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.     

A model for personalized in vivo analysis of human immune responsiveness

Studies of human immune diseases are generally limited to the analysis of peripheral blood lymphocytes of heterogeneous patient populations. Improved models are needed to allow analysis of fundamental immunologic abnormalities predisposing to disease and in which to assess immunotherapies. Immunodeficient mice receiving human fetal thymus grafts and fetal CD34(+) cells intravenously produce robust human immune systems, allowing analysis of human T cell development and function. However, to use humanized mice to study human immune-mediated disorders, immune systems must be generated from adult hematopoietic cells. Here, we demonstrated robust immune reconstitution in mice with hematopoietic stem cells (HSCs) aspirated from bone marrow of adults with type 1 diabetes (T1D) and healthy control volunteers. In these humanized mice, cryopreservation of human leukocyte antigen allele-matched fetal thymic tissue prevented allogeneic adult HSC rejection. Newly generated T cells, which included regulatory T cells (T(regs)), were functional and self-tolerant and had a diverse repertoire. The immune recognition of these mice mimicked that of the adult CD34(+) cell donor, but the T cell phenotypes were more predominantly "na?ve" than those of the adult donors. HSCs from T1D and control donors generated similar numbers of natural T(regs) intrathymically; however, peripheral T cells from T1D subjects showed increased proportions of activated or memory cells compared to controls, suggesting possible HSC-intrinsic differences in T cell homeostasis that might underlie immune pathology in T1D. This "personalized immune" mouse provides a new model for individualized analysis of human immune responses that may provide new insights into not only T1D but also other forms of immune function and dysfunction as well.     

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.     

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.

     

Alloreactivity but Failure to Reject Human Islet Transplants by Humanized Balb/c/Rag2−/−gc−/− Mice

A human islet transplant can cure patients with type 1 diabetes. A drawback of islet transplantation is the life‐long immunosuppressive medication, often associated with severe side effects. Moreover, in spite of the immunosuppressive therapy, islets are lost in the majority of transplanted patients over time. An improved small animal model for studies on human islet allograft rejection mechanisms and the development of new measures for its prevention is clearly warranted. Here, we evaluated the potential of Balb/cRag2^?/?γc^?/? mice carrying a human‐like immune system (so‐called humanized mice) as a tool for studies on the rejection of transplanted human islets. Human T cells from Balb/cRag2^?/?γc^?/? mice, which as neonates had been transplanted with CD34⁺ human cord blood haematopoietic stem cells (HIS mice), proliferated in response to allogeneic human dendritic cells, but failed to reject a human islet allograft transplanted to the renal subcapsular space as assessed by immunohistochemistry and analysis of human serum C‐peptide levels. Histological analysis revealed that few if any T cells had migrated to the grafted tissue. These observations question the usefulness of the HIS mouse model for studies on human islet allograft rejection mechanisms and highlight the need for further improvements.     

Rapid evolution of HIV-1 to functional CD8⁺ T cell responses in humanized BLT mice

The development of mouse/human chimeras through the engraftment of human immune cells and tissues into immunodeficient mice, including the recently described humanized BLT (bone marrow, liver, thymus) mouse model, holds great promise to facilitate the in vivo study of human immune responses. However, little data exist regarding the extent to which cellular immune responses in humanized mice accurately reflect those seen in humans. We infected humanized BLT mice with HIV-1 as a model pathogen and characterized HIV-1-specific immune responses and viral evolution during the acute phase of infection. HIV-1-specific CD8(+) T cell responses in these mice were found to closely resemble those in humans in terms of their specificity, kinetics, and immunodominance. Viral sequence evolution also revealed rapid and highly reproducible escape from these responses, mirroring the adaptations to host immune pressures observed during natural HIV-1 infection. Moreover, mice expressing the protective HLA-B*57 allele exhibited enhanced control of viral replication and restricted the same CD8(+) T cell responses to conserved regions of HIV-1 Gag that are critical to its control of HIV-1 in humans. These data reveal that the humanized BLT mouse model appears to accurately recapitulate human pathogen-specific cellular immunity and the fundamental immunological mechanisms required to control a model human pathogen, aspects critical to the use of a small-animal model for human pathogens.     

Development of a new humanized mouse model to study acute inflammatory arthritis

ABSTRACT: BACKGROUND: Substantial advances have been generated in understanding the pathogenesis of rheumatoid arthritis (RA). Current murine models of RA-like disease have provided great insights into the molecular mechanism of inflammatory arthritis due to the use of genetically deficient or transgenic mice. However, these studies are limited by differences that exist between human and murine immune systems. Thus, the development of an animal model that utilizes human immune cells, will afford the opportunity to study their function in the initiation and propagation of inflammatory arthritis. METHODS: One to two-day old irradiated NOD-scid IL2rgammanull (NSG) mice were reconstituted with human CD34+ cord blood stem cells. Leukocytes were analyzed by flow cytometry and circulating antibodies were determined by ELISA. Arthritis was induced by injecting complete Freund's adjuvant into knee or ankle joints. Mice were also treated with the TNF inhibitor, Etanercept, or PBS and joints were analyzed histologically. RESULTS: Humanized mice were established with high reconstitution rates and were able to spontaneously produce human immunoglobulins as well as specific IgG in response to immunization. Intraperitoneal injection of thioglycolate or injection of complete Freund's adjuvant into joints resulted in migration of human immune cells to the injected sites. Arthritic humanized mice treated with Etanercept had markedly less inflammation, which was associated with decreased total numbers of human CD45+ cells, including human lymphocytes and neutrophils. CONCLUSIONS: The humanized mouse model is a new model to study inflammatory arthritis disease using human leukocytes without rejection of engrafted tissue. Future studies may adapt this system to incorporate RA patient cord blood and develop a chimeric animal model of inflammatory arthritis using genetically predisposed immune cells.     

Regulatory T cells: customizing for the clinic

Immune-suppressive cellular immunotherapy requires large numbers of antigen-specific regulatory T cells (T(reg) cells), lymphocytes that suppress certain immune responses. Together, three papers in this issue of Science Translational Medicine describe protocols for the ex vivo expansion of human T(reg) cells and assess the immune-suppressive function of ex vivo-manipulated T(reg) cells after transfer into humanized mouse disease models. Along with recent phase I clinical trial results, these new data provide a platform for clinical use of T(reg) cells as personalized therapeutic agents for the treatment of autoimmune diseases, graft-versus-host disease, and transplant rejection.     

人源化小鼠及其在病毒感染研究中的应用

人源化小鼠是指将人体细胞或组织移植入免疫缺陷小鼠,即保证不会产生免疫排斥,又可保证移植入的人体细胞/组织在小鼠体内作为合适的基质,适用于人体病原的感染及增殖,相较于普通动物模型可以获得更接近人体的较为可信的实验数据.目前人源化小鼠已经在病毒感染机制、诱导的免疫应答、抗病毒药物筛选及疫苗研发、基因治疗等方面的研究中获得应用,尤其对一些高度宿主特异性的病毒如HIV、HCV、DENV具有重大的研究价值.     

Humanized mouse models of HIV infection

Because of the limited tropism of HIV, in vivo modeling of this virus has been almost exclusively limited to other lentiviruses, such as simian immunodeficiency virus, that reproduce many important characteristics of HIV infection. However, there are significant genetic and biological differences among lentiviruses and some HIV-specific interventions are not effective against other lentiviruses in nonhuman hosts. For these reasons, much emphasis has recently been placed on developing alternative animal models that support HIV replication and recapitulate key aspects of HIV infection and pathogenesis in humans. Humanized mice, CD34+ hematopoietic progenitor cell transplanted immunodeficient mice, and in particular mice also implanted with human thymus/liver tissue (bone marrow liver thymus mice) that develop a functional human immune system, have been the focus of a great deal of attention as possible models to study virtually all aspects of HIV biology and pathogenesis. Humanized mice are systemically reconstituted with human lymphoid cells, offering rapid, reliable, and reproducible experimental systems for HIV research. Peripheral blood of humanized mice can be readily sampled longitudinally to assess reconstitution with human cells and to monitor HIV replication, permitting the evaluation of multiple parameters of HIV infection such as viral load levels, CD4+ T-cell depletion, immune activation, as well as the effects of therapeutic interventions. Of high relevance to HIV transmission is the extensive characterization and validation of the reconstitution with human lymphoid cells of the female reproductive tract and of the gastrointestinal tract of humanized bone marrow liver thymus mice that renders them susceptible to both vaginal and rectal HIV infection. Other important attributes of all types of humanized mice include: (i) their small size and cost that make them widely accessible; (ii) multiple cohorts of humanized mice can be made from multiple human donors and each cohort has identical human cells, permitting control of intragenetic variables; (iii) continuous de novo production of human immune cells from the transplanted CD34+ cells within each humanized mouse facilitates long-term experiments; (iv) both primary and laboratory HIV isolates can be used for experiments; and (v) in addition to therapeutic interventions, rectal and vaginal HIV prevention approaches can be studied. In summary, humanized mice can have an important role in virtually all aspects of HIV research, including the analysis of HIV replication, the evaluation of HIV restriction factors, the characterization of successful biomedical HIV prevention strategies, the evaluation of new treatment regimens, and the evaluation of novel HIV eradication strategies.     

Antibody-functionalized peptidic membranes for neutralization of allogeneic skin antigen-presenting cells

We report herein application of an in situ material strategy to attenuate allograft T cell responses in a skin transplant mouse model. Functionalized peptidic membranes were used to impede trafficking of donor antigen-presenting cells (dAPCs) from skin allografts in recipient mice. Membranes formed by self-assembling peptides (SAPs) presenting antibodies were found to remain underneath grafted skins for up to 6 days. At the host–graft interface, dAPCs were targeted by using a monoclonal antibody that binds to a class II major histocompatibility complex (MHC) molecule (I-A^d) expressed exclusively by donor cells. Using a novel cell labeling near-infrared nanoemulsion, we found more dAPCs remained in allografts treated with membranes loaded with anti-I-A^d antibodies than without. In vitro, dAPCs released from skin explants were found adsorbed preferentially on anti-I-A^d antibody-loaded membranes. Recipient T cells from these mice produced lower concentrations of interferon-gamma cultured ex vivo with donor cells. Taken together, the data indicate that the strategy has the potential to alter the natural course of rejection immune mechanisms in allogeneic transplant models.     

CD4+ helper T cells are required for resistance to a highly metastatic murine tumor

A role of CD4+ T helper cells in induction of tumor transplant rejection leading to complete regression of a highly metastatic DBA/2 mouse lymphoma was analyzed. Using an anti-CD4 monoclonal antibody (GK1.5) which eliminates T helper cells in vivo and in vitro, we found that CD4+ cells are required for tumor resistance in syngeneic DBA/2 mice or allogeneic but major histocompatibility complex-identical B10.D2 mice. In contrast, in allogeneic C57BL/6 mice tumor rejection was independent of CD4+ cells. An analogous requirement for immune CD4+ cells for in vitro induction of CD8+ tumor-specific cytotoxic T cells was found in these respective strains. The requirement for immune CD4+ cells in vitro could be replaced by recombinant interleukin 2. These results demonstrate a role of CD4+ regulatory T cells and T-T cell cooperation in the induction of anti-tumor immunity and tumor rejection, and point to possible therapeutic interventions in the afferent phase of anti-tumor immune responses.     

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.     

Lung transplant acceptance is facilitated by early events in the graft and is associated with lymphoid neogenesis

Early immune responses are important in shaping long-term outcomes of human lung transplants. To examine the role of early immune responses in lung rejection and acceptance, we developed a method to retransplant mouse lungs. Retransplantation into T-cell-deficient hosts showed that for lungs and hearts alloimmune responses occurring within 72 h of transplantation are reversible. In contrast to hearts, a 72-h period of immunosuppression with costimulation blockade in primary allogeneic recipients suffices to prevent rejection of lungs upon retransplantation into untreated allogeneic hosts. Long-term lung acceptance is associated with induction of bronchus-associated lymphoid tissue, where Foxp3⁺ cells accumulate and recipient T cells interact with CD11c⁺ dendritic cells. Acceptance of retransplanted lung allografts is abrogated by treatment of immunosuppressed primary recipients with anti-CD25 antibodies. Thus, events contributing to lung transplant acceptance are established early in the graft and induction of bronchus-associated lymphoid tissue can be associated with an immune quiescent state.     

Human regulatory T cells with alloantigen specificity are more potent inhibitors of alloimmune skin graft damage than polyclonal regulatory T cells

Graft rejection by the immune system is a major cause of transplant failure. Lifelong immunosuppression decreases the incidence of graft rejection; however, nonspecific immunosuppression results in increased susceptibly to infection and cancer. Regulatory T cells (T(regs)), which suppress the activation of the immune system and induce tolerance, are currently under evaluation for use in clinical transplantation. Ex vivo expanded polyclonal T(regs) that are introduced into transplant recipients alter the balance of T effector cells to T(regs); however, experimental data suggest that alloantigen-specific T(regs) would be more effective at preventing graft rejection. We have developed a method to enrich alloantigen-specific human T(regs) based on the coexpression of activation markers, CD69 and CD71. These T(regs) could be readily expanded in vitro and demonstrated potent antigen-specific suppression. In a humanized mouse model of alloimmune-mediated injury of human skin grafts, alloantigen-specific T(regs) resulted in a significant reduction in clinically relevant indicators of dermal tissue injury when compared with polyclonal T(regs), restoring a histology comparable to healthy skin. This method of human allospecific T(reg) selection should be scalable to the clinic. The improved in vivo efficacy of alloantigen-specific T(regs) over polyclonal T(regs) shown here suggests that generating "customized" T(regs) with defined anti-donor allospecificities may improve current practice in clinical immunotherapy.     

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.     

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.     

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.     

IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice

Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common γ-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common γ-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID γ-chain(-/-) (NSG); and Rag2(-/-) γ-chain(-/-) (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34(+) cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common γ-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.     

Fetal islet xenotransplantation in rodents and primates

β cell replacement in IDDM by transplantation of either isolated adult islets of Langerhans or of proliferating immature islet tissue from fetal pancreas are potential ways of curing this disease. Because of the dearth of human cadaver donors adult allogeneic islets are scarce and in most Western societies availability of human fetal tissue of suitable maturity is also uncommon. The use of xenogeneic islets from domestic species already widely used for human consumption, e.g. pigs, could overcome this scarcity but xenogeneic tissues are faced with major problems of graft rejection. Hyperacute rejection (HAR) is the main cause of destruction of immediately vascularised xenografts and is caused by the interaction of natural cross-reactive antibodies with donor endothelial cells. Neovascularized islet grafts do not have donor EC as the target for HAR and are not subjected to this problem but are still acutely rejected. The mechanism of this destruction is still poorly understood but is clearly T cell dependent. However, current immunosuppression that is usually adequate for control of allograft rejection generally does not prevent xenograft rejection. A better understanding of the ways in which xenoantigens are recognised and of the nature of the immune response they initiate is fundamental to the development of appropriate strategies for the safe and effective control of xenograft rejection. The studies summarized herein describe the response of mice and primates to a challenge with fetal pig pancreas grafts. The rejection response that develops is different from that seen against a challenge with fetal allogeneic islets. Although the xenograft response is highly T cell dependent the actual effectors of graft damage appear to be different from those that provoke allograft destruction and include macrophages and granulocytes, particularly eosinophils, and possibly non-classical T cells.