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, 37 (10), 1163-1173

Precision Mouse Models With Expanded Tropism for Human Pathogens

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Precision Mouse Models With Expanded Tropism for Human Pathogens

Angela Wahl et al. Nat Biotechnol.

Abstract

A major limitation of current humanized mouse models is that they primarily enable the analysis of human-specific pathogens that infect hematopoietic cells. However, most human pathogens target other cell types, including epithelial, endothelial and mesenchymal cells. Here, we show that implantation of human lung tissue, which contains up to 40 cell types, including nonhematopoietic cells, into immunodeficient mice (lung-only mice) resulted in the development of a highly vascularized lung implant. We demonstrate that emerging and clinically relevant human pathogens such as Middle East respiratory syndrome coronavirus, Zika virus, respiratory syncytial virus and cytomegalovirus replicate in vivo in these lung implants. When incorporated into bone marrow/liver/thymus humanized mice, lung implants are repopulated with autologous human hematopoietic cells. We show robust antigen-specific humoral and T-cell responses following cytomegalovirus infection that control virus replication. Lung-only mice and bone marrow/liver/thymus-lung humanized mice substantially increase the number of human pathogens that can be studied in vivo, facilitating the in vivo testing of therapeutics.

Conflict of interest statement

COMPETING INTERESTS

P.A.D. is an inventor of the acoustic angiography imaging technique, and a co-founder of SonoVol, Inc., a company which has licensed this patent.

Figures

Fig. 1:
Fig. 1:. Subcutaneous implantation of human lung tissue into immunodeficient mice results in the development of readily accessible ectopic human lung implants.
(a) Subcutaneous human lung implant (n=1 LoM imaged, black arrow). (b) B-mode ultrasound imaging of a human lung implant (blue circle) under the skin. (c) Volume (cm3) of human lung implants (n=12, filled circles) in mice determined from B-mode ultrasound images. (d-e) In vivo visualization of human lung implant blood vessel vascularization by acoustic angiography (n=12 implants analyzed). (d) Acoustic angiography image showing blood vessels encapsulating (left) and within (right) a human lung implant. A red dashed line (left panel) indicates the location of the implant cross-section depicted in the right panel. (e) Three-dimensional rendering of vascularization (red) of a human lung implant (blue). (f) Gross structure of an excised human lung implant (n=1) two months post-surgery exhibiting blood vessel vascularization (black arrows). (g) Histologic sections of human lung implants (n=6 implants analyzed) showing presence of airways, ciliated epithelium, alveolar structures, cartilage and associated blood vessels (images: top panel 5X, bottom panels 100X). (h) Immunofluorescence staining for human epithelial, endothelial and mesenchymal cells in a human lung implant (n=3 implants analyzed, left panels) and mouse lung (n=1 mouse lung analyzed, right panels) (positive cells: red, nuclei: blue). Immunofluorescence staining for cytokeratin 19 (magenta) and (i) cilia or (j) club cells (green, scale bars: 20 µm) in a human lung implant (n=4 implants analyzed). (k) AB-PAS staining for mucous secretions (blue, scale bars: 20 µm) in a human lung implant (n=4 implants analyzed). In c, horizontal lines represent mean ± s.e.m. Scales bars in b and d: 2 mm. In g and h, scale bars shown for 5X (1 mm), 10X (50 µm), and 100X (100 µm) images.
Fig. 2:
Fig. 2:. LoM are susceptible to infection with a broad range of emerging and clinically relevant human pathogens.
(a) MERS-CoV titer in infected lung implants (105 PFU dose: n=8, filled squares; 104 PFU dose: n=4, open squares). (b) MERS-CoV-infected cells (green) co-stained for human epithelial (cytokeratin 19), endothelial (CD34) or mesenchymal (vimentin) cell markers (magenta) in a lung implant (n=1, images: 40X). (c) ZIKV-RNA copies in infected lung implants (104 FFU dose: n=4, filled upward triangles; 103 FFU dose: n=4, open upward triangles). (d) ZIKV-infected cells (green) co-stained for human epithelial or mesenchymal cell markers (magenta) in a lung implant (n=1, images: 40X). (e) BCG colony forming units (CFU) (4×104 CFU dose: n=3, closed diamonds; 4×102 CFU dose: n=3, open diamonds) and (f) Ziehl-Neelsen acid-fast staining for BCG (positive: pink) in lung implants (n=2). Top and bottom images: 100X. (g) Number of RSV-GFP positive cells in lung implants (n=6, closed downward triangles). (h) RSV-infected cells (green) co-stained for a human epithelial cell marker (magenta) in a lung implant (n=1, image: 40X) (i) Co-staining for RSV-infected cells (green) and ciliated cells (magenta) (top) and isotype control antibody (green) and ciliated cells (magenta) (bottom panel) in a human lung implant (n=4 analyzed, images: 100X). (j) AB-PAS staining for mucus (blue) in a naïve control (n=4 analyzed, top) and RSV-infected (n=4 analyzed, bottom) lung implant (scale bars: 100 µm). (k) HCMV-DNA levels (open circles) in lung implants at 4 (n=4), 7 (n=4), 14 (n=5) and 21 (n=3) days post TB40/E exposure. (l) HCMV immediate early (IE), early (E) and late (L) protein expression in lung implants (n=1 per time point, images: 40X, positive cells: brown). (m) HCMV-infected cells (green) co-stained for a human mesenchymal cell marker (magenta) in a lung implant (n=1, image: 40X). (n) Ganciclovir (GCV) was administered to LoM daily (100 mg/kg) for 17 days starting two days prior to HCMV TB40/E exposure. HCMV-luciferase activity in lung implants of GCV-treated LoM (orange boxes; n=6) and untreated control LoM (white boxes; days 4–21, n=7 and day 25, n=6) was compared with a two-tailed Mann-Whitney test. The median (horizontal line), upper and lower quartiles (box ends) and minimum to maximum values (whiskers) are shown. In a-k, n=number of lung implants analyzed and horizontal lines represent mean ± s.e.m. In b, d, f, h, i, l and m scale bars shown for 40X (50 µm) and 100X (20 µm) images. In b, d, h and m the bottom left inset image represents the area indicated with a dashed box and nuclei are stained blue.
Fig. 3:
Fig. 3:. Systemic reconstitution of BLT-L humanized mice with human innate and adaptive immune cells.
(a) Construction of BLT-L humanized mice. (b) Human hematopoietic cells (hCD45+) in peripheral blood of BLT-L mice (n=26 mice, 5 cohorts). Colors indicate BLT-L mouse cohorts. (c) Immunofluorescence staining for human epithelial, endothelial and mesenchymal cells in a human lung implant of a BLT-L mouse (n=4 mice analyzed, positive cells: red, nuclei: blue). Images shown are at 10X magnification (scale bars: 50 µm). (d) Human hematopoietic (hCD45) cells including dendritic cells (hCD11c), macrophages (hCD68), B cells (hCD20), NK/NK T cells (hCD56), and T cells (hCD3, hCD4 and hCD8) in the human lung implant (n=3 mice analyzed, positive cells: brown). Images shown are at 40X magnification (scale bars: 50 µm). (e) Levels of hCD45 cells including human myeloid cells (hCD33), B cells (hCD19) and T cells (hCD3) as well as the ratio of human CD4:CD8 T cells in the human lung implants (open circles) of BLT-L mice (hCD45, hCD33 and hCD3 analysis n=18 implants and hCD19 and hCD4:CD8 analysis, n=15 implants). (f) Human CD45+ cells in tissues of BLT-L mice (n=4 mice analyzed) by flow cytometry. GI: gastrointestinal. FRT: female reproductive tract. In b and e, horizontal lines represent mean ± s.e.m.
Fig. 4:
Fig. 4:. HCMV infection induces robust, sustained humoral and T cell responses that control virus replication in BLT-L mice.
(a) HCMV TB40/E-luciferase activity in lung implants of LoM (blue boxes; n=6 implants) and BLT-L mice (yellow boxes; n=6 implants). Baseline (BL): luminescence measured pre-exposure. The median (horizontal line), upper and lower quartiles (box ends) and minimum to maximum values (whiskers) are shown. Two-tailed Mann-Whitney test. (b) HCMV-specific IgM in the plasma of HCMV-exposed BLT-L mice (TB40/E: BLT-L5 and BLT-L6, ADrUL131: BLT-L 9) and five naïve control BLT-L mice. Mean values (horizontal lines) and technical replicates are shown for each mouse. Dashed line: threshold for seropositivity defined from naïve mice. (c) HCMV-specific CD8+ T cells detected by pentamer-reactivity in the lung implant (H LNG) of one BLT-L mouse 12 days post-exposure. (d) Pentamer-reactive HCMV-specific CD8+ T cells in the lung implant 21 days post-exposure (left). Overlay of pentamer-reactive cells (red) on PMA/ionomycin stimulated CD8+ T cells (gray) from one BLT-L mouse is shown (right). (e) IFN-γ ELISpot reactivity to commonly targeted CD8+ T cell epitopes in human infection. Mean values (horizontal lines) and technical replicates are shown for two BLT-L mice. SFU: spot forming units. (f and g) IFN-γ ELISpot reactivity to the HCMV Immediate Early 1 (IE1) protein. Mean values (horizontal lines) and technical replicates are shown for six BLT-L mice. (e-g) ELISpot data are background subtracted. Criteria for positivity: ≥2x mean of replicate negative control wells and >50 SFU/million CD3+ T cells. (h) IE1-specific IFN-γ+CD107a+ CD8+ T cells detected 125 days post-HCMV exposure in one BLT-L mouse. (e-h) Cells were isolated from the (e, f and h) liver (LIV) or (g) mouse lung (M LNG). (b-h) BLT-L cohorts are indicated in parentheses. (c-h) BLT-L mice were exposed to HCMV strains TB40/E or AD169 as indicated. Full details of immune reactivity for each BLT-L mouse are indicated in Supplementary Tables 4 and 5.
Fig. 5:
Fig. 5:. HCMV re-exposure further promotes antibody induction and class switching and boosts HCMV specific T cell responses in BLT-L mice.
HCMV-specific (a) IgM and (b) IgG in naïve BLT-L mice (n=5) and following single or 3 or 4 exposures to HCMV TB40/E (IgM n=15 single and 11 re-exposed mice, IgG n= 4 single and 11 re-exposed mice) or AD169 (IgM n=8 single and 13 re-exposed mice, IgG n=4 single and 11 re-exposed mice). Negative (open diamonds) and positive (filled diamonds) standards (STD). Shown is the mean of three technical replicates per mouse. Dashed line: threshold for seropositivity defined from naïve controls. (c) HCMV re-exposure induced IE1-specific TNF-α/IFN-γ double positive CD4+ T cells at the site of inoculation, the human lung implant, and systemically in the liver. Contour plots exampling HCMV-IE1 specific CD8+ T cell IFN-γ production and CD107a degranulation in a BLT-L mouse. (d) CD8+ T cells were isolated from combined tissues of TB40/E (top panels representative of measurements in four independent BLT-L mice) or AD169 (bottom panels representative of measurements in 11 independent BLT-L mice) exposed mice, stimulated and then assessed using intracellular cytokine staining. (e) Summary graph showing background subtracted IE1-specific CD8+ T cell responses (IFN-γ production) in mice receiving repeated inoculations of TB40/E or AD169. (f) Repeated TB40/E (top panels representative of measurements in four independent BLT-L mice) and AD169 (bottom panels representative of measurements in four independent BLT-L mice) exposure induced high frequencies of HCMV-specific CD8+ T cells as determined by pentamer reactivity. Full details of immune reactivity for each multiple exposed BLT-L mouse are indicated in Supplementary Tables 7 and 8.

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