Study Subjects. Patients with TB were diagnosed at Dr. F. Muñiz or P. Piñero Hospitals (Buenos Aires, Argentina) based on clinical and radiological data together with the identification of acid-fast bacilli (AFB) in sputum. First peripheral blood samples were collected before one week of anti-tuberculosis therapy administration and second samples were obtained between 14 and 21days of anti-TB treatment. All patients had received anti-tuberculosis (anti-TB) regular therapy for drug sensitive Mtb strains. Bacillus Calmette-Guerin (BCG) vaccinated healthy control individuals lacking a history of TB (HD) participated in this study.
Peripheral blood was collected in heparinized tubes from each participant after obtaining a written informed consent for the collection of samples and the subsequent analysis. All methods were carried out in accordance with relevant guidelines and regulations.
The protocols conducted in this work were approved by the Ethics Committees of Muñiz and Piñero Hospitals.
Exclusion criteria and classification of patients. The exclusion criteria were carried out as previously described36. Briefly, individuals participating of the study were 18 - 60 years old and had no history of diseases affecting the immune system, such as HIV infection, treatment with immunosuppressive drugs,a recent diagnosis of cancer, hepatic or renal disease, pregnancy, or positive serology for other viral (e.g., hepatitis A, B or C), or bacterial (e.g., leprosy, syphilis) infections. Subjects with anticoagulant medication orbleeding disorders that might be at an increased risk of bleeding during the procedure of obtaining the sample were excluded from the study.
Individuals with latent infection were excluded from the present study by using the QuantiFERON-TB Gold Plus kit (Qiagen, Germany, USA).
TB Patients were classified as high responders (HR-TB) or low responders (LR-TB), based on their in vitro lymphocyte responses to a whole cell lysate of M. tuberculosis (Mtb-Ag) as previously described26. Briefly, HR-TB patients are individuals displaying significant proliferative responses, IFN-γ production and an increased percentage of SLAMF1 positive cells after Mtb-Ag stimulation; whereas LR-TB patients exhibit low proliferative responses, IFN-γ release and SLAMF1 positive cells. LR-TB patients had more severe pulmonary disease compared with HR individuals.
In the studied population, no differences regarding age distribution or sex were found (Table 2).
Antigen. In vitro stimulation of peripheral blood mononuclear cells (PBMC) was performed with a cell lysate from the virulent Mycobacterium tuberculosis strain H37Rv, prepared by probe sonication (Mtb-Ag) (BEI Resources, NIAID, NIH: Mycobacterium tuberculosis, Strain H37Rv, Whole Cell Lysate, NR-14822).
Cell preparation and reagents. PBMC were isolated by centrifugation on Ficoll-Hypaque (Amersham Biosciences, NJ, USA). Flow cytometry assays were performed after PBMC were washed with PBS plus 1% BSA and 0.1% NaN3 and resuspended in staining buffer (PBS plus 1% Fetal Bovine Serum (FBS)). Viability was determined by exclusion of trypan blue (≥ 98%). PBMC were also cultured (1 × 106 cells/mL), with or without Mtb-Ag (10 μg/mL) with RPMI 1640 medium (Gibco, MD, USA) supplemented with 1% L-glutamine, 1% penicillin/streptomycin, and 10% FBS (Gibco, MD, USA) during 48 h. Then, levels of IFN-γ were measured using a commercial ELISA kit (Human IFN-γ ELISA MAX Standard Kit, BioLegend, USA) following the manufacturer´s instructions.
Flow cytometry. PBMC from TB patients and HD were stained with specific fluorophore-marked antibodies against CD14 (FITC, clone HCD14, BioLegend, USA) and CD16 (APC, clone 3G8, BioLegend, USA) for differentiating monocyte subsets. MDSC classification was performed staining PBMC with specific fluorophore-marked antibodies against CD14 (FITC, clone HCD14, BioLegend, USA), CD11b (PE, clone ICRF44, BioLegend, USA), CD15 (PE/Cy7, clone HI98, BioLegend, USA), and HLA-DR (APC, clone L243, BioLegend, USA). Negative control samples were incubated with irrelevant isotype matched monoclonal antibody (BioLegend, USA).
Gating strategies are shown in the representative density plots of figures.
Cells were incubated for 30 min with the specific antibodies at room temperature in the dark and two washes were made with washing buffer. Samples were analyzed on a FACSAria II flow cytometer (BD Biosciences, CA, USA).
Proliferation index. PBMC were stimulated with Mtb-Ag for five days and cells were pulsed with [3H]TdR (1 μCi/well) and harvested 16 hours later. [3H]TdR incorporation was measured in a liquid scintillation counter as counts per minute (c.p.m). Proliferation index for each individual was calculated as (c.p.m. after Mtb-Ag stimulation)/(c.p.m. after culturing with medium).
Statistical analysis. Analysis of variance (ANOVA) and post hoc Tukey´s multiple comparisons test were used as indicated in figure legends. The Mann–Whitney U test was used to analyze differences between groups. For categorical variables, the Chi-square (and Fisher´s exact) test for homogeneity was performed to compare proportions of subjects between groups. In the graphs each symbol represents an individual and the horizontal lines indicate the mean ± standard error of the mean (SEM). Correlations were calculated using the non-parametric Spearman correlation test. Receiver operating characteristic (ROC) curve analysis was performed to analyze the predictive value of the frequencies of CD14+CD16+ and M-MDSC cells populations, calculating the area under the curve (AUC) and the 95% confidence interval (CI). Analyses were performed using GraphPad Prism 8.0.2 software. P < 0.05 was considered statistically significant.