PBMCs were isolated by standard Ficoll density gradient centrifugation using Leucosep® tubes (Greiner, Bio-one, Alphen aan den Rijn, The Netherlands). PBMCs were collected and stored in liquid nitrogen until use. Recombinant proteins were produced as described previously 56. In short, PCR was used to amplify the selected Mtb H37Rv genes from genomic H37Rv DNA. The PCR products were cloned using Gateway Technology (Invitrogen, San Diego, CA, USA) and were subsequently sequenced. Escherichia coli this website strain BL21 (DE3) was used
to over-express Mtb proteins. Recombinant proteins were further purified as described previously 56. All recombinant proteins were tested in quality control assays including, size and purity check, determination of residual endotoxin levels as well as non-specific T-cell stimulation and cellular toxicity in lymphocyte stimulation assays 55. PPD (batch RT49) was purchased from Statens Serum Institute (Copenhagen, Denmark). Synthetic peptides were synthesized as previously described 57. Peptides from Mtb DosR antigens Rv1733c, Rv2029c, Rv2031c and control antigen Ag85B were 20-mers peptides with 10 aa overlap, except peptides 20–22 of Ag85B which were 15-mers with 10 aa overlap (Table S1A–D). The 20-mer peptides of Rv1733c and Rv2029c were elongated with two lysine (K) residues at the C-terminal to improve solubility. The HLA-A*0201-restricted,
HIV-1 p17 Gag77–85 epitope (SLYNTVATL) Selleck PI3K Inhibitor Library was used as control peptide 58. T-cell phenotype analysis was performed as previously described 59. In brief, PBMCs were stimulated for 16 h with protein (10 μg/mL) or peptide pools (5 μg/mL) in the presence of co-stimulatory antibodies anti-CD28/anti-CD49d (Sanquin, The Netherlands and BD Biosciences respectively). After Sclareol 4–6 h, Brefeldin A (3 μg/mL; Sigma) was added to the culture. Cell surface staining was performed for the following
markers; CD3-PB, CD4-PercP/Cy5.5, CD8-AmCyan, CD45RA-PE/Cy5, CD25-APC/Cy7 and CCR7-PE/Cy7. Subsequently, intracellular markers were stained with IFN-γ-Alexa700, TNF-α-APC, IL-2-PE and CD69-FITC (BD Biosciences) using Intrastain kit (Dako Cytomation, Denmark). Samples were acquired on an LSRII. CD4+ and CD8+ populations of ≥2×105 events were analyzed using FlowJo (Treestar, Ashland, OR, USA) and SPICE software (provided by Dr. Mario Roederer, Vaccine Research Center, NIAID, NIH, USA). Boolean gate analysis was used to study the different single, double and polyfunctional CD4+ and CD8+ T cells. Proliferation was measured using carboxy-fluorescein diacetate, succinimidyl ester (CFSE) dilution and flow cytometry. PBMCs from study subjects were thawed, washed and labeled with CFSE (Molecular Probes, Leiden, The Netherlands) at a final concentration of 5 μM for 10 min at 37°C. Washed, counted and viable cells were seeded in triplicates in 96-well round-bottom plates at a concentration of 1.