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

NSG-A2-BLT mice mount adaptive immune responses to BCG infection. NSG-A2-BLT mice were infected i.v. with BCG at a dose of 1 × 105 CFU per mouse. One month later mice were sacrificed, and T cell recall response of splenocytes was measured by ELISPOT (n = 6 for infected NSG-A2-BLT mice and n = 2 for uninfected NSG-A2-BLT mice) (A) and ELISA (n = 3 for infected NSG-BLT mice) (B). Culture conditions and antigenic stimulation are described in Methods. Abbreviations: CFP, Culture filtrate proteins; WC, Heat-killed whole BCG; PMA, Phorbol 12-myristate 13-acetate; Iono, ionomycin. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: NSG-A2-BLT mice mount adaptive immune responses to BCG infection. NSG-A2-BLT mice were infected i.v. with BCG at a dose of 1 × 105 CFU per mouse. One month later mice were sacrificed, and T cell recall response of splenocytes was measured by ELISPOT (n = 6 for infected NSG-A2-BLT mice and n = 2 for uninfected NSG-A2-BLT mice) (A) and ELISA (n = 3 for infected NSG-BLT mice) (B). Culture conditions and antigenic stimulation are described in Methods. Abbreviations: CFP, Culture filtrate proteins; WC, Heat-killed whole BCG; PMA, Phorbol 12-myristate 13-acetate; Iono, ionomycin. *p < 0.05.

Mentions: To evaluate the utility of NSG-A2-BLT mice as an animal model for TB, we assessed the response of engrafted human immune cells to infection with BCG. NSG-A2 and NSG-A2-BLT mice were infected with BCG intravenously (i.v.) at a dose of 1 × 105 colony-forming units (CFU) and euthanized four weeks post-infection to measure bacterial load in the lung, and human adaptive immune responses. Splenocytes from infected NSG-A2-BLT mice responded to ex vivo restimulation with mycobacterial antigens by producing IFN-γ as measured by ELISPOT, while splenocytes from uninfected NSG-A2-BLT mice did not produce measurable IFN-γ in response to any of these antigens (Figure 1A). Splenocytes from BCG infected mice had a stronger response to activation by PMA/ionomycin than splenocytes from uninfected mice, indicating an expansion of non-antigen-specific Th1 cells from infected NSG-A2-BLT mice. Splenocytes from infected NSG-A2-BLT mice did not produce IFN-γ without antigenic stimulation. Unlike the ELISPOT result, the levels of IFN-γ secreted into media in response to ex vivo antigenic stimulation measured by ELISA were not statistically higher than in the media control (Figure 1B). Splenocytes stimulated with PMA/ionomycin produced large amounts of IFN-γ, indicating the presence of functionally competent T cells capable of secreting IFN-γ. To determine if NSG-A2-BLT mice acquired the capacity to restrict BCG replication, we compared lung bacillary load in NSG-A2-BLT mice and NSG-A2 mice 4 weeks after BCG infection. Rather than demonstrating enhanced control of BCG, the lungs of NSG-A2-BLT mice had higher CFU counts than the non-engrafted NSG-A2 controls (Figure 2). This unexpected result implies that the engrafted human immune cells are unable to kill intracellular bacilli, instead providing an environment more conducive for replication than host murine macrophages.


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 mount adaptive immune responses to BCG infection. NSG-A2-BLT mice were infected i.v. with BCG at a dose of 1 × 105 CFU per mouse. One month later mice were sacrificed, and T cell recall response of splenocytes was measured by ELISPOT (n = 6 for infected NSG-A2-BLT mice and n = 2 for uninfected NSG-A2-BLT mice) (A) and ELISA (n = 3 for infected NSG-BLT mice) (B). Culture conditions and antigenic stimulation are described in Methods. Abbreviations: CFP, Culture filtrate proteins; WC, Heat-killed whole BCG; PMA, Phorbol 12-myristate 13-acetate; Iono, ionomycin. *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: NSG-A2-BLT mice mount adaptive immune responses to BCG infection. NSG-A2-BLT mice were infected i.v. with BCG at a dose of 1 × 105 CFU per mouse. One month later mice were sacrificed, and T cell recall response of splenocytes was measured by ELISPOT (n = 6 for infected NSG-A2-BLT mice and n = 2 for uninfected NSG-A2-BLT mice) (A) and ELISA (n = 3 for infected NSG-BLT mice) (B). Culture conditions and antigenic stimulation are described in Methods. Abbreviations: CFP, Culture filtrate proteins; WC, Heat-killed whole BCG; PMA, Phorbol 12-myristate 13-acetate; Iono, ionomycin. *p < 0.05.
Mentions: To evaluate the utility of NSG-A2-BLT mice as an animal model for TB, we assessed the response of engrafted human immune cells to infection with BCG. NSG-A2 and NSG-A2-BLT mice were infected with BCG intravenously (i.v.) at a dose of 1 × 105 colony-forming units (CFU) and euthanized four weeks post-infection to measure bacterial load in the lung, and human adaptive immune responses. Splenocytes from infected NSG-A2-BLT mice responded to ex vivo restimulation with mycobacterial antigens by producing IFN-γ as measured by ELISPOT, while splenocytes from uninfected NSG-A2-BLT mice did not produce measurable IFN-γ in response to any of these antigens (Figure 1A). Splenocytes from BCG infected mice had a stronger response to activation by PMA/ionomycin than splenocytes from uninfected mice, indicating an expansion of non-antigen-specific Th1 cells from infected NSG-A2-BLT mice. Splenocytes from infected NSG-A2-BLT mice did not produce IFN-γ without antigenic stimulation. Unlike the ELISPOT result, the levels of IFN-γ secreted into media in response to ex vivo antigenic stimulation measured by ELISA were not statistically higher than in the media control (Figure 1B). Splenocytes stimulated with PMA/ionomycin produced large amounts of IFN-γ, indicating the presence of functionally competent T cells capable of secreting IFN-γ. To determine if NSG-A2-BLT mice acquired the capacity to restrict BCG replication, we compared lung bacillary load in NSG-A2-BLT mice and NSG-A2 mice 4 weeks after BCG infection. Rather than demonstrating enhanced control of BCG, the lungs of NSG-A2-BLT mice had higher CFU counts than the non-engrafted NSG-A2 controls (Figure 2). This unexpected result implies that the engrafted human immune cells are unable to kill intracellular bacilli, instead providing an environment more conducive for replication than host murine macrophages.

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