Human T lymphocytes,bearing T cell receptor(TCR)γδ,play an important role in anti-tumor/microbe immune responses although they only account for a small proportion in human T lymphocyte pool.However,to date,only few antigens recognized by humanγδT cells have been defined.To better understandγδT cell function,a crucial issue is to identify more ligands forγδT cell.The present study focuses on that how to use a new technical strategy to identify protein ligands recognized byγδT cell.It is also the key point to understand the principles governing TCRγδspecificity and diversity and to reveal the biological functions ofγδT cell.The antigen-binding site of TCR is formed primarily by six complementarity determining regions(CDRs).Sequence diversity in antigen receptors is not evenly distributed among all six CDRs,but is highly concentrated in one or two CDR3.It is supposed that the primary structure of CDR3δ,due to its similarity to V_H CDR3 of BCR in gene composition,could serve as the key determinant of specificity in antigen binding by the TCRγδ.This hypothesis has been confirmed.The binding activity of a synthesized CDR3δpeptide OT3,derived from tumor infiltrating lymphocytes(TILs) in ovarian epithelial carcinoma(OEC),was analyzed through a series of in vitro binding assays including OT3 peptide-mediated SPR assay,immunofluorescence assays,enzyme immunoassay,and OT3 peptide competition test.OT3 grafted-Ig was also employed to repeat major tests.OT3 and OT3 grafl-Ig shared a similar binding specificity to target cells or tissues,suggesting that CDR3δplayed a determinant role in antigen recognizing of TCRγδand OT3 peptide could be employed as a specific probe to identify putative protein ligands for TCRγδ.Based on the binding specificity of OT3 peptide,three technical strategies were used to identify TCRγδ-recognized proteins.First is to pan OT3 specific 12-peptides in a 12-mer random peptide phage-displayed library with OT3 peptide;seconed is to scan OT3 specific proteins in a human ovarian cancer lambda cDNA library with OT3 peptide;third is to identify OT3 peptide-binding proteins within an OEC tumor cell line SKOV3 cell total protein extracts by affinity chromatography and liquid chromatography-electrospray ionization tandem mass spectrometry(LC-ESI MS/MS) analysis.With the first strategy, three OT3-specific 12-peptides were gained,which not only bind toγδT cells,but also functionally activateγδT cells in vitro and could be served as epitopes for TCRγδ.But there were no matched human proteins to the liner sequence of these functional epitopes in Basic local alignment search tool(BLAST) analysis,suggesting that they would be epitopes of other species.High specificity and powerful binding were needed for scanning a cDNA library and the second strategy failed.Only the third immuno-biochemical strategy worked and with this strategy,we successfully identified three candidate tumor associated antigen for TCRγδ,including pyruvate kinase 3,human mutS homolog 2 (hMSH2) and heat shock protein(HSP) 60.Identification of HSP60 confirms the validity of this method because HSP60 is an identified ligand for TCRγδ.Pyruvate kinase 3 and hMSH2,which is very important in DNA mismatch repair,may be new protein ligands recognized by TCRγδ.According to analysis of MS,hMSH2 showed possible sequence characteristics as ligand for TCRγδ.In present study,we put forward lots of validation works on the role of hMSH2.RT-PCR results indicated that there seemed to be mutations in the mRNA of hMSH2 in OEC cell line SKOV3 cells.A soluble form of hMSH2 in SKOV3 culture supernatant and a 60kD shortened hMSH2 were found in Western blotting.There were surface expressions of the mutational hMSH2 on the surface of SKOV3 cells and several other tumor cell lines,including Hela,803,HepG2,NCI-H520 and Daudi.Ectopically expressed hMSH2 were also detected in tumor tissues.In addition,the cytotoxicity of Vδ2γδT cells,but not Vδ1 ones against SKOV3 cells,could be blocked by anti-hMSH2 antibody partially.Meanwhile,five recombinant proteins have been expressed and purified,including the normal full-length of hMSH2,hMSH2 segments containing the first two domains near N terminal or the last two domains near C terminal and hMSH2 N/C segments with dot-mutations respectively.All five recombinant hMSH2 proteins not only bind to Vδ2γδT cells,but also functionally trigger the proliferation of Vδ2γδT cells in vitro.Tested proteins accelerated the activation cytokines(for example Interferon-γ) secretion of Vδ2γδT cells and the cytotoxicity of Vδ2γδT cells to SKOV3 cells also be promoted by recombinant hMSH2 proteins.Moreover,full length of hMSH2 recombinant protein was successfully expressed in baculovirus expression system holding more activity than that expressed by E.coli.It laid a foundation for the further researches on hMSH2.Taken together,our findings:First,confirmed the critical role of CDR3δin antigen binding specificity of TCRγδ. The antigen recognition of TCRγδprimarily depends on CDR3δand synthetic CDR3δpeptide is an excellent probe for new ligands of TCRγδ.Second,put forward a novel,effective and simple immuno-biochemical strategy to identify protein ligands recognized byγδT cells based on CDR3δpeptide.Third,reported at first time that ectopically expressed hMSH2 in malignant situation might become a novel biomarker on tumor cells for Vδ2γδT cells.The ectopic expression of hMSH2 on tumor cells may alert the immune system on the early stage of tumor pathogenesis.Last,proved baculovirus expression system is a powerful and versatile eukaryotic expression system.The insect cell,unlike bacterium,is capable of performing many of the post-translational modifications that are required for forming biologically active proteins.In the four achievements above,the second and third achievements make more senses. The immuno-biochemical strategy solved a difficult problem puzzled immunology for a long time and provided a novel,effective and simple technique to identify ligands for TCRγδ.The discovery of ectopically expressed hMSH2 suggested a new pathway for immunosurveillance ofγδT cell.Ectopically expressed hMSH2 would be an important target molecule in tumor immunotherapy. |