Supplementary Information
2. Materials and methods
2.1 Antibodies and Reagents
Anti-CD8-eFluorTM 450, anti-CD4-PerCP-Cy5.5, anti-IFN-γ-PE, Human IFN-γ ELISPOT antibody pair were from BD Biosciences (NJ, USA). pET-32a (+), genomic DNA of M. tb H37Rv and Eschericbia coli BL21(D3) were conserved in our laboratory. Plasmid Purification Kits, Ni Sepharose High Performance Purification Columns, Ficoll-Paque™ Plus, 96-well plates, IMDM culture medium, isopropyl-1-thio-β-galactopyranoside (IPTG), BCA Protein Assay Reagent, LPS detection ELISA Kit, endonucleases of Nde I and BamH I were purchased from Beijing Tiangen Biotechnology Co. Ltd. (Beijing, China). Phytohemagglutinin (PHA), tween-20, dimethyl sulphoxide (DMSO), bovine serum albumin (BSA) were from Sigma-Aldrich. QuantiFERON-TB Gold In-Tube (QFT) test was from Cellestis (VIC, Australia). 14-plex cytokines/chemokines kit was from Millipore (Billerica, MA, USA). Purified protein derivative (PPD) was from Beijing Xiangrui Biotechnology Co. Ltd. (Beijing, China). PrimeSTAR HS DNA Polymerase was from Takara, Japan. FITC-conjugated anti-human immunoglobulin G2a (IgG2a), APC-conjugated IgG1 and Horseradish peroxidase (HRP)-conjugated IgG purchased from Invitrogen (Shanghai, China).
2.2 Study population
This study was approved by institutional human experimentation committee in Kunming University of Science and Technology (Kunming, China). Study subjects meeting all requirements of adult volunteers (>18 years), well knowing purposes and procedures of the study, providing written informed consent, were included in this study. By screening, 21 individuals with LTBI, 33 patients with active pulmonary tuberculosis (PTB), and 17 healthy individuals (HI, controls) were recruited to detect cellular and humoral responses against a recombinant Rv2660c protein.
LTBI cases were identified by the QFT test with non-TB patients positive and were asymptomatic volunteers. The PTB patients were diagnosed by bacterial culture for M. tb, Ziehl-Neelsen staining of sputum, apparent symptom and chest radiography evidence. The healthy individuals were determined that the people had no any TB clinical symptom, and had no clinical examination evidence of microbiological detection from respiratory tract, and chest X-ray.
2.3 Preparation of recombinant proteins
Primers of sense: 5’- GC CAT ATG CTA GTG AAA CTG GTT CAA T -3’ (Nde I) and antisense: 5’- TT GGA TCC GTG ATA GCG GGC GTC GAC CA -3’(BamH I) were designed according to Rv2660c gene sequence, respectively. Gene of Rv2660c was amplified from genomic DNA of M. tb H37Rv strain using PCR with PrimeSTAR HS DNA Polymerase. After purification, the PCR product was digested with NdeⅠand BamHⅠ, and cloned into corresponding sites of pET-32a (+) vector. E. coli BL21 (DE3) PlysS was chosen for T7-polymerase based over expression of Rv2660c. After DNA sequencing, the transformant was cultured in LB medium containing ampicine (50μg/ml) at 37°C. This culture was grown with shaking (180rpm/min) until the OD600nm reached approximately 0.6, recombinant protein was induced with 0.5mM IPTG and cultured for another 6 h. Cells were harvested by centrifugation at 4°C and lysed by sonication. Soluble Rv2660c protein was purified by Ni Sepharose High Performance Purification Columns according to the manufacturer’s instructions. By this method, the recombinant Rv2660c protein was able to be effectively purified and presented in a native condition. The molecular weight of the protein was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified protein was concentrated by ultrafiltration through Amicon Centrifugal Filter Devicesand, and was stored at -80°C. The protein concentration was determined by BCA Protein Assay Reagent with bovine serum albumin as the standard. The residual endotoxin of recombinant Rv2660c protein was measured by LPS detection ELISA Kit. In order to highlight the antigenicity of Rv2660c in LTBI human population, the ESAT-6 (Early Secretory Antigen Target 6) protein prominently expressed in active M. tb was also prepared by the same methods for a control antigen.
2.4 Isolation and culture of peripheral blood mononuclear cells
Blood samples from PTB patients were obtained from vein within 14 d of the initial treatment. The peripheral blood mononuclear cells (PBMCs) from LTBI cases, PTB patients, and healthy individuals were isolated by Ficoll-Paque™ Plus gradient centrifugation according to the manufacturer’s instructions. To remove remaining erythrocytes from the cell pellets, cells were re-suspended in ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3 and 0.1 mM Na2EDTA, pH 7.3) and then incubated for 5 min on ice. PBS was added to the lysis buffer, the cells were washed with PBS twice and then suspended in IMDM cell culture medium with 10% human AB serum. The PBMCs were cultured for the following experiments.
2.5 Enzyme-linked immunosorbent spot assay
According to the manufacturer’s instructions, the Human IFN-γ ELISpot pair, Streptavidin-HRP ELISpot, and AEC (3-amino-9-ethylcarbazole) Substrate were sequentially added. MultiScreen 96-well plates were pre-coated with anti-human IFN-γ antibody. 1×105 PBMCs were suspended in DMEM medium containing 10% human AB serum with 200μl of final volume, and were added into the 96-well plates. The PBMCs were co-cultured with stimulators of recombinant Rv2660c protein (final concentration: 10μg/ml), recombinant ESAT-6 protein (10μg/ml), PPD (10μg/ml), or phytohaemagglutinin (PHA, 10μg/ml) for 20 h. After stimulation culture, the number of spot forming cells (SFC) was counted under dissecting microscope by three investigators who have no prior knowledge of the experimental information. The result was expressed as SFC/106 cells, the assays ≥ 250 SFC/106 PBMCs against PHA was considered valid. The experiment was repeated three times for each PBMC sample, and the results were expressed as mean±SE.
2.6 Percentages of CD4+T and CD8+ T cells producing IFN-γ
The isolated PBMCs were suspended in DMEM medium containing 10% human AB serum, and were added into 96-well plates with 1×105/ 200μl/well. The cells were co-cultured with stimulators of recombinant Rv2660c protein (final concentration: 10μg/ml), recombinant ESAT-6 protein (10μg/ml), PPD (10μg/ml), DMEM medium (negative control) in 95% relative humidity, 5% CO2, at 37°C. After 72 h culture with stimulators, the PBMCs were harvested to detect the percentages of CD4+ T and CD8+ T cells producing IFN-γ. Four hours before culture termination, cells were treated with brefeldin A. After the treatment, cells were blocked with blocking buffer (containing 0.05% NaN3 and 2% human pooled serum) for 20 min at 4°C, and then stained with Anti-CD8-eFluorTM 450, anti-CD4-PerCP-Cy5.5, or PBS (negative controls) for 1 h, respectively. After that, cells were fixed with 4% paraformaldehyde for 30 min, and then treated with permeabilization buffer (containing 0.01% BSA and 0.05% tween-20). PE conjugated anti-human IFN-γ was added and incubated for 40 min at room temperature. After a washing step, the percentages of CD8+ T and CD4+ T cells producing IFN-γ were measured by flow cytometry (FACSCalibur, BD, Franklin Lakes, NJ, USA), and the data was analyzed by FlowJo software. The experiment was repeated three times for each PBMC sample. The results were expressed as mean±SE.
2.7 Measurement of cytokines and chemokines
Culture supernatants of PBMCs stimulated with recombinant Rv2660c protein, recombinant ESAT-6 protein, PPD and DMEM medium (negative controls) were collected. The production of cytokines and chemokines in the supernatants was detected by 14-plex cytokines / chemokines kit including IFN-γ, IP-10, MIP-1alpha (MIP-1a), MIP-1β, TNF-α, MCP-1, RANTES, IL-2, IL-4, IL-5, IL-10, IL-13, IL-15 and IL-17, according to the manufacturer’s instructions. In brief, the filter plates were pre-wetted, and then were added with 25μl of samples, standards and controls, respectively. The plates were sealed and incubated for 16 h at 4°C under shaking at 250 rpm, then were washed and were added with 25μl of detection antibody. Under shaking, the plates were incubated in the dark at room temperature for 1 h. Then, 25μl of PE-conjugated streptavidin was added to incubate for 1 h under shaking at room temperature. After a washing step, 150μl of the sheath fluid was added. After sealing and mixing, the plates were immediately read on the Bioplex 200 analyzer (Bio-Rad, USA). Three repeated experiments were done for each sample, the results were expressed as mean ± SE.
2.8 Antibody responses to recombinant Rv2660c protein
Serum samples from LTBI cases, PTB patients and healthy individuals were obtained by centrifugalization, respectively, and were stored at -20°C until use. An indirect Enzyme-linked Immunosorbent Assay (iELISA) was used to detect the antibody responses. ELISA plates were coated overnight at 4°C with recombinant Rv2660c protein, recombinant ESAT-6 protein, PPD (final concentration: 10µg/ml), and PBS (negative control), respectively. The plates were then washed with PBST buffer (0.1M PBS containing 0.05% Tween-20, pH 7.2) for three times and blocked 90 min at 37°C with 5% skim milk in PBS (pH 7.2). After three times washing, the plates were incubated with twofold serial dilution of test sera for 45 min at 37°C. After washing, the plates were added with FITC-conjugated IgG2a, APC-conjugated IgG1 and HRP-conjugated IgG at a dilution of 1:1500, 1:1500 and 1:2000, and incubated for 1h at 37°C, respectively. Then, 50μl fresh substrate consisting of 0.07% orthophenylenediamine and 0.05% hydrogen peroxide in citric acid phosphate buffer (pH 5.0) was added into wells of plates, and kept in dark for 15min. Added 50μl of 3M sulfuric acid to stop the reaction, and read in an automated plate reader (Bio-Rad, USA) at 490 nm. Three repeated tests were done for each serum sample, the results were expressed as mean±SE.
2.9 Statistical analysis
Experimental data represented mean ± SE. The statistical analyses were made by SPSS13.0 software. One-way ANOVA was used for multiple group comparisons. Differences were considered significant for P-values ≤ 0.05.
Table 1 Characteristics of study projects
LTBI (n=21)n (%) / PTB (n=33)
n (%) / HI (n=17)
n (%) / Total (n=71)
n (%)
Age, years
median (range) / 63.6 (23~82) / 68.5 (32~85) / 61.4 (28~86) / 64.5(23~86)
Female sex / 10 (47.62) / 16 (48.48) / 9 (52.94) / 35 (49.30)
LTBI: individuals who were positive tested by QFT but had no any clinical evidence of active TB. PTB: patients with active pulmonary TB confirmed by microbiological examination. HI: healthy individuals who had no any TB evidence of clinical symptom, microbiological examination, and chest X-ray.