The CDR3δ1 Sequence Analysis Of Tumor-infiltration γδl Lymphocyte (γδ1TIL) And The Molecular Basis For TCRγ/δ1 CDR3δ1 Binding With Tumor Antigens

γδT lymphocytes, as a minor T lymphocyte subset in peripheral blood, play an important role in anti-infection immunity, anti-tumor immunity, immunoregulation and autoimmunity and become a hot spot. However, compared withαβT lymphocytes whose functions and structures were well understood, little was known about the structural basis ofγδTCR for its antigen recognition. Moreover, compared with y982TCR, studies on Vδ1 in human epithelial mucosa were quite less from peripheral blood in the mechanism and structural basis of antigen recognition.In the past several years, our research group has performed a series of systemic studies on the ligands of TCRy982 and the molecular basis for TCRγ982. binding with tumor antigens, and achieved some promising results which indicated that CDR3δacts as a crutial factor for the specific TCRy9δ2 antigen recognition [1].This current study is aimed to pursue the role of another subset ofγδT cells,δ1T cells, in tumor immunity and the molecular mechanism of antigen recognition of Vδ1. The first part is the CDR3δ1 sequence analysis of tumor-infiltrationγδ1 lymphocyte (γδ1TIL).Firstly, we successfully cultured 16 cases of tumor infiltration lymphocytes (γδTILs) from 14 solid tumor tissues and 2 ascites by immobilized antibody amplification in vitro. After 3 to 4 weeks of culturing,γδTIL grows in a dominant manner (up to 98%), andδ1 subtype was the main subtype of the culturedγδTILs.CDR3δ1 gene fragments were analyzed through sequence analysis following RT-PCR. Characteristics of CDR3δ1 sequence were found as follows:The master sequences of CDR3δ1 inγδTILs from same tissue sources but different individuals tend to be same though differences still exist. While master sequences of CDR3δ1 from different tissues are different. And this master sequence of CDR381 inγδILs from different donors but the same tissue source is relatively shorter. These results showed that the CDR3δ1 sequences inγδTILs recognizing tumors with same tissue sources have limited diversity, indicating the surface antigens from tumors with the same tissue sources also have limited diversity. One common sequence from three gastric cancerγδ1TILs called GTM is chosen as the basis of the following study.Western blot was performed to detect binding molecules in total protein extracted from BGC-823 (gastric cancer cell lines), CDR3δ1 (GTM) was used as probe. and two bands were found at the positions of 66-90KD and above 90KD respectively. This suggested that there might be some proteins recognized by CDR3δ1. However, we didn’t successfully isolate CDR381-binding proteins from BGC-823 total protein extract by GTM-affinity column chromatography.Analysis ofγδTIL phenotypes amplified in vitro showed that NKG2D. CD8. GranzymeA and TLR8 are highly expressed onγδTILs, while CD4. GITR. CTLA-4 and CD 127 are little expressed. The results of cytotoxic experiments performed in gastric W2-γδTIL and renal S2-γδTIL showed that both W2-γδTIL and S2-γδTIL had cytotoxic effects on tumor cells. The data of cytokine detection showed that allγδTILs have a high secreting level of IL-6 and the level of GM-CSF secreted byγδ1TILs is clearly higher than that ofαβTILs.The second part of this paper is to explore functions of different domains of CDR3δ1 on antigen recognition. In this part, three technical strategies including synthesized CDR3δ1 peptides, CDR3δ1-grafted TCR fusion proteins and cells transfected withδ1-CDR3(GTM)-y4 were used to investigate the structural basis for CDR381 specifically binding with tumor antigens.The binding of CDR3δ1 peptide GTM, and it mutants in V(GTM-Vm), D(GTM-Dm) and J(GTM-Jm) region to tumor cells or tissues was detected by flow cytometry, confocal, ELISA, SPR and immnohistochemistry. The data showed that the binding activity of GTM-Vm, or GTM-Jm except GTM-Dm was significantly lower than that of GTM, suggesting that the conserved flanking domains of CDR3δ1 V and J play critical roles in antigen binding.To further confirm the results, we expressed CDR3δ1-grafted TCR fusion proteins, y4-Fc/δ1-CDR3(GTM)-Fc and its three mutated fusion proteins y4-Fc/δ1-CDR3(GTM-Vm)-Fc, y4-Fc/δ1-CDR3(GTM-Dm)-Fc and y4-Fc/δ1-CDR3(GTM-Jm)-Fc. The trends of binding between fusion proteins and tumor cells or tissues were consistent with those detected from synthetic peptides, which further confirmed the hypothesis that the interaction of TCRγ4δ1 with tumor antigens depended on its conversed flanking sequences.Finally, in order to simulate the molecular structure of antigen recognition byγδ1TIL in cell level and detect function of different domains of CDR3δ1 in tumor antigen recognition, complete y4 andδ1 chains were transfected into J.RT3-T3.5 cell to establish cell lines which express different CDR3δ1-grafted TCR dimer on membrane.γδTCR expression on cells transfected withδ1-CDR3(GTM)-y4 andδ1-CDR3(GTM-Dm)-y4 was detected, while noγδTCR expression was found on cells transfected withδ1-CDR3 (GTM-Vm)-y4 andδ1-CDR3(GTM-Jm)-y4 by FACS assay. Even though the random-mutated amino acids were changed into alanines, no expression was detected as well. The functional study is under way.Comprehensively, conclusions are drawn as followings:1.16 tumor-infiltrationγδlymphocytes (γδTILs) were cultured successfully in vitro by optimizing the culture conditions. The main subtypeγδTIL amplificated in vitro isδ1. Andγδ1TILs have cytotoxic effects on tumor cells in vitro.2. The CDR3δ1 sequence ofγδTIL cultured in vitro has the characteristics of relative dominance. The CDR3δ1 sequences ofγδTILs from the same tissue sources but different individuals trend to be the same. One common CDR3δ1 master sequence from 3 gastric cancerγδTILs called GTM was chosen as the basis of the following structural study.3. By the way of random mutation, it is shown that the conserved flanking regions of CDR3δ1(V and J region) play a critical role in antigenic binding forγδ1TCR in the level of both CDR3δ1 peptide andγ4δ1T-Fc fusion protein.4. We successfully construct a platform to express TCRγ4δ1 on J.RT3-T3.5 cells via lentivirus expression system, which can be used to simulateγδ1TIL and study the molecular basis its tumor recognition in cellular level. The function experiment of transfection cells is still ongoing.