Study On Immune Mechanism Of Foxp3 + Regulatory T Cells Regulating Tuberculosis

Part1. Study on cellular immune responses of Mycobacerium tuberculosis infectionObjective To evaluated the T cell phenotypic distributions and TB antigen-specific immune responses in the peripheral blood of populations with different TB infection status. And to further compare the T cell subsets and TB antigen-specific immune responses of between the peripheral blood and the lesion site of tuberculous pleurisy. Methods One hundred and twenty-five participants were enrolled in this study including ATB patient (n=46), LTBI subjects (n=34), and HC (n=45). IGRAs were employed for screening LTBI subjects. The whole blood cell surface staining, intracellular cytokine staining (ICS), flow cytometry were used to detect T cells phenotypic distributions in the peripheral blood mononuclear cells (PBMC) and tuberculous pleural effusion mononuclear cells (PFMC). PPD or phosphorylated antigen HMBPP were employed as stimulators for detection of Mycobacterium tuberculosis antigen-specific T cells cell functions. The comparisons among different groups were done by Mann-Whitney and Kruskal-Wallis test.Results The absolute numbers (cells/μL) of CD3+, CD3+CD4+, CD3+CD8+and CD3+Vy2+Vδ2+T cells in ATB group trended lower, among which, CD3+CD4+T cells decreased more obviously. Importantly, PPD-specific CD3+CD4+T cells ATB patients was significantly lower than that in LTBI subjects (P=0.039). On the other hand, the vast majority of cells in the PFMCs were CD3+T cells and the ratio of CD3+CD4+/CD3+CD8+T cells in PFMCs were significantly higher than that in PBMCs. However, the CD3+Vy2+Vδ2+T-cell subsets were significantly lower in PFMCs than that in PBMCs. Furthermore, the PPD-specific CD4+T cells in PFMCs accounted for79.43%and CD8+T cells accounted for11.39%, which indicated that αβT cell subsets play a major role in the anti-TB immune response in the lesion sites of tuberculosis infection.Conclusion The cellular immue responses were impaired in ATB patients. The inhibition of CD3+CD4+T cell function may be a key factor in the activation of LTBI. Part2. Role of Foxp3+Tregs in impaired anti-tuberculosis immunity in active tuberculosisObjective To evaluate the role of Foxp3+Tregs in impaired anti-tuberculosis immunity in active tuberculosis. And to compare the number and function of Foxp3+Tregs between circulating and the lesion site of tuberculosis infection.Methods A total of93participants were recruited including ATB patients (n=42), LTBI subjects (n=18) and HC (n=33). IGRAs were employed for screening LTBI subjects. Whole blood surface staining, ICS, flow cytometry and realtime-PCR were used to detect Foxp3+Tregs levels and the CTLA-4expressions among the three groups. The comparisons among different groups were done by Mann-Whitney and Kruskal-Wallis test.Results Higher levels of Foxp3+Tregs were found in ATB group (5.91±1.86%), compared to LTBI group (4.34±1.16%) and HC group (4.62±1.43%)(P=0.012and P=0.0008, respectively). While there were no differences of IL-10levels among three groups (P=0.26). We further found that CTLA-4levels in Tregs were higher in ATB group (22.85±10.90%) than those in LTBI(6.99±2.51%) and HC(9.54±4.48%) groups (both P<0.0001). In patients with tuberculous pleurisy, Foxp3+Tregs and the CTLA-4expressions on Tregs in PFMCs were found both higher than those in PBMCs (P=0.008and0.002, respectively).Conclusion Foxp3+Tregs cells negatively regulate the immune responsein active Mycobacterium tuberculosis infection. CTLA-4maybe the key molecule mediated the inhibition of anti-tuberculosis instead of IL-10. Part3. The mechanism of Foxp3+Tregs in the immunopathogenesis of active Mycobacterium tuberculosis infectionObjective To explore the mechanism of Foxp3+Tregs in active Mycobacterium tuberculosis infection.Methods Totally33sputum smear-positive untreated patients with active pulmonary tuberculosis were recruited, of which22cases (66.7%) were male and mean age was42.4years. Flow cytometry, Realtime-PCR and Luminex’s xMAP technology were employed to detect the antigen-specific immune responses. Magnetic active cell sorting(MACS) was used to isolate CD4+CD25+CD127dim/-Tregs and CD4+CD25-Tresp cells in the peripheral blood mononuclear cells(PBMCs). Tregs and Tresp cells were mixed cultured with different proportions upon PPD stimulation with or without CTLA-4blockade. ICS and CFSE lymphocyte proliferation inhibition experiments were used to detecte the cytokine levels and proliferation of Foxp3+Tregs and CD4+CD25-Tresp cells. The comparisons among different groups were done by Mann-Whitney and Kruskal-Wallis test..Results With PPD stimulation, IFN-y secretion of Tresp and Tresp cells proliferation were both significantly inhibited by Tregs in vitro (P=0.0004and P=0.043, respectively). Furthermore, the inhibition was significanly attenuated by adding CTLA-4blockade in the medium(P=0.048), which indicated that CTLA-4blockade could abrogate the suppressive function of Foxp3+Tregs in vitro. Levels of interferon-y and interleukin-2in the supernatant and their mRNA expressions were decreased by adding Tregs and significantly up-regulatedby adding CTLA-4blockade in the cell cuture system. However, there were no significant differences of mRNA levels of IL-10and TGF-β before and after adding CTLA-4blockade.Conclusion The inhibiton of Foxp3+Tregs on anti-tuberculosis immune response in active Mycobacterium tuberculosis infection is probably through CTLA-4molecule. Blocking CTLA-4pathway could reversed the suppressive effect of Foxp3+Tregs on the Mycobacterium tuberculosis antigen-specific cellular immunity. Thus, CTLA-4is expected to be the novel target of active TB immunotherapy.