Current advances in humanized mouse models

Humanized mouse models that have received human cells or tissue transplants are extremely useful in basic and applied human disease research. Highly immunodeficient mice, which do not reject xenografts and support cell and tissue differentiation and growth, are indispensable for generating additional appropriate models. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγ^null gene (e.g., NOD/SCID/γc^null and Rag2^nullγc^null mice). These strains show not only a high rate of human cell engraftment, but also generate well-differentiated multilineage human hematopoietic cells after human hematopoietic stem cell (HSC) transplantation. These humanized mice facilitate the analysis of human hematology and immunology in vivo. However, human hematopoietic cells developed from HSCs are not always phenotypically and functionally identical to those in humans. More recently, a new series of immunodeficient mice compensates for these disadvantages. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γc^null and Rag2^nullγc^null mice. In this review, we describe the current knowledge of human hematopoietic cells developed in these mice. Various human disease mouse models using these humanized mice are summarized.     

Humanized NOG mice as a model for tuberculosis vaccine‐induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis

The humanized mouse model has been developed as a model to identify and characterize human immune responses to human pathogens and has been used to better identify vaccine candidates. In the current studies, the humanized mouse was used to determine the ability of a vaccine to affect the immune response to infection with Mycobacterium tuberculosis. Both human CD4⁺ and CD8⁺ T cells responded to infection in humanized mice as a result of infection. In humanized mice vaccinated with either BCG or with CpG‐C, a liposome‐based formulation containing the M. tuberculosis antigen ESAT‐6, both CD4 and CD8 T cells secreted cytokines that are known to be required for induction of protective immunity. In comparison to the C57BL/6 mouse model and Hartley guinea pig model of tuberculosis, data obtained from humanized mice complemented the data observed in the former models and provided further evidence that a vaccine can induce a human T‐cell response. Humanized mice provide a crucial pre‐clinical platform for evaluating human T‐cell immune responses in vaccine development against M. tuberculosis.     

High activation and skewed T cell differentiation are associated with low IL-17A levels in a hu-PBL-NSG-SGM3 mouse model of HIV infection

The humanized NOD/SCID/IL-2 receptor γ-chain^null (NSG) mouse model has been widely used for the study of HIV pathogenesis. Here, NSG mice with transgenic expression of human stem cell factor (SCF), granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 (NSG-SGM3) were injected with peripheral blood leukocytes (PBL mice) from two HIV-infected (HIV⁺) patients who were under anti-retroviral therapy (ART; referred as HIV⁺ mice) or one HIV-seronegative healthy volunteer (HIV⁻). Such mice are either hu-PBL-NSG-SGM3 HIV⁺ or HIV⁻ mice, depending on the source of PBL. The kinetics of HIV replication and T cell responses following engraftment were evaluated in peripheral blood and secondary lymphoid tissues. High HIV replication and low CD4 : CD8 ratios were observed in HIV⁺ mice in the absence of anti-retroviral therapy (ART). Consistent with high activation and skewed differentiation of T cells from the HIV-infected donor, HIV⁺ mice exhibited a higher T cell co-expression of human leukocyte antigen D-related (HLA-DR) and CD38 than HIV⁻ mice, as well as a shifted differentiation to a CCR7⁻CD45RA⁺ terminal effector profile, even in the presence of ART. In addition, HIV replication and the activation/differentiation disturbances of T cells were associated with decreased plasma levels of IL-17A. Thus, this hu-PBL-NSG-SGM3 mouse model recapitulates some immune disturbances occurring in HIV-infected patients, underlying its potential use for studying pathogenic events during this infection.     

MHC class II-deficient mice allow functional human CD4+ T-cell development

Humanized mouse models have been developed to study cell-mediated immune responses to human pathogens in vivo. How immunocompetent human T cells are selected in a murine thymus in such humanized mice remains poorly explored. To gain insights into this mechanism, we investigated the differentiation of human immune compartments in mouse MHC class II-deficient immune-compromised mice (humanized Ab0 mice). We observed a strong reduction in human CD4⁺ T-cell development but despite this reduction Ab0 mice had no disadvantage during Epstein–Barr virus (EBV) infection. Viral loads were equally well controlled in humanized Ab0 mice compared to humanized NSG mice, and improved T-cell recognition of autologous EBV-transformed B cells was observed, especially with respect to cytotoxicity. MHC class II blocking experiments with CD4⁺ T cells from humanized Ab0 mice demonstrated MHC class II restriction of lymphoblastoid cell line recognition. These findings suggest that a small number of CD4⁺ T cells in humanized mice can be solely selected on human MHC class II molecules, presumably expressed by reconstituted human immune cells, leading to improved effector functions.     

Human lymphohematopoietic reconstitution and immune function in immunodeficient mice receiving cotransplantation of human thymic tissue and CD34+ cells

Small animal models with functional human lymphohematopoietic systems are highly valuable for the study of human immune function under physiological and pathological conditions. Over the last two decades, numerous efforts have been devoted towards the development of such humanized mouse models. This review is focused on human lymphohematopoietic reconstitution and immune function in humanized mice by cotransplantation of human fetal thymic tissue and CD34⁺ cells. The potential use of these humanized mice in translational biomedical research is also discussed.     

Dengue virus infection and immune response in humanized RAG2(-/-)gamma(c)(-/-) (RAG-hu) mice

Dengue viral (DENV) pathogenesis and vaccine studies are hampered by the lack of an ideal animal model mimicking human disease and eliciting an adaptive human immune response. Although currently available animal models have been very useful in dissecting some key aspects of disease pathogenesis, a major limitation with these is the lack of a human immune response. In this study, we sought to overcome this difficulty by utilizing a novel mouse model that permits multi-lineage human hematopoiesis and immune response following transplantation with human hematopoietic stem cells. To generate immunocompetent humanized mice, neonatal RAG2(-/-)gamma(c)(-/-) mice were xenografted with human CD34+ hematopoietic stem cells, resulting in de novo development of major functional cells of the human adaptive immune system. To evaluate susceptibility to dengue viral infection, humanized mice were challenged with DEN-2 serotype. Viremia lasting up to 3 weeks was detected in infected mice with viral titers reaching up to 10(6.3) RNA copies/ml. Fever characteristic of dengue was also noted in infected mice. Presence of human anti-dengue antibodies was evaluated using an antibody capture ELISA. Anti-dengue IgM was first detected by 2 weeks post-infection followed by IgG at 6 weeks. Sera from some of the infected mice were also found to be capable of dengue virus neutralization. Infected mouse sera showed reactivity with the viral envelope and capsid proteins in immunoprecipitation assay. These results demonstrate for the first time that humanized mice are capable of dengue viral primary human immune responses thus paving the way for new dengue immunopathogenesis and vaccine studies.     

Cell-secreted Gp96-Ig-peptide complexes induce lamina propria and intraepithelial CD8+ cytotoxic T lymphocytes in the intestinal mucosa

Induction of mucosal immunity is critical for protection from enteric pathogens. Heat shock protein gp96 is one of the primary peptide and protein chaperones located in the endoplasmic reticulum. We reported previously that a cell-secreted gp96-Ig fusion protein (gp96-Ig) mediated strong systemic, antigen-specific CD8-CTL expansion in vivo. We now evaluate the mucosal immune response to stimulation by secreted gp96 using allogeneic NIH-3T3 transfected with ovalbumin (OVA) and gp96-Ig. A single intraperitoneal NIH-3T3-OVA-gp96-Ig immunization caused significant homing of OVA-specific TCR transgenic CD8 cells (OT-I) to Peyer's patches, to the intraepithelial compartment and to the lamina propria. Intraperitoneal immunization with cells secreting gp96-Ig provided stronger mucosal immunity than the same dose instilled vaginally or rectally or injected subcutaneously or intradermally. Our results provide the first evidence that cell-based gp96-Ig-secreting vaccines may serve as a potent modality to induce mucosal immunity.     

Parameters for establishing humanized mouse models to study human immunity: Analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the IL2rγ mutation

“Humanized” mouse models created by engraftment of immunodeficient mice with human hematolymphoid cells or tissues are an emerging technology with broad appeal across multiple biomedical disciplines. However, investigators wishing to utilize humanized mice with engrafted functional human immune systems are faced with a myriad of variables to consider. In this study, we analyze HSC engraftment methodologies using three immunodeficient mouse strains harboring the IL2rγ^null mutation; NOD-scid IL2rγ^null, NOD-Rag1^null IL2rγ^null, and BALB/c-Rag1^null IL2rγ^null mice. Strategies compared engraftment of human HSC derived from umbilical cord blood following intravenous injection into adult mice and intracardiac and intrahepatic injection into newborn mice. We observed that newborn recipients exhibited enhanced engraftment as compared to adult recipients. Irrespective of the protocol or age of recipient, both immunodeficient NOD strains support enhanced hematopoietic cell engraftment as compared to the BALB/c strain. Our data define key parameters for establishing humanized mouse models to study human immunity.     

PRO 140 Monoclonal Antibody to CCR5 Prevents Acute Xenogeneic Graft-versus-Host Disease in NOD-scid IL-2Rynull Mice

Graft-versus-host disease (GVHD) is a prevalent and potentially lethal complication of hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic GVHD are important tools used to study the human immune response in vivo. Here we used NOD-scid IL-2Ry^null mice (NSG) transplanted with human bone marrow stem cells to evaluate the role of immune cell engraftment in the production of acute GVHD. PRO 140, a humanized monoclonal antibody targeting the chemokine receptor, CCR5, was used to evaluate its influence on bone marrow cell engraftment and modulation of acute GVHD. We evaluated the kinetics of engraftment by determining the percentage and absolute numbers of human CD45⁺ cells and CD3⁺ T cells from peripheral blood, spleen, and bone marrow in treated and control mice. With a dosing schedule of 2?mg of test or control antibody administered i.p. twice weekly, PRO 140–treated mice showed no signs of GVHD throughout the 70-day study period and gained weight until they were killed at 70 days for flow cytometry analysis. Control mice started losing weight after 25 days, showed classic signs of GVHD (ruffled fur, lethargy, severe hunching), and all were killed by day 54. The percentage and absolute numbers of human CD45⁺ cells in peripheral blood increased in both groups of mice throughout the 50-day comparison period and was lower in the PRO 140–treated mice at day 50. There was no difference in human CD45⁺ cells detected in bone marrow from control and PRO 140–treated killed mice. At this time point 76.1% and 68.2% of the hematopoietic cells from peripheral blood and from bone marrow, respectively, were of human lineage and 14.9% and 28%, respectively, were of mouse origin. With a schedule using 10-fold less dose of antibody (.2?mg i.p. twice weekly), PRO 140 still significantly modulated acute GVHD in terms of both weight loss and survival times, but no mice from either control or test group survived. By targeting the CCR5 chemokine receptor, PRO 140 modulated acute GVHD in a dose-response fashion in this xenogeneic mouse model without significantly altering engraftment.     

Human allograft rejection in humanized mice: a historical perspective

Basic research in transplantation immunology has relied primarily on rodent models. Experimentation with rodents has laid the foundation for our basic understanding of the biological events that precipitate rejection of non-self or allogeneic tissue transplants and supported the development of novel strategies to specifically suppress allogeneic immune responses. However, translation of these studies to the clinic has met with limited success, emphasizing the need for new models that focus on human immune responses to allogeneic tissues. Humanized mouse models are an exciting alternative that permits investigation of the rejection of human tissues mediated by human immune cells without putting patients at risk. However, the use of humanized mice is complicated by a diversity of protocols and approaches, including the large number of immunodeficient mouse strains available, the choice of tissue to transplant and the specific human immune cell populations that can be engrafted. Here, we present a historical perspective on the study of allograft rejection in humanized mice and discuss the use of these novel model systems in transplant biology.     

HIV-1 immunopathogenesis in humanized mouse models

In recent years, the technology of constructing chimeric mice with humanized immune systems has markedly improved. Multiple lineages of human immune cells develop in immunodeficient mice that have been transplanted with human hematopoietic stem cells. More importantly, these mice mount functional humoral and cellular immune responses upon immunization or microbial infection. Human immunodeficiency virus type I (HIV-1) can establish an infection in humanized mice, resulting in CD4⁺ T-cell depletion and an accompanying nonspecific immune activation, which mimics the immunopathology in HIV-1-infected human patients. This makes humanized mice an optimal model for studying the mechanisms of HIV-1 immunopathogenesis and for developing novel immune-based therapies.     

Heat‐Shock Protein gp96 Enhances T Cell Responses and Protective Potential to Bacillus Calmette‐Guérin Vaccine

The commonly used Bacillus Calmette‐Guérin (BCG) vaccine only induces moderate T cell responses and is less effective in protecting against pulmonary tuberculosis (TB) in adults and ageing populations. Thus, developing new TB vaccine candidates is an important strategy against the spread of Mycobacterium tuberculosis. Here, we demonstrated that immunization with heat‐shock protein gp96 as an adjuvant led to a significantly increased CD4⁺ and CD8⁺ T cell response to a BCG vaccine. Secretion of the Th1‐type cytokines was increased by splenocytes from gp96‐immunized mice. In addition, adding gp96 as an adjuvant effectively improved the protection against intravenous challenge with Mycobacterium bovis BCG in mice. Our study reveals the novel property of gp96 in boosting the vaccine‐specific T cell response and its potential use as an adjuvant for BCG vaccines against mycobacterial infection.     

Comprehensive Analysis of the Activation and Proliferation Kinetics and Effector Functions of Human Lymphocytes,and Antigen Presentation Capacity of Antigen-Presenting Cells in Xenogeneic Graft-Versus-Host Disease

Xenogeneic graft-versus-host disease (GVHD) models in highly immunodeficient mice are currently being used worldwide to investigate human immune responses against foreign antigens in vivo. However, the individual roles of CD4⁺ and CD8⁺ T cells, and donor/host hematopoietic and nonhematopoietic antigen-presenting cells (APCs) in the induction and development of GVHD have not been fully investigated. In the present study, we comprehensively investigated the immune responses of human T cells and the antigen presentation capacity of donor/host hematopoietic and nonhematopoietic APCs in xenogeneic GVHD models using nonobese diabetic/Shi-scid-IL2rg^null mice. CD4⁺ T cells and, to a lesser extent, CD8⁺ T cells individually mediated potentially lethal GVHD. In addition to inflammatory cytokine production, CD4⁺ T cells also supported the activation and proliferation of CD8⁺ T cells. Using bone marrow chimeras, we demonstrated that host hematopoietic, but not nonhematopoietic, APCs play a critical role in the development of CD4⁺ T cell-mediated GVHD. During early GVHD, we detected 2 distinct populations in memory CD4⁺ T cells. One population was highly activated and proliferated in major histocompatibility complex antigen (MHC)^+/+ mice but not in MHC^?/? mice, indicating alloreactive T cells. The other population showed a less activated and slowly proliferative status regardless of host MHC expression, and was associated with higher susceptibility to apoptosis, indicating nonalloreactive T cells in homeostasis-driven proliferation. These observations are clinically relevant to donor T cell response after allogeneic hematopoietic stem cell transplantation. Our findings provide a better understanding of the immunobiology of humanized mice and support the development of novel options for the prevention and treatment for GVHD.     

Co-transplantation of fetal bone tissue facilitates the development and reconstitution in human B cells in humanized NOD/SCID/IL-2Rγnull (NSG) mice

Background  

In terms of the function and reconstitution efficacy of human immune cells, co-transplantation of human fetal tissues, such as thymus and liver, with CD34⁺ hematopoietic stem cells (HSCs) has potential advantages in the generation of humanized mice.     

Induction of human humoral immune responses in a novel HLA-DR-expressing transgenic NOD/Shi-scid/γcnull mouse

Mounting evidence has demonstrated that NOD-Shi/scid/γc(null) (NOG) mice are one of the most suitable mouse strains for humanized mouse technologies, in which various human cells or tissues can be engrafted without rejection and autonomously maintained. We have characterized and analyzed various features of the human immune system reconstituted in NOG mice by transplanting human hematopoietic stem cells (hu-HSC). One of the problems of the quasi-immune system in these hu-HSC NOG mice is that the quality of immune responses is not always sufficient, as demonstrated by the lack of IgG production in response to antigen challenge. In this study, we established a novel transgenic NOG sub-strain of mice bearing the HLA-DRA and HLA-DRB1:0405 genes, which specifically expresses HLA-DR4 molecules in MHC II-positive cells. This mouse strain enabled us to match the haplotype of HLA-DR between the recipient mice and human donor HSC. We demonstrated that T-cell homeostasis was differentially regulated in HLA-matched hu-HSC NOG mice compared with HLA-mismatched control mice, and antibody class switching was induced after immunization with exogenous antigens in HLA-matched mice. This novel mouse strain improves the reconstituted human immune systems that develop in humanized mice and will contribute to future studies of human humoral immune responses.     

Heat shock fusion protein gp96-Ig mediates strong CD8 CTL expansion in vivo

PROBLEM: As shown previously, gp96-Ig peptide complexes secreted by an ovalbumin transfected tumor (EG7) mediate strong, specific tumor immunity through a CD4 T cell independent CD8+ CTL response. In this study, we set out to develop a system to quantitatively determine the CD8 CTL response to gp96-Ig and to evaluate the influence of an established wild type tumor. METHODS: Secreted heat shock protein gp96-Ig was constructed by replacement of the endoplasmic reticulum retention signal with the Fc portion of IgGI, transfected into EG7 (EG7-gp96-Ig) and used to induce CD8+ CTL expansion in vivo. Adoptively transferred, ovalbumin specific T-cell receptor (TCR) transgenic CD8+ cells (OT-1) responded with clonal expansion to the immunization with EG7-gp96-Ig. OT-1 expansion was quantitated with K(b-peptide)-tetramers by flow cytometry. RESULTS: In response to primary immunization with EG7-gp96-Ig, OT-1 expand from an initial frequency of 0.5 to 25% of all CD8 cells, and to 50% of all CD8 cells after a booster immunization. Endogenous ovalbumin specific CD8 cells also expand strongly. Antigen specific effector function was measured by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) for interferon-gamma (IFN-gamma). While effector function was strongly induced by secreted gp96-Ig, not all expanded OT-1 produce IFN-gamma. EG7 does not cause OT-1 expansion, but rather induces anergy. If OT-1 are transferred into wild type EG7 tumor bearing mice to induce anergy of OT-1, immunization with EG7-gp96-Ig can partly overcome unresponsiveness. CONCLUSIONS: We conclude that secreted gp96-Ig is a powerful mediator of specific CD8+ CTL responses in vivo. Secretory gp96 mimics release of gp96 by damaged or necrotic cells that is able to activate dendritic cells without CD4 help. Gp96-Ig associated peptides have not been selected by binding to major histocompatibility complex (MHC). Specific immunization by secreted gp96-Ig therefore is expected to occur also in allogeneic settings.     

Engineering humanized mice for improved hematopoietic reconstitution

Humanized mice are immunodeficient animals engrafted with human hematopoietic stem cells that give rise to various lineages of human blood cells throughout the life of the mouse. This article reviews recent advances in the generation of humanized mice, focusing on practical considerations. We discuss features of different immunodeficient recipient mouse strains, sources of human hematopoietic stem cells, advances in expansion and genetic modification of hematopoietic stem cells, and techniques to modulate the cytokine environment of recipient mice, in order to enhance reconstitution of specific human blood lineage cells. We highlight the opportunities created by new technologies and discuss practical considerations on how to make best use of the widening array of basic models for specific research applications.     

cAIMP administration in humanized mice induces a chimerization-level-dependent STING response

It is well understood that the STING signalling pathway is critical for generating a robust innate immune response to pathogens. Human and mouse STING signalling pathways are not identical, however. For example, mice lack IFI16, which has been proven important for the human STING pathway. Therefore, we investigated whether humanized mice are an appropriate experimental platform for exploring the human STING signalling cascade in vivo. We found that NOG mice reconstituted with human cord blood haematopoietic stem cells (humanized NOG mice) exhibit human STING signalling responses to an analogue of the cyclic di-nucleotide cGAMP. There was an increase in the proportions of monocytes in the lungs of mice receiving cGAMP analogue. The most robust levels of STING expression and STING-induced responses were observed in mice exhibiting the highest levels of human chimerization. Notably, differential levels of STING in lung versus spleen following cGAMP analogue treatment suggest that there are tissue-specific kinetics of STING activation and/or degradation in effector versus inductive sites. We also examined the mouse innate immune response to cGAMP analogue treatment. We detected that mouse cells in the immunodeficient NOG mice responded to the cGAMP analogue and they do so with distinct kinetics from the human response. In conclusion, humanized NOG mice represent a valuable experimental model for examining in vivo human STING responses.     

Perforin is required for innate and adaptive immunity induced by heat shock protein gp96

Tumor-secreted gp96-Ig is highly immunogenic and triggers CD8 T cell-mediated tumor rejection. In vivo secreted gp96-Ig and gp96-myc cause NK activation and clonal expansion of specific CD8(+) CTL in wild-type and in Fas-ligand-deficient (gld) mice but not in perforin- (PKO) or IFN-gamma-deficient (GKO) mice. Transfer of perforin-competent NK cells restores the ability of PKO mice to clonally expand CD8 CTL in response to gp96-Ig. The data demonstrate an essential role for perforin-mediated functions in the activation of innate and adaptive immunity by heat shock protein gp96-peptide complexes. Crosspresentation of antigens by heat shock proteins seems to require a perforin-dependent positive feedback loop between NK and DC for both sustained NK activation and clonal CTL expansion. The studies also explain how depressed NK activity in patients with tumors or after viral infections could diminish CTL responses.     

Humanized mouse as an appropriate model for accelerated global HIV research and vaccine development: current trend

Humanized mouse models currently have seen improved development and have received wide applications. Its usefulness is observed in cell and tissue transplant involving basic and applied human disease research. In this article, the development of a new generation of humanized mice was discussed as well as their relevant application in HIV disease. Furthermore, current techniques employed to overcome the initial limitations of mouse model were reviewed. Highly immunodeficient mice which support cell and tissue differentiation and do not reject xenografts are indispensable for generating additional appropriate models useful in disease study, this phenomenom deserves emphases, scientific highlight and a definitive research focus. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγ^null gene (e.g. NOD/SCID/γc^null and Rag2^nullγc^null mice) through various genomic approaches. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γc^null and Rag2^nullγc^null mouse backgrounds. The application of these techniques serves as a quick and appropriate mechanistic model for basic and therapeutic investigations of known and emerging infections.