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Precision mouse models with expanded tropism for human pathogens

Nature Biotechnology volume 37, pages 1163–1173 (2019)Cite this article

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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.

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Fig. 1: Subcutaneous implantation of human lung tissue into immunodeficient mice results in the development of readily accessible ectopic human lung implants.
Fig. 2: LoM are susceptible to infection with a broad range of emerging and clinically relevant human pathogens.
Fig. 3: Systemic reconstitution of BLT-L humanized mice with human innate and adaptive immune cells.
Fig. 4: HCMV infection induces robust, sustained humoral and T-cell responses that control virus replication in BLT-L mice.
Fig. 5: HCMV re-exposure further promotes antibody induction and class switching and boosts HCMV-specific T-cell responses in BLT-L mice.

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The data generated are available from corresponding authors on reasonable request.

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Acknowledgements

This work was supported by NIH grants no. AI103311 (N.J.M.), no. AI123811 (N.J.M.), no. AI110700 (R.S.B.), no. AI100625 (R.S.B.), no. P30 AI027763 (N.G.), no. AI113736 (R.J.P.), no. T32 HL069768 (I.G.N.), no. AI123010 (A.W.), no. AI111899 (J.V.G.), no. AI140799 (J.V.G.), no. MH108179 (J.V.G.), no. CA189479 (P.A.D.) and no. CA170665 (P.A.D) and the North Carolina University Cancer Research Fund (N.J.M.). This work was also supported by the UNC Center for AIDS Research (CFAR) (grant no. P30 AI050410). We thank K. Arend for the generation of HCMV virus stocks used in these experiments. We thank P. Collins and M. Peeples for recombinant RSV expressing GFP and C. O’Connor for recombinant HCMV TB40/E expressing luciferase. We thank G. Clutton for input on the analysis of antigen-specific T-cell responses. The authors thank members of the Garcia laboratory for technical assistance. We thank technicians at the UNC Animal Histopathology Core, The Marsico Lung Institute Tissue and Procurement Core, and the Department of Comparative Medicine. We also thank J. Schmitz and technicians at the UNC Clinical Microbiology/Immunology Laboratories. We thank J. Nelson and technicians at the UNC CFAR Virology Core Laboratory and K. Mollan at the UNC CFAR Biostatistics Core. The authors thank M.T. Heise, L.J. Picker and J.P. Ting for manuscript advice and helpful discussions.

Author information

Author notes

  1. Christian R. Aguilera-Sandoval

    Present address: BD Life Sciences, San Jose, CA, USA

  2. These authors contributed equally: Chandrav De, Maria Abad Fernandez.

Authors and Affiliations

  1. Division of Infectious Diseases, International Center for the Advancement of Translational Science, Center for AIDS Research, University of North Carolina, School of Medicine, Chapel Hill, NC, USA

    Angela Wahl, Chandrav De, Rachel A. Cleary, Claire E. Johnson, Nathaniel J. Schramm, Christian R. Aguilera-Sandoval & J. Victor Garcia

  2. Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC, USA

    Maria Abad Fernandez, Erik M. Lenarcic, Yinyan Xu, Laura M. Rank, Heather A. Vincent, Wes Sanders, Allison Boone, Ralph S. Baric, Raymond J. Pickles, Miriam Braunstein, Nathaniel J. Moorman & Nilu Goonetilleke

  3. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA

    Erik M. Lenarcic, Heather A. Vincent, Wes Sanders & Nathaniel J. Moorman

  4. Department of Epidemiology, University of North Carolina, Chapel Hill, NC, USA

    Adam S. Cockrell & Ralph S. Baric

  5. Joint Department of Biomedical Engineering, University of North Carolina and North Carolina State University, Chapel Hill, NC, USA

    Isabel G. Newsome & Paul A. Dayton

  6. Marsico Lung Institute, University of North Carolina, Chapel Hill, NC, USA

    Allison Boone & Raymond J. Pickles

  7. Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    William H. Hildebrand

  8. UNC HIV Cure Center, University of North Carolina, Chapel Hill, NC, USA

    Nilu Goonetilleke

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  1. Angela Wahl
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Contributions

A.W. inoculated mice with HCMV; collected and processed PB and tissues from HCMV-exposed mice; performed experiments with PB and tissue cells and analyzed the data; performed the flow cytometric and IHC analysis of naïve LoM and BLT-L mice; performed the human cytokine/chemokine analysis; inoculated mice with BCG and collected tissues from BCG-exposed mice; conceived and designed experiments; contributed to data interpretation, data presentation and manuscript writing; and conceived, designed and coordinated the study, the preparation of the manuscript and its revision. C.D. inoculated mice with HCMV; performed the GCV experiment, the in vivo imaging of mice and neutralization assay; collected and processed PB and tissues from HCMV-exposed mice; performed experiments with PB and tissue cells and analyzed the data; performed the human cytokine/chemokine analysis; inoculated mice with RSV; and collected and processed tissues and measured RSV-GFP expression by flow cytometry. M.A.F. performed experiments with PB and tissue cells and analyzed the data; standardized, designed, performed and analyzed immunological assays; and contributed to data interpretation, data presentation and manuscript writing. E.M.L. performed experiments with PB and tissue cells and analyzed the data and performed the HCMV real-time PCR analyses. Y.X. performed experiments with PB and tissue cells and analyzed the data and standardized, designed, performed and analyzed immunological assays. A.S.C. produced stocks of MERS-CoV; inoculated mice with MERS-CoV and collected tissues from MERS-CoV infected animals; conceived and designed experiments; and contributed to data interpretation, data presentation and manuscript writing. R.A.C. performed dual immunofluorescence staining of infected LoM. C.E.J. performed immunofluorescence staining of animals, collected tissues from BCG-exposed mice and performed the acid-fast staining. N.J.S. produced stocks of ZIKV, inoculated mice with ZIKV, and collected and processed tissue from ZIKV-infected mice. L.M.R. processed and cultured tissue homogenate to determine the number of the BCG c.f.u. and the analyzed data. I.G.N. performed the ultrasound and acoustic angiography imaging and analysis. H.A.V. and W.S. performed experiments with PB and tissue cells and analyzed the data and performed the HCMV transcriptome analysis. C.R.A.S. collected and processed PB and tissues from HCMV-exposed mice. A.B. performed immunofluorescence staining of animals. W.H.H. helped conceive and design experiments. P.A.D., R.S.B., N.J.M. and N.G. conceived and designed experiments and contributed to data interpretation, data presentation and manuscript writing. R.J.P. provided stocks of RSV-GFP; performed the immunofluorescent analysis of tissues sections for RSV infection and AB-PAS staining, and the analysis of human lung implant structures; conceived and designed experiments; and contributed to data interpretation, data presentation and manuscript writing. M.B. processed and cultured tissue homogenate to determine the number of the BCG c.f.u. and the analyzed data; conceived and designed experiments; and contributed to data interpretation, data presentation and manuscript writing. J.V.G. conceived and designed experiments and contributed to data interpretation, data presentation and manuscript writing; and conceived, designed and coordinated the study, the preparation of the manuscript and its revision.

Corresponding authors

Correspondence to Angela Wahl or J. Victor Garcia.

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Competing interests

P.A.D. is an inventor of the acoustic angiography imaging technique, and a cofounder of SonoVol, Inc., a company that has licensed this patent.

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Integrated supplementary information

Supplementary Figure 1 Histological analysis of the structure and cellular composition of donor matched human lung tissue pre- and post-implantation.

The structure and cellular composition of human lung tissue pre-implantation (n=2 analyzed) and donor matched LoM lung implant harvested 2 months post-implantation (n=4 analyzed) were analyzed by (a) H&E and (b) immunofluorescent staining. In b, co-staining was performed for epithelial cells (cytokeratin 19, magenta) and cilia (alpha-acetylated tubulin, green) or club cells (CC10, green). Arrows show cuboidal cells lining alveoli. In a, scale bars are 100 µm (left panel) and 200 µm (right panel). In b, scale bars are 200 µm (left panels) and 100 µm (right panels).

Supplementary Figure 2 Presence of mouse cells in the human lung implants of LoM and NSG mouse lung.

Immunohistochemical staining for murine epithelial cells, endothelial cells, hematopoietic cells in LoM human lung implants (n=3 analyzed, top panels) and the mouse lung (n=1 analyzed, bottom panels). Images: 10X, scale bars: 100 µm, positive cells: brown. m, mouse.

Supplementary Figure 3 Presence of human immune cells in human lung tissue pre- and post-implantation.

Immunohistochemical staining for human hematopoietic (hCD45) cells including macrophages (hCD68), dendritic cells (hCD11c), B cells (hCD20) and T cells (hCD3) in human donor matched lung tissue pre-implantation (n=1 analyzed, left panels) and two months post-implantation (n=1 analyzed, right panels). Images: 10X, scale bars: 100 µm, positive cells: brown.

Supplementary Figure 4 In vivo gene expression profile of HCMV-infected LoM is consistent with lytic replication.

Total RNA was extracted from human lung implants harvested from HCMV TB40/E infected LoM 14 days post exposure (n=2 TB40/E infected implants). Double stranded cDNA ((ds)cDNA) was generated from ribosomal RNA (rRNA) depleted total RNA. HCMV (ds)cDNA was enriched with custom designed biotinylated probes spanning both strands of the entire HCMV genome and sequenced using next generation sequencing. High quality reads were aligned to the HCMV genome, and viral expression was quantified in read per kilobase per million (rpkm). Values show read counts per gene normalized to gene length read (rpkm).

Supplementary Figure 5 Reconstitution of the peripheral blood of BLT-L mice with human innate and adaptive immune cells.

Levels of (a) human hematopoietic cells (hCD45) including (b) human myeloid cells (hCD33), B cells (hCD19) and T cells (hCD3) as well as the (c) levels of CD4+ (hCD4) and CD8+ (hCD8) T cells and (d) ratio of human CD4:CD8 T cells in the peripheral blood of BLT-L mice (n=11, filled circles). Horizontal lines represent mean ± s.e.m.

Supplementary Figure 6 Levels of human immune cells in the human lung implants and mouse lung of BLT-L mice.

Levels of (a) human hematopoietic cells (hCD45) including (b) human myeloid cells (hCD33), B cells (hCD19) and T cells (hCD3) in the human lung implants (circles; hCD45, hCD33, and hCD3 n=18, hCD19 n=15) and mouse lung (squares, n=11) of BLT-L mice. (c) Levels of CD4+ (hCD4) and CD8+ (hCD8) T cells and (d) ratio of human CD4:CD8 T cells in the human lung implants (circles, n=15) and mouse lungs (squares, n=11) of BLT-L mice. (e) Human CD4+ and CD8+ T cell activation (CD38+HLA-DR+) levels in the human lung implant (circles, n=7) and mouse lung (squares, n=4) of BLT-L mice. Horizontal lines represent mean ± s.e.m. Human immune cell levels in the human lung implants and mouse lung were compared with a two-tailed Mann-Whitney test.

Supplementary Figure 7 Systemic presence of human immune cells in BLT-L mice.

(a-c) The memory phenotype of human T cells in the human lung implants of BLT-L mice (n=4 BLT-L mice, one lung implant per animal). (a) Percent of CD4+ (filled circles) and CD8+ (filled squares) human T cells expressing a memory phenotype (CD45RO+). (b) Percent of memory (CD45RO+) CD4+ (circles) and CD8+ (squares) human T cells expressing an effector memory (Tem, CCR7neg, closed symbols) or central memory (Tcm, CCR7+, open symbols) phenotype. (c) Percent of memory (CD45RO+) CD4+ (filled circles) and CD8+ (filled squares) T cells that are tissue-resident (TRM, CD69+). (d) Flow cytometry gating scheme. Regions identify the following human cell populations: RI (live cells), RII (human hematopoietic cells), RIII (T cells), RIV (CD8+ T cells), RV (CD4+ T cells), RVI (memory CD8+ T cells), RVII (CD8+ Tem), RVIII (CD8+ Tcm), RIX (CD8+ TRM), RX (memory CD4+ T cells), RXI (CD4+ Tem), RXII (CD4+ Tcm) and RXIII (CD4+ TRM). In a-c, horizontal lines represent mean ± s.e.m. (e) Human hematopoietic (hCD45) cells including dendritic cells (hCD11c), macrophages (hCD68), B cells (hCD20) and T cells (hCD3, hCD4 and hCD8) in lymphoid (spleen and lymph nodes) and non-lymphoid (liver and mouse lung) of BLT-L mice by immunohistochemical staining (positive cells: brown). Images shown are at 20X magnification and represent three BLT-L mice (scale bars: 100 µm).

Supplementary Figure 8 Increased plasma human cytokine and chemokine levels in BLT-L mice following HCMV exposure.

Levels of human GM-CSF IFN-γ, IL-6, IL-8, MDC, IP-10, GRO and MCP-1 in the PB plasma of BLT-L mice (n=10 mice, filled circles) pre and 4 days after HCMV TB40/E inoculation. A value of 3.2 pg/ml was graphed for measurements below the limit of detection of the assay (3.2 pg/ml, shown with a dashed line). Human cytokine and chemokine levels pre and post HCMV inoculation were compared with a two-tailed Wilcoxon matched-pairs signed rank test.

Supplementary Figure 9 In vivo replication of HCMV AD169 in LoM human lung implants.

(a) HCMV-DNA levels in LoM human lung implants at 4, 7, 14, 21 and 28 days post AD169 exposure (day 4: n=3 implants, days 7, 14, 21 and 28: n=4 implants, filled squares). Horizontal lines represent mean ± s.e.m. (b) HCMV immediate early (IE), early (E) and late (L) proteins in the human lung implant of an AD169-infected LoM 21 days post-exposure (n=1 lung implant analyzed, positive cells: brown). Images shown are at 40X magnification (scale bars: 50 µm). Positive cells in the bottom panel are indicated with black arrows.

Supplementary Figure 10 Plasma of HCMV-exposed BLT-L mice contains HCMV neutralizing activity.

Heat-inactivated plasma from naïve (n=1, open blue circles) and repeatedly HCMV TB40/E exposed BLT-L mice (n=6, filled symbols) was incubated with HCMV TB40/E expressing RFP for 1 h prior to the addition of virus to epithelial cells (ARPE-19). Epithelial cells were incubated at 37 °C with the virus/plasma mixture for 2 h at which time the virus/plasma mixture was removed and fresh media added. Shown is the number of HCMV TB40/E-RFP+ epithelial cells 72 h post-infection in quadruplicate wells. Horizontal lines represent mean ± s.e.m. The percent reduction in TB40/E RFP+ cells compared to wells infected with HCMV pre-treated with naïve control plasma is shown in the table.

Supplementary Figure 11 Gating strategies for the identification of HCMV-specific human T cell responses in BLT-L mice by intracellular cytokine staining (ICS) and pentamer staining.

Representative flow cytometry plots indicating the gating used to detect (a) human CD8+ T cells expressing IFN-γ and CD107a and (b) human CD4+ T cells expressing IFN-γ and TNFα by ICS. (c) Representative flow cytometry plots indicating the gating strategy used to detect HLA class1a-restricted HCMV-specific human CD8+ T cells by pentamer staining.

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Supplementary Information

Supplementary Figs. 1–11 and Supplementary Tables 1–10

Reporting Summary

Supplementary Video 1: Vascularization of human lung implants

Three-dimensional rendering of vascularization (red) of a human lung implant (blue).

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Wahl, A., De, C., Abad Fernandez, M. et al. Precision mouse models with expanded tropism for human pathogens. Nat Biotechnol 37, 1163–1173 (2019). https://doi.org/10.1038/s41587-019-0225-9

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