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.     

Humanized mice: novel model for studying mechanisms of human immune-based therapies

The lack of relevant animal models is the major bottleneck for understanding human immunology and immunopathology. In the last few years, a novel model of humanized mouse has been successfully employed to investigate some of the most critical questions in human immunology. We have set up and tested in our laboratory the latest technology for generating mice with a human immune system by reconstituting newborn immunodeficient NOD/SCID-γ ^−/−_c mice with human fetal liver-derived hematopoietic stem cells. These humanized mice have been deemed most competent as human models in a thorough comparative study with other humanized mouse technologies. Lymphocytes in these mice are of human origin while other hematopoietic cells are chimeric, partly of mouse and partly of human origin. We demonstrate that human CD8 T lymphocytes in humanized mice are fully responsive to our novel cell-based secreted heat shock protein gp96^HIV-Ig vaccine. We also show that the gp96^HIV-Ig vaccine induces powerful mucosal immune responses in the rectum and the vagina, which are thought to be required for protection from HIV infection. We posit the hypothesis that vaccine approaches tested in humanized mouse models can generate data rapidly, economically and with great flexibility (genetic manipulations are possible), to be subsequently tested in larger nonhuman primate models and humans.

     

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.     

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.     

Novel immunological approach to asses donor reactivity of transplant recipients using a humanized mouse model

In organ transplantation, a reproducible and robust immune-monitoring assay has not been established to determine individually tailored immunosuppressants (IS). We applied humanized mice reconstituted with human (hu-) peripheral blood mononuclear cells (PBMCs) obtained from living donor liver transplant recipients to evaluate their immune status. Engraftment of 2.5 × 10⁶ hu-PBMCs from healthy volunteers and recipients in the NSG mice was achieved successfully. The reconstituted lymphocytes consisted mainly of hu-CD3⁺ lymphocytes with predominant CD45RA⁻CD62L^lo T_EM and CCR6⁻CXCR3⁺CD4⁺ Th1 cells in hu-PBMC-NSG mice. Interestingly, T cell allo-reactivity of hu-PBMC-NSG mice was amplified significantly compared with that of freshly isolated PBMCs (p < 0.05). Furthermore, magnified hu-T cell responses to donor antigens (Ag) were observed in 2/10 immunosuppressed recipients with multiple acute rejection (AR) experiences, suggesting that the immunological assay in hu-PBMC-NSG mice revealed hidden risks of allograft rejection by IS. Furthermore, donor Ag-specific hyporesponsiveness was maintained in recipients who had been completely weaned off IS (n = 4), despite homeostatic proliferation of hu-T cells in the hu-PBMC-NSG mice. The immunological assay in humanized mice provides a new tool to assess recipient immunity in the absence of IS and explore the underlying mechanisms to maintaining operational tolerance.     

Foxp3 and Treg cells in HIV-1 infection and immuno-pathogenesis

FoxP3⁺CD4⁺CD25⁺ regulatory T (Treg) cells are implicated in a number of pathologic processes including elevated levels in cancers and infectious diseases, and reduced levels in autoimmune diseases. Treg cells are activated to modulate immune responses to avoid over-reactive immunity. However, conflicting findings are reported regarding relative levels of Treg cells during HIV-1 infection and disease progression. The role of Treg cells in HIV-1 diseases (aberrant immune activation) is poorly understood due to lack of a robust model. We summarize here the regulation and function of Foxp3 in Treg cells and in modulating HIV-1 replication. Based on recent findings from SIV/monkey and HIV/humanized mouse models, a model of the dual role of Treg cells in HIV-1 infection and immuno-pathogenesis is discussed.     

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.     

An in vivo model of allergic inflammation: pulmonary human cell infiltrate in allergen-challenged allergic Hu-SCID mice

CB.17 severe combined immunodeficient (SCID) mice were used to establish a model of allergic pulmonary inflammation. SCID mice were intraperitoneally reconstituted with 10⁷ peripheral blood mononuclear cells (PBMC) from patients sensitive to Dermatophagoides pteronyssinus (Dpt) and 2 weeks later were exposed to Dpt aerosols. After Dpt nebulization, SCID mice engrafted with PBMC from Dpt-sensitive patients developed a specific human IgE response as well as an intense pulmonary infiltrate of human cells. In contrast, SCID mice reconstituted with PBMC from patients allergic to grass pollen or from nonallergic donors failed to produce IgE or to exhibit a similar inflammatory response. The level of the IgE production was dependent on the IgE level of the allergic donor. In the lungs of the same allergic SCID mice, 2 months after Dpt inhalation, the cell infiltrate contained CD45⁺, CD45RO⁺, CD20⁺ and HLA-DR⁺ human cells. They were located in perivascular and peribronchial areas and disseminated in the mouse lung parenchyma. Moreover, mRNA IL-5⁺ cells and eosinophils were found in peribronchial infiltrates. The observations indicate that humanized allergic SCID mice may develop, after nebulization with the relevant allergen, immune reactions similar to those observed in man and suggest that SCID mice may represent a useful model to analyze the regulatory mechanisms of IgE-associated allergic diseases.     

Minocycline attenuates HIV‐1 infection and suppresses chronic immune activation in humanized NOD/LtsZ‐scidIL‐2Rγnull mice

More than a quarter of a century of research has established chronic immune activation and dysfunctional T cells as central features of chronic HIV infection and subsequent immunodeficiency. Consequently, the search for a new immunomodulatory therapy that could reduce immune activation and improve T‐cell function has been increased. However, the lack of small animal models for in vivo HIV study has hampered progress. In the current study, we have investigated a model of cord blood haematopoietic progenitor cells (CB‐HPCs) ‐transplanted humanized NOD/LtsZ‐scidIL‐2Rγ^null mice in which progression of HIV infection is associated with widespread chronic immune activation and inflammation. Indeed, HIV infection in humanized NSG mice caused up‐regulation of several T‐cell immune activation markers such as CD38, HLA‐DR, CD69 and co‐receptor CCR5. T‐cell exhaustion markers PD‐1 and CTLA‐4 were found to be significantly up‐regulated on T cells. Moreover, increased plasmatic levels of lipopolysaccharide, sCD14 and interleukin‐10 were also observed in infected mice. Treatment with minocycline resulted in a significant decrease of expression of cellular and plasma immune activation markers, inhibition of HIV replication and improved T‐cell counts in HIV‐infected humanized NSG mice. The study demonstrates that minocycline could be an effective, low‐cost adjunctive treatment to regulate chronic immune activation and replication of HIV.     

Establishing humanized mice using stem cells: maximizing the potential

Studies on physiology and pathology as they relate to the immune system draw heavily upon rodent models. With the increasing impetus provided by initiatives in translational medicine, the demand for ever more sophisticated, 'humanized' murine models is greater than ever. However, the design and implementation of studies in such mice is far from trivial. Here we provide a technical perspective on the increasing interest in developing humanized mice. We give examples of primary data starting with the routine procurement of human donor material, through CD34(+) cell purification prior to engraftment to injection into immunocompromised mice. Our goal is to provide practical advice to the many investigators who may be commencing or considering such studies.     

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.     

High diversity of the immune repertoire in humanized NOD.SCID.γc−/− mice

The diversity of the human immune repertoire and how it relates to a functional immune response has not yet been studied in detail in humanized NOD.SCID.γc^?/? immunodeficient mice. Here, we used a multiplex PCR on genomic DNA to quantify the combinatorial diversity of all possible V–J rearrangements at the TCR‐β chain and heavy chain Ig locus. We first show that the combinatorial diversity of the TCR‐β chain generated in the thymus was well preserved in the periphery, suggesting that human T cells were not vastly activated in mice, in agreement with phenotypic studies. We then show that the combinatorial diversity in NOD.SCID.γc^?/? mice reached 100% of human reference samples for both the TCR and the heavy chain of Ig. To document the functionality of this repertoire, we show that a detectable but weak HLA‐restricted cellular immune response could be elicited in reconstituted mice after immunization with an adenoviral vector expressing HCV envelope glycoproteins. Altogether, our results suggest that humanized mice express a diversified repertoire and are able to mount antigen‐specific immune responses.     

Long‐term human immune system reconstitution in non‐obese diabetic (NOD)‐Rag (–)‐γ chain (–) (NRG) mice is similar but not identical to the original stem cell donor

The murine immune system is not necessarily identical to it human counterpart, which has led to the construction of humanized mice. The current study analysed whether or not a human immune system contained within the non‐obese diabetic (NOD)‐Rag1^null‐γ chain^null (NRG) mouse model was an accurate representation of the original stem cell donor and if multiple mice constructed from the same donor were similar to one another. To that end, lightly irradiated NRG mice were injected intrahepatically on day 1 of life with purified cord blood‐derived CD34⁺ stem and progenitor cells. Multiple mice were constructed from each cord blood donor. Mice were analysed quarterly for changes in the immune system, and followed for periods up to 12 months post‐transplant. Mice from the same donor were compared directly with each other as well as with the original donor. Analyses were performed for immune reconstitution, including flow cytometry, T cell receptor (TCR) and B cell receptor (BCR) spectratyping. It was observed that NRG mice could be ‘humanized’ long‐term using cord blood stem cells, and that animals constructed from the same cord blood donor were nearly identical to one another, but quite different from the original stem cell donor immune system.     

A Hitchhiker's guide to humanized mice: new pathways to studying viral infections

Humanized mice are increasingly appreciated as an incredibly powerful platform for infectious disease research. The often very narrow species tropism of many viral infections, coupled with the sometimes misleading results from preclinical studies in animal models further emphasize the need for more predictive model systems based on human cells rather than surrogates. Humanized mice represent such a model and have been greatly enhanced with regards to their immune system reconstitution as well as immune functionality in the past years, resulting in their recommendation as a preclinical model by the US Food and Drug Administration. This review aims to give a detailed summary of the generation of human peripheral blood lymphocyte‐, CD34⁺ haematopoietic stem cell‐ and bone marrow/liver/thymus‐reconstituted mice and available improved models (e.g. myeloid‐ or T‐cell‐only mice, MISTRG, NSG‐SGM3). Additionally, we summarize human‐tropic viral infections, for which humanized mice offer a novel approach for the study of disease pathogenesis as well as future perspectives for their use in biomedical, drug and vaccine research.     

Attenuated immune control of Epstein–Barr virus in humanized mice is associated with the multiple sclerosis risk factor HLA-DR15

Immune responses to Epstein–Barr virus (EBV) infection synergize with the main genetic risk factor HLA-DRB1*15:01 (HLA-DR15) to increase the likelihood to develop the autoimmune disease multiple sclerosis (MS) at least sevenfold. In order to gain insights into this synergy, we investigated HLA-DR15 positive human immune compartments after reconstitution in immune-compromised mice (humanized mice) with and without EBV infection. We detected elevated activation of both CD4⁺ and CD8⁺ T cells in HLA-DR15 donor-reconstituted humanized mice at steady state, even when compared to immune compartments carrying HLA-DRB1*04:01 (HLA-DR4), which is associated with other autoimmune diseases. Increased CD8⁺ T cell expansion and activation was also observed in HLA-DR15 donor-reconstituted humanized mice after EBV infection. Despite this higher immune activation, EBV viral loads were less well controlled in the context of HLA-DR15. Indeed, HLA-DR15-restricted CD4⁺ T cell clones recognized EBV-transformed B cell lines less efficiently and demonstrated cross-reactivity toward allogeneic target cells and one MS autoantigen. These findings suggest that EBV as one of the main environmental risk factors and HLA-DR15 as the main genetic risk factor for MS synergize by priming hyperreactive T-cell compartments, which then control the viral infection less efficiently and contain cross-reactive CD4⁺ T cell clones.     

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.     

Functional engraftment of human peripheral T and B cells and sustained production of autoantibodies in NOD/LtSzscid/IL‐2Rγ−/− mice

NOD/LtSzscid/IL‐2Rγ^?/? (NSG) mice have advantages in establishing humanized mouse models. However, transferring human PBMCs into these mice often causes lethal GVH disease. In this study, we discovered an improved method for the engraftment of normal or pathological human PBMCs into NSG mice and examined the subsequent induction of specific immune responses. We sequentially transferred human CD4⁺ memory T (Tm) and B cells obtained from PBMCs of healthy adults or patients with autoimmune diseases into NSG mice. Removing naïve CD4⁺ T cells from the transferred PBMCs allowed successful engraftment without lethal GVH disease. The transferred Tm cells were found to reside mainly in the spleen and the lymphoid nodules, where they expressed MHC class II molecules and produced cytokines, including IL‐21. Surprisingly, the transferred B cells were also well maintained in the lymphoid organs, underwent de novo class‐switch recombination, and secreted all isotypes of human Igs at significant levels. Moreover, transferring patient‐derived Tm and B cells resulted in sustained production of IgM‐rheumatoid factor and antiaminoacyl transfer RNA synthetase Abs in these mice. These results suggest that transfer of Tm and B cells derived from human PBMCs into NSG mice could be a useful method for the study of human autoimmune mechanisms.     

Origin and ontogeny of lung macrophages: from mice to humans

Macrophages are tissue-resident myeloid cells with essential roles in host defense, tissue repair, and organ homeostasis. The lung harbors a large number of macrophages that reside in alveoli. As a result of their strategic location, alveolar macrophages are critical sentinels of healthy lung function and barrier immunity. They phagocytose inhaled material and initiate protective immune responses to pathogens, while preventing excessive inflammatory responses and tissue damage. Apart from alveolar macrophages, other macrophage populations are found in the lung and recent single-cell RNA-sequencing studies indicate that lung macrophage heterogeneity is greater than previously appreciated. The cellular origin and development of mouse lung macrophages has been extensively studied, but little is known about the ontogeny of their human counterparts, despite the importance of macrophages for lung health. In this context, humanized mice (mice with a human immune system) can give new insights into the biology of human lung macrophages by allowing in vivo studies that are not possible in humans. In particular, we have created humanized mouse models that support the development of human lung macrophages in vivo. In this review, we will discuss the heterogeneity, development, and homeostasis of lung macrophages. Moreover, we will highlight the impact of age, the microbiota, and pathogen exposure on lung macrophage function. Altered macrophage function has been implicated in respiratory infections as well as in common allergic and inflammatory lung diseases. Therefore, understanding the functional heterogeneity and ontogeny of lung macrophages should help to develop future macrophage-based therapies for important lung diseases in humans.     

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.     

Generation of hematopoietic humanized mice in the newborn BALB/c-Rag2null Il2rγnull mouse model: a multivariable optimization approach

Hematopoietic humanized mice generated via transplantation of human hematopoietic stem cells (hHSCs) into immunodeficient mice are a valuable tool for studying development and function of the human immune system. This study was performed to generate a protocol that improves development and quality of humanized mice in the BALB/c-Rag2(null)Il2rγ(null) strain, testing route of injection, in vitro culture and freezing of hHSCs, types of cytokines in the culture, and co-injection of lineage-depleted CD34(-) cells. Specific hHSC culturing conditions and the addition of support cells were found to increase the frequency, and human hematopoietic chimerism, of humanized mice. The optimized protocol resulted in BALB/c-Rag2(null)Il2rγ(null) humanized mice displaying more consistent human hematopoietic and lymphoid engraftment. Thus, hematopoietic humanized mice generated on a BALB/c immunodeficient background represent a useful model to study the human immune system.