A volume of 100 μl of cell suspension was added to 96-well plates (Costar, Cambridge, MA, USA), and the Ag85A protein was added to the wells in triplicate at the final concentration of 5 μg/ml. After 72-h incubation, cell-free supernatants were harvested and were screened for the presence of IFN-γ and IL-4 with an ELISA detection system (Jingmei, Biotech) according to the manufacturer’s instruction. Intracellular IFN-γ measurement using flow CT99021 cell line cytometry.  Splenocytes from vaccinated

mice were cultured at 2.5 × 106/ml in 24-well tissue culture plates (Nunclon, Roskilde, Denmark) in the presence of 5 μg of Ag85A protein/ml for 3 days. Brefeldin A (Sigma) was added to the cultures for the last 5 h to prevent secretion of the intracellular cytokines.

One million cells from each group were first incubated with fluorescein isothiocyanate-conjugated ABT-737 anti-CD4 Ab (clone RM4 to 4 PharMingen, San Jose, CA, USA) or CD8 Ab for 30 min at 4 °C. Cells were then washed, fixed with 4% paraformaldehyde and permeabilized with phosphate-buffered saline containing 0.1% saponin. To label intracellular IFN-γ, cells were incubated with phycoerythrin-conjugated anti-IFN-γ Ab (clone XMG1.2; PharMingen) for 1 h at room temperature, washed and acquired on a cytofluorometer (FACSCALIBUR; BD, Mountain View, CA, USA). Lymphocytes were gated by their forward- and side light-scattering properties, and 100,000 cells were acquired in the lymphocyte gate. Analysis was performed by cell quest all software (BD, San Jose, CA, USA). Cytotoxicity assay of T cell [22].  Spleen cells adjusted to a concentration of 107/ml from in vivo-primed mice were co-cultured with mitomycin (10 μg/ml)-treated target cells (P815-Ag85A, 5 × 105/ml) in a 10-ml

cell suspension in RPMI 1640 for 5 days at 37 °C in 5% CO2. Twenty units per millilitre recombinant murine IL-2 (Biosource, Camarillo, CA, USA) was also added to the cell solution for 5 days. The P815 cell was used as a negative control. To measure the specific lysis of the target cells, we used the lactate dehydrogenase (LDH) release assay, which yields highly similar results as the standard chromium release assay, but does not require the use of radioisotopes. In 96-well round-bottom plates, effector cells were incubated with target cells at different E/T ratio for 4 h in phenol red-free RPMI 1640 containing 2% BSA, 2 mm glutamine, and 1% penicillin and streptomycin. After centrifuging the plates at 250 g for 10 min, 100 μl per well of the supernatant was then transferred to 96-well plates, and lysis was determined by measuring LDH release using cytotoxicity detection kit (Roche Molecular Biochemicals). The released LDH converted the added substrate tetrazolium salt into red formazan product, and the amount of colour is proportional to the number of lysed cells. The absorbance values from supernatants were recorded at OD 492 nm on an ELISA microplate reader.