The Molecular Structural Foundation Of Antigen Recognition For Complementary Determinant Region (CDR) 3 In δ2 Chain Of T Cell Receptor

Recently,γδT cells,which had been considered as a member of natural immunity, play an important role in the field of anti-infection immunity,anti-tumor immunity, immuneregulation and autoimmunity.But until now,little was known about the structural basis withinγδTCR antigen recognition and little progress had been made on the verification ofγδTCR antigen.The study suggested that CDR3 ofδchain was the important position for antigen recognition.In recent years,our groups selected the CDR3 ofδ2 chain(CDR3δ) as a research target and did some investigation about the interaction machnism of CDR3 peptide and CDR3 grafted Ig with tumor cell,tumor tissue and tumor cell protein extract. Our results suggested that CDR3δwas the crucial position for antigen recognition.In this paper,through the reconstruction ofγδTCR transfectant cell,we set up a technique system using CDRδgrafed TCRγδexpressed on cell membrane.The article focuses on elucidating the key structural basis for specificity ofγδTCR,especially the molecular mechanism betweenγδTCR CDR3 and its ligands and providing new method for searching antigen epitope ofγδT cell.We conducted the experiments and got some results shown as follows:1.The molecular structural foundation of antigen recognition of CDR3δ1) The construction oftransfectant cells expressingγδTCR with specific CDR3 sequenceThe full-lengthγ9 andδ2 chain containing specific CDR3 sequence were transfected into J.RT3-T3.5 cell.The transfectant cells expressingγδTCR with specific CDR3 sequence were successfully constructed and they could mimicγδTCR to recognize specific antigen. 2)γδT cells recognize tumor cells via CDR3δregionThe transfectant cells expressingγδTCR with OT3(OT3γδTCR cells) have the cytotoxicity effect on many tumor cells and the cytotoxicity effect could be blocked by functional grade purified anti-humanγδTCR.In addition,when the transfectant cells were stimulated by tumor cell protein extract,the production of IL-2 for OT3γδTCR cells was higher than that of the irrelative control and mock control.Meanwhile,the production of IL-2 between cells transfected byγ9 and OT3δ2 chains and single OT3δ2 chain had no significant difference.These data strongly indicated that the OT3γδTCR cells could specifically bind the molecules expressed in tumors andγδT cells recognize tumor cells mainly via CDR3δ2 region.3) Specific binding of CDR3δto antigens depends upon its flanking sequencesIn order to further explore the important recognition region ofδ2CDR3 to tumor cells, we construct transfectant cells with different domain variants of OT3,respectively.That is VmOT3γδTCR,DmOT3γδTCR and JmOT3γδTCR cells.After the transfectant cells were stimulated by tumor cells or tumor cell protein extracts,the production of IL-2 was detected to evalute the reactivity of various transfectant cells to tumor cell.In contrast to production of IL-2 by OT3γδTCR transfectant cells,VmOT3γδTCR and JmOT3γδTCR transfectant cells secret lower level of IL-2 after the stimulation by the protein extract of HO8910,Hela and SKOV3 cells and HSP70,N teminal(MNS) and C terminal(MCS) of hMSH2.DmOT3γδTCR transfectant cells secret equivalent level of IL-2.For cytotoxicity assay,under the condition of different ratio of effector cells and target cells,the cytotoxicity of the VmOT3γδTCR and JmOT3γδTCR transfectant cells to SKOV3 cells were lower than that of OT3γδTCR transfectant cells,while the cytotoxicity of DmOT3γδTCR transfectant cells decreased indistinctively.The results demonstrated that specific binding of OT3γδTCR transfectant cells to tumor cells and cell protein extracts depends upon its flanking sequences.4) Isoleucine residue at position 97 in CDR3 of the TCRδ2 chain was not sufficient to account for the recognition mechanisms ofγδT cell to protein antigen We construct OT10γδTCR transfectant cells,whose 97 position ofδ2 chain was a hydrophobic amino acid,isoleucine.In order to investigate the role of isoleucine at theδ97 position in tumor antigen recognition,we mutated it to leucine(δ97L),serine(δ97S) and glutamic acid(δ97D),respectively.In contrast to the higher level of IL-2 by OT10γδTCR transfectant cells,δ97LγδTCR transfectant cells secret lower level of IL-2,δ97S andδ97DγδTCR transfectant cells secrete little of IL-2 when these transfectant cells were stimuated by non-peptide antigen.Meanwhile,the amino acid substitution of isoleucine at positionδ97 did not change the response ofγδTCR transfectant cell to tumor cells.The data suggested that isoleucine residue at position 97 in CDR3 of the TCRδ2 chain was not sufficient to account for recognition mechanisms ofγδT cell to protein antigen,which was different from non-peptide antigen reactiveγδT cell clone.2.A new strategy for the identification of tumor antigenic epitopesWe develop a new strategy to identify the epitopes specific forγδT cells using OT3γδTCR transfectant cells based on subtractive screening in vitro with a phage display 12 peptide library.Two preponderant peptides were selected.The results from binding analysis and functional test suggested that the antigenic epitopes,which was fished by screening phage display peptide libraries using OT3γδTCR transfectant cells,could be recognized by humanγδT cells.This new strategy had been confirmed reasonable by our experiments.In conclusion,this study had demonstrated that CDR3δplayed a central part within the antigen binding,which was to determine the specificity of antigen binding.Besides, our results had also suggested for the first time that the V and J fragments,which lied in the flanking sequence of CDR3δdomain,determined the specific binding between CDR3δand epitope,which might contribute to explaining the limited specificity repertoire inγδT cells.Furthermore,by using the OT3γδTCR transfectant cells as probes and based on the subtractive screening method,for the first time,we had developed a new strategy to identify the epitopes bound specifically withγδT cells within a 12 peptides phage display library.And this new strategy had been confirmed reasonable by our experiments.With this new strategy,new way could be found on screening of tumor associate antigen(TAA) or tumor specific antigen(TSA),which could be identified byγδT cells.Thus more and more biomarkers or therapeutic target could be provided for the diagnosis and immunotherapy towards tumor and more stable theory basis could be set within the field of application and exploration ofγδT cells on anti-tumor treatments.In addition,we identified ULBP4 and RAET1G2 splice variants including ULBP4-Ⅰ, ULBP4-Ⅱ,ULBP4-Ⅲand RAET1G3,which could be recognized by human NKG2D.