Decreased T helper 17 cells in tuberculosis is associated with increased percentages of programmed death ligand 1, T helper 2 and regulatory T cells

This prospective study was conducted at National Taiwan University Hospital from January 2014 to August 2016. Patients aged ≥20 years who were diagnosed with active TB were recruited. Active TB was diagnosed by cultures positive for Mycobacterium tuberculosis or a typical pathology of Mycobacterium tuberculosis infection or suspicious radiographic findings plus a positive response to empirical TB treatment [4, 13]. In addition, we recruited age- and sex-matched controls with negative sputum cultures for mycobacteria. Patients with human immunodeficiency virus (HIV) infection, autoimmune diseases under regular chemotherapy, and those with a bleeding tendency that increased the risk of blood sampling were excluded.

The Research Ethics Committee of National Taiwan University Hospital approved this study (IRB No: 201312043RINB). All of the participants provided written informed consent, and the methods were carried out in accordance with the approved guidelines.

Peripheral blood from the enrolled subjects was sampled into heparin-containing tubes. Mononuclear cells were immediately isolated using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Sweden), and were then suspended in medium containing RPMI-1640 (Life Technologies; USA), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin (Life Technologies, USA). We isolated lymphocytes by negative selection using CD14-positive selection system (MACS system, Miltneyi Biotec Inc.) if PBMCs was more than 5 x 10⁶ cell/mm³. Otherwise, we used PBMCs for further experiments for avoiding cells loss during lymphocyte selection. The intracellular cytokine responses in CD14-negative lymphocytes were similar to those in PBMCs, which were lower than those in peripheral blood leukocytes (Additional file 1: Figure S1, supplement file). All cells were immediately frozen using a CELL-BANKER system (ZENOAQ, Japan) following the manufacturer’s instructions. The cells were then stored at -80 °C and defrosted within days of the scheduled experiments. Before the experiments, viable cells were counted using a Scepter™ 2.0 Handheld Automated Cell Counter (Millipore Corporation, Billerica, MA, USA).

Total cellular RNA was extracted from peripheral blood lymphocytes after 6 h of stimulation with phorbol 12-myristate 13-acetate (50 uM) and Ionomycin (10 uM) using a Direct-zol™ RNA MiniPrep kit (Zymo Research, CA, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized using an iScriptTM cDNA Synthesis kit (Life Science Research, USA). A real-time polymerase chain reaction (PCR) with iQ SYBR Green Supermix (BIO-RAD, Singapore) was performed with 4.5 ng of cDNA using a Bio-Rad MyiQ Single-Color Real-Time PCR Detection System, and analyzed using Bio-Rad iQ5 Optical System 2.0 (Bio-Rad, CA). PCR mixtures were denatured at 95 °C for 5 min, followed by 40 cycles of 15 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C for amplification. The sense and antisense primers of IFN-γ (forward, 5’-ATT CGG TAA CTG ACT TGA ATG TCC-3’; reverse, 5’-CTC TTC GAC CTC GAA ACA GC-3’), IL-17 (forward, 5’-ATC TCC ACC GCA ATG AGG AC-3’; reverse, 5’-GTG G.AC AAT CGG GGT GACAC-3’), and GAPDH (forward, 5’-CCT CAA GAT CAT CAG CAA TG-3’; reverse, 5’-CAC GAT ACC AAA GTT GTC AT -3’) were applied. The mRNA expression level of each target gene was normalized to the respective GAPDH expression.

The PBMCs or peripheral blood lymphocytes were cultivated in 96-well plates in 300 μl of RPMI 1640 medium with 10% Fetal bovine serum and 1% penicillin-streptomycin (2 x 10⁵ cells per well). Phorbol 12-myristate 13-acetate (50 ng/ml, TOCRIS, USA), and ionomycin calcium salt (1 μM/ml, Sigma-Aldrich, USA) were added to enhance the levels of cytokines because intracellular cytokine staining yielded a very low percentage (Additional file 1: Figure S1, supplement file) at baseline without stimulation. Protein transport inhibitor (BD Bioscience, USA) was added to the co-culture. The peripheral blood lymphocytes were retrieved after 6 h of reaction and measured using flow cytometry (FACSVerse, BD Biosciences, USA). We measured the level of CD4⁺ T cells using anti-CD4-APC, and stained different types of T cells with anti-IFN-γ-PerCP, anti-IL-4-PerCP, anti-17A-PE, anti-CD25-FITC, anti-Foxp3-PerCP, anti-TNF-α-PerCP antibodies (BD Biosciences, CA, USA). Anti-PD-1-PEcy7.0 (used together with anti-IFN and anti-IL-17A), anti-PD-1-PE (used for other staining antibodies), anti-PD-L1-FITC, and anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4)-PEcy7.0 (eBiosciences, USA) were used to examine the percentages of PD-1, PD-L1 and CTLA-4 on T cells. Data were analyzed using BD FACSuite V software (BD, Biosciences, USA). We discriminated the lymphocyte population using forward scatter (FSC) and side scatter (SSC). Within the group, we gated the subgroups of different T lymphocyte and measured the percentage of PD-1, PD-L1 and CTLA-4 respectively.

Clinical data including age, sex, co-morbidities, radiographic findings and laboratory data at enrollment were recorded in a standardized case report form with default options. Inter-group differences were analyzed using the Student’s t-test or Mann-Whitney U test for numerical variables, where appropriate. The chi-square test was used for categorical variables. Statistical significance was set at p < 0.05. All analyses were performed using SPSS version 19.0 (Chicago, IL).