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 5-Fluoracil in vivo 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 H 89 mw 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 Ribonucleotide 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).