The CDR3?1 Sequence Analysis Of Tumor-infiltration ??l Lymphocyte (??1TIL) And The Molecular Basis For TCR?/?1 CDR3?1 Binding With Tumor Antigens

??T 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 with??T lymphocytes whose functions and structures were well understood, little was known about the structural basis of??TCR for its antigen recognition. Moreover, compared with y982TCR, studies on V?1 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 TCR?982. binding with tumor antigens, and achieved some promising results which indicated that CDR3?acts as a crutial factor for the specific TCRy9?2 antigen recognition [1].This current study is aimed to pursue the role of another subset of??T cells,?1T cells, in tumor immunity and the molecular mechanism of antigen recognition of V?1. The first part is the CDR3?1 sequence analysis of tumor-infiltration??1 lymphocyte (??1TIL).Firstly, we successfully cultured 16 cases of tumor infiltration lymphocytes (??TILs) from 14 solid tumor tissues and 2 ascites by immobilized antibody amplification in vitro. After 3 to 4 weeks of culturing,??TIL grows in a dominant manner (up to 98%), and?1 subtype was the main subtype of the cultured??TILs.CDR3?1 gene fragments were analyzed through sequence analysis following RT-PCR. Characteristics of CDR3?1 sequence were found as follows:The master sequences of CDR3?1 in??TILs from same tissue sources but different individuals tend to be same though differences still exist. While master sequences of CDR3?1 from different tissues are different. And this master sequence of CDR381 in??ILs from different donors but the same tissue source is relatively shorter. These results showed that the CDR3?1 sequences in??TILs 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 cancer??1TILs 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), CDR3?1 (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 CDR3?1. However, we didn't successfully isolate CDR381-binding proteins from BGC-823 total protein extract by GTM-affinity column chromatography.Analysis of??TIL phenotypes amplified in vitro showed that NKG2D. CD8. GranzymeA and TLR8 are highly expressed on??TILs, while CD4. GITR. CTLA-4 and CD 127 are little expressed. The results of cytotoxic experiments performed in gastric W2-??TIL and renal S2-??TIL showed that both W2-??TIL and S2-??TIL had cytotoxic effects on tumor cells. The data of cytokine detection showed that all??TILs have a high secreting level of IL-6 and the level of GM-CSF secreted by??1TILs is clearly higher than that of??TILs.The second part of this paper is to explore functions of different domains of CDR3?1 on antigen recognition. In this part, three technical strategies including synthesized CDR3?1 peptides, CDR3?1-grafted TCR fusion proteins and cells transfected with?1-CDR3(GTM)-y4 were used to investigate the structural basis for CDR381 specifically binding with tumor antigens.The binding of CDR3?1 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 CDR3?1 V and J play critical roles in antigen binding.To further confirm the results, we expressed CDR3?1-grafted TCR fusion proteins, y4-Fc/?1-CDR3(GTM)-Fc and its three mutated fusion proteins y4-Fc/?1-CDR3(GTM-Vm)-Fc, y4-Fc/?1-CDR3(GTM-Dm)-Fc and y4-Fc/?1-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 TCR?4?1 with tumor antigens depended on its conversed flanking sequences.Finally, in order to simulate the molecular structure of antigen recognition by??1TIL in cell level and detect function of different domains of CDR3?1 in tumor antigen recognition, complete y4 and?1 chains were transfected into J.RT3-T3.5 cell to establish cell lines which express different CDR3?1-grafted TCR dimer on membrane.??TCR expression on cells transfected with?1-CDR3(GTM)-y4 and?1-CDR3(GTM-Dm)-y4 was detected, while no??TCR expression was found on cells transfected with?1-CDR3 (GTM-Vm)-y4 and?1-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-infiltration??lymphocytes (??TILs) were cultured successfully in vitro by optimizing the culture conditions. The main subtype??TIL amplificated in vitro is?1. And??1TILs have cytotoxic effects on tumor cells in vitro.2. The CDR3?1 sequence of??TIL cultured in vitro has the characteristics of relative dominance. The CDR3?1 sequences of??TILs from the same tissue sources but different individuals trend to be the same. One common CDR3?1 master sequence from 3 gastric cancer??TILs 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 CDR3?1(V and J region) play a critical role in antigenic binding for??1TCR in the level of both CDR3?1 peptide and?4?1T-Fc fusion protein.4. We successfully construct a platform to express TCR?4?1 on J.RT3-T3.5 cells via lentivirus expression system, which can be used to simulate??1TIL and study the molecular basis its tumor recognition in cellular level. The function experiment of transfection cells is still ongoing.