Enrichment Of Regulatory T Cells In Blood Of Patients With Multidrug-resistant Tuberculosis

Background Tuberculosis(TB),caused by Mycobacterium tuberculosis(Mtb),remains one of the world's largest infectious diseases.Although progress has been made to reduce global incidence of drug-susceptible tuberculosis,the identification and spread of multidrug-resistant TB(MDR-TB)during the past decade threaten to undermine TB control and elimination.MDR-TB is defined as resistance to the two key first-line anti-TB drugs,isoniazid(INH)and rifampicin(RMP/RIF).It has been widely recognized that the development of active TB is related to the host immune status.Despite decades of intensive study,the immune response to Mtb is poorly understood,reflecting the complicated interaction between the immune system's capacity to reliably eradicate Mtb and the ability of the bacteria to escape from this protective immune response to sustain infection.The host protection against Mtb consists principally of Mtb-specific Th1-type IFN-?-producing CD4~+ and CD8~+ effector T cells.Although this response helps to limit bacterial replication and dissemination in vivo,it also causes significant immunopathology.The immune system itself has regulatory mechanisms to limit such immunopathologic effects of the cellular immune response.Among suppressor T cells,regulatory T cells(Treg cells)mediate peripheral tolerance by actively suppressing effector T cells and inhibiting immune-mediated tissue damage.Due to the immunosuppressive nature of Treg cells,it is not surprising that some microbial pathogens make use of these cells to evade protective immunity.The impaired responsiveness of peripheral blood T cells in patients with TB suggests that Treg cells could also be a key regulators of immune response in tuberculosis.But the character of Treg cells in pulmonary TB is still not obvious.Furthermore,few reports have focused on Tregs in the immune response of human MDR-TB.Therefore,in the present study we seek to further demonstrate the numbers and functions of Tregs,and expression of signaling molecules in these cells from peripheral blood of patients with MDR-TB,in order to investigate the immune regulatory mechanisms of human response to Mtb,especially MDR-TB infection.Methods and Results Part 1 The expression of regulatory T cells in peripheral blood of MDR-TB patients Methods: Patients with drug-sensitive tuberculosis(S-TB),MDR-TB and healthy controls(HCs)were recruited into this study.Levels of IL-10 and TGF-?1 from peripheral blood were measured using ELISA assay,and levels of CD4~+CD25~+Foxp3~+ Treg cells and CD4~+CD25~+CD127~-Treg cells were measured using Flow Cytometry.Results: MDR-TB patients produced significantly higher IL-10 level in their serum than S-TB patients(p<0.05),and the two groups secreted much more IL-10 than HC groups(p<0.05,respectively).The expression of TGF-? displayed the same trend.A higher proportion of CD4~+CD25~+Foxp3~+ Treg cells and CD4~+CD25~+CD127~-Treg cells were observed in TB patients.Furthermore,we detected a marked increased percentage of CD4~+CD25~+Foxp3~+ Tregs and CD4~+CD25~+CD127~-Treg cells in MDR-TB patients than that in S-TB patients.A positive correlation between IL-10 / TGF-?1 levels and CD4~+CD25~+Foxp3~+ Treg frequency was found.Conclusion: The increased Treg prevalence in MDR-TB is likely a response to persistent bacterial burden and chronic ongoing inflammatory state.Part 2 The functional characteristics of CD4~+CD25~+ Treg cells derived from PBMCs of TB patients Methods: Samples were sorted for separation of CD4~+CD25~+ and CD4~+CD25~-T cells by use of the immunomagnetic bead separation method.For detection of Treg suppressive function,CD4~+CD25~-Teffs were cocultured with autologous Tregs,and proliferation was analyzed by flow cytometry to determine the dilution of CFSE fluorescence intensity.Treg-Depleted PBMCs were cultured to measure IFN-? concentration in response to BCG stimulation.Results: Tregs down-regulated proliferation of Teffs in TB patients.Undivided proliferation of CFSE-labeled Teff cells were markedly increased when cocultured with autologous Treg cells at a 1:1 ratio.Depletion of CD4~+CD25~+ Treg cel Is from PBMCs increased IFN-? concentration in response to BCG stimulation.Conclusion: Tregs down-regulated proliferation of Teffs in TB patients,suggesting that Tregs are functionally active in TB.Part 3 The expression of CTLA-4,PD-1,ICOS on Treg cells from PBMCs of TB patients Methods: PBMCs of the three cohorts were analyzed for surface expression of PD-1,CTLA-4 or ICOS on CD4~+CD25~+Treg cells.Treg-depleted PBMCs of TB patients were cultured in the presence of neutralizing antibody to PD-1 and BCG.Level of CD4~+CD25~+Foxp3~+ Treg cells was then measured using Flow Cytometry.Results: CTLA-4 and PD-1 expression were significantly up-regulated in Tregs of TB patients,especially in that of MDR-TB patients(p<0.05,compared to S-TB group).The percentage of ICOS~+ Tregs in PBMCs was also elevated in TB patients than in HCs(p<0.05).Nonetheless,we observed a higher percentage of the mean ICOS~+ Tregs in MDR-TB group compared to that of S-TB group.However,no statistically significant difference between the two groups was found.What's more,CTLA-4~+,PD-1~+,ICOS~+ Treg frequency all displayed high degree of correlation with frequency of Foxp3~+ Tregs in MDR-TB patients.Anti-PD-1 reduced BCG-induced expansion of Tregs.Conclusion: Accumulation of cells with this phenotypes may play an important role in immune dysregulation in TB.Summary we detected an increased frequency of circulating Tregs in MDR-TB patients that suppress Teff cells proliferation,and express altered cell surface phenotypes.Tight correlation between the MDR status and persistence of Tregs suggest the important role of Mtb driven expansion and maintenance of Tregs in human tuberculosis.The data from our study would provide evidence for further investigations regarding the mechanism of immunity and allow a better understanding of the immunopathogenesis of MDR-TB.