The Characteristics Of The Immune Repertoire Of ?? T Cell TCR In The Peripheral Blood Of Pulmonary Tuberculosis Patients And The Molecular Mechanism Of ?? T Cell Recognition Of Polypeptide/protein Antigens

Tuberculosis(TB)caused by Mycobacterium tuberculosis(Mtb),the top infectious disease killer worldwide,remains a severe global threat to human health.China is one of the high TB burden countries.The control of TB lacks new diagnostics,new drugs,and new vaccines;of these,effective vaccines are of vital importance to the prevention of TB.Currently,the only vaccine that is available and widely used against TB is bacille Calmette-Guerin(BCG).which can effectively protect children against active TB diseases,such as TB meningitis,but its efficacy in protecting adults and the aging population against pulmonary tuberculosis is highly variable.Therefore,safer and more effective vaccines against TB are urgently needed.To develop efficient TB vaccines,the mechanism of immune protection against TB must be identified.The ?? T cells are thought to play a critical role in anti-mycobacterial immunity,the activation of ?? T cells appear rapidly following Mtb infection,and ?? T cells provide protective immunity against Mtb infection with the combined properties of both innate and adaptive immunity.Vaccines for Mtb based on ?? T cells provide a novel approach to TB control.Originally,phosphoantigens were reported to activate ?? T cells,and were considered to be the main ?? T cell receptor(TCR)-recognized antigens.However,only a subset of the phosphoantigen-responsive ?? T cells mediate protective tuberculosis immunity.Based on this research,we established a reverse genetics strategy and successfully identified BCG and Mtb-derived protein antigens in our previous studies,which were not only recognized by ?? TCR,but could also activate?? T cells and exhibit cytolytic effector function against BCG-infected cells.Thus,this thesis focused on two questions.Firstly,in our previous strategy,we used PCR and Sanger sequencing to find preponderant complementarity-determining region 3(CDR3)sequences in pulmonary tuberculosis patients.However,due to the limitation of the throughput of this method,we only analyzed 102-03 sequences and obtained limited information about ?? TCR.In this study,to investigate the profile of the ?? T cell TCR repertoire and to identify more preponderant CDR3 sequences in tuberculosis patients,we analyzed the expressed T cell receptor y chain(TRG)and T cell receptor ? chain(TRD)repertoire of circulating ?? T cells that were isolated from people with latent tuberculosis infection(LTBI)and active pulmonary tuberculosis(TB).Compared with the healthy controls(HC),we found some changes in the TRG and TRD repertoire of the TB patients:the CDR3? tended to be more polyclonal;the length of CDR3y had a trend to be longer;the frequency of TRGV9 and TRGJP usage increased;and the ?? T cells expressing CDR3 sequences using a V?9-J?P rearrangement expanded significantly during Mtb infection.Additionally,we found some new preponderant y8 TCR CDR3 sequences in the LTBI group,the TB group,and the LTBI&TB group,which are expected to be more representative than the preponderant CDR3 sequences we obtained in our previous studies and could be used to screen novel Mtb-derived ligands of ?? TCR.Secondly,we want to study the molecular mechanisms by which the ?? T cell receptors recognized the peptide/protein antigens with the structural biology methods.After we tried the different expression systems,including the prokaryotic expression system,insect baculovirus expression vector system,and transient protein expression in mammalian cells,we expressed the ?? TCR in HEK293S GnTI-cells for large-scale expression of the protein for structural studies.The purified ?? TCR proteins were used for initial screening with the rapid screening kits.We got two initial crystallization conditions for the y8 TCR,one of them had no diffraction,and the other one had a promising diffraction with 4 A and is undertaken to optimization now.We got three initial crystallization conditions for the ?? TCR/peptide complex;they are also undertaken to optimization now.All of the crystals are waiting for the X-ray diffraction to collect data.Taken together,this study comprehensively characterized the TRG and TRD repertoire of TB patients,and provided some information about the preponderant CDR3 sequences of specifically recognized Mtb-derived antigens,which might help to develop new vaccines and adjuvants against TB.In addition,the molecular mechanisms by which the ?? T cell receptors recognized the peptide/protein antigens are still relying on the crystal structures with high resolution.