on the cell surface, and the function of DC. This finding suggests that small peptides do not directly participate in genetic information transmission when DC are loaded with peptides. Our research provides evidence that peptide-pulsed DC may be employed therapeutically in clinic.