Establishment And Optimization Of Flow Cytometry-based Diagnositic Method For Tuberculosis

Tuberculosis (TB) is one of the infectious diseases harmful to human health, which is especially serious in our country. In TB prevention and control, the biggest challenge is how to diagnose the early tuberculosis infections in a rapid and accurate way. At present, the gold standard in tuberculosis diagnosis is cultivation of Mycobacterium tuberculosis, which is not helpful for the early diagnosis. The traditional immunological diagnostic method tuberculin skin test (TST) has some limitations too, such as TST may give false-positive results or can’t detect the patients because of weak immunity. In this study, we applied a flow cytometric assay to identify CD4+T lymphocytes specific for Mycobacterial antigens ESAT-6/CFP-10. By using this approach, we monitored active TB and non-TB (NT) patients. At the same time we analyzed the results obtained with both T-SPOT and the flow cytometry test in order to establish whether the latter test could be an additional tool in the diagnosis of active patients. In addition, co-stimulatory antibodies were added to PBMC with TB specific-antigen in order to optimize the method. Finally, we make an overall evaluation the TB infection donors with ESAT-6/CFP-10 skin test by flow cytometry in a clinical study.1. Establishment of a flow cytometry-based diagnostic method for TuberculosisA total of 91 donors were recruited from the Public Health Clinical Center in Shanghai from July 2014 to March 2015. The donors were divided into 2 different groups:71 active TB patients and 20 NTB patients. We analysed cytokine-secreting CD4+T lymphocytes specific for ESAT-6/CFP-10 and PPD by flow cytometry-based intracellular cytokine staining. At the same time we compared flow cytometry results with T-SPOT results in order to establish whether the former test could be an additional tool in the diagnosis of active patients. The results showed that the frequency of ESAT-6/CFP-10-specific IFN-γ+CD4+T cells was significantly higher in subjects with active TB. Similar results were obtained by IL-2+CD4+T and TNF-α+ CD4+T cells. From the data we can conclude that the flow cytometric assay (83%) has a better sensitivity than T-SPOT (74.6%) for the diagnosis of TB infection. On the basis of the TB specific-antigen, the method has a good specificity and a ROC curve was constructed with different cut-offs of percentage of ES AT-6/CFP-10-specific IFN-y secreting cells. The area under the curve (AUC) is 0.868, and arrow indicates the optimal cut-off (0.017%) with the best combination of sensitivity and specificity. The above results indicated that the identification by flow cytometry of antigen-specific T lymphocytes on antigen stimulation of PBMC could be an effective tool in the diagnosis of TB infection.2. Optimization of flow cytometry-based diagnostic method for TuberculosisSignals from co-stimulatory and inhibitory molecules play positive and negative roles in T cell activation, respectively. A stimulatory antibody against CD28 (aCD28) was commonly used in detecting Mycobacterial antigen-specific T cell responses. To improve the sensitivity, a blocking antibody against inhibitory molecule PD-1 (aPD-1) and a stimulatory antibody against SLAM (aSLAM) were added to PBMC with TB specific antigens. In addition, a stimulatory antibody against CD49d (aCD49d) was used as an additional group. The study involved 26 patients with tuberculosis, and higher frequencies of TB specific IFN-γ-producing T cell on stimulating with aCD28/aPD-1/aSLAM and aCD28/aCD49d were observed in8 patients, while no effects were observed on the background. However, we did not find obvious increase in other subjects. According to this result, we think that inhibition of PD-1 signaling pathways and stimulation with aSLAM may be helpful for improving T cell function in TB patients. Due to low sample numbers, additional experiments should be performed with more patients in order to determine whether it helps to improve the sensitivity of diagnosis of tuberculosis by flow cytometry.3. Evaluation of T cell responses in TB patients inoculated with recombinant M.tb ESAT-6/CFP-10In this study, a intracellular cytokines detection technique was used to evaluate the T cell response in TB patients who accepted recombinant M.tb ESAT-6/CFP-10 skin test. We found that the percentage of TB antigen-specific IFN-γ+/IL-2+CD4+T cells have significantly decreased responses, but TNF-α+CD4+T cell present no change 3 days after TB antigen inoculation. In addition, the percentage of TB antigen-specific IFN-γ+/IL-2+/TNF-α+CD8+T cell also showed a decreased response, but did not achieve significance. We also anlaysed poly functional T cell responses and found that TB antigen and PPD-specific IFN-y+TNF-a+IL-2+CD4+T cells significantly decreased, while IFN-γ TNF-α+ IL-2- CD4+T cells significantly increased after antigen inoculation. In addition, TB antigen-specific IFN-γ+TNF-α+IL-2’CD8+T cells and IFN-γ+TNF-α-IL-2-CD8+T cells also increased. The data made a quantitative description about cellular immune responses of TB patients before and after TB antigen skin test, which provided important information for clinical research.