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Preparation and Application of CD4~+αβ TCR Tetramers and HLA-DR/peptide C5Tetramers

Author: XieDan
Tutor: ZhangKouXing
School: Sun Yat-sen University
Course: Internal Medicine
Keywords: Mycobacterium tuberculosis tuberculosis tetramer CD14~+monocytes andmacrophages CD4~+T cells
CLC: R392
Type: Master's thesis
Year: 2012
Downloads: 67
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Abstract


Background and objective:Tuberculosis is a kind of chronic infectious disease based on the respiratorysystem caused by tuberculosis' dissertation">Mycobacterium tuberculosis, which threatens human healthand spreads by respiratory passage mainly. The incidence of tuberculosis hadsignificantly decreased because of the application of the vaccine BCG (M.borisBacille Caimette-Guerin) worldwide. But the incidence and mortality of theMTB has increased these years because BCG can not make the body get thelifetime immunity, the infection of HIV, the increase of floatingpopulation and the rising of multi-drug resistant TB.Early detection and diagnosis of TB is the key to reduce the TB spreadand to control the TB infection. Due to the lack of specific clinicalmanifestation of tuberculosis, the laboratory diagnosis of TB is the main ways and means to find the source of infection and is the important basisfor diagnosing and treating TB. The smear test of sputum and mycobacterialculture is the most common method for the detection of Mycobacteriumtuberculosis. The smear test of sputum is simple, but the positive rate is very low.Isolating culture for MTB, as the gold standard for diagnosis TB, takes long time.Both smear test of sputum and mycobacterial culture are difficult to meetclinical needs. In recent years, with the rapid development of biotechnology andmolecular biology, based on improving existing diagnostic techniques, a number ofnew diagnosic TB techniques have been introduced. But they have some limitations.Sensitive PCR methods are available for MTB detection, but they require additionalequipment and trained personnel, which have restrained its application in low-incomeareas. Interferon release test (interferon gamma release assays, IGRA) is basedon memory effective T cells release IFN-γ when MTB reinfect, whichquantitively detect the level of IFN-γ to determine TB infection orlatent tuberculosis infection (LTBI) with high specificity and sensitivity.IGRA has been used in Britain, the United States, Japan and other countries todetect active TB, LTBI, to differentiate TB infection from BCG vaccination, and todetect drug-resistant TB infection. But the IGRA is too expensive to use in developedcountries. To solve the limitation of existing clinical laboratory diagnostic methods, itis necessary to need more specific and sensitive new diagnostic methods to detect thesystemic and local infection of TB infection patients and AIDS-relatedtuberculosis patients.MTB is an intracellular pathogenic bacterium and mainly houses in themonocyte–macrophages. Anti-TB immune response is mainly depended onT cells-mediated immunity. After MTB infected human body, theantigen-presenting cells (APC) process the antigens, and formedMHC/peptide complexes with the MHC molecules on the surface of the APC.TCRs specifically recognize HLA/peptide complexes, and then mediate aseries of immune responses. Therefore, the interaction between TCR and HLA/peptide can be used for testing antigen-specific T-cells. However,the interaction is of very low affinity, too weak for these peptide-loadedMHC molecules to be useful directly in detecting peptide-specific T cells.Several studies have shown that when soluble MHC-peptide complexes aremultimerized, they can achieve much higher avidity for the TCR on the Tcell surface. To solve this problem, Altman set up the soluble MHC/peptidetetramer assay with biotin-avidin cascade principle in1996, whichgreatly increased the intermolecular affinities and stability forMHC/peptide-TCR binding, successfully applied to the detection ofantigen-specific T cells and provided a new method for the in vitroobservation and quantification of antigen-specific T cells.Tetramer is based on the TCR specific recognition and binding MHC-peptidecomplexes on the surface of antigen-presenting cells and target cells. Currently,MHC-peptide tetramers have been used to isolate, clone, active and directquantitatively detect antigen-specific T cells, as well as functional analyze and epitopeanalyze these antigen-specific T cells, and to explore the interaction between TCRand MHC. Conversely, according to the tetramer principle based on the specific andrestricted recognition and binding between MHC-peptide complexes and TCR,soluble TCR tetramer can be constructed to recognize specific MHC-peptidecomplexes and facilitate the study of relationship between T cells and APCs and thedesign of immunotherapeutic and effective vaccines of infectious diseases, tumors,autoimmune diseases and so on. In recent years, MHC class I and MHCclassⅡtetramers have been used to research tuberculosis, to detect the MTBantigen-specific T cells for determining and immunization monitoring active andlatent TB,which have got some positive results. To date, few lab uses constructedTCR α/β tetramer to analyze MTB specific peptide/HLA complex. Therefore, in ourprevious research, several kinds of stable Drosophila S2cells lines which couldsteadily secret CD4~+α/β TCR complex monomers or HLA DR/peptide C5complexes monomers had been constructed. The secreting complex monomers can be used to construct the corresponding CD4~+α/β TCR tetramers andHLA-DR/peptide C5tetramers respectively. The CD4~+α/β TCR tetramers can beused to detect the MTB specific APCs. And the HLA DR/peptide C5tetramerscan be used to detect the MTB-antigen specific CD4~+T cells.The objective of this study: The five kinds CD4~+αβ TCR tetramers by usingbiotinylated monomers expressed and purified from constructed stable DrosophilaSchneider2cell (S2cell) lines were prepared. The PE-labeled CD4~+αβ TCRtetramers were used to co-stain with S2cell lines expressing MTB peptide/HLA-DRcomplexes on the cell membrane, and also were used to detect tetramer-boundCD14~+monocytes and macrophages in the peripheral blood mononuclear cells(PBMC) of pulmonary tuberculosis (PTB) patients and other control groups by flowcytometric analysis. And the FITC-labeled CD4~+αβ TCR tetramers were used toexamine tetramer-bound CD14~+monocytes and macrophages, and MTBantigen-specific and tetramer-bound cells by in situ staining. According to the aboveresults of flow cytometry and in situ staining, the specificity and sensitivity of fivekinds CD4~+αβ TCR tetramers in detecting MTB infections could be assessed. Thefour kinds HLA-DR/peptide C5tetramers by using biotinylated monomersexpressed and purified from constructed stable S2cell lines were prepared. In orderto confirm the specificity and sensitivity of four kinds HLA-DR/peptide C5tetramers in detecting MTB infections and to determine the four kinds tetramercould monitor PTB development and recovery, The four tetramers were used todetect tetramer-bound CD4~+T cells in PBMC of PTB patients and other controlgroups by flow cytometric analysis, Also the above tetramers were used to examinetetramer-positive CD4~+T cells at different time point of PTB patients during theuntreatment and treatment by flow cytometry.Materials and methods:1. The stable S2cell lines which cotransfected with vectors that carries the TCR αchain and TCR β chain were cultured, and monomer of recombinant CD4~+αβ TCR were purified using histidine affinity chromatographic column. After identified by dotblot, the monomers were tetramized by adding SAV-PE or SAV-FITC at8:1molarratio.2. PE labeled CD4~+αβ TCR tetramers were co-stained with S2cell linesexpressing MTB peptide/HLA DR complexes on the cell surface by flow cytometry.3. PE labeled CD4~+αβ TCR tetramers were used to detect tetramer-bound CD14~+monocytes and macrophages in the PBMC of PTB patients and other control groupsby flow cytometric analysis.4. The FITC-labeled CD4~+αβ TCR tetramers were used to detect tetramer-boundCD14~+monocytes and macrophages, and MTB antigen-specific and tetramer-positivecells by in situ staining.5. The four kinds HLA-DR/peptide C5tetramers by using biotinylated monomersexpressed and purified from constructed stable S2cell lines were prepared.6. PE labeled HLA DR/peptide C5tetramers were used to detect the percentage oftetramer-positive CD4~+T cells in PBMCs of TB patients, umbilical cord blood,non-TB infected patients by flow cytometry.7. Detecting the tetramer-positive CD4~+T cells at different time point ofPTB patients with different HLA DR/peptide C5tetramers during the untreatmentand treatment.Results:1. CD4~+αβ TCR monomers were purified and identified. And the monomers weremade into tetramers and labeled with PE or FITC.2. The flow cytometry results of PE labeled CD4~+αβ TCR tetramers co-stainingwith7kinds MTB peptide/HLA-DR cell lines and anti human HLA-DR antibody:The NO.1TCR tetramer was well binding with S2cell lines expressingC14/HLA-DR*1504on the cell membrane. The NO.2TCR tetramer was well bindingwith S2cell lines expressing C5/HLA-DRB1*1501and E6/HLA-DRB1*0803on thecell membrane. The NO.3TCR tetramer was well binding with S2cell linesexpressing C5/HLA-DRB1*1501and C14/HLA-DRB1*1602on the cell membrane. The NO.4TCR tetramer was well binding with S2cell lines expressingC5/HLA-DRB1*1501on the cell membrane. The NO.5TCR tetramer was wellbinding with S2cell lines expressing E6/HLA-DRB1*0803on the cell membrane.But all of the five kinds CD4~+αβ TCR tetramers were few binding S2cell linesexpressing E7/DRB1*0901, E6/DRB1*1504and C14/DRB1*0818on the cellmembrane.3. PE labeled CD4~+αβ TCR tetramer and anti CD14-FITC antibody co-stainingwith PBMCs from TB patients, non-TB infected patients, healthy person andumbilical cord blood specimen, after detection with flow cytometry and statisticalanalysis, came to conculosion as bellows:a. There were statistic test differences of the percentage of tetramer-bound CD14~+monocytes and macrophages between the samples of TB patients and control donorswith five CD4~+α/β TCR tetramers respectively(p<0.01).b. The statistical analysis showed that the percentage of tetramer-bound CD14~+monocytes and macrophages had no difference between NO.1and NO.2TCRtetramer when detecting with the same35TB patients(p=0.53).c. The statistical analysis showed that the percentage of tetramer-bound CD14~+monocytes and macrophages had no difference between NO.3, NO.4and NO.5TCRtetramer when detecting with the same12TB patients(p=0.815).4. The result of in situ staining with five FITC-labeled CD4~+α/β TCR tetramers,tetramer-bound CD14~+monocytes and macrophages, and MTB antigen-specific andtetramer-bound cells were positive in PTB tissue and negative in control pneumoniatissue.5. HLA-DR/peptide C5monomers were purified and identified.And themonomers were made into tetramers and labeled with PE.6. PE labeled HLA-DR/peptide C5tetramer and anti CD4-FITC antibodyco-staining with PBMCs from TB patients, non-TB infected patients and umbilicalcord blood specimen, after detection with flow cytometry and statistical analysis,came to conclusion as bellows:a. There were statistic test differences of the percentage of tetramer-bound CD4~+T cells between the samples of TB patients and control donors with four kindsHLA-DR/peptide C5tetramers respectively(p<0.01).b. There were statistic test differences of the percentage of tetramer-bound CD4~+Tcells between TB patients and non-TB infected patients with NO.6, NO.7, NO.8HLA-DR/peptide C5tetramer(p<0.01).c. The statistical analysis showed that the percentage of tetramer-bound CD4~+Tcells had no difference between NO.6, NO.7and NO.8HLA-DR/peptide C5tetramerwhen detecting with the same39TB patients(p<0.01). There were statistic testdifferences of the percentage of tetramer-positive CD4~+T cells between NO.5andNO.6, NO.7, NO.8tetramer when detecting with the same39TB patients(p=0.639).7. Four kinds HLA-DR/peptide C5tetramers detected the percentage oftetramer-positive CD4~+T cells were high at pre-treatment, after treatment thepercentage were getting down even at the normal level in four TB patients.Conclusion:1. Five kinds of CD4~+αβ TCR monomers and four kinds HLA-DR/C5peptidemonomers were purified and identified as identifying method by dot blot. And all thefive kinds CD4~+αβ TCR monomers and four kinds HLA-DR/C5peptidemonomers were successfully made into tetramers.2. The CD4~+αβ TCR tetramers could detect MTB infections and were specific todetect MTB infections by flow cytometric analyses and in situ staining, which laid alaboratory foundation in the diagnosis and immune mechanism research of TB byusing TCR tetramers.3. There were no sensitivity differences in detecting MTB infections betweenNO.1and NO.2CD4~+αβ TCR tetramer. Also no sensitivity difference was seen indetecting MTB infections with NO.3, NO.4and NO.5CD4~+α/β TCR tetramer.4. The specificity of NO.6, NO.7, NO.8HLA-DR/C5peptide tetramers indetecting MTB infections by flow cytometric analysis could be seen. No sensitivitydifference was seen in detecting MTB infections with NO.6, NO.7and NO.8HLA-DR/C5peptide tetramer. The specificity and sensitivity was low with NO.5 HLA-DR/C5peptide tetramer detecting MTB infections.5. The HLA-DR/C5peptide tetramers could be used to track peptide-specificCD4~+T cells in the PBL of TB patients during the untreatment and treatment, it issignificant for monitoring TB development and recovery.

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