Figure 1.
Production of the humanized BLT mouse to study TB.
NOD/SCID/γcnull (NSG) mice were engrafted with human fetal liver and thymus, and supplemented with CD34+ cells. Shown in A, flow cytometry analysis displaying side scatter (SSC) and forward scatter (FSC) characteristics (Gate 1) of isolated peripheral blood from a representative BLT mouse twelve weeks post-engraftment. Plots 2–4 are the gating strategy for selection of cells expressing human CD45 pan leukocyte marker, the corresponding isotype control (IgG1 PE Cy7), and the CD3+ population subgate. B, percentage of the gated cells expressing markers for T cell subsets (CD4, CD8), NK cells (CD3−CD56+) and monocyte/macrophages (CD14+). C, average leukocyte % among gated CD45 cells in four groups of reconstituted BLT mice (n = 44) used for the subsequent studies (Fig. 2–8). D shows the expression of antigen presenting cells (APC) markers relevant to antigen presentation (HLA-DR) and T cell activation (CD40, CD80, CD86) expressed by peripheral blood monocyte/macrophages.
Figure 2.
Functional potential of splenic T cell populations in the BLT mouse.
Spleens were disrupted to single-cell suspensions and activated with control (media), antibodies to human CD3/CD28, or rIL-15 (15 ng/ml) for 5 days. Following activation, the surface expression of cellular phenotype markers (CD45, CD3, CD4, and CD8) and intracellular proteins (Ki67, granulysin, perforin, and IFN-γ) was detected using flow cytometry. Shown in A are side scatter and forward scatter characteristics of isolated splenocytes (Gate 1), gating strategy to enable analysis of human CD45+ and CD3+ cell populations and individual T cell (CD4 and CD8) subset gating. B, increase in % of human CD45+ cells within the isolated splenocytes following 5 d activation with recombinant IL-15 (15 ng/ml) (upper plots) and activation-induced increase in expression of the proliferation marker Ki67 (lower plots) by treatment displayed as a color dot plot overlay. C, expression of effector molecules (granulysin, perforin, IFN-γ) shown by dot plot overlays demonstrating inducible expression upon activation with CD3/CD28 (red) and rIL-15 (blue) compared to non-activated cells (black). Data shown in A, B, and C is from a representative mouse (n = 3).
Figure 3.
Progression of M.tb infection in humanized BLT mice.
Animals were infected i.n. with CFU tdTomato H37RV M.tb and in-vivo imaging (IVIS) performed at weekly time points. Shown in A is the fluorescent intensity signal from a group of animals infected with 106 CFU represented with a pseudocolor scale ranging from yellow (most intense) to dark red (least intense) and IVIS images of BLT mice; across: non-infected control mouse and M.tb infected BLT mice at 1, 2, and 4 weeks p.i. B, bacterial burden (CFU) are shown per milligram (mg) of tissue from individual animals (n = 3 per time point) at 2, 3, and 4 weeks p.i.
Figure 4.
Lung and liver pathology in M.tb-infected humanized mice.
M.tb-infected humanized mice have lung and liver pathology consistent with development of TB. Animals were infected i.n. with 106 CFU tdTomato H37RV M.tb after verification of appropriate reconstitution with human leukocytes. Shown are images captured by brightfield microscopy following staining of infected tissues using hematoxylin and eosin (H&E) and acid-fast stain (Ziehl-Neelson) to detect acid-fast bacilli (AFB). The images show tissue damage and inflammation localized to M.tb bacilli in formalin-fixed tissue sections of BLT mice infected i.n. with 106 cfu tdTomato M.tb H37Rv. Images are representative of mice sacrificed at 2, 3, and 4 wk p.i. described in Fig. 3. Shown in A is the lung tissue pathology visualized by H&E staining (left panels, 4X). Localization and burden of bacilli are shown in right panels (40X) from the region indicated in the H&E image, as visualized by acid fast and hematoxylin staining. B, liver tissue pathology at 2, 3, and 4 weeks p.i. (H&E 10X, AFB 40X of indicated region).
Figure 5.
TB in humanized mice infected with a low dose of M.tb.
Animals were infected i.n. with 250 CFU tdTomato H37RV M.tb after verification of appropriate reconstitution with human leukocytes. Shown are images captured by brightfield microscopy following analysis of lung and liver from a representative animal (n = 11). The images demonstrate tissue damage and inflammation (hematoxylin and eosin, H&E) localized to M.tb bacilli (acid fast bacilli, AFB) in formalin-fixed tissue sections from lung and liver of BLT mice sacrificed at 6–8 weeks p.i. Shown in A are gross lung lobes (left panels) and cross sections of whole lung stained with H&E, captured using a stereomicroscope. B, Lung tissue pathology visualized by H&E staining (left panels) and AFB (right panels). Top panels (20X) demonstrate bronchial obstruction in the lung and the large numbers of bacteria within the obstruction. Middle panels (20X) show cholesterol crystal deposits (black arrow) observed in large granulomas at later stages (≥6 wk) of infection. Bottom panels (4X) shows center of large, coalescing granulomas characterized by necrosis and lack of AFB. C, shown is liver tissue pathology (left panel, 10X) and AFB (right panel, 40X) in the indicated region). Images are from lung and liver of a representative animal (n = 6).
Figure 6.
Human T cells are recruited to and organize at lung granulomas and sites of inflammation following M.tb infection.
Animals were infected i.n. with 250 colony forming units (CFU) tdTomato H37RV M.tb following establishment of human immune cell populations. Formalin fixed paraffin embedded tissue sections were cut, dewaxed and stained with antibody to human CD3. Marker expression was visualized with Fast Red substrate and images captured by brightfield microscopy. A, shown is the localization of T cells relative to a granuloma periphery and center (top left; 4X, top right; 10X). Enlarged images of T cell staining in the indicated areas are shown in the bottom panels (40X). (B) Human T cells in portal tracts and sites of inflammation in the liver (top panel, 10X) and an enlarged area showing orchestration of T cells around an inflammatory focus (bottom panel, 20X). Shown are representative images (n = 3) of animals sacrificed at 7 wk p.i.