(A) Comparison of M. tuberculosis growth in lungs of aerosol-infected or dermal-infected Nos2^–/– mice. CFUs from homogenates were enumerated on days 1, 14, 28, and 56 p.i. (mean ± SEM; n = 5). The “†” indicates the time point at which aerosol-infected Nos2^–/– mice succumb to infection. (B) H&E staining of Nos2^–/– lung tissue at days 28 and 56 p.i. demonstrated presence of nonnecrotizing granulomas that were undetectable in WT mice at day 56 (original magnification, ×20). Nonnecrotizing granulomas in Nos2^–/– lungs stained positively with anti-CD3 and anti-F4/80 mAbs, demonstrating presence of T cells and macrophages, respectively. These structures resembled nonnecrotizing granulomas observed during active human tuberculosis stained for T cells and macrophages with anti-CD3 and anti-CD68, respectively. (C) Cells purified from lungs of infected mice at days 14 and 28 p.i. were stimulated for 6 hours with ESAT-6 peptide, stained for surface CD4, intracellular IFN-γ, and TNF-α, and analyzed by FACS. Panels show cells purified from mice after dermal or aerosol infection. Numbers show percentages of CD4⁺ IFN-γ⁺ TNF-α⁺ cells out of total CD4⁺-gated T cells ± SEM (n = 3). Despite reduced bacterial load compared with aerosol-infected mice, dermal-infected mice prime robust CD4⁺ T cell responses against ESAT-6 that were detectable in the lung by day 14 p.i. (D) Lung CFUs from TNF-α– or IFN-γ–blocked and control Nos2^–/– mice at days 1–56 p.i. (mean ± SEM; n = 5). TNF-α– or IFN-γ–blocked Nos2^–/– mice at days 28 and 56 p.i. had significantly elevated CFUs compared with control mice. *P < 0.05.