*2.8. IFN-*γ *ELISpot Analysis*

The Enzyme-linked immune absorbent spot (ELISpot) assay was performed using the commercial murine IFN-γ ELISpot kit (Mabtech, Nacka Strand, Sweden) according to the manufacturer's instructions. The ELISpot plates (MSISP4510, 96-well plates with polyvinylidene difluoride membranes, Millipore, USA) were 70% EtOH treated and coated with purified anti-mouse interferon-γ (IFN-γ) capture monoclonal antibody diluted in phosphate-buffered saline (PBS) to a final concentration of 5 μg/mL at 4 ◦C overnight. Then, 250,000 fresh splenocytes were added to each well and stimulated with 17 peptide pools containing a total of 147 15 mer overlapping peptides (OLP) spanning the HTI sequence, at a concentration of 10 μg/mL per peptide. Tuberculin purified protein derivative (PPD, AJ vaccines, Copenhagen, Denmark,) at a concentration of 5 μg/mL was used to assess TB-specific responses. All the samples and controls were plated in duplicate wells. ELISpot assays were incubated for 16 h at 37 ◦C, 5% CO2. The plates were subsequently washed 5× with PBS, incubated for 2 h with a biotinylated anti-IFN-γ monoclonal antibody (mAb) diluted in PBS 2% Fetal Calf Serum (FCS) to a final concentration of 2 μg/mL, washed 5× in PBS, and incubated with the streptavidin–alkaline phosphatase conjugate in PBS 2% FCS. Then, plates were washed 5× with PBS before incubating with 100 μL of 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) substrate solution (Sigma-Aldrich, St Louis, MO, USA). After 5–10 min, the plates were washed with tap water, dried, and the resulting spots counted using an ELISPOT reader (Autoimmune Diagnostika GmbH, Strassberg, Germany). For each animal, the mean of background responses was subtracted individually from all the wells to enable a comparison of the IFN-γ spot forming cells (SFC)/106 between groups. To define positive responses, a threshold was defined as at least five spots per well, and responses exceeding the mean number of spots in negative control wells plus three standard deviations of the negative control wells.
