Detection Of C5-specific CD4~+t-lymphoctyes In Peripheral Bloods And Pathological Tissues From Patients With Pulmonary Tuberculosis By Using Tetramer Technology

Background：Tuberculosis(TB) is a kind of chronic disease induced by Mycobacteriumtuberculosis(MTB), and also is a kind of systemic disease．Every organ of the bodycould be suffered from TB, such as lung, kidney, liver, stomach, brain, intestine,bladder, skin, testis and bone, especially pulmonary infection. The epidemic rate oftuberculosis had significantly decreased because of the application of the vaccineBCG worldwide, various chemical drugs and popularity of chemical therapy. Inrecent years, due to movement of population, rising of multi-drug resistant TB andinfection of human immunodeficiency virus with tuberculosis, the prevalence oftuberculosis increased globally. Whether infected individual falls ill or not iscodetermined by immune condition of MTB-infected host, quantity and toxicity of thebacteria. MTB is one kind of intra-cellular bacterium. The main type of immuneresponse to MTB is T-lymphoctyes, especially CD4~+T cells-mediatedimmunity．When MTB infects human body,antigen-presenting cells(APCs), especiallymononuclear macrophages phagocytose, process, and then present the antigen to HLAmolecules on the surface of the APC and form peptide／HLA complexes.T-lymphoctyes via T-lymphoctye receptor(TCR)particularly recognize and combine the antigen presented by mononuclear-macrophage, This process is limited by theMajor Histocompatibility Complex(MHC)and finally form TCR-peptide-MHCcomplex. And then mediate a series of immune responses. The interaction betweenTCR and peptide／MHC is of very low affinity, causing difficulties in detectingpeptide-specific T-lymphoctyes. Altman sets up peptide／MHC tetramer assay by theprinciple of Biotin-Avidin cascade amplification in1996, thereby greatly increasingthe intermolecular affinities and stability for MHC／peptide-TCR binding．This toughproblem has successfully been solved.Currently, a number of peptide／MHCI tetramer complexes have successfullybeen applied to detect activity of antigen specific CTL, effect of the vaccine andresearch in immunology. Conventionally, genetic engineering technology is used tomake peptide／MHCI tetramer complexes. First of all, β2microglobulin and thebiotinylated heavy chain of MHC Class I(a chain)that15amino acids of BirAenzyme substrate peptide is added at the C terminal of the chain are separately madethrough genetic engineering technology. Then, a chain, β2microglobulin and specificantigen are incubated together to form MHC-peptide complexes, After thebiotinylation by BirA enzyme, the biotinylated peptide／MHC complex is bound tostreptavidin at4:1ratio to form peptide／MHCI tetramer. However, MHC Class IImolecules is a heterodimer consisted of β chains. Since both α and β chains arecharacterized by polymorphism, they can not combine peptide well when they arefolded in vitro. Thus, it is more complex to prepare MHC Class II tetramers.According to Novak’s research, the α and β chains of MHC Class II molecules werefused with linker and leucine zipper-protein, at the same time, the β chain wascombined with BSP at C-terminus. After high expression, purification andbiotinylation, the biotinylated molecules were then incubated with peptide in highconcentration for72hours to generate peptide／MHC II complexes at37℃.Eventually, the complexes were incubated with streptavidin to form MHC class IIpeptide complexes tetramers.The two kinds of stable Drosophila S2cells lines (C5/HLA-DRB1*090102andC5/HLA-DRB1*130201) which can steadily secrete C5/HLA-DRB1complex monomers had been constructed before the study. C5, an amino acid sequence derivedfrom CFP10, had been proved that it is a broad-spectrum polypeptide reacted withCD4~+T-lymphoctyes by ELISPOT. C5/HLA-DRB1*090102andC5/HLA-DRB1*130201belong to HLA-DRB1alleles and respectively encodedifferent HLA-DRB1molecule.Objective：1. The tetramers were used to detect in situ stain the slices of tuberculosisgranuloma tissues to examine combining the C5-specific T-lymphoctyes under thelaser confocal microscope.2. The tetramers were used to detect the proportion of C5-specificCD4~+T-lymphoctyes in peripheral blood mononuclear cell of tuberculosis patients byflow cytometry.Content and methods：Two types of PE-labeled C5/HLA-DRB1(C5/HLA-DRB1*090102andC5/HLA-DR B1*130201) tetramers by using biotinylated monomers expressed andpurified from constructed stable Drosophila Schneider2cell (S2cell) lines wereprepared. The tetramers labeled with FITC were prepared and used to in situ costainlung tissue slices of PTB patients with mouse anti-human CD4and examine C5/HLA-DRB1CD4~+T-lymphoctyes under the laser confocal microscope. The tetramerswere respectively used to examine tetramer-positive (C5-specific) CD4~+T-lymphoctyes in the PBL of PTB patients by anti-human CD4-FITC costaining andflow cytometric analysis. The PBLs of non-TB patients with pulmonary infection andhealthy adults were used as controls. Nonparametric Wilcoxon test in analysis ofvariance were conducted with SPSS16.0software.Results：In the study, the Drosophila S2cells which express the secretary biotinylated C5 ／HLA-DRB1complexes monomers were induced．And then the monomers whichwere identified by dot blot and SDS-PAGE were purified and concentrated, then, thebiotinylated C5／HLA-DRB1complex tetramers were made. We have generatedtwo different HLA-DR gene types of tetramers：comparation and detection wereperformed by using two tetramers respectively for pulmonary tuberculosis patientsbloods, non-tuberculosis patients blood and healthy donors blood. The medians ofC5-specific CD4~+T-lymphoctyes from PBLs of patients with pulmonary tuberculosis,non-TB patients with pulmonary infection and healthy adults were respectively0.15%,0.09%, and0.03%by using C5/HLA-DRB1*090102tetramer, while the medianswere respectively0.19%,0.10%, and0.04%by using C5/HLA-DRB1*130201tetramer. The medians of peptide C5-specific CD4~+T-lymphocytes in PTB patientswere much higher than control bloods (p﹤0.05). There was no significant differencebetween the two tetramers (p>0.05). C5-specific CD4~+T-lymphoctyes were observedin lung tissue slices of PTB patients by in situ tetramer staining. From the perspectiveof tetramer, the results that the same TB patients were detected by using abovetetramers respectively were statistically analyzed. There were no significantdifferences detected by two kinds of tetramers. Application of two kinds of tetramersin situ tetramer staining to detect histopathology tissues can be used to directlyobserve the ratio, localization and distribution of C5-specific CD4~+T-lymphocytesrespond to MTB peptide C5. The C5-specific proportion of CD4~+T-lymphoctyes wasobserved in the slice of pulmonary TB granuloma, while not was observed in the sliceof non-TB patients with pulmonary infection for control. Therefore, the tests of twokinds of C5／HLA-DRB1tetramers are specific for histopathologic examination ofTB patients.Conclusion：1．The purified secretary proteins expressed by S2cells were identified as C5／HLA-DRB1complexes monomers by Dot blot and SDS-PAGE.2．The two kinds of prepared tetramers could be used to in situ staining of pathological slide of tuberculosis patients．3． The two kinds of prepared C5／HLA-DRB1tetramers could be used to detect theproportion of C5-specific CD4~+T-lymphocytes of blood of tuberculosis patients,which paves a way for further study and application.