Current humanized mouse models for studying human immunology and HIV-1 immuno-pathogenesis

A robust animal model for “hypothesis-testing/mechanistic” research in human immunology and immuno-pathology should meet the following criteria. First, it has well-studied hemato-lymphoid organs and target cells similar to those of humans. Second, the human pathogens establish infection and lead to relevant diseases. Third, it is genetically inbred and can be manipulated via genetic, immunological and pharmacological means. Many human-tropic pathogens such as HIV-1 fail to infect murine cells due to the blocks at multiple steps of their life cycle. The mouse with a reconstituted human immune system and other human target organs is a good candidate. A number of human-mouse chimeric models with human immune cells have been developed in the past 20 years, but most with only limited success due to the selective engraftment of xeno-reactive human T cells in hu-PBL-SCID mice or the lack of significant human immune responses in the SCID-hu Thy/Liv mouse. This review summarizes the current understanding of HIV-1 immuno-pathogenesis in human patients and in SIV-infected primate models. It also reviews the recent progress in the development of humanized mouse models with a functional human immune system, especially the recent progress in the immunodeficient mice that carry a defective gammaC gene. NOD/SCID/gammaC^−/− (NOG or NSG) or the Rag2^−/−gammaC^−/− double knockout (DKO) mice, which lack NK as well as T and B cells (NTB-null mice), have been used to reconstitute a functional human immune system in central and peripheral lymphoid organs with human CD34⁺ HSC. These NTB-hu HSC humanized models have been used to investigate HIV-1 infection, immuno-pathogenesis and therapeutic interventions. Such models, with further improvements, will contribute to study human immunology, human-tropic pathogens as well as human stem cell biology in the tissue development and function in vivo.     

SCID-hu小鼠：HIV研究的小型动物模型

长期以来缺乏病毒体内感染的小动物模型是制约HIV-1研究取得突破性进展的一大障碍。研究者将胎儿的胸腺和胎肝组织移植到重症联合免疫缺陷(SCID)小鼠体内,构建了SCID-hu(Thy／Liv)人鼠嵌合模型。该模型具备正常功能性的人造血器官“Thy／Liv”,较真实地模拟了HIV-1感染人胸腺后的状况,是研究HIV-1体内感染较成功且很有潜力的嵌合鼠模型。SCID-hu(Thy／Liv)模型的构建使得在小型动物体内研究HIV的某些致病机制、临床前评价各种先导药物的体内抗HIV活性、评价新的治疗方案及寻求合适的基因治疗等成为可能,为在体内研究人造血系统和免疫系统的病理生理机能及人干细胞基因治疗提供了有力的工具,有广泛的应用前景。     

Preparation and Maintenance of SCID-hu Mice for HIV Research

The SCID-hu mouse bearing a functional human thymic implant can be easily infected with HIV. Infection results in virus replication and relatively rapid depletion of CD4⁺human thymocytes, resulting in a pathologic profile similar to that seen in the thymus of HIV-infected humans. The use of the SCID-hu model for HIV research requires protection of the animals from opportunistic infections and protection of the operators from human pathogens. This discussion describes reliable methods of animal care and surgical procedures to meet these needs.     

IFN-α-Induced Upregulation of CCR5 Leads to Expanded HIV Tropism In Vivo

Chronic immune activation and inflammation (e.g., as manifest by production of type I interferons) are major determinants of disease progression in primate lentivirus infections. To investigate the impact of such activation on intrathymic T-cell production, we studied infection of the human thymus implants of SCID-hu Thy/Liv mice with X4 and R5 HIV. X4 HIV was observed to infect CD3⁻CD4⁺CD8⁻CXCR4⁺CCR5⁻ intrathymic T-cell progenitors (ITTP) and to abrogate thymopoiesis. R5 HIV, by contrast, first established a nonpathogenic infection of thymic macrophages and then, after many weeks, began to replicate in ITTP. We demonstrate here that the tropism of R5 HIV is expanded and pathogenicity enhanced by upregulation of CCR5 on these key T-cell progenitors. Such CCR5 induction was mediated by interferon-α (IFN-α) in both thymic organ cultures and in SCID-hu mice, and antibody neutralization of IFN-α in R5 HIV-infected SCID-hu mice inhibited both CCR5 upregulation and infection of the T-cell progenitors. These observations suggest a mechanism by which IFN-α production may paradoxically expand the tropism of R5 HIV and, in so doing, accelerate disease progression.     

Surgical Methods for Full-Thickness Skin Grafts to Induce Alopecia Areata in C3H/HeJ Mice

Alopecia areata is a cell-mediated autoimmune disease of humans and many domestic and laboratory animal species. C3H/HeJ inbred mice spontaneously develop alopecia areata at a low frequency (approximately 20% by 12 mo of age). Transferring full-thickness skin grafts from affected, older mice to young mice of the same strain reliably reproduces alopecia areata, thus enabling investigators to study disease pathogenesis or intervention with a variety of therapeutic approaches. We here describe in detail how to perform full-thickness skin grafts and the follow-up procedures necessary to consistently generate mice with alopecia areata. These engrafted mice can be used to study the pathogenesis of cell-mediated autoimmune disease and for drug-efficacy trials. This standard protocol can be used for many other purposes when studying abnormal skin phenotypes in laboratory mice.Abbreviations: AA, alopecia areata; DEBR, Dundee experimental bald ratAlopecia areata (AA) is a nonscarring, cell-mediated, autoimmune disease that causes hair loss in humans. At any time, between 0.05% and 0.1% of people express some form of the disease.^3,19,20 Hair loss has been characterized as patchy (alopecia areata), total loss on the top of the head (alopecia totalis), or total loss of all body hair (alopecia universalis). Progress in understanding the pathogenesis and genetics of AA as well as the means to develop and test new therapies was severely hampered until the development of a spontaneous mouse (C3H/HeJ) disease model that very closely mimics the adult-onset form of AA.^23,26 In addition to the laboratory mouse, several other species have been proposed as models for AA, but most are poorly characterized or not readily available. These include hair loss syndromes in dogs, cats, horses, cattle, and nonhuman primates and even a feather-loss syndrome in chickens.¹¹ The Dundee experimental bald rat (DEBR) also has many features of AA.^15-17C3H/HeJ mice develop a spontaneous, complex polygenic, AA-like hair loss.^25,30 Mouse AA undergoes stages of waxing and waning in terms of clinically evident areas of alopecia, and the extent of alopecia varies greatly between subjects, thus complicating the use of these spontaneous models as drug-screening tools. Full-thickness skin grafts initially were used as a tool to decipher whether the inflammation seen histologically was driving the skin lesions or whether the skin abnormalities caused changes that resulted in localized, chronic inflammation.¹⁰ To this end, we grafted affected skin to severely immunodeficient (Prkdc^scid) mutant mice congenic on the C3H/HeJ background and to histocompatible C3H/HeJ mice of the same sex as the donor. We found that full-thickness skin grafts could be used to initiate AA in histocompatible recipients in a controlled and predictable manner. Hair regrew in the immunodeficient mice, but it regrew white rather than agouti,¹⁰ a feature also seen in human AA and in injured mouse skin because of damage to melanocyte stem cells.¹³ Both the spontaneous and graft-induced forms of this mouse model have been used extensively to test hypotheses regarding disease mechanisms and responses to various treatments and to refute the association of AA with suspected infectious or antigenic challenges.^5,9,22,27 This graft-initiated mouse model is now readily available as individual mice or for contract drug-efficacy trials (The Jackson Laboratory, West Sacramento, CA; http://jaxmice.jax.org/services/alopecia_areata.html;http://jaxmice.jax.org/library/notes/504/504b.html).We here describe how to perform full-thickness skin grafts in mice, to enable investigators to reliably reproduce this AA model system in their own laboratories.     

Practical Murine Hematopathology: A Comparative Review and Implications for Research

Hematologic parameters are important markers of disease in human and veterinary medicine. Biomedical research has benefited from mouse models that recapitulate such disease, thus expanding knowledge of pathogenetic mechanisms and investigative therapies that translate across species. Mice in health have many notable hematologic differences from humans and other veterinary species, including smaller erythrocytes, higher percentage of circulating reticulocytes or polychromasia, lower peripheral blood neutrophil and higher peripheral blood and bone marrow lymphocyte percentages, variable leukocyte morphologies, physiologic splenic hematopoiesis and iron storage, and more numerous and shorter-lived erythrocytes and platelets. For accurate and complete hematologic analyses of disease and response to investigative therapeutic interventions, these differences and the unique features of murine hematopathology must be understood. Here we review murine hematology and hematopathology for practical application to translational investigation.Abbreviations: GEM, genetically engineered mouse; NMB, new methylene blue; nRBC, nucleated RBC; RDW, RBC distribution width; TNCC, total nucleated cell countHematology is an important adjunct to both clinical medicine and biomedical research, with more than 1700 currently funded NIH projects¹⁰⁹ and more than 3400 research articles published over the past 5 years using mouse models.¹²⁰ There are now more than 6000 genetically engineered mouse (GEM) models of disease, with 500 new GEM created each year at the Jackson Laboratory alone, and several large projects are underway to thoroughly phenotype each new mutant mouse strain (https://www.komp.org/).^13,176 A mouse tumor database (http://tumor.informatics.jax.org/mtbwi/index.do) is available to provide information regarding mouse models of human cancer, and the Mouse Phenome Database at the Jackson Laboratory provides links to phenotypic data for many GEM models (http://phenome.jax.org/).⁸ The defined components to complete the phenotyping of GEM models have been recently reviewed.^13,157,176 In addition, 21 inbred strains of mice are commonly used for investigations into such topics as response to infectious and genetically induced disease and dietary and pharmacologic therapies. These commonly used laboratory mouse strains have, for example, inherent differences in immunology or iron trafficking, which can affect research outcomes.^16,47,137 These interstrain differences are important to recognize and understand as a component of effective study design and prior to strain selection for laboratory investigations, especially when hematologic responses to disease need to be considered.^13,16,137For any appropriately designed experiment, concurrent age-, sex-, and strain-matched control mice must be included to accurately compare the effects of a disease, genetic manipulation or therapeutic intervention;^13,155 alternatively, individual mice can be used as their own controls in some studies. Several important guidelines exist to ensure that appropriate numbers of experimental and control mice are incorporated into a study design to maximize statistical power yet minimize waste.^{13,40-42,71,72,176} During and between studies, consistent blood collection methods are essential for accurate comparative analyses. Species-appropriate hematologic instrumentation and timely analysis of fresh blood are necessary to minimize preanalytic hematologic errors.^3,37,71 Especially important for mice and their restricted available blood volume are the use of practical, accurate, species-specific, and up-to-date hematologic methods.Here we comprehensively review murine hematology and hematopathologic responses to disease in the context of biomedical research, discovery, and phenotyping studies. To maximize the opportunity for detecting phenotypes, disease, and responses to therapeutic interventions in mice, we focus on providing a practical summary of methods and analysis for accurate hematologic studies and on describing the morphologic assessment of mouse hematopathology in peripheral blood and bone marrow in ways that will be useful to those—veterinarians and researchers alike—who work with murine species.     

Cell Population Analyses During Skin Carcinogenesis

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2^YFP mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER⁺ and R26-SmoM2^YFP+ mice in this protocol); 2) inducing SmoM2^YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.     

Proliferation of functional human natural killer cells with anti‐HIV‐1 activity in NOD/SCID/Jak3null mice

Natural killer cells, a critical component of the innate immune system, eradicate both virus‐infected cells and tumor cells through cytotoxicity and secretion of cytokines. Human NK cell research has largely been based on in vitro studies because of the lack of appropriate animal models. In this study, a selective proliferation model of functional human NK cells was established in NOD/SCID/Jak3^null (NOJ) mice transplanted with peripheral blood mononuclear cells (PBMC) and K562 cells. The antiviral effects of NK cells were evaluated by challenging this mouse model with HIV‐1. The percentage of intracellular p24⁺ T cells and the amount of plasma p24 was decreased compared with NOJ mice transplanted with PBMC. Our findings indicate that NK cells have an anti‐HIV‐1 effect through direct cytotoxicity against HIV‐1‐infected cells. These mice provide an important model for evaluating human NK function against human infectious diseases such as HIV‐1 and malignancies.     

Impact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease

Background aimsGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects.MethodsThe ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed.ResultsInjection of 200 × 10⁶ human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM₁ antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 10⁶ MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 10⁶ human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 10⁶ MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs.ConclusionsInjection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models.     

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures,Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability ({"type":"clinical-trial","attrs":{"text":"NCT01151124","term_id":"NCT01151124"}}NCT01151124 and {"type":"clinical-trial","attrs":{"text":"NCT02117635","term_id":"NCT02117635"}}NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycER^TAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3’UTRs by dual reporter plasmid transfection and dual luciferase assay.     

Further improvements of the P. falciparum humanized mouse model

Background

It has been shown previously that it is possible to obtain growth of Plasmodium falciparum in human erythrocytes grafted in mice lacking adaptive immune responses by controlling, to a certain extent, innate defences with liposomes containing clodronate (clo-lip). However, the reproducibility of those models is limited, with only a proportion of animals supporting longstanding parasitemia, due to strong inflammation induced by P. falciparum. Optimisation of the model is much needed for the study of new anti-malarial drugs, drug combinations, and candidate vaccines.

Materials/Methods

We investigated the possibility of improving previous models by employing the intravenous route (IV) for delivery of both human erythrocytes (huRBC) and P. falciparum, instead of the intraperitoneal route (IP), by testing various immunosuppressive drugs that might help to control innate mouse defences, and by exploring the potential benefits of using immunodeficient mice with additional genetic defects, such as those with IL-2Rγ deficiency (NSG mice).

Results

We demonstrate here the role of aging, of inosine and of the IL-2 receptor γ mutation in controlling P. falciparum induced inflammation. IV delivery of huRBC and P. falciparum in clo-lip treated NSG mice led to successful infection in 100% of inoculated mice, rapid rise of parasitemia to high levels (up to 40%), long-lasting parasitemia, and consistent results from mouse-to-mouse. Characteristics were closer to human infection than in previous models, with evidence of synchronisation, partial sequestration, and receptivity to various P. falciparum strains without preliminary adaptation. However, results show that a major IL-12p70 inflammatory response remains prevalent.

Conclusion

The combination of the NSG mouse, clodronate loaded liposomes, and IV delivery of huRBC has produced a reliable and more relevant model that better meets the needs of Malaria research.     

Low immunogenicity of allogeneic human umbilical cord blood-derived mesenchymal stem cells in vitro and in vivo

Evaluation of the immunogenicity of human mesenchymal stem cells (MSCs) in an allogeneic setting during therapy has been hampered by lack of suitable models due to technical and ethical limitations. Here, we show that allogeneic human umbilical cord blood derived-MSCs (hUCB-MSCs) maintained low immunogenicity even after immune challenge in vitro. To confirm these properties in vivo, a humanized mouse model was established by injecting isolated hUCB-derived CD34+ cells intravenously into immunocompromised NOD/SCID IL2γnull (NSG) mice. After repeated intravenous injection of human peripheral blood mononuclear cells (hPBMCs) or MRC5 cells into these mice, immunological alterations including T cell proliferation and increased IFN-γ, TNF-α, and human IgG levels, were observed. In contrast, hUCB-MSC injection did not elicit these responses. While lymphocyte infiltration in the lung and small intestine and reduced survival rates were observed after hPBMC or MRC5 transplantation, no adverse events were observed following hUCB-MSC introduction. In conclusion, our data suggest that allogeneic hUCB-MSCs have low immunogenicity in vitro and in vivo, and are therefore “immunologically safe” for use in allogeneic clinical applications.     

A germline-competent embryonic stem cell line from NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice

The NOD.Cg-Prkdc ^scid Il2rg ^tm1Wjl /SzJ mouse strain, commonly known as NSG (for NOD SCID Gamma) is severely immunodeficient and thus is an excellent recipient for xenografts, and in particular for engrafting human tumor cells and human hematopoietic stem cells. In the latter case, these cells give rise to many human hematopoetic lineages in their NSG hosts, resulting in recapitulation of many of the features of a human immune system. However, the immune system of these ??humanized mice?? (huMice) is not completely functional, in part because of a lack of expression of necessary human cytokines and HLA molecules by NSG host tissues. In order to facilitate the genetic modification of this strain in order to improve the huMouse model, we have created germline competent ES cells of this strain in which such modifications can be carried out.     

An improved pre-clinical patient-derived liquid xenograft mouse model for acute myeloid leukemia

Background

Xenotransplantation of patient-derived AML (acute myeloid leukemia) cells in NOD-scid Il2rγ ^null (NSG) mice is the method of choice for evaluating this human hematologic malignancy. However, existing models constructed using intravenous injection in adult or newborn NSG mice have inferior engraftment efficiency, poor peripheral blood engraftment, or are difficult to construct.

Methods

Here, we describe an improved AML xenograft model where primary human AML cells were injected into NSG newborn pups intrahepatically.

Results

Introduction of primary cells from AML patients resulted in high levels of engraftment in peripheral blood, spleen, and bone marrow (BM) of recipient mice. The phenotype of engrafted AML cells remained unaltered during serial transplantation. The mice developed features that are consistent with human AML including spleen enlargement and infiltration of AML cells into multiple organs. Importantly, we demonstrated that although leukemic stem cell activity is enriched and mediated by CD34⁺CD117⁺ subpopulation, CD34⁺CD117^? subpopulation can acquire CD34⁺CD117⁺ phenotype through de-differentiation. Lastly, we evaluated the therapeutic potential of Sorafenib and Regorafenib in this AML model and found that periphery and spleen AML cells are sensitive to these treatments, whereas BM provides a protective environment to AML.

Conclusions

Collectively, our improved model is robust, easy-to-construct, and reliable for pre-clinical AML studies.

     

Irradiated Compared with Nonirradiated NSG Mice for the Development of a Human B-Cell Lymphoma Model

NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice are a superior strain for the engraftment of human tumors, as they provide an ideal model to explore the potency, toxicity, and dosage of therapeutic drugs. Although whole-body nonlethal irradiation is often performed to enhance engraftment, the need for irradiation to establish a human B-cell lymphoma model using the NSG strain has not been addressed. In the current study, a mouse model of B-cell lymphoma was established by intravenous injection of human B-cell lymphoma Z138 cells into mice with and without irradiation. Tumor development, signs of engraftment, survivability of engrafted mice, histopathology, and immunohistochemistry were evaluated. Potential sex-associated variations in the model were assessed also. Irradiation of NSG mice did not enhance tumor cell engraftment, and nonirradiated animals had increased survivability. Mice with irradiation survived for a median of 27 d before being euthanized due to signs of morbidity, whereas those without irradiation had a median survival of 35 d. Both irradiated and nonirradiated mice were normal in activity until 3 wk after the injection of cells. At that time, the mice started to show signs of lymphoma including ruffled fur, decreased activity, and hindlimb paralysis. There were no significant differences in evaluated parameters between male and female mice. Therefore, we conclude that a model of B-cell lymphoma can successfully be established by using Z138 cells in nonirradiated male and female NSG mice.Abbreviations: NHL, nonHodgkin lymphoma; NSG, NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJThe National Cancer Institute defines nonHodgkin lymphoma (NHL) as cancer of lymphocytes, and it affects various organs of the immune system, including lymph nodes, spleen, and bone marrow. The several different forms of NHL include slow-progressing, fast-progressing, B-cell, and T-cell types.⁶ Mantle cell lymphoma is a rare type of aggressive B-cell lymphoma (occurring in about 6% of lymphoma patients in the United States²) and is extremely difficult to treat. Patients with mantle cell lymphoma are treated with chemotherapeutic drugs, radiotherapy and transplantation of bone marrow,⁷ but the lymphoma relapses after 3 to 4 y in nearly 50% of the patients.⁴ Therefore, it is essential to develop strategies for enhancing the therapeutic options in patients with B-cell lymphoma and specific drugs that can cure the disease or prevent its relapse. Because animal experiments enable the preclinical testing of promising therapeutics for subsequent evaluation in humans, the development of an appropriate animal model is crucial.Mice engrafted with human tumors act as a model for testing various therapeutic drugs for their potency, toxicity, and dosing.¹ Severe immunodeficient mice (SCID) mice have widely been used to disseminate tumor cells in vivo,¹³ where the cells are engrafted via intravenous injection.^10,24 These mice have been used to develop a mouse model for human Burkitt lymphoma (a type of B-cell lymphoma) by using the Daudi cell line or SU-DHL-4 cells.²³ In these experiments, hindlimb paralysis and solid tumor development were observed as characteristic signs of lymphoma in the engrafted mice.^9,23 Whereas one group observed hindlimb paralysis even without irradiation of mice, the other did not see this development in any of their nonirradiated animals.²³ However, irradiation altered the pattern of tumor growth and the animals’ responses to various chemotherapeutic drugs. It also led to variations in the animals’ immune status in general and made them more susceptible to thyomas.²³ Therefore, whether to irradiate mice prior to the injection of B-cell lymphoma cells has been a topic of debate.The development of NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice has provided a valuable tool for the development of a B-cell lymphoma model, because they lack mature B and T cells and various cytokines such as IL2, 4, 7, 9, 15, and 21, leading to impaired development of NK cells.^14,20 Some studies have shown that irradiating mice prior to the injection of various tumor cells enhances the engraftment and growth rate of the tumor,^3,21 but immunocompromised mice, especially NSG mice, are known to be sensitive to irradiation and subsequently may manifest increased morbidity and mortality.¹² In addition, the irradiation process can cause considerable distress in mice, and many institutions and IACUC require close monitoring and special care of mice after irradiation.¹⁶ In our lab, mice routinely are provided with nutritional and fluid supplements and are placed on heating pads after irradiation, to prevent dehydration and death. Given irradiation''s potential negative effect on animal health and the irradiation-associated variations reported in similar animal models, the elimination of irradiation may yield a less stressful and more reliable model for NHL.The objective of the current study was to compare irradiated and nonirradiated NSG mice as a model for a specific type of B-cell NHL, mantle cell lymphoma. We evaluated engraftment, the development of clinical signs, and survival and potential sex-associated differences in each of those parameters in both irradiated and nonirradiated NSG mice injected with Z138 (mantle cell lymphoma) cells.     

Induction of Robust Cellular and Humoral Virus-Specific Adaptive Immune Responses in Human Immunodeficiency Virus-Infected Humanized BLT Mice

The generation of humanized BLT mice by the cotransplantation of human fetal thymus and liver tissues and CD34⁺ fetal liver cells into nonobese diabetic/severe combined immunodeficiency mice allows for the long-term reconstitution of a functional human immune system, with human T cells, B cells, dendritic cells, and monocytes/macrophages repopulating mouse tissues. Here, we show that humanized BLT mice sustained high-level disseminated human immunodeficiency virus (HIV) infection, resulting in CD4⁺ T-cell depletion and generalized immune activation. Following infection, HIV-specific humoral responses were present in all mice by 3 months, and HIV-specific CD4⁺ and CD8⁺ T-cell responses were detected in the majority of mice tested after 9 weeks of infection. Despite robust HIV-specific responses, however, viral loads remained elevated in infected BLT mice, raising the possibility that these responses are dysfunctional. The increased T-cell expression of the negative costimulator PD-1 recently has been postulated to contribute to T-cell dysfunction in chronic HIV infection. As seen in human infection, both CD4⁺ and CD8⁺ T cells demonstrated increased PD-1 expression in HIV-infected BLT mice, and PD-1 levels in these cells correlated positively with viral load and inversely with CD4⁺ cell levels. The ability of humanized BLT mice to generate both cellular and humoral immune responses to HIV will allow the further investigation of human HIV-specific immune responses in vivo and suggests that these mice are able to provide a platform to assess candidate HIV vaccines and other immunotherapeutic strategies.An ideal animal model of human immunodeficiency virus (HIV) infection remains elusive. Nonhuman primates that are susceptible to HIV infection typically do not develop immunodeficiency (63), and although the simian immunodeficiency virus (SIV) infection of rhesus macaques has provided many critically important insights into retroviral pathogenesis (30), biological and financial considerations have created some limitations to the wide dissemination of this model. The great need for an improved animal model of HIV itself recently has been underscored by the disappointing results of human trials of MRKAd5, an adenovirus-based HIV type 1 (HIV-1) vaccine. This vaccine was not effective and actually may have increased some subjects'' risk of acquiring HIV (53). In the wake of these disappointing results, there has been increased interest in humanized mouse models of HIV infection (54). The ability of humanized mouse models to test candidate vaccines or other immunomodulatory strategies will depend critically on the ability of these mice to generate robust anti-HIV human immune responses.Mice have provided important model systems for the study of many human diseases, but they are unable to support productive HIV infection, even when made to express human coreceptors for the virus (7, 37, 52). A more successful strategy to humanize mice has been to engraft human immune cells and/or tissues into immunodeficient severe combined immunodeficiency (SCID) or nonobese diabetic (NOD)/SCID mice that are unable to reject xenogeneic grafts (39, 42, 57). Early versions of humanized mice supported productive HIV infection and allowed investigators to begin to address important questions in HIV biology in vivo (23, 40, 43-45). More recently, human cord blood or fetal liver CD34⁺ cells have been used to reconstitute Rag2^−/− interleukin-2 receptor γ chain-deficient (γ_c^−/−) and NOD/SCID/γ_c^−/− mice, resulting in higher levels of sustained human immune cell engraftment (27, 29, 61). These mice have allowed for stable, disseminated HIV infection (2, 4, 24, 65, 67), including mucosal transmission via vaginal and rectal routes (3). These mice recently have been used to demonstrate an important role for Treg cells in acute HIV infection (29) and to demonstrate that the T-cell-specific delivery of antiviral small interfering RNA is able to suppress HIV replication in vivo (31). These mice also have demonstrated some evidence of adaptive human immune responses, including the generation of HIV-specific antibody responses in some infected mice (2, 65), and some evidence of humoral and cell-mediated responses to non-HIV antigens or pathogens (24, 61). Most impressively, Rag2^−/− γ_c^−/− mice reconstituted with human fetal liver-derived CD34⁺ cells have generated humoral responses to dengue virus infection that demonstrated both class switching and neutralizing capacity (32). In spite of these advances, however, these models have not yet been reported to generate de novo HIV-specific cell-mediated immune responses, which are considered to be a crucial arm of host defense against HIV infection in humans.In contrast to humanized mouse models in which only human hematopoietic cells are transferred into immunodeficient mice, the surgical implantation of human fetal thymic and liver tissue has been performed in addition to the transfer of human hematopoietic stem cells (HSC) to generate mice in which human T cells are educated by autologous human thymic tissue rather than by the xenogeneic mouse thymus. Melkus and colleagues refer to mice they have reconstituted in this way as NOD/SCID-hu BLT (for bone marrow, liver, and thymus), or simply BLT, mice (41). We previously referred to mice that we have humanized in a similar way as NOD/SCID mice cotransplanted with human fetal thymic and liver tissues (Thy/Liv) and CD34⁺ fetal liver cells (FLC) (33, 60) but now adopt the designation BLT mice as well. BLT mice demonstrate the robust repopulation of mouse lymphoid tissues with functional human T lymphocytes (33, 41, 60) and can support the rectal and vaginal transmission of HIV (13, 59). Further, BLT mice demonstrate antigen-specific human immune responses against non-HIV antigens and/or pathogens (41, 60). The ability of these mice to generate human immune responses against HIV, however, has not yet been reported. In this study, we investigated whether the provision of autologous human thymic tissue in BLT mice generated by the cotransplantion of human fetal Thy/Liv tissues and CD34⁺ FLC would allow for the maturation of human T cells in humanized mice capable of providing improved cellular responses to HIV as well as providing adequate help for improved humoral responses. To describe the cells contributing to human immune responses in BLT mice, we also characterized the phenotypes of multiple subsets of T cells, B cells, dendritic cells (DCs), and monocytes/macrophages present in uninfected humanized mice. The generation of robust HIV-directed human cellular and humoral immune responses in these mice would further demonstrate the ability of humanized mice to provide a much needed platform for the evaluation of HIV vaccines and other novel immunomodulatory strategies.     

Xenogeneic Graft-versus-Host-Disease in NOD-scid IL-2Rγ(null) Mice Display a T-Effector Memory Phenotype

The occurrence of Graft-versus-Host Disease (GvHD) is a prevalent and potentially lethal complication that develops following hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic-GvHD based upon immunodeficient strains injected with human peripheral blood mononuclear cells (PBMC; "Hu-PBMC mice") are important tools to study human immune function in vivo. The recent introduction of targeted deletions at the interleukin-2 common gamma chain (IL-2Rγ(null)), notably the NOD-scid IL-2Rγ(null) (NSG) and BALB/c-Rag2(null) IL-2Rγ(null) (BRG) mice, has led to improved human cell engraftment. Despite their widespread use, a comprehensive characterisation of engraftment and GvHD development in the Hu-PBMC NSG and BRG models has never been performed in parallel. We compared engrafted human lymphocyte populations in the peripheral blood, spleens, lymph nodes and bone marrow of these mice. Kinetics of engraftment differed between the two strains, in particular a significantly faster expansion of the human CD45(+) compartment and higher engraftment levels of CD3(+) T-cells were observed in NSG mice, which may explain the faster rate of GvHD development in this model. The pathogenesis of human GvHD involves anti-host effector cell reactivity and cutaneous tissue infiltration. Despite this, the presence of T-cell subsets and tissue homing markers has only recently been characterised in the peripheral blood of patients and has never been properly defined in Hu-PBMC models of GvHD. Engrafted human cells in NSG mice shows a prevalence of tissue homing cells with a T-effector memory (T(EM)) phenotype and high levels of cutaneous lymphocyte antigen (CLA) expression. Characterization of Hu-PBMC mice provides a strong preclinical platform for the application of novel immunotherapies targeting T(EM)-cell driven GvHD.     

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts^1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (http://sitemaker.umich.edu/pletcherlab/software). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.     

Induction of γδT cells from HSC-enriched BMCs co-cultured with iPSC-derived thymic epithelial cells

T cells bearing γδ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of γδT cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse γδT cells by co-culturing HSC-enriched bone marrow cells (HSC-eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC-eBMCs and iTECs cultured with IL-2 + IL-7 for up to 21 days induced CD45.1⁺ γδT cells that expressed a broad repertoire of Vγ and Vδ T-cell receptors. Notably, the induced lymphocytes contained few or no αβT cells, NK1.1⁺ natural killer cells, or B220⁺ B cells. Adoptive transfer of the induced γδT cells to leukemia-bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human γδT cells for clinical applications.