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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.

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NSG-A2-BLT mice develop lung lesions in response to BCG infection. Mice were infected as indicated in Figure 1, and sacrificed for histopathological examination. Lungs were inflated in 10% formalin, and embedded in paraffin for making thin sections. A. H&E stains on NSG-A2, NSG-A2-BLT, and C57BL/6 mice (100× and 20×, scale bars = 100 μm and 500 μm, respectively). Blue arrows indicate BCG lesions. B. Serial lung sections from NSG-A2-BLT mice infected with BCG were stained for H&E, Acid Fast bacilli, and immunohistochemistry for human CD68, human CD4, and human CD8. Immunostaining was visualized by colorimetric detection. Red arrows indicate BCG bacilli (200×, scale bars = 50 μm). C. Serial lung sections from NSG-A2-BLT and C57BL/6 mice infected with BCG were stained with human-specific antibodies against CD4, CD8, and CD68 followed by secondary antibody labeled with Alexa 555, and examined by fluorescence microscopy (200×, scale bar = 50 μm). Images are representative of tissues from four animals.
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Figure 3: NSG-A2-BLT mice develop lung lesions in response to BCG infection. Mice were infected as indicated in Figure 1, and sacrificed for histopathological examination. Lungs were inflated in 10% formalin, and embedded in paraffin for making thin sections. A. H&E stains on NSG-A2, NSG-A2-BLT, and C57BL/6 mice (100× and 20×, scale bars = 100 μm and 500 μm, respectively). Blue arrows indicate BCG lesions. B. Serial lung sections from NSG-A2-BLT mice infected with BCG were stained for H&E, Acid Fast bacilli, and immunohistochemistry for human CD68, human CD4, and human CD8. Immunostaining was visualized by colorimetric detection. Red arrows indicate BCG bacilli (200×, scale bars = 50 μm). C. Serial lung sections from NSG-A2-BLT and C57BL/6 mice infected with BCG were stained with human-specific antibodies against CD4, CD8, and CD68 followed by secondary antibody labeled with Alexa 555, and examined by fluorescence microscopy (200×, scale bar = 50 μm). Images are representative of tissues from four animals.

Mentions: Human granulomas are not reproduced in murine models of TB [5]. We sought to determine whether engrafted human immune cells develop lesions resembling human granulomas. Lung tissue sections were prepared for H&E, Ziehl-Neelsen (“acid-fast”), and immunostaining for human immune cell markers (Figure 3). H&E staining revealed inflammatory lesions in the lungs of BCG-infected NSG-A2-BLT mice (Figure 3A). No lesions were found in uninfected NSG-A2-BLT mice (data not shown). Non-engrafted NSG-A2 mice infected with BCG had myeloid cell aggregates that were smaller and less frequent than the lesions in NSG-A2-BLT mice. When compared with lesions in immunocompetent C57BL/6 mice infected with BCG in parallel, the lesions of NSG-A2-BLT mice were less frequent but comparable in size, and appeared to be more compact (Figure 3A). Ziehl-Neelsen staining in NSG-A2-BLT mouse lung sections showed that bacilli were scattered throughout the lung, inside and outside areas of inflammation, indicating that the lesions did not prevent spreading of infection. To evaluate human cell recruitment, serial lung sections from NSG-A2-BLT mice were stained with human-specific antibodies against CD4, CD8, and CD68 followed by colorimetric (Figure 3B) and fluorescent detection (Figure 3C). The lesions of NSG-A2-BLT mice contained abundant CD68+ human macrophages, but few to no detectable human CD4+ and CD8+ cells. All three antibodies produced negligible signals in lung sections from BCG-infected C57BL/6 mice, confirming their specificity for human antigens (Figure 3C). No caseous necrosis was observed. Collectively, the lesions induced by BCG in NSG-A2-BLT mice were less well developed than human granulomas.


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)

NSG-A2-BLT mice develop lung lesions in response to BCG infection. Mice were infected as indicated in Figure 1, and sacrificed for histopathological examination. Lungs were inflated in 10% formalin, and embedded in paraffin for making thin sections. A. H&E stains on NSG-A2, NSG-A2-BLT, and C57BL/6 mice (100× and 20×, scale bars = 100 μm and 500 μm, respectively). Blue arrows indicate BCG lesions. B. Serial lung sections from NSG-A2-BLT mice infected with BCG were stained for H&E, Acid Fast bacilli, and immunohistochemistry for human CD68, human CD4, and human CD8. Immunostaining was visualized by colorimetric detection. Red arrows indicate BCG bacilli (200×, scale bars = 50 μm). C. Serial lung sections from NSG-A2-BLT and C57BL/6 mice infected with BCG were stained with human-specific antibodies against CD4, CD8, and CD68 followed by secondary antibody labeled with Alexa 555, and examined by fluorescence microscopy (200×, scale bar = 50 μm). Images are representative of tissues from four animals.
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Related In: Results  -  Collection

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Figure 3: NSG-A2-BLT mice develop lung lesions in response to BCG infection. Mice were infected as indicated in Figure 1, and sacrificed for histopathological examination. Lungs were inflated in 10% formalin, and embedded in paraffin for making thin sections. A. H&E stains on NSG-A2, NSG-A2-BLT, and C57BL/6 mice (100× and 20×, scale bars = 100 μm and 500 μm, respectively). Blue arrows indicate BCG lesions. B. Serial lung sections from NSG-A2-BLT mice infected with BCG were stained for H&E, Acid Fast bacilli, and immunohistochemistry for human CD68, human CD4, and human CD8. Immunostaining was visualized by colorimetric detection. Red arrows indicate BCG bacilli (200×, scale bars = 50 μm). C. Serial lung sections from NSG-A2-BLT and C57BL/6 mice infected with BCG were stained with human-specific antibodies against CD4, CD8, and CD68 followed by secondary antibody labeled with Alexa 555, and examined by fluorescence microscopy (200×, scale bar = 50 μm). Images are representative of tissues from four animals.
Mentions: Human granulomas are not reproduced in murine models of TB [5]. We sought to determine whether engrafted human immune cells develop lesions resembling human granulomas. Lung tissue sections were prepared for H&E, Ziehl-Neelsen (“acid-fast”), and immunostaining for human immune cell markers (Figure 3). H&E staining revealed inflammatory lesions in the lungs of BCG-infected NSG-A2-BLT mice (Figure 3A). No lesions were found in uninfected NSG-A2-BLT mice (data not shown). Non-engrafted NSG-A2 mice infected with BCG had myeloid cell aggregates that were smaller and less frequent than the lesions in NSG-A2-BLT mice. When compared with lesions in immunocompetent C57BL/6 mice infected with BCG in parallel, the lesions of NSG-A2-BLT mice were less frequent but comparable in size, and appeared to be more compact (Figure 3A). Ziehl-Neelsen staining in NSG-A2-BLT mouse lung sections showed that bacilli were scattered throughout the lung, inside and outside areas of inflammation, indicating that the lesions did not prevent spreading of infection. To evaluate human cell recruitment, serial lung sections from NSG-A2-BLT mice were stained with human-specific antibodies against CD4, CD8, and CD68 followed by colorimetric (Figure 3B) and fluorescent detection (Figure 3C). The lesions of NSG-A2-BLT mice contained abundant CD68+ human macrophages, but few to no detectable human CD4+ and CD8+ cells. All three antibodies produced negligible signals in lung sections from BCG-infected C57BL/6 mice, confirming their specificity for human antigens (Figure 3C). No caseous necrosis was observed. Collectively, the lesions induced by BCG in NSG-A2-BLT mice were less well developed than human granulomas.

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