Identification Of Antigenic Epitope Peptides Recognized By Human γδT Cells & Establishment And Characterization Of Healthy Donor's γδT Cell Clones

γδT cells play an important role in anti-infection and anti-tumor immune responses, but only a few antigens recognized byγδT cells have been identified and antigenic peptide epitopes recognized byγδT cells were no reports so far.Therefore,it is necessary to study the antigenic epitope peptides recognized byγδT cells and thus clarify the structural basis of activation ofγδT cells.On the other hand,activatedγδT cells exert effector functions on tumor cells directly via cytotoxicity or indirectly via producing powerful cytokines.Once antigenic epitope peptides which could activateγδT cells were obtained,it can provide an effective means of activatingγδT cells in vitro and in vivo.To this end,the first scientific question we concerned is whether there exist antigenic epitope recognized by humanγδT cells? If there exist,whether the ability of stimulatingγδT cells of tandem epitope peptides was superior to that of single epitope peptide? Therefore,in the first part of my thesis,we identified the epitope peptides recognized byγδT cells.In previously study,we synthesized three CDR3-peptide OT1,OT2 and OT3 according to the gene sequence ofγδTCR CDR3 derived from tumor infiltrating lymphocyte(TIL) s in ovarian epithelial carcinoma(OEC),and then we used the OT1, OT2 and OT3 as probe in an attempt to identify possible epitopes forγδT cells by screening a Ph.D.-12^TM Phage Display Peptide Library.We obtained seven peptides (EP1～EP7) recognized by probes as the candidates of epitope peptides,and these peptides could trigger the cytotoxicity ofγδT cells to tumor cells.However,perhaps because of their small molecular weight,the ability of activatingγδT cells of these peptides is weak. In order to enhance the functions of candidate epitope peptides,we tried to construct tandem peptides to improve their stimulation activity.The specific strategy for the experiment is to borrow a GST carrier protein and adopt a tandem-connection strategy to construct expression vectors of GST-epitope fusion protein,and the function of recombinant GST-epitope fusion protein would be verified to further clarify the strategy's feasibility.The results of the present study were summarized as follows:Firstly,we synthesized these seven candidate epitope peptides and investigated their biological functions in vitro.The results confirmed that these peptides were able to induce significant proliferation response of humanγδT cells and could selectively trigger the growth ofγδT cells in vitro.Furthermore,we performed a MTT experiment to validate that cytotoxicity ofγδT cells against SKOV3 cells was promoted after pre-incubated with these peptides.All the results indicate that these peptides have the feature of antigen epitopes recognized byγδT cells,which provided a basis to further clarify the activation structure ofγδT cells.Secondly,we constructed four types of GST-epitope fusion proteins expression vectors containing single epitope or tandem epitopes by using isocaudamer technique.The results confirmed that the GST-epitope fusion proteins could bind to probe OT3, OT3(V)-Fc andγδTCR specially as shown by the analysis of ELISA and flow cytometry. Subsequently,we did CFSE,MTT assay,semi-quantity RT-PCR and ELISA to investigate the potential effect of GST- epitope fusion proteins to.γδT cells.The results of CFSE and MTT indicated that these GST-epitope fusion proteins could promote the proliferation ofγδT cells.Moreover,after pre-incubated with GST-epitope fusion proteins,significant cytotoxicity of.γδT cells to tumor cell lines was observed in a dose-dependant manner.In addition,the expression level of IL-2,IFN-γ,TNF-α,FasL and gramB were also found to be increased.Meanwhile,there were significant difference between GST-tandem epitope fusion protein groups and GST-single epitope fusion protein group,demonstrating that the function of epitopes were enhanced by tandem connected.Finally,the function of GST-epitope fusion proteins was further analyzed through experimental immunotherapy in subcutaneous tumor bearing nude mice models.Three intraperitoneal injections of.γδT cells pre-incubated with GST-EPt7 greatly decreased the sizes of SKOV3 tumors and prolonged the survival of tumor-bearing nude mice, significantly different from PBS control.In conclusion,for the first time,we systematically verified the existence of epitope peptides for.γδT cells;confirmed that the activation of tandem epitope peptides were superior to that of single epitope through the construction of four types of GST-epitope fusion proteins and functional tests,which clarify the strategy's feasibility of enhancing the activation of epitope peptides by tandem connected.These results provide a new means for activatingγδT cells in tumor immunotherapy.γδT cells,a special group of T cells,are an indispensable bridge linking innate immunity and specific immunity.AsγδT cells account for only small proportion in the peripheral blood,it is relatively cumbersome to separate and purify,and hinder the in-depth study of the structure and function ofγδT cells.Therefore,establishment ofγδT cell clones in vitro,especiallyγδT cell single clones,will help us in-depth study of its antigen recognition,cell activation and functions.Therefore,in the second part of my thesis,we established healthy donor'sγδT cell clones.In this study,we firstly explored the freezing and thawing condition ofγδT cells. Secondly,γδT cells were cloned by limiting dilution after positive choice,with ⁶⁰Co irradiated allogeneic PBMC as feeder cells.Flow cytometry analysis and molecular biology technique were then used to identifyγδT cells clones,and MTT assay was used to verify their cytotoxicity.Results demonstrated that fiveγδT clones have been established. The subtype of these five clones is all Vγ9 Vδ2 and the expression of CD4 and CD8 molecule were negative.Among these five clones,two clones were monoclonal,and these two clones are identical,the amino acid sequence of VδCDR3 is ACDTIPGDLVRADKLIFGKG and the amino acid sequence of VγCDR3 is ALWEVGKLGKKIKVFGPG;other threeγδT clones are oligoclonal.All of these five clones could kill tumor cell line SKOV3.Functional study of these fiveγδT cell clones is now in process.In conclusion,in the second part of the thesis,we successfully established five normal peripheral bloodγδT cell clones,which laid a solid foundation for further study onγδT cells.