Study About The Mechanism Of T-cell Activation And The TCR-T Immunotherapy

T-cell receptor(TCR)-engineered T-cell(TCR-T)immunotherapy is a kind of adoptive cell transfer therapy(ACT).The gene-modified TCRs can specifically identify the tumor associated antigen(TAA)derived peptides presented by major histocompatibility complex(MHC)so that the activated T cells can kill tumor cells specifically.The graduation thesis embraced two topics around TCR-T cells.Briefly,the first topic named "The role of the FG loop in TCR ? chain constant region in T cell function",which was intended to explore the mechanism of T cell activation.The second topic named "Identification of an HLA-A*24:02-restricted ?-fetoprotein signal peptide-derived antigen and its specific T-cell receptor for T-cell immunotherapy".Topic 1:Manifest research revealed that the extracellular information of antigen ligation is transmitted into intracellular activation signaling via TCR-CD3 interactions during the initiation of T cell activation.However,the mechanism of the transmission process remains incomplete.The FG loop in TCR ? chain constant domain(TCR C?FG loop)possesses highly structured features.Compared with immunoglobulin Fab CH1,the TCR C? region includes a unique elongated and solvent-exposed fragment,C? FG loop,which is located in the rigid area of the interface between V? and C?domain.Besides,a cavity formed by C? FG loop together with Ca CD loop and Ca EF loop is hypothesized to accommodate one CD3? subunit.In addition,hydrogen bonds and a hydrophobic core are formed by several amino acid residues in FG loop.Therefore,the FG loop is hypothesized to participate in T cell function.However,the precise role of C? FG loop in the T cell function remains unclear.To explore the influence of FG loop on T cell function,we inserted a flexible peptide comprised of 24 amino acid residues in C? FG loop or C? DE loop.The analysis about the homology modeling showed that the flexible peptide is most likely to disturb the FG loop conformation while antigen ligation.Our exploration is developed on two aspects,physical characteristics analysis and biological functional assays.To analyze the physical characteristics of purified TCR,we construct the prokaryotic expression vectors and express inclusion bodies,and the refolded TCR was purified by ion exchange and gel filtration.The DSC analysis and BIAcore analysis of purified TCR indicate that the insertion of flexible peptide show slightly influence on stability and almost no influence on affinity to pMHC.So,the insertion of flexible peptide can be exploited in the functional exploration of FG loop by T cell function assays.To analyze the function of FG loop during T cell activation,we obtained TCR-T cells by transfecting T cells with prepared lentivirus.The results of ELISpot and LDH cytotoxic assay showed that insertion of flexible peptide in TCR C? FG loop or DE loop does not influence obviously on T cell function.Finally,several speculations about the function of C? FG loop in TCR-CD3 extracellular interaction upon antigen ligation are listed according to the obtained results.One speculation is that FG loop may not be involved in the TCR-CD3 extracellular interaction upon antigen ligation.The other possibility is that,only several residues is linked with TCR-CD3 extracellular interaction in the initial signaling transmission process.However,only several amino acid residues are significant in FG loop,these key residues may not be disturbed upon flexible peptide insertion.Besides,the FG loop may participate in the TCR-CD3 interactions in a very flexible conformation,therefore,the inserted flexible peptide may not disturb its normal effect.Finally,the FG loop may play a part in T cell function with the complete loop,irrelevant with its changeable conformation.This study would make a supplement to TCR-CD3 extracellular interaction.Topic 2:Hepatocellular carcinoma(HCC)is the most common type of liver cancer with limited treatments.Asia has the highest HCC incidence rates;China accounts for over 50%of all HCC cases worldwide.T-cell receptor(TCR)-engineered T-cell immunotherapies specific for human leukocyte antigen(HLA)-A*02:01-restricted ?-fetoprotein(AFP)peptide have shown encouraging results in clinics.HLA-A*24:02 is more common than HLA-A*02:01 in Asian countries,including China.Here we identified a novel HLA-A*24:02-restricted peptide KWVESIFLIF(AFP2-11)located in AFP signal peptide domain by mass spectrometric analysis of HLA-bound peptides from HepG2 cells.A TCR(KWV3.1)specific for AFP2-11-HLA-A*24:02 was isolated from peripheral blood mononuclear cells of a healthy donor.The binding affinity of soluble KWV3.1 to its antigen was determined to be?55?M,within the affinity range of native TCRs for self-antigens.KWV3.1-transfected T cells could specifically activate and kill AFP2-11 pulsed T2-A24 cells and AFP+/HLA-A*24:02+tumor cell lines,demonstrating that AFP2-11 can be naturally presented on the surface of AFP+tumor cell lines.The newly identified antigenic peptide can provide a novel target for immunotherapeutic strategies for patients with AFP+/HLA-A*24:02+HCC.