| Tuberculosis is an infectious disease caused by the Mycobacterium tuberculosis(MTB),which is mainly transmitted through the respiratory tract and causes about 10million new cases and 1.4 million deaths each year,making it the second leading cause of death globally.Cell immunity mediated by T cells plays a key role in controlling MTB infection after the organism is infected with MTB.However,in chronic MTB disease,T cell exhaustion occurs when T cells are continuously exposed to large amounts of antigens.The main manifestations are the gradual decrease of proliferation ability,the decrease of the secretion level of pro-inflammatory factors,and the overexpression of some inhibitory molecules.The higher the load of MTB and the longer the duration of infection,the deeper the T cell exhaustion.In the in vitro experiment of MTB infection,peripheral blood mononuclear cells from patients with chronic tuberculosis also showed exhaustion state,and the exhaustion state of peripheral blood mononuclear cells could be reversed after blocking the Programmed death-1(PD-1)and T cell immunoglobulin domain and mucin domain-3(Tim-3)receptor pathways.The results showed that the cytokine secretion and other functions were restored and the ability to control the growth of MTB was enhanced.The incidence of tuberculosis(TB)remains high,and even worse,the incidence of multidrug-resistant tuberculosis(MDR-TB)continues to climb which has increased the burden of TB control.Therefor MDR-TB remains a major global concern.Under the action of long-term chronic infection,the immune function of MDR-TB patients is low.So far,the response of human T cells to MDR-TB infection remains unclear.Therefore,in this study,we evaluated the inhibitory receptor in peripheral blood and MDR-TB patients protective cytokine expression,through the analysis of healthy people,clinical sensitivity tuberculosis patients(the drug sensitive tuberculosis,DS-TB)as well as the function of peripheral blood T cell exhaustion MDR-TB patients related indicators,as well as several immune effector cytokine expression,to explore drug resistance pulmonary tuberculosis patients expressed t-cell exhaustion is immune to its effects.Objective:To analyze whether T cells are depleted in patients with multidrug-resistant tuberculosis(MDR-TB),and the effect of T cell exhaustion on the expression of immune effect in patients with drug-resistant tuberculosis(DS-TB),so as to find a new therapeutic target to restore the function of T cells in patients with DS-TB.Method:38 DS-TB patients,20 MDR-TB patients and healthy control volunteers(HD)who were hospitalized in Shanghai Pulmonary Hospital were collected,and peripheral blood mononuclear cells were isolated.1.PBMC was stimulated by M.tuberculosis H37Rv lysates with a mass concentration of 10μg/m L for 72 hours.PD-1,Tim-3 and lymphocyte Activation gene-3 on CD4~+T and CD8~+T cells were detected by flow cytometry.The expression level of Lag-3 and the secretion of interferon-gamma(IFN-γ),Tumor necrosis factor-alpha(TNF-α)and interleukin-2(IL-2)were observed to study the exhaustion state of T cells.2.We blocked the Tim-3and PD-1pathways with anti-human PD-1and anti-human Tim-3 antibody,either alone or combination during the M.tuberculosis H37Rv lysates infection.Using ELISA to detect T lymphocyte culture supernatant of IL-2,IFN-γ,TNF-αlevels,to further verify the impairment of T cell function,the reversal of T cell exhaustion by PD-1 and Tim-3 blocking antibody was studiedResults:1.After stimulated by MTB H37RV antigen,the expression level of PD-1on CD4~+T cells and CD8~+T in MDR-TB group was significantly higher than that in HD group and DS-TB group(P<0.0001,P<0.001),but there was no statistical difference between the DS-TB group and the HD group(P>0.05).2.After MTB H37RV antigen stimulation,the expression level of Tim-3 on CD4~+T cells in MDR-TB group was significantly higher than that in HD group and DS-TB group(P<0.001,P<0.0001),however,there was no statistical difference between the DS-TB group and the HD group(P>0.05).Compared with HD group and DS-TB group,the expression of Tim-3 on CD8~+T cells in MDR-TB group was significantly increased(P<0.0001,P<0.001),however,there was no significant difference between the DS-TB group and the HD group(P>0.05).3.After MTB H37RV antigen stimulation,the expression level of Lag-3 on CD4~+T cells and CD8~+T in MDR-TB group increased compared with HD group and DS-TB group,but there was no significant difference among all groups(P>0.05).4.After the MTB H37RV antigen stimulation,the expression level of IL-2 in CD4~+T cells of MDR-TB group and DS-TB group was significantly higher than that of HD group(P<0.01,P<0.0001),compared with the DS-TB group,the expression level of IL-2 in CD4~+T cells in the MDR-TB group showed a downward trend,and the difference was not statistically significant.Concordant with CD4~+T cells,the expression level of IL-2 in CD8~+T cells in MDR-TB group and DS-TB group was significantly increased compared with HD group(P<0.0001),compared with the DS-TB group,the expression level of IL-2 in CD4~+T cells in the MDR-TB group showed a downward trend,and the difference was not statistically significant.5.After stimulation with MTB H37RV antigen,the expression of IFN-γin CD4~+T cells of DS-TB group was significantly higher than that of HD group(P<0.05),however,there was no significant difference in the expression of IFN-γin CD4~+T cells between the MDR-TB group and the previous two groups(P>0.05).Compared with HD group,the expression of IFN-γin CD8~+T cells of DS-TB group and MDR-TB group was significantly increased(P<0.0001,P<0.05),there was no significant difference in IFN-γexpression in CD8~+T cells between the two groups(P>0.05).6.After MTB H37RV antigen stimulation,the expression of TNF-αin CD4~+T cells of DS-TB group was significantly higher than that of HD group and MDR-TB group,respectively(P<0.0001,P<0.01),there was no significant difference between HD group and MDR-TB(P>0.05).There was no significant difference in the expression of TNF-αin CD8~+T cells among all groups(P>0.05).7.Compared with MTB+Iso and MTB+α-Tim-3 groups,IL-2 secretion level in the supernatant of MTB+α-PD-1+α-Tim-3 group was significantly increased in HD group(P<0.05).In MDR-TB group,compared with MTB+Iso and MTB+α-PD-1group,the secretion of IL-2 in cell supernatant of MTB+α-PD-1+α-Tim-3 group was significantly increased(P<0.0001,P<0.05),however,there were no significant differences among different treatment methods in the DS-TB group.8.Compared with the Control group,the secretion of IFN-γin the supernatant of HD group was significantly increased(P<0.01),however,there was no significant difference in the ability of antigen-specific T lymphocytes to secrete IFN-γamong other groups.Compared with the Control group,the secretion of IFN-γin cell supernatant was significantly increased(P<0.001),compared with the MTB+Iso group,the secretion of IFN-γin the supernatant of MTB+α-PD-1+α-Tim-3 group was significantly increased(P<0.01),however,there were no significant differences among different treatment methods in the DS-TB group(P>0.05).9.Compared with the Control group,the secretion of TNF-αin the supernatant of HD group was significantly increased(P<0.0001),however,there was no significant difference in the ability of antigen-specific T lymphocytes to secrete TNF-αamong other groups.The results of DS-TB and MDR-TB were consistent with those of HD group.Conclusion:This study confirmed that T cell exhaustion occurs in MDR-PTB patients,the expression of T cell immune effect was partially reversed after the administration of T cell inhibitory receptor.Blocking PD-1/Tim-3 may be a therapeutic target for MDR-PTB patients. |
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