The Mechanism On Antigen Recognition Of γδ T Cells: Specificity Of TCRδ₂-CDR3 Binding To Antigen And A New Strategy For The Identification Of Tumor Antigenic Epitopes

γδT cells, likeαβT cells and B cells, generate a diverse repertoire of antigen-recognition receptors through somatic rearrangement of V, D, and J gene segments. It has been convincingly demonstrated thatαβT cells andγδT cells have different functional roles in the immune system, yet the identity of endogenous ligands forγδT cells is unclear and little is known about the molecular basis of lingand recognition through their specificγδTCR. In addition,γδT cells exhibit several unique features in tumor immunology. They use simple and fast recognition molecule patterns that directly recognize tumor antigens without Ag presentation and MHC restriction. They are able to recognize a wider variety of tumor antigens via different pathway and exert effector functions on tumor cells directly via cytotoxicity or indirectly via producing powerful cytokines. So,γδT cells are supposed to be a new candidate for adoptive immunotherapy for cancer patients. As we know, tumor associate antigen (TAA) or tumor specific antigen (TSA) is able to selectively induce activation and expansion ofγδT cells in vivo. So, finding TAA or TSA may be another feasible strategy for obtaining sufficient forγδT cells specific tumor antigen. But, only a few tumor antigens recognized byγδT cells have been identified so far. Screening tumor-associated antigen recognized byγδT cells using transfectional and soluble TCRγδhasn't gotten any satisfactory results.In order to elucidate the key structural basis for specificity ofγδT cell receptor (TCR), especially the interactive molecular mechanism betweenγδTCR CDR3 and its ligands and contribution of intermediate variable sequences and two-side conserved flanking sequences within CDR3 to receptor-ligand binding. We analyzed the binding activities of synthesized TCRVδ₂ complementarity determining regions (CDR) 3 peptides with Vδ₂CDR3 to target tumor cells, tissues or antigens via biospecific interaction analysis, enzyme immunoassay and immunofluorescence assays. In addition, we develop a new strategy to identify the epitopes specific forγδT cells using TCRδ₂-CDR3 derived from tumor infiltrating lymphocytes with TCRγδ-expressing phenotype based on the binding interaction ofγδTCR with antigen is similar to that of antibody. We conducted the experiments and got some results shown as follows:1. The structural basis on specificity of TCRδchain binding to antigen1) The characteristic of amino acid sequences of CDR3δWe analyzed the amino acid sequence deduced from V_δcDNA fromγδTILs in OEC tissues. Flanking regions of CDR3δcontained conserved "CA" at the N terminus and conserved "FGKG" at the C terminus. In contrast, the inner regions of CDR3δwere composed of variable sequences, in which common motifs were hardly observed.2) Synthesized CDR3δpeptides specifically bound to target cellsFirstly, we synthesized two CDR3δpeptides, OT3 and OT1 prompted by our previous finding thatγδT lymphocytes could suppressed the growth of SKOV3 cells in nude mice.We further analyzed systematically the interaction of the CDR3δpeptide with different tumor cells or tumor specimens by immunohistochemistry, flow cytometry and confocal microscopy using a biotin-streptavidin labeling system. Our data show that the CDR3δpeptides from the OECγδTIL can bind the OEC tissue but non-binding to normal ovary tissue. However, the peptides cross-reacted with other types of carcinomas including gastric, hepatic and colonic caricinomas, which means at least this peptide is tumor specific. These results suggested thatγδT cells can recognize antigens expressed by dysregulated cells of epithelium origin, consistent with the hypothesis thatγδT cells respond to antigens commonly expressed on tumor cells or tumor associated antigens (TAA). These data strongly indicated that the synthesized peptide CDR3δcould specifically bind to the molecules expressed in tumors.3) Specific binding of CDR3δpeptide to antigens depends upon its flanking sequencesTo determine which motif of CDR3δis the key determinant for antigen binding, we synthesized control peptides of OT3 by replacing V, D, J and V J segments of OT3 with random arrangement amino acid sequences of same length, respectively. Bindings of these synthesized control peptides to target cells, tissues or ligands were examined by confocal microscopy, immunohistochemistry or SPR, respectively.Our data indicate that flanking sequences constituted by V and J segments are more important to the recognition of antigens, since the mutation of V and J portion of the CDR3δpeptides almost abolished their binding to tumor cells or tissues, while mutation of N-D-N residues merely weakened the binding.2. A new strategy for the identification of tumor antigenic epitopes1) MTT was used to evaluate CDR3 synthesized peptide blocking cytotoxicity ofγδT cells in vitroWe performed a MTT experiment to evaluate whetherγδTCR had the similar binding specificity with synthesized CDR3δpeptides. The results showed that cytotoxicity ofγδT cells against SKOV3 cells was inhibited after blocking with CDR3 synthesized peptides.2) Screening the phage display library by CDR3 peptideLast, the antigenic epitopes we fished by screening phage display peptide libraries using CDR3 peptides are able to induce significant proliferative response of humanγδT cells and could selectively trigger the growth ofγδT cells in vitro. The results confirmed our strategy of rapid and specific identification for tumor antigenic epitopes based on interaction between TCRγδCDR3 and epitopes.In conclusion, our results showed that the flanking sequence of CDR3δdomain, a conserved part, takes a dominant responsibility for ligand-binding specificity, which might contribute to explaining the limited specificity repertoire inγδT cells. The synthesized peptide CDR3δcould specifically bind to the molecules expressed in tumors and CDR3 synthesized peptides could inhibit cytotoxicity ofγδT cells against target cells. Furthermore, the function assay of antigen epitope peptides screened with CDR3δpeptides identify the strategy of identification of an antigenic epitope derived from OEC based on interaction between TCRγδCDR3 and epitopes is reasonable. According to this strategy, we could screen high affinity epitopes, which are available for immunotheragy not only for OEC but also for other carcinomas.