Mtb (strain Erdman) infections were performed via the aerosol route as described previously [13 (link)]. Infections were performed using a Glas-Col (Terre Haute, IN) full body inhalation exposure system. Mice received an inoculation dose of 25–75 CFU/mouse, as measured by plating undiluted lung homogenate within 24 hours of infection. At different times post-infection, mice were euthanized, organs were aseptically removed, individually homogenized in the FastPrep24 (MP Biomedicals, Santa Ana, CA, USA), and viable bacteria were enumerated by plating 10-fold serial dilutions of organ homogenates onto 7H11 agar plates (Hardy Diagnostics, Santa Maria, CA, USA). Mtb (strain H37Rv) in vitro infections of macrophages and DCs were performed as described previously [52 (link)]. H37Rv was grown and prepared as described [53 (link)]. Bacteria were counted in a Petroff-Hausser counter (Hausser Scientific, Horsham, PA, USA) and added to macrophages or DCs at an intended multiplicity of infection (MOI) of 5–10 for three hours. Cultures were washed three times to remove extracellular bacteria, and T cells were added the same day. For CFU measurement, cells were lysed with 1% Triton X-100/PBS and lysate from quadruplicate conditions on d0 and d5 post-infection, and 5-fold dilutions were plated on Middlebrook 7H11 agar plates (Hardy Diagnostics).
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