The Jackson Laboratory
Bar Harbor, ME
Investigates human immunological diseases and malignancy through the development and leveraging of novel humanized mouse models
We previously described a recessive mouse mutation named curly-bare (cub). Homozygotes are characterized by a hairless phenotype. The cub mutation was mapped to a locus on distal Chr 11, and a dominant genetic modifier of the cub phenotype (Mcub) was mapped to a 10 cM interval on Chr 5. A single copy of the dominant Mcub allele in combination with the cub/cub genotype results in a full, wavy coat rather than the hairless coat of cub/cub mcub/mcub mice. As a consequence of ear notching the cub/cub mice, we discovered that this mutation resulted in accelerated wound healing. DNA sequencing of cub/cub mice identified a 12,681 base pair (bp) deletion in the Rhbdf2 gene, which results in loss of exons 2–6, but the remaining exons were expressed. We subsequently confirmed splicing of exons 1 to 7. The cub gene henceforth named Rhbdf2cub. The wild type Rhbdf2 gene encodes an inactive rhomboid protease iRhom2, one of a family of enzymes containing a long cytosolic N-terminus and a dormant peptidase domain of unknown function. Inactive rhomboids (iRhoms) are highly conserved but proteolytically inactive intramembrane proteins that are characterized by a long cytosolic N-terminal domain, a conserved cysteine-rich inactive rhomboid homology domain (IRHD), and a dormant proteolytic site lacking an active-site serine residue within the peptidase domain. iRhom2 has been implicated in epithelial regeneration and cancer growth through constitutive activation of EGFR signaling. However, little is known about the physiological substrates for iRhom2 or the molecular mechanisms underlying these functions. Wild type iRhom2 is a short-lived protein whose stability can be increased by select mutations in the N-terminal domain. In turn, these stable variants function to augment the secretion of EGF family ligands, including amphiregulin. In vivo, N-terminal iRhom2 mutations induce accelerated wound healing as well as accelerated tumorigenesis, but do not drive spontaneous tumor development. This work underscores the physiological prominence of iRhom2 in controlling EGFR signaling events involved in wound healing and neoplastic growth, and yields insight into the function of key iRhom2 domains. The Rhbdf2cub modifier gene (Mcub) is a loss-of-function mutation in Areg, a gene that encodes the autocrine keratinocyte growth factor amphiregulin. This mutation was henceforth referred to as AregMcub. Recent reports show a role for iRhoms in EGFR-mediated human neoplastic growth. Missense mutations in RHBDF2 have been shown to cause tylosis with human esophageal cancer. Although the mechanisms underlying the pathogenesis of cancer are evolving, these studies further strengthen the link between iRhoms and EGFR signaling.
Homozygosity for a new spontaneous mouse mutation named "thrombocytopenia and cardiomyopathy" (trac) results in thrombocytopenia, dilated cardiomyopathy, and infertility. A/J-trac/trac mice show a precipitous drop in platelet numbers and increases in platelet volume by 4 weeks of age. By 2-3 months of age, trac/trac mice have a 20-fold decrease in platelet number, a 3-fold increase in platelet volume, and a greatly increased bleeding time. Blood smears showed abnormally large platelets and megakaryocytoid cells. Increased numbers of megakaryocytes were present in bone marrow, spleen, and lungs. Ultrastructural studies of trac/trac megakaryocytes showed a poorly developed demarcation system and a failure to form platelet territories. To identify the responsible gene, we produced a fine-structure genetic map of a 5-megabase interval containing the trac locus on mouse chromosome 17. We found a G-to-A mutation at base 1435 (refseq nm031884) of Abcg5 (ATP-binding cassette sub-family G, member 5). This G>A base change results in a tryptophan codon (UGG) at amino acid position 463(uniprot) being changed to a premature stop codon (UAG). ABCG5 (sterolin-1) functions as part of a heterodimer, with ABCG8, that regulates plant sterol uptake. The trac/trac mutant mice have greatly elevated plasma levels of plant sterols. When placed on a phytosterol-free diet, the thrombocytopenia was reversed. Recent studies have shown that Mediterranean macrothrombocytopenia is caused by mutations in ABCG5 or ABCG8. Analyses of CD3+ and Mac-3+ cells and stainable collagen in hearts of Abcgtrac/Abcg5trac mice showed inflammation and myocyte degeneration beginning postweaning and progression to marked dilative and fibrosing cardiomyopathy by 140 days. Transmission electron microscopy (TEM) demonstrated myocyte vacuoles consistent with swollen endoplasmic reticulum (ER). Myocytes with cytoplasmic glycogen and irregular actinomyosin filament bundles formed mature intercalated disks with normal myocytes suggesting myocyte repair. Identification of the molecular basis of the mouse Abcg5trac mutation provides a new model for studying the role of phytosterols in pathogenic changes in the hematopoietic, cardiovascular and reproductive systems.
Since the discovery of the "nude" mouse more than 40 years ago, investigators have attempted to model human tumor growth in immunodeficient mice. Here, we summarize how the field has advanced over the ensuing years owing to improvements in the murine recipients of human tumors. These improvements include the discovery of the scid mutation and development of targeted mutations in the recombination-activating genes 1 and 2 (Rag1(null), Rag2(null)) that severely cripple the adaptive immune response of the murine host. More recently, mice deficient in adaptive immunity have been crossed with mice bearing targeted mutations designed to weaken the innate immune system, ultimately leading to the development of immunodeficient mice bearing a targeted mutation in the gene encoding the interleukin 2 (IL2) receptor common γ chain (IL2rg(null), also known in humans as cytokine receptor common subunit γ). The IL2rg(null) mutation has been used to develop several immunodeficient strains of mice, including the NOD-scid IL2rg(null) (NSG) strain. Using NSG mice as human xenograft recipients, it is now possible to grow almost all types of primary human tumors in vivo, including most solid tumors and hematological malignancies that maintain characteristics of the primary tumor in the patient. Programs to optimize patient-specific therapy using patient-derived xenograft tumor growth in NSG mice have been established at several institutions, including The Jackson Laboratory. Moreover, NSG mice can be engrafted with functional human immune systems, permitting for the first time the potential to study primary human tumors in vivo in the presence of a human immune system.
Stromal cells and the extracellular environment are vital to human tumors, influencing growth and response to therapy. Human tumor cell lines lack stroma and transplantation into immunodeficient mice does not allow meaningful analyses of the effects of stroma on tumor cell growth. Studies of xenografts of primary human tumor fragments in nude mice and in early scid mouse models were constrained by poor tumor growth accompanied by host-versus-graft reactivity, dramatically altering tumor architecture and tumor microenvironment. In contrast, severely immunodeficient NOD-scid and NOD-Rag1 (null) strains carrying the IL2rg (null) mutation (NSG and NRG) support the growth of many types of human primary tumor. We compared the take rate, growth and architectural preservation of 10 clinically distinct primary human colon cancers in NOD-scid, NOD-Rag1 (null) , NSG and NRG mice and determined the contribution of mouse and human cells to the stroma during tumor proliferation and expansion in secondary hosts and tumor response to treatment with 5-fluorouracil (5-FU). NSG and NRG mice more readily support growth of human primary colon tumor fragments than do NOD-scid, NOD-Rag1 (null) mice and maintain tumor architectural integrity in the primary recipient and through subsequent transplant generations. The human colon tumors were responsive to treatment with 5-FU. Human stromal cells in the primary graft were replaced by mouse-derived fibroblasts in a dynamic process during subsequent passages. Thus, human colon cancer xenografts propagated in NSG and NRG mice maintain structural fidelity while replacing human stromal cells with murine stromal cells.
Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rg(null) mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.
Despite an initial response to chemotherapy, most patients with ovarian cancer eventually progress and succumb to their disease. Understanding why effector T cells that are known to infiltrate the tumor do not eradicate the disease after cytoreduction is critically important to the development of novel therapeutic strategies to augment tumor immunity and improve patient outcomes. Such studies have been hampered by the lack of a suitable in vivo model. We report here a simple and reliable model system in which ovarian tumor cell aggregates implanted intraperitoneally into severely immunodeficient NSG mice establish tumor microenvironments within the omentum. The rapid establishment of tumor xenografts within this small anatomically well-defined site enables the recovery, characterization, and quantification of tumor and tumor-associated T cells. We validate here the ability of the omental tumor xenograft (OTX) model to quantify changes in tumor cell number in response to therapy, to quantify changes in the tumor vasculature, and to demonstrate and study the immunosuppressive effects of the tumor microenvironment. Using the OTX model, we show that the tumor-associated T cells originally present within the tumor tissues are anergic and that fully functional autologous T cells injected into tumor-bearing mice localize within the tumor xenograft. The transferred T cells remain functional for up to 3 days within the tumor microenvironment but become unresponsive to activation after 7 days. The OTX model provides for the first time the opportunity to study in vivo the cellular and molecular events contributing to the arrest in T cell function in human ovarian tumors.
The study of human-specific infectious agents has been hindered by the lack of optimal small-animal models. More recently development of novel strains of immunodeficient mice has begun to provide the opportunity to utilize small-animal models for the study of many human-specific infectious agents. The introduction of a targeted mutation in the IL2 receptor common gamma chain gene (IL2rg(null)) in mice already deficient in T and B cells led to a breakthrough in the ability to engraft hematopoietic stem cells, as well as functional human lymphoid cells and tissues, effectively creating human immune systems in immunodeficient mice. These humanized mice are becoming increasingly important as pre-clinical models for the study of human immunodeficiency virus-1 (HIV-1) and other human-specific infectious agents. However, there remain a number of opportunities to further improve humanized mouse models for the study of human-specific infectious agents. This is being done by the implementation of innovative technologies, which collectively will accelerate the development of new models of genetically modified mice, including; i) modifications of the host to reduce innate immunity, which impedes human cell engraftment; ii) genetic modification to provide human-specific growth factors and cytokines required for optimal human cell growth and function; iii) and new cell and tissue engraftment protocols. The development of "next generation" humanized mouse models continues to provide exciting opportunities for the establishment of robust small-animal models to study the pathogenesis of human-specific infectious agents, as well as for testing the efficacy of therapeutic agents and experimental vaccines.
The objective of this study was to develop an immunodeficient mouse model that spontaneously develops hyperglycemia to serve as a diabetic host for human islets and stem cell derived beta cells in the absence or presence of a functional human immune system. We backcrossed the Ins2Akita mutation onto the NOD-Rag1null IL2rgnull strain and determined 1) spontaneous development of hyperglycemia, 2) ability of human islets, mouse islets and dissociated mouse islet cells to restore euglycemia, 3) the generation of a human immune system following engraftment of human hematopoietic stem cells, and 4) the ability of the humanized mice to reject human islet allografts. We confirmed the defects in innate and adaptive immunity and the spontaneous development of hyperglycemia conferred by the IL2rgnull, Rag1null, and Ins2Akita genes in NOD-Rag1null IL2rgnull Ins2Akita (NRG-Akita) mice. Mouse and human islets restored NRG-Akita mice to normoglycemia. Insulin-positive cells in dissociated mouse islets required to restore euglycemia in chemically diabetic NOD-scid IL2rgnull and spontaneously diabetic NRG-Akita mice were quantified following transplantation via the intra-pancreatic and subrenal routes. Engraftment of human hematopoietic stem cells in newborn NRG-Akita and in NRG mice resulted in equivalent human immune system development in a normoglycemic or chronically hyperglycemic environment, with some engrafted NRG-Akita mice capable of rejecting human islet allografts. NRG-Akita mice provide a model system for validation of the function of human islets and human adult stem cell, embryonic stem cell, or induced pluripotent stem cell-derived beta cells in the absence or presence of an alloreactive human immune system.
Graft-versus-host disease (GVHD) is a life-threatening complication of human allogeneic hematopoietic stem cell transplantation. NOD-scid IL2rgnull (NSG) mice injected with human PBMC engraft at high levels and develop a robust xenogeneic (xeno) GVHD, which reproduces many aspects of the clinical disease. Here we show that enriched and purified human CD4 T cells readily engraft in NSG mice and mediate xeno-GVHD, although with slower kinetics as compared to injection of whole PBMC. Moreover purified human CD4 T cells engraft in NSG mice that do not express murine MHC class II (NSG-Abo) but do not induce GVHD, demonstrating the importance of murine MHC class II in the xeno-response. Injection of purified human CD4 T cells into a newly developed NSG mouse strain that expresses human HLA-DR4 but not murine class II (NSG-Abo DR4) induces an allogeneic GVHD characterized by weight loss, fur loss, infiltration of human cells in skin, lung and liver and a high level of mortality. The ability of human CD4 T cells to mediate an allo-GVHD in NSG-Abo DR4 mice suggests that this model will be useful to investigate GVHD pathogenesis and to evaluate human specific therapies.
Type 1 diabetes (T1D) is characterized by the destruction of the insulin-producing β-cells of pancreatic islets. Genetic and environmental factors both contribute to T1D development. Viral infection with enteroviruses is a suspected trigger for T1D, but a causal role remains unproven and controversial. Studies in animals are problematic because of species-specific differences in host cell susceptibility and immune responses to candidate viral pathogens such as coxsackie B virus (CVB). In order to resolve the controversial role of viruses in human T1D, we developed a viral infection model in immunodeficient mice bearing human islet grafts. Hyperglycemia was induced in mice by specific ablation of native β-cells. Human islets, which are naturally susceptible to CVB infection, were transplanted to restore normoglycemia. Transplanted mice were infected with CVB4 and monitored for hyperglycemia. Forty-seven percent of CVB4-infected mice developed hyperglycemia. Human islet grafts from infected mice contained viral RNA, expressed viral protein, and had reduced insulin compared to grafts from uninfected mice. Human-specific gene expression profiles in grafts from infected mice revealed the induction of multiple interferon stimulated genes. Thus, human islets can become severely dysfunctional with diminished insulin production following CVB infection of β-cells, resulting in diabetes.
The development of small-animal models that elicit human immune responses to dengue virus (DENV) is important since prior immunity is a major risk factor for developing severe dengue disease. This study evaluated anti-DENV human antibody (hAb) responses generated from immortalized B cells after DENV-2 infection in NOD-scid IL2rγnull mice that were co-transplanted with human fetal thymus and liver tissues (BLT-NSG mice). DENV-specific human antibodies predominantly of the IgM isotype were isolated during acute infection and in convalescence. We found that while a few hAbs recognized the envelope protein produced as a soluble recombinant, a number of hAbs only recognized epitopes on intact virions. The majority of the hAbs isolated during acute infection and in immune mice were serotype-cross-reactive and poorly neutralizing. Viral titers in immune BLT-NSG mice were significantly decreased after challenge with a clinical strain of dengue. DENV-specific hAbs generated in BLT-NSG mice share some of the characteristics of Abs isolated in humans with natural infection. Humanized BLT-NSG mice provide an attractive preclinical platform to assess the immunogenicity of candidate dengue vaccines.