The status of donor cancer tissues affects the fate of patient-derived colorectal cancer xenografts in NOG mice

Patient-derived xenografts (PDXs) of tumors are increasingly becoming important tools for translational research in oncology. The NOD.Cg-Prkdc^scid Il2rg^tm1Sug/Jic (NOG) mouse is an efficient host for PDXs. Thus as a basis for future development of methods to obtain PDXs from various disease types, we have studied the factors that affect the outcome of transplantation of human colorectal cancer in NOG mice. Of the original donor cases examined, 73% had successful engraftment. The outcome of donor-matched tissues was consistent in most cases, and was thought to show that the condition of the host did not affect engraftment. Next we analyzed the tumor aggressiveness in terms of histology grade of the original tumor and found that they were related to engraftment. Detailed histopathological examination of the transplanted tissues strongly indicated that lymphocytes engrafted with the tumor cells affect engraftment. As a factor related to transplantation of lymphocytes, we studied the human IgG concentration in the serum of tumor-bearing mice, but there was no tendency for higher concentrations to result in unsuccessful engraftment. Finally, we studied the type, density and location of T cells in the original donor tissue to determine the immune contexture and found that the unsuccessful engraftment cases tended to have an adequate or coordinated immune contexture compared to successful engraftment cases. From these results, we concluded that the aggressiveness and the T cell infiltration of the original tumor affect the outcome of transplantation in the NOG mouse.     

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.     

Characterization of EBV-related Lymphoproliferative Lesions Arising in Donor Lymphocytes of Transplanted Human Tumor Tissues in the NOG Mouse

Human tumor tissue line models established in the severely immunodeficient NOD.Cg-Prkdc^scid Il2rg^tm1Sug/Jic (NOD/Shi-scid, IL-2Rγ^null or NOG) mouse are important tools for oncology research. During the establishment process, a lymphoproliferative lesion (LPL) that replaces the original tumor cells in the site of transplantation occurs. In the present study, we studied the impact of the LPL on the establishment process and the characteristics of the lesion, investigated the systemic distribution of the lesion in the mouse, and evaluated the potential of a simple identification method. The incidence of the lesion varied among tumor types, and the lesion was found to be the leading cause of unsuccessful establishment with gastric and colorectal cancer. The lesion consisted of a varying population of proliferating lymphoid cells that expressed CD20. The cells were positive for Epstein-Barr virus (EBV)-related antigens, and EBV DNA was detected. There was systemic distribution of the lesion within the NOG mouse, and the most consistent gross finding was splenomegaly. Additionally, identification of LPL-affected cases was possible by detecting splenomegaly in the 1st and 2nd generation mice at necropsy. From our findings the lesion was judged to arise from EBV-infected B cells originating from the donor, and monitoring splenomegaly at necropsy was thought effective as a simple method for identifying the lesion at an early stage of the establishment process.     

Establishment of a humanized model of liver using NOD/Shi-scid IL2Rgnull mice

Severely immunodeficient NOD/Shi-scid IL2Rg^null (NOG) mice are used as recipients for human tissue transplantation, which produces chimeric mice with various types of human tissue. NOG mice expressing transgenic urokinase-type plasminogen activator in the liver (uPA-NOG) were produced. Human hepatocytes injected into uPA-NOG mice repopulated the recipient livers with human cells. The uPA-NOG model has several advantages over previously produced chimeric mouse models of human liver: (1) the severely immunodeficient NOG background enables higher xenogeneic cell engraftment; (2) the absence of neonatal lethality enables mating of homozygotes, which increased the efficacy of homozygote production; and (3) donor xenogeneic human hepatocytes could be readily transplanted into young uPA-NOG mice, which provide easier surgical manipulation and improved recipient survival.     

NOD-Rag2null IL-2Rγnull Mice: An Alternative to NOG Mice for Generation of Humanized Mice

We have developed NOD-Rag2^null IL-2Rγ^null (NR2G) mice similar to NOD-scidIL-2Rγ^null (NOG) mice that are known as an excellent host to generate humanized mice. To evaluate the usefulness of NR2G mice as a host for humanized mice, the engraftment rates and differentiation of human cells after human hematopoietic stem cell (HSC) transplantation were compared among NR2G, NOG, and NOD-scid mice. For this purpose, the appropriate irradiation doses to expand the niche for human stem cells in the bone marrow were first determined. As a result, 8 and 2.5 Gy in adult, and 4 and 1 Gy in newborn NR2G and NOG mice, respectively, were found to be appropriate. Next, 5 × 10⁴ human umbilical cord blood CD34⁺ cells were intravenously inoculated into irradiated adult or newborn of the immunodeficient mice. These HSC transplantation experiments demonstrated that both NR2G and NOG mice showed high engraftment rates compared with NOD-scid mice, although NOG mice showed a slightly higher engraftment rate than that for NR2G mice. However, no difference was found in the human cell populations differentiated from HSCs between NR2G and NOG mice. The HSC transplantation experiments to adults and newborns of two immunodeficient mice also revealed that the HSC transplantation into newborn mice resulted in higher engraftment rate than those into adults. These results showed that NR2G mice could be used as an alternative host to NOG mice to generate humanized mice.     

Low Dose Focused Ultrasound Induces Enhanced Tumor Accumulation of Natural Killer Cells

Natural killer (NK) cells play a vital antitumor role as part of the innate immune system. Efficacy of adoptive transfer of NK cells depends on their ability to recognize and target tumors. We investigated whether low dose focused ultrasound with microbubbles (ldbFUS) could facilitate the targeting and accumulation of NK cells in a mouse xenograft of human colorectal adenocarcinoma (carcinoembryonic antigen (CEA)-expressing LS-174T implanted in NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice) in the presence of an anti-CEA immunocytokine (ICK), hT84.66/M5A-IL-2 (M5A-IL-2). Human NK cells were labeled with an FDA-approved ultra-small superparamagnetic iron oxide particle, ferumoxytol. Simultaneous with the intravenous injection of microbubbles, focused ultrasound was applied to the tumor. In vivo longitudinal magnetic resonance imaging (MRI) identified enhanced accumulation of NK cells in the ensonified tumor, which was validated by endpoint histology. Significant accumulation of NK cells was observed up to 24 hrs at the tumor site when ensonified with 0.50 MPa peak acoustic pressure ldbFUS, whereas tumors treated with at 0.25 MPa showed no detectable NK cell accumulation. These clinically translatable results show that ldbFUS of the tumor mass can potentiate tumor homing of NK cells that can be evaluated non-invasively using MRI.     

Establishment of a new model of human multiple myeloma using NOD/SCID/gammac(null) (NOG) mice

We developed a new experimental animal model of human multiple myeloma using immunodeficient NOD/SCID/gammac(null) (NOG) mice. A human myeloma cell line, U266, was intravenously inoculated into 20 NOG mice, all of which developed hind leg paralysis and distress around 6 weeks after transplantation. Pathological studies showed that only the bone marrow was infiltrated with U266 cells, and no cells were present in other organs. Osteolytic lesions in cortical bones and loss of trabecular bones were prominent in U266-transplanted NOG mice. In contrast, U266 cells were not detected in CB17scid or NOD/SCID mice 6 weeks after intravenous inoculation. Human IgE, produced by U266 cells, was detected in the serum of U266-transplanted NOG mice by ELISA. The results indicated that this hu-myeloma NOG model might be useful for studying the pathogenesis of myeloma and related osteolytic lesions, and are suggestive of its applicability to the future development of new drugs.     

Pathogenesis of murine astrovirus in experimentally infected mice

Astroviruses are often associated with gastrointestinal diseases in mammals and birds. Murine astrovirus (MuAstV) is frequently detected in laboratory mice. Previous studies on MuAstV in mice did not report any symptoms or lesions. However, little information is available regarding its pathogenicity in immunodeficient mice. Therefore, in this study, we experimentally infected germ-free NOD.Cg-Prkdc^scidIl2rg^tm1Sug/ShiJic (NOG) mice, which are severely immunodeficient, with MuAstV. Germ-free mice were used for experimental infection to eliminate the effects of intestinal bacteria. Mice in each group were then necropsied and subjected to PCR for MuAstV detection, MuAstV RNA quantification in each organ, and histopathological examination at 4 and 28 days post inoculation (DPI). Tissue samples from the small intestine were examined by transmission electron microscopy. No symptoms or abnormalities were detected in any mice during necropsy. The MuAstV concentration was highest in the lower small intestine, where it increased approximately 8-fold from 4 to 28 DPI. Transmission electron microscopy revealed circular virus particles of approximately 25 nm in diameter in the cytoplasm of the villous epithelial cells of the lower small intestine. Histopathological examination did not reveal any abnormalities, such as atrophy, in the intestinal villi. Our results suggest that MuAstV proliferates in the villous epithelial cells of the lower small intestine and has weak pathogenicity.     

Hepatitis B Virus Infection and Immunopathogenesis in a Humanized Mouse Model: Induction of Human-Specific Liver Fibrosis and M2-Like Macrophages

The mechanisms of chronic HBV infection and immunopathogenesis are poorly understood due to a lack of a robust small animal model. Here we report the development of a humanized mouse model with both human immune system and human liver cells by reconstituting the immunodeficient A2/NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ mice with human HLA-A2 transgene) with human hematopoietic stem cells and liver progenitor cells (A2/NSG-hu HSC/Hep mice). The A2/NSG-hu HSC/Hep mouse supported HBV infection and approximately 75% of HBV infected mice established persistent infection for at least 4 months. We detected human immune responses, albeit impaired in the liver, chronic liver inflammation and liver fibrosis in infected animals. An HBV neutralizing antibody efficiently inhibited HBV infection and associated liver diseases in humanized mice. In addition, we found that the HBV mediated liver disease was associated with high level of infiltrated human macrophages with M2-like activation phenotype. Importantly, similar M2-like macrophage accumulation was confirmed in chronic hepatitis B patients with liver diseases. Furthermore, gene expression analysis showed that induction of M2-like macrophage in the liver is associated with accelerated liver fibrosis and necrosis in patients with acute HBV-induced liver failure. Lastly, we demonstrate that HBV promotes M2-like activation in both M1 and M2 macrophages in cell culture studies. Our study demonstrates that the A2/NSG-hu HSC/Hep mouse model is valuable in studying HBV infection, human immune responses and associated liver diseases. Furthermore, results from this study suggest a critical role for macrophage polarization in hepatitis B virus-induced immune impairment and liver pathology.     

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.     

An improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rγnull mice allows HIV replication and development of anti-HIV immune responses

Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγ(null) (NSG) and NOD/SCID/IL2Rγ(null) (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.     

Assessment of Benzene-Induced Hematotoxicity Using a Human-Like Hematopoietic Lineage in NOD/Shi-scid/IL-2R��null Mice

Despite recent advancements, it is still difficult to evaluate in vivo responses to toxicants in humans. Development of a system that can mimic the in vivo responses of human cells will enable more accurate health risk assessments. A surrogate human hematopoietic lineage can be established in NOD/Shi-scid/IL-2Rγ^null (NOG) mice by transplanting human hematopoietic stem/progenitor cells (Hu-NOG mice). Here, we first evaluated the toxic response of human-like hematopoietic lineage in NOG mice to a representative toxic agent, benzene. Flow cytometric analysis showed that benzene caused a significant decrease in the number of human hematopoietic stem/progenitor cells in the bone marrow and the number of human leukocytes in the peripheral blood and hematopoietic organs. Next, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice (Mo-NOG mice). A comparison of the degree of benzene-induced hematotoxicity in donor-derived hematopoietic lineage cells within Mo-NOG mice indicated that the toxic response of Hu-NOG mice reflected interspecies differences in susceptibilities to benzene. Responses to the toxic effects of benzene were greater in lymphoid cells than in myeloid cells in Mo-NOG and Hu-NOG mice. These findings suggested that Hu-NOG mice may be a powerful in vivo tool for assessing hematotoxicity in humans, while accounting for interspecies differences.     

Defucosylated anti-CCR4 monoclonal antibody exercises potent ADCC-mediated antitumor effect in the novel tumor-bearing humanized NOD/Shi-scid, IL-2Rγnull mouse model

Purpose  There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities. Thus, the first aim of this study was to establish a human tumor-bearing mouse model in which human immune cells can engraft and mediate ADCC, but where the endogenous mouse immune cells cannot mediate ADCC. The second aim was to evaluate ADCC mediated in these humanized mice by the defucosylated anti-CC chemokine receptor 4 (CCR4) monoclonal antibody (mAb) which we have developed and which is now in phase I clinical trials. Experimental design  NOD/Shi-scid, IL-2Rγ^null (NOG) mice were the recipients of human immune cells, and CCR4-expressing Hodgkin lymphoma (HL) and cutaneous T-cell lymphoma (CTCL) cell lines were used as target tumors. Results  Humanized mice have been established using NOG mice. The chimeric defucosylated anti-CCR4 mAb KM2760 showed potent antitumor activity mediated by robust ADCC in these humanized mice bearing the HL or CTCL cell lines. KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in HL-bearing humanized mice. Conclusions  Anti-CCR4 mAb could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. In addition, using our humanized mice, we can perform the appropriate preclinical evaluation of many types of antibody based immunotherapy.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

Activation of Notch1 promotes development of human CD8 single positive T cells in humanized mice

Notch1 mutations are found in more than 50% of human T cell acute lymphoblastic leukemia (T-ALL) cells. However, the functions of Notch1 for human T cell development and leukemogenesis are not well understood. To examine the role of Notch1, human hematopoietic stem cells (HSCs), which had been transduced with a constitutively active form of Notch1 (ICN1), were transplanted into severely immunodeficient NOD/Shi-scid-IL2rγ^null (NOG) mice. We found that the great majority of the ICN1-expressing hematopoietic cells in the bone marrow expressed surface markers for T cells, such as CD3, CD4, and CD8, and that this T cell development was independent of the thymus. Accordingly, phenotypically mature CD8⁺ single positive (SP) T cells were observed in the spleen. Furthermore, T-ALL developed in one NOG recipient mouse out of 26 that had been secondary transferred with the T cells developed in the first NOG mice. These results indicate that Notch1 signaling in HSCs promotes CD8⁺ SP T cell development, and that T cell leukemogenesis may require additional oncogenic factors other than Notch1 activation.     

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) ^{1, 2} or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) ³. Both models were mainly utilized for the study of HIV infection.One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant ⁴. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency ^5-8.We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC ^{7, 9}. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (http://jaxmice.jax.org/research/immunology/005557-housing.html).     

In vivo assay of human NK-dependent ADCC using NOD/SCID/γc (NOG) mice

Monoclonal antibodies are essential to the success of molecularly targeted therapies. Recently, numerous therapeutic antibodies have been developed for various diseases, including cancer and autoimmune diseases. Experimental systems to effectively evaluate these candidate antibodies are urgently needed. One of the mechanisms used by antibodies to kill tumor cells is antibody-dependent cellular cytotoxicity (ADCC), in which natural killer cells (NK) are the main mediator. The capacity to induce ADCC has conventionally been assessed in the human-mouse xeno-graft model, in which human peripheral blood mononuclear cells (PBMC), containing NK cells along with antibodies, are administered to tumor-bearing immunodeficient mice. However, contamination from other cellular populations often affects tumor growth, making it difficult to evaluate the antibody’s effect. In this study, we established a new NK-dependent ADCC assay model using a supra-immunodeficient strain of mice, NOD/SCID/γc^null (NOG). Our model system simply consisted of three elements: isolated human NK cells, a Burkitt’s lymphoma cell line (Daudi), and an anti-CD20 antibody (Rituximab). In this experimental setting, human NK cells from healthy donors retained their killing activity and suppressed the growth of Daudi cells in NOG mice when they were administered along with Rituximab. This system, therefore, is useful for evaluating the in vivo function of human NK cells.     

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.     

Cardiovascular adaptations of pregnancy in T and B cell-deficient mice

The pathophysiology of gestational hypertensive disorders is incompletely defined. T lymphocytes are implicated. Both T and natural killer (NK) cells express RAS and, in implantation sites, NK cells are highly enriched. We hypothesized that T cells and/or NK cells contribute to circulatory control during pregnancy. Using radiotelemetry of arterial pressure, heart rate, and activity, mice without T and B cells (genotypes BALB/c-Rag2(-/-) and NOD.scid) were examined at baseline and across pregnancy. These strains differ in NK cell competency, with Rag2(-/-) being normal and NOD.scid impaired. Circulatory features differed between these inbred strains. Rag2(-/-); had blood pressure responses to pregnancy that did not differ from congenic normal mice. NOD.scid had higher midgestational blood pressure compared with normoglycemic NOD mice (3-5 mm Hg greater than NOD; P < 0.004). In comparison to controls, both T and B strains had much higher heart rates after first trimester that did not remit until parturition (>30 bpm greater than control; P < 0.0001). NOD.scid had additional anomalies, including 90% depletion of circulating NK cells and elevated (57%) proliferation of uterine NK cells within implantation sites. These data demonstrate immune control of midgestational heart rate and suggest NK cells contribute to midpregnancy regulation of mean arterial pressure.     

A Novel Mouse Model for Stable Engraftment of a Human Immune System and Human Hepatocytes

Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2^-/- IL-2Rγc^-/- NOD.sirpa uPA^tg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20–50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.