For mycobacterial CFP, the membrane was probed with rabbit polyclonal antibodies made against M. tuberculosis CFP (BEI Resources, NR-13809) and then incubated with goat anti-rabbit HRP-conjugated IgG as described above. IT-12 and NR-13809 were obtained from Colorado State University, Colorado, USA, under the TB Vaccine Testing

and Research Material Contract. In exosome-priming experiments, mice were immunized via an i.n. route with a final injection volume of 30 μL (15 μL/nostril) as described previously [21]. Briefly, five mice per group were anaesthetized with isoflurane and administered with PBS alone or with purified exosomes isolated from CFP-treated or untreated macrophages, at a dose of 20 μg/mouse or 40 μg/mouse. The mice were immunized three times at an interval GSK1120212 clinical trial of 2 weeks. Two weeks after final exosome vaccination, mice were sacrificed and used to measure antigen-specific T-cell activation and 4 weeks after final vaccination, a separate set of mice were infected with M. tuberculosis. As a positive control, M. bovis BCG (1 × 106 CFU/mouse, Pasteur JAK inhibitor strain) was given i.n. as a single dose 8 weeks prior to M. tuberculosis infection. For BCG priming and exosome boosting experiments, five mice per group were first s.c. immunized with a single dose of M. bovis BCG (1 × 106 CFU/mouse, Pasteur strain) in 50

μL of PBS and subsequently rested for 8 months before boosting. Exosome booster immunization was administrated twice i.n. at 2-week intervals as described above. Another set of BCG-vaccinated mice were also boosted with BCG i.n. at 1 × 106 CFU at the same time as the first exosome boost vaccination. Mice were sacrificed to measure antigen-specific immune

responses or infected with M. tuberculosis H37Rv as described for the exosome-priming experiments. Six weeks following the final vaccination of exosomes, mice were challenged with M. tuberculosis H37Rv using an Inhalation Exposure System (Glas-Col, Terre Haute, IN, USA). Four M. tuberculosis infected mice per group were humanely sacrificed 1 day after infection to determine the bacterial load in the lungs and spleens. The amount of M. tuberculosis used in NADPH-cytochrome-c2 reductase the infection was calculated to give approximately 50 to 150 CFU/lung in mice. For all other infections, mice were euthanized 6 weeks after mycobacterial challenge and the lungs and spleens were removed and homogenized in PBS containing 0.05% v/v Tween-80. The tissue homogenate was appropriately diluted in the same buffer, and then 50 μL of the diluted homogenate was spread on Middlebrook 7H11 agar plates with 10% OADC, 0.5% glycerol and 0.05% Tween-80, and containing a cocktail of fungizone (Hyclone) and PANTA (polymixin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin; BD, Sparks, MD, USA).