Limits...
Engrafted human cells generate adaptive immune responses to Mycobacterium bovis BCG infection in humanized mice.

Lee J, Brehm MA, Greiner D, Shultz LD, Kornfeld H - BMC Immunol. (2013)

Bottom Line: However, the levels of secreted IFN-γ, determined by ELISA, were not significantly elevated by antigenic stimulation.The lesions did not resemble granulomas typical of human TB.The lack of protection was associated with low IFN-γ levels and limited numbers of T cells recruited to the lesions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA. jinhee.lee@umassmed.edu.

ABSTRACT

Background: Currently used mouse models fail to fully reflect human immunity to tuberculosis (TB), which hampers progress in research and vaccine development. Bone marrow-liver-thymus (BLT) mice, generated by engrafting human fetal liver, thymus, and hematopoietic stem cells in severely immunodeficient NOD/SCID/IL-2Rγ(-/-) (NSG) mice, have shown potential to model human immunity to infection. We engrafted HLA-A2-positive fetal tissues into NSG mice transgenically expressing human leukocyte antigen (HLA)-A2.1 (NSG-A2) to generate NSG-A2-BLT mice and characterized their human immune response to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection to assess the utility of this model for investigating human TB.

Results: NSG-A2-BLT mice were infected intravenously with BCG and the immune response of engrafted human immune cells was characterized. After ex vivo antigenic stimulation of splenocytes, interferon (IFN)-γ-producing cells were detected by ELISPOT from infected, but not uninfected NSG-A2-BLT mice. However, the levels of secreted IFN-γ, determined by ELISA, were not significantly elevated by antigenic stimulation. NSG-A2-BLT mice were susceptible to BCG infection as determined by higher lung bacillary load than the non-engrafted control NSG-A2 mice. BCG-infected NSG-A2-BLT mice developed lung lesions composed mostly of human macrophages and few human CD4+ or CD8+ T cells. The lesions did not resemble granulomas typical of human TB.

Conclusions: Engrafted human immune cells in NSG-A2-BLT mice showed partial function of innate and adaptive immune systems culminating in antigen-specific T cell responses to mycobacterial infection. The lack of protection was associated with low IFN-γ levels and limited numbers of T cells recruited to the lesions. The NSG-A2-BLT mouse is capable of mounting a human immune response to M. tuberculosis in vivo but a quantitatively and possibly qualitatively enhanced effector response will be needed to improve the utility of this model for TB research.

Show MeSH

Related in: MedlinePlus

Human macrophages are the dominant mononuclear cells in the lesions that promote BCG growth. A. Lung sections prepared from NSG-A2-BLT mice infected with BCG as described in Figure 1 were stained with FITC-labeled anti-human CD68 and FITC-labeled anti-mouse F4/80 (200×, scale bars = 50 μm) (A). Lung sections were stained with anti-PPD antibody followed by secondary antibody labeled with Alexa 555. DAPI was used for counter staining (200×, scale bars = 50 μm) (B). Images are representative of tissues from four animals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3924189&req=5

Figure 4: Human macrophages are the dominant mononuclear cells in the lesions that promote BCG growth. A. Lung sections prepared from NSG-A2-BLT mice infected with BCG as described in Figure 1 were stained with FITC-labeled anti-human CD68 and FITC-labeled anti-mouse F4/80 (200×, scale bars = 50 μm) (A). Lung sections were stained with anti-PPD antibody followed by secondary antibody labeled with Alexa 555. DAPI was used for counter staining (200×, scale bars = 50 μm) (B). Images are representative of tissues from four animals.

Mentions: We hypothesized that the lack of protection and higher BCG growth in NSG-A2-BLT mice relative to non-engrafted NSG-A2 mice was due to residual mouse-origin macrophages concealing BCG from human adaptive immune responses despite the expression of human HLA-A2 in both strains. To test that hypothesis, we compared the relative abundance of mouse and human macrophages in the lungs of NSG-A2-BLT mice. Mouse and human macrophages were visualized by staining with anti-mouse F4/80 and anti-human CD68 antibody. Without infection, the frequencies of mouse and human macrophages in the lungs were comparable and low (Figure 4A). On the other hand, human macrophages outnumbered murine macrophages in the lesions of BCG-infected mice (Figure 4A). When lung sections were stained with anti-PPD to visualize BCG bacilli, a large number of BCG bacilli were found in the lung lesions (Figure 4B). This observation suggests that human macrophages recruited to the site of infection support BCG growth better than murine macrophages. In C57BL/6 mice infected with BCG in parallel with NSG-A2-BLT mice, there were much fewer BCG bacilli found in the lung lesions due to efficient adaptive immune responses (Figure 4B). Thus, NSG-A2-BLT mice likely lack efficient T cell effector function at the site of infection.


Engrafted human cells generate adaptive immune responses to Mycobacterium bovis BCG infection in humanized mice.

Lee J, Brehm MA, Greiner D, Shultz LD, Kornfeld H - BMC Immunol. (2013)

Human macrophages are the dominant mononuclear cells in the lesions that promote BCG growth. A. Lung sections prepared from NSG-A2-BLT mice infected with BCG as described in Figure 1 were stained with FITC-labeled anti-human CD68 and FITC-labeled anti-mouse F4/80 (200×, scale bars = 50 μm) (A). Lung sections were stained with anti-PPD antibody followed by secondary antibody labeled with Alexa 555. DAPI was used for counter staining (200×, scale bars = 50 μm) (B). Images are representative of tissues from four animals.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3924189&req=5

Figure 4: Human macrophages are the dominant mononuclear cells in the lesions that promote BCG growth. A. Lung sections prepared from NSG-A2-BLT mice infected with BCG as described in Figure 1 were stained with FITC-labeled anti-human CD68 and FITC-labeled anti-mouse F4/80 (200×, scale bars = 50 μm) (A). Lung sections were stained with anti-PPD antibody followed by secondary antibody labeled with Alexa 555. DAPI was used for counter staining (200×, scale bars = 50 μm) (B). Images are representative of tissues from four animals.
Mentions: We hypothesized that the lack of protection and higher BCG growth in NSG-A2-BLT mice relative to non-engrafted NSG-A2 mice was due to residual mouse-origin macrophages concealing BCG from human adaptive immune responses despite the expression of human HLA-A2 in both strains. To test that hypothesis, we compared the relative abundance of mouse and human macrophages in the lungs of NSG-A2-BLT mice. Mouse and human macrophages were visualized by staining with anti-mouse F4/80 and anti-human CD68 antibody. Without infection, the frequencies of mouse and human macrophages in the lungs were comparable and low (Figure 4A). On the other hand, human macrophages outnumbered murine macrophages in the lesions of BCG-infected mice (Figure 4A). When lung sections were stained with anti-PPD to visualize BCG bacilli, a large number of BCG bacilli were found in the lung lesions (Figure 4B). This observation suggests that human macrophages recruited to the site of infection support BCG growth better than murine macrophages. In C57BL/6 mice infected with BCG in parallel with NSG-A2-BLT mice, there were much fewer BCG bacilli found in the lung lesions due to efficient adaptive immune responses (Figure 4B). Thus, NSG-A2-BLT mice likely lack efficient T cell effector function at the site of infection.

Bottom Line: However, the levels of secreted IFN-γ, determined by ELISA, were not significantly elevated by antigenic stimulation.The lesions did not resemble granulomas typical of human TB.The lack of protection was associated with low IFN-γ levels and limited numbers of T cells recruited to the lesions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA. jinhee.lee@umassmed.edu.

ABSTRACT

Background: Currently used mouse models fail to fully reflect human immunity to tuberculosis (TB), which hampers progress in research and vaccine development. Bone marrow-liver-thymus (BLT) mice, generated by engrafting human fetal liver, thymus, and hematopoietic stem cells in severely immunodeficient NOD/SCID/IL-2Rγ(-/-) (NSG) mice, have shown potential to model human immunity to infection. We engrafted HLA-A2-positive fetal tissues into NSG mice transgenically expressing human leukocyte antigen (HLA)-A2.1 (NSG-A2) to generate NSG-A2-BLT mice and characterized their human immune response to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection to assess the utility of this model for investigating human TB.

Results: NSG-A2-BLT mice were infected intravenously with BCG and the immune response of engrafted human immune cells was characterized. After ex vivo antigenic stimulation of splenocytes, interferon (IFN)-γ-producing cells were detected by ELISPOT from infected, but not uninfected NSG-A2-BLT mice. However, the levels of secreted IFN-γ, determined by ELISA, were not significantly elevated by antigenic stimulation. NSG-A2-BLT mice were susceptible to BCG infection as determined by higher lung bacillary load than the non-engrafted control NSG-A2 mice. BCG-infected NSG-A2-BLT mice developed lung lesions composed mostly of human macrophages and few human CD4+ or CD8+ T cells. The lesions did not resemble granulomas typical of human TB.

Conclusions: Engrafted human immune cells in NSG-A2-BLT mice showed partial function of innate and adaptive immune systems culminating in antigen-specific T cell responses to mycobacterial infection. The lack of protection was associated with low IFN-γ levels and limited numbers of T cells recruited to the lesions. The NSG-A2-BLT mouse is capable of mounting a human immune response to M. tuberculosis in vivo but a quantitatively and possibly qualitatively enhanced effector response will be needed to improve the utility of this model for TB research.

Show MeSH
Related in: MedlinePlus