The Experimental Study Of Predominant Th1 Type Cellular Immune Response Induced By Antigen Ag85B Under Regulation Of α-GalCer In Active Osteoarticular Tuberculosis Patients

Objective: To select the preponderant antigen and observe Th1 cellular immune response induced by preponderant antigen that stimulated by iNKT cell activator(α-GalCer)in active osteoarticular tuberculosis patients.Methods: 1.We collected the peripheral blood of active osteoarticular TB patients,latent tuberculosis infection,and healthy people as control.The level of serum cytokines(IFN-γ,IL-2,IL-4 and IL-10)in each group was detected by enzyme-linked immunosorbent assay(ELISA).The proportion of CD4+IFN-γ+ cells in peripheral blood mononuclear cell(PBMC)was detected by flow cytometry.2.We cultivated the PBMC of active osteoarticular TB patients with Ag85 B,ESAT-6,CFP-10 and PPD respectively.The level of cytokines(IFN-γ,IL-2,IL-4 and IL-10)and the proportion of CD4+IFN-γ+ cells in peripheral blood mononuclear cell were detected.The expression of T-bet and GATA-3 were detected by quantitative PCR and western blot.3.Ag85 B antigen and α-GalCer were added in peripheral blood mononuclear lymphocyte of active osteoarticular TB patients jointly,the level of cytokines(IL-12,IFN-γ,IL-2,IL-4 and IL-10)and the proportion of CD4+IFN-γ+ cells in peripheral blood mononuclear cell were detected.The protein expression level of JAK/STAT pathway was detected by Real time PCR and Western blot.Results: 1.The levels of IFN-γ and IL-2 were significantly decreased in active osteoarticular tuberculosis patients serum,compared with latent tuberculosis infection group and healthy people group,similar situation deteceted in the proportion of CD4+IFN-γ+ cells.However,the levels of Th2 type cytokines(IL-4 and IL-10)were increased in Active osteoarticular TB group.2.The concentration values of IFN-γ in Ag85 B antigen group and ESAT-6 antigen group were obviously higher than PPD group,while there were no significant differences between CFP-10 antigen group and PPD group.The level of IL-2 in Ag85 B group was higher than the PPD group,while no statistical differences were detected in ESAT-6 and CFP-10 groups compared to PPD group(P>0.05).On the other hand,the concentration levels of IL-4 and IL-10 in experimental antigens(Ag85B,ESAT-6 and CFP-10)were not statistically significant different from PPD group.The proportion of CD4+IFN-γ+ cells and the expression of T-bet in Ag85 B group were higher than the other groups,while the differences of GATA-3 was not significant.3.The secretion of Th1 cytokines(IFN-γ,IL-2)was increased significantly under stimulation of α-GalCer/Ag85 B,while Th2 type cytokines(IL-4,IL-10)reduced.The secretion of IL-12 was increased significantly under stimulation of α-GalCer/Ag85 B group.The results revealed that the expression of Jak2,Tyk2,STAT1 and STAT4 was significantly up-regulated in α-GalCer/Ag85 B group compared with other groups.However,the expressions of Jak1,Jak3 and STAT6 was not statistical different in all the groups.Conclusion: The secretion of Th1 cytokine was insufficient in active osteoarticular TB patients;Ag85B antigen can enhance Th1 type cellular immune response by up-regulating T-bet;α-GalCer and Ag85 B antigen increase Th1 type cellular immune response,which was associated with the activation of JAK/STAT signaling pathway by enhancing the production of IL-12 and IFN-γ.