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.     

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.     

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.

     

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.     

Delayed onset of graft‐versus‐host disease in immunodeficent human leucocyte antigen‐DQ8 transgenic,murine major histocompatibility complex class II‐deficient mice repopulated by human peripheral blood mononuclear cells

Haematopoietic humanization of mice is used frequently to study the human immune system and its reaction upon experimental intervention. Immunocompromised non‐obese diabetic (NOD)‐Rag1^–/– mice, additionally deficient for the common gamma chain of cytokine receptors (γc) (NOD‐Rag1^–/– γc^–/– mice), lack B, T and natural killer (NK) cells and allow for efficient human peripheral mononuclear cell (PBMC) engraftment. However, a major experimental drawback for studies using these mice is the rapid onset of graft‐versus‐host disease (GVHD). In order to elucidate the contribution of the xenogenic murine major histocompatibility complex (MHC) class II in this context, we generated immunodeficient mice expressing human MHC class II [human leucocyte antigen (HLA)‐DQ8] on a mouse class II‐deficient background (Aβ^–/–). We studied repopulation and onset of GVHD in these mouse strains following transplantation of DQ8 haplotype‐matched human PBMCs. The presence of HLA class II promoted the repopulation rates significantly in these mice. Virtually all the engrafted cells were CD3⁺ T cells. The presence of HLA class II did not advance B cell engraftment, such that humoral immune responses were undetectable. However, the overall survival of DQ8‐expressing mice was prolonged significantly compared to mice expressing mouse MHC class II molecules, and correlated with an increased time span until onset of GVHD. Our data thus demonstrate that this new mouse strain is useful to study GVHD, and the prolonged animal survival and engraftment rates make it superior for experimental intervention following PBMC engraftment.     

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.     

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.     

Anti‐PR3 immune responses induce segmental and necrotizing glomerulonephritis

Wegener's granulomatosis (WG) is a life‐threatening autoimmune vasculitis that affects lungs, kidneys and other organs. A hallmark of WG is the presence of classic anti‐neutrophil cytoplasmic antibodies (c‐ANCA) against self‐proteinase 3 (PR3). Little is known about the role of these antibodies and PR3‐specific immune responses in disease development. In this study, we demonstrate that PR3‐specific autoimmune responses are pathogenic in non‐obese diabetic (NOD) mice with an impaired regulatory arm of the immune response. Immunization of autoimmunity prone NOD mice with rmPR3 (recombinant mouse PR3) in complete Freund's adjuvant (CFA) resulted in high levels of c‐ANCA, without detectable disease development. However, when splenocytes from these immunized mice were transferred into immunodeficient NOD–severe combined immunodeficiency (SCID) mice, the recipient mice developed vasculitis and severe segmental and necrotizing glomerulonephritis. No disease developed in NOD–SCID mice that received splenocytes from the CFA‐alone‐immunized donors (controls), indicating that disease development depends upon PR3‐specific immune responses. In contrast to the pathology observed in NOD–SCID mice, no disease was observed when splenocytes from rmPR3‐immunized C57BL/6 mice were transferred into immunodeficient C57BL/6‐RAG‐1^–/– mice, suggesting that complex and probably multi‐genetic factors play a role in the regulation of disease development.     

人源化小鼠异位肺癌动物模型的构建

目的：探讨一种可用于研究肺癌免疫的人源化小鼠动物模型。方法：采用人外周血单个核细胞输注非肥胖糖尿病/严重联合免疫缺陷小鼠体内建立人源化小鼠,在此小鼠模型基础上再构建异位肺癌荷瘤模型。结果：10只人源化小鼠异位荷瘤模型全部构建成功,免疫重构的小鼠外周血及脾脏中均可检测到大量人CD3＋T、CD4＋T、CD8＋T细胞。人源化荷瘤小鼠肿瘤组织内可发现人CD3＋T、CD4＋T、CD8＋T淋巴细胞浸润。结论：该模型为我们了解肺癌发生发展与免疫系统的关系,也为研究免疫治疗干预措施等提供了有价值的工具。     

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.     

Human T cell development in the liver of humanized NOD/SCID/IL-2Rγ(null)(NSG) mice generated by intrahepatic injection of CD34(+) human (h) cord blood (CB) cells

In this study, we explore the possibility of human T cell development in the liver of humanized mice generated by intrahepatic injection of CD34(+) hCB cells into conditioned NOD/SCID/IL-2Rγ(null)(NSG) newborn mice. The intrahepatic injection of CD34(+) hCB cells led to effective reconstitution of human myeloid and lymphoid lineage cells. In contrast to the previously reported Rag2(-/-)γ(c)(-/-) humanized mice, interestingly, the thymus function of humanized NSG mice was markedly reduced in terms of its size and cell contents, whereas the livers of humanized NSG mice profoundly contained double-positive (DP), hCD4 and hCD8 single positive (SP), hCD34(+)hCD38(lo)hCD1a(-) (TSP), hCD34(+)hCD38(hi)hCD1a(-) (ETP), and hCD34(+)hCD38(+)hCD1a(+) (pre-T cells) cells. Furthermore, immunostaining of the liver revealed that human T cells were co-localized with hDCs. Taken together, our results demonstrate that the intrahepatic injection of hCD34(+) hCB cells can facilitate human T cell development in the livers of humanized NSG mice.     

Human immune system development and survival of non‐obese diabetic (NOD)‐scid IL2rγnull (NSG) mice engrafted with human thymus and autologous haematopoietic stem cells

Immunodeficient mice bearing targeted mutations in the IL2rg gene and engrafted with human immune systems are effective tools for the study of human haematopoiesis, immunity, infectious disease and transplantation biology. The most robust human immune model is generated by implantation of human fetal thymic and liver tissues in irradiated recipients followed by intravenous injection of autologous fetal liver haematopoietic stem cells [often referred to as the BLT (bone marrow, liver, thymus) model]. To evaluate the non‐obese diabetic (NOD)‐scid IL2rγ^null (NSG)–BLT model, we have assessed various engraftment parameters and how these parameters influence the longevity of NSG–BLT mice. We observed that irradiation and subrenal capsule implantation of thymus/liver fragments was optimal for generating human immune systems. However, after 4 months, a high number of NSG–BLT mice develop a fatal graft‐versus‐host disease (GVHD)‐like syndrome, which correlates with the activation of human T cells and increased levels of human immunoglobulin (Ig). Onset of GVHD was not delayed in NSG mice lacking murine major histocompatibility complex (MHC) classes I or II and was not associated with a loss of human regulatory T cells or absence of intrathymic cells of mouse origin (mouse CD45⁺). Our findings demonstrate that NSG–BLT mice develop robust human immune systems, but that the experimental window for these mice may be limited by the development of GVHD‐like pathological changes.     

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.     

An improved model to study tumor cell autonomous metastasis programs using MTLn3 cells and the Rag2<Superscript>−/−</Superscript> γc<Superscript>−/−</Superscript> mouse

The occurrence of metastases is a critical determinant of the prognosis for breast cancer patients. Effective treatment of breast cancer metastases is hampered by a poor understanding of the mechanisms involved in the formation of these secondary tumor deposits. To study the processes of metastasis, valid in vivo tumor metastasis models are required. Here, we show that increased expression of the EGF receptor in the MTLn3 rat mammary tumor cell-line is essential for efficient lung metastasis formation in the Rag mouse model. EGFR expression resulted in delayed orthotopic tumor growth but at the same time strongly enhanced intravasation and lung metastasis. Previously, we demonstrated the critical role of NK cells in a lung metastasis model using MTLn3 cells in syngenic F344 rats. However, this model is incompatible with human EGFR. Using the highly metastatic EGFR-overexpressing MTLn3 cell-line, we report that only Rag2^−/−γc^−/− mice, which lack NK cells, allow efficient lung metastasis from primary tumors in the mammary gland. In contrast, in nude and SCID mice, the remaining innate immune cells reduce MTLn3 lung metastasis formation. Furthermore, we confirm this finding with the orthotopic transplantation of the 4T1 mouse mammary tumor cell-line. Thus, we have established an improved in vivo model using a Rag2^−/− γc^−/− mouse strain together with MTLn3 cells that have increased levels of the EGF receptor, which enables us to study EGFR-dependent tumor cell autonomous mechanisms underlying lung metastasis formation. This improved model can be used for drug target validation and development of new therapeutic strategies against breast cancer metastasis formation.     

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.     

ORIGINAL ARTICLE: Development of Human Graafian Follicles Following Transplantation of Human Ovarian Tissue into NOD/SCID/γcnull Mice

Problem Transplantation of human ovarian cortex into host mice may permit various kinds of challenges in reproductive medicine. A novel immunodeficient mouse strain (NOD/SCID/γcnull: NOG) has been developed as a host of transplantation of human tissue. Method of study Human ovarian cortex was transplanted into various sites of NOG mice and human follicular development was examined by immunohistochemistry. Results Transplantation of human ovarian tissue into NOG mice resulted in approximately similar tissue survival and follicle growth as did transplantation into non‐obese diabetic‐severe combined immunodeficient mice. The human Graafian follicule from NOG mouse expressed the same steroidogenic enzymes as observed in human Graafian follicles, which developed in the human body. The NOG mice’s ovarian bursa was better placed for transplantation than the back skin or kidney capsule. Conclusion These results represent the successful generation and biological confirmation of the human Graafian follicles from the human ovarian cortex in the NOG mice.     

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.     

Histopathological characteristics of human non-tumor thyroid tissues in a long-term model of adenomatous goiter xenografts in the NOD/Shi-scid,IL-2Rγnull mouse

There is a growing need for modeling the human thyroid to link data obtained from animals to humans because of its sensitivity to radiation exposure and endocrine disruption chemicals. In a scid mouse model produced by transplanting human thyroid tissues, leakiness and thymic lymphoma that occurs spontaneously in the scid mouse can complicate the interpretation of experimental results. Considering that the NOD.Cg-Prkdc^scid Il2rg^tm1Sug/Jic mouse (NOD/Shi-scid, IL-2Rγ^null or NOG mouse) may be a better host because this strain has low incidence of leakiness and thymic lymphoma, we have evaluated the potential of a model that allows long-term observation of non-tumor human thyroid tissues in this mouse. We transplanted tissues of human adenomatous goiter into NOG mice and examined the tissues histopathologically. The morphology of human adenomatous goiter tissues was maintained from 24 to 44 weeks after transplantation in NOG mice with no noted differences between donor-matched tissues or the weeks after transplantation. The tissues expressed thyroglobulin protein and mRNA as well as thyroperoxidase. Endothelial cells originating from human were found in the transplanted tissues and were thought to be a characteristic of this model. The intactness of the tissues before transplantation was found to affect the rate of tissue engraftment. From the present results we have concluded that transplanted thyroid tissues in NOG mice maintain the histopathological characteristics of their origin for long terms. Therefore this model was thought feasible for toxicity evaluation.