The Changes Of Vδ1-8Subsets Of γδ T Lymphocytes In Peripheral Blood Of Tuberculosis Patients

BackgroundTuberculosis （TB） is one of the most common infectious diseases of harming topeople’s health, but the pathogenesis and the immune mechanism of TB infection havenot been fully elucidated. Previous studies have shown that CD4+T cells and CD8+Tcells of αβ T cells, and macrophages play a key role in the protec immunity totuberculosis infection. In recent years, a lot of research results confirmed that the γδ Tcells also play an important role in the anti mycobacterium tuberculosis infectionimmunity. γδT cells are unique compared with αβT cells. Many studies have shownthat γδT cells have different characteristics of in anti-infection and antitumor immunity,and in specific immune response regulation, autoimmune disease and allergic disease.γδT cells produce variety of cytokines and chemokines. In anti-infection immunity,especially in the early immune against mycobacterium tuberculosis,it may play animportant role. According to the δ chain，γδT cells have eight different subsets, normalpeople are given priority to Vδ2subset. Some studies have found that the proportion ofthe Vδ1and Vδ2subsets will change significantly in some disease. We also observedthe same phenomenon at the early stages of the tuberculosis study. We focus onscientific problems are: distribution of Vδ1-8subsets; obvious difference distributioneach of the healthy adults and TB. And then discusses Vδ1-8subsets may play differentroles in TB infection, especially Vδ1and Vδ2subsets.ObjectiveTo investigate of the distribution of Vδ1-8subsets of peripheral blood in healthy adultand TB in our country. Through the comparison, to explore Vδ1-8subsets, especiallyVδ1and Vδ2subsets may play different roles in tuberculosis.MethodsA certain amount of10ml venous blood in heparin anticoagulant tube from healthyadults and tuberculosis person, which can blend incomplete1:1with1640cultures.Choose peripheral blood mononuclear cell by the density gradient centrifugation with the lymphocyte separation medium. With PBS (phosphate buffer) after washing, adjustthe cell concentration5×106/ml—1×107/ml.1、Flow cytometry instrument testThe cell suspension, adding anti CD3-FITC10μl, anti CD3-APC10μl, antiVδ1-PE10μl,antγδ-TC10mu l, antiVδ1-FITC10μl, antiVδ2-PE10μl,surface after dyeing, withFACSCalibur flow cytometry instrument (Becton Dickinson company, the UnitedStates). Data analysis by Cell Quest softwareof BD company.To calculate the respectivepercentage of the Vδ1+， Vδ2+cell,and Vδ1－/Vδ2－(i.e. Vδ3-8+) cells in γδT cells.2、Fluorescence quantitative RT-PCR assayTaking the cells suspension using Trizol cracking after extraction of RNA and NanoVuePlus ultramicro protein nucleic acid detector (GE Healthcare company) to identify thequality of RNA, with RevertAid pass the First Strand cDNA short Kit Kit (Fermentas)according to the standard after add sample in cDNA on reverse transcription PCR, thencDNA by standard addition SYBR Green Reagents fluorescent reagent, addStepOnePlus real-time fluorescent quantitative PCR (AB Applied Biosystems), usingQuantitation-comparative(CT) Fast method, reaction inside for GAPDH(glyceraldehyde-3-phosphate dehydrogenase). Data from the δ1-8gene cycle numberand difference of internal after2-conversion gene expression is obtained, and usingstatistical method to compute the mean and standard deviation.Results1、 In36cases of healthy adults，the proportion of δ2subsets of γδT cells is the highestby flow cytometry instrument test. Detecting theδ1-8genes by RT-PCR, the relativeexpression quantity of δ2gene is0.0955±0.006, accounting for57.96%of the totalamount. Other genes arranged according to the relative expression quantity frommore to less, followed by δ3、δ1、δ7、δ8、δ4、δ5、δ6.2、 In10cases of TB patients, most of γδT cells are mainly composed of δ1subsets byflow cytometry instrument test. Detecting theδ1-8genes by RT-PCR, the relativeexpression quantity of δ1gene is0.01723±0.0001, accounting for46.60%of thetotal amount. The relative expression quantity of δ2gene is0.0042±0.0007,accounting for11.44%of the total amount. Other genes arranged according to therelative expression quantity from more to less, followed byδ5、δ6、δ4、δ7、δ3、δ8. 3、Statistical validation correlation coefficient respectively, in healthy adults group: theR2is between0.941to0.985of Vδ1group,Vδ2group and Vδ1-/Vδ2-(i.e. Vδ3-8+)group. In TB patients group: the R2is between0.967to0.990of Vδ1group,Vδ2groupand Vδ1-/Vδ2-(i.e. Vδ3-8+) group.(P<0.05).Conclusions1、 In36cases of healthy adults，detecting theδ1-8genes by flow cytometry instrumenttest and RT-PCR, which conclude the distribution ofδ1-8genes: the proportion ofvδ2subsets of γδT cells is the highest, accounting for57.96%of the total amount.Other genes arranged according to the relative expression quantity from more to less,followed by δ3、δ1、δ7、δ8、δ4、δ5、δ6.2、 Compared with healthy adults, in10cases of TB patients, most of γδT cells aremainly composed of δ1subsets by flow cytometry instrument test. Detectingtheδ1-8genes by RT-PCR, the proportion of vδ1subsets of γδT cells is the highest,accounting for46.60%of the total amount. Other genes arranged according to therelative expression quantity from more to less, followed byδ5、δ6、δ4、δ7、δ3、δ8.3、 The results are consistent of flow cytometry assay and Real-time RT-PCR assaysand express strong linear positive correlation on experimental methodology. It hasbeen verified by statistical correlation coefficient respectively. Statistical validationof the different subsets of healthy adults group and the TB group, R2is between0.941to0.990(P <0.050)4、The changes of gene expression and proportion of Vδ1-8compared to healthyadults with TB patients, which shows that Vδ2cells in TB patients weresignificantly less healthy adult, and percentage of Vδ1cells is relatively higher. Wecan conclude that γδT cells play the role of the early days of tuberculosis infection,especially Vδ2subset may be dominant. The Vδ2cells in TB patients group wasobviously less than healthy adults group, which may be due to the cells in the TBinfection caused by the consumption. Through the test data, we also found that therelative expression gene of Vδ1-8of TB patients group is significantly lessthanhealthy adults group, which may be related to immune function decline,lymphopenia.