Humanized mouse as an appropriate model for accelerated global HIV research and vaccine development: current trend

Humanized mouse models currently have seen improved development and have received wide applications. Its usefulness is observed in cell and tissue transplant involving basic and applied human disease research. In this article, the development of a new generation of humanized mice was discussed as well as their relevant application in HIV disease. Furthermore, current techniques employed to overcome the initial limitations of mouse model were reviewed. Highly immunodeficient mice which support cell and tissue differentiation and do not reject xenografts are indispensable for generating additional appropriate models useful in disease study, this phenomenom deserves emphases, scientific highlight and a definitive research focus. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγ^null gene (e.g. NOD/SCID/γc^null and Rag2^nullγc^null mice) through various genomic approaches. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γc^null and Rag2^nullγc^null mouse backgrounds. The application of these techniques serves as a quick and appropriate mechanistic model for basic and therapeutic investigations of known and emerging infections.     

Parameters for establishing humanized mouse models to study human immunity: Analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the IL2rγ mutation

“Humanized” mouse models created by engraftment of immunodeficient mice with human hematolymphoid cells or tissues are an emerging technology with broad appeal across multiple biomedical disciplines. However, investigators wishing to utilize humanized mice with engrafted functional human immune systems are faced with a myriad of variables to consider. In this study, we analyze HSC engraftment methodologies using three immunodeficient mouse strains harboring the IL2rγ^null mutation; NOD-scid IL2rγ^null, NOD-Rag1^null IL2rγ^null, and BALB/c-Rag1^null IL2rγ^null mice. Strategies compared engraftment of human HSC derived from umbilical cord blood following intravenous injection into adult mice and intracardiac and intrahepatic injection into newborn mice. We observed that newborn recipients exhibited enhanced engraftment as compared to adult recipients. Irrespective of the protocol or age of recipient, both immunodeficient NOD strains support enhanced hematopoietic cell engraftment as compared to the BALB/c strain. Our data define key parameters for establishing humanized mouse models to study human immunity.     

Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex

Immunodeficient non-obese diabetic (NOD)-severe combined immune-deficient (scid) mice bearing a targeted mutation in the gene encoding the interleukin (IL)-2 receptor gamma chain gene (IL2rγ^null) engraft readily with human peripheral blood mononuclear cells (PBMC). Here, we report a robust model of xenogeneic graft-versus-host-like disease (GVHD) based on intravenous injection of human PBMC into 2 Gy conditioned NOD-scid IL2rγ^null mice. These mice develop xenogeneic GVHD consistently (100%) following injection of as few as 5 × 10⁶ PBMC, regardless of the PBMC donor used. As in human disease, the development of xenogeneic GVHD is highly dependent on expression of host major histocompatibility complex class I and class II molecules and is associated with severely depressed haematopoiesis. Interrupting the tumour necrosis factor-α signalling cascade with etanercept, a therapeutic drug in clinical trials for the treatment of human GVHD, delays the onset and progression of disease. This model now provides the opportunity to investigate in vivo mechanisms of xenogeneic GVHD as well as to assess the efficacy of therapeutic agents rapidly.     

Roles for NK Cells and an NK Cell-Independent Source of Intestinal Gamma Interferon for Innate Immunity to Cryptosporidium parvum Infection

A gamma interferon (IFN-γ)-dependent innate immune response operates against the intestinal parasite Cryptosporidium parvum in T- and B-cell-deficient SCID mice. Although NK cells are a major source of IFN-γ in innate immunity, their protective role against C. parvum has been unclear. The role of NK cells in innate immunity was investigated using Rag2^−/− mice, which lack T and B cells, and Rag2^−/− γ_c^−/− mice, which, in addition, lack NK cells. Adult mice of both knockout lines developed progressive chronic infections; however, on most days the level of oocyst excretion was higher in Rag2^−/− γ_c^−/− mice and these animals developed morbidity and died, whereas within the same period the Rag2^−/− mice appeared healthy. Neonatal mice of both mouse lines survived a rapid onset of infection that reached a higher intensity in Rag2^−/− γ_c^−/− mice. Significantly, similar levels of intestinal IFN-γ mRNA were expressed in Rag2^−/− and Rag2^−/− γ_c^−/− mice. Also, infections in each mouse line were exacerbated by treatment with anti-IFN-γ neutralizing antibodies. These results support a protective role for NK cells and IFN-γ in innate immunity against C. parvum. In addition, the study implies that an intestinal cell type other than NK cells may be an important source of IFN-γ during infection and that NK cells may have an IFN-γ-independent protective role.Cryptosporidiosis is an infectious diarrheal disease that affects different types of vertebrates, including mammals (3). The etiological agent is the monoxenous protozoan parasite Cryptosporidium, which belongs to the Apicomplexa. One species, Cryptosporidium hominis, may have a predilection for infecting humans, while a morphologically similar parasite, Cryptosporidium parvum, readily infects both cattle and humans (3). The cryptosporidia of mammals invade intestinal epithelial cells, where they multiply asexually to produce merozoites that infect more cells. Eventually, merozoites may undergo differentiation into gamonts that form new oocysts, containing four sporozoites, and the oocysts transmit infection to new hosts by the fecal-oral route. The clinical phase of cryptosporidiosis normally lasts a few days but may persist and become fatal in immunocompromised hosts (2).Studies of protective host immune responses to Cryptosporidium indicate that elimination of infection involves adaptive immunity and, in particular, requires the presence of CD4⁺ T cells. AIDS patients with low CD4⁺ cell counts have shown increased susceptibility to cryptosporidial infection and high rates of morbidity and mortality, while resolution of AIDS-associated infection following anti-human-immunodeficiency-virus drug treatment coincided with the partial recovery of intestinal CD4⁺ T-cell counts (2, 23). Mice with a CD4⁺ T-cell deficiency were found to be incapable of clearing C. parvum infection (1), and similarly, depletion of these cells from immunocompetent animals with specific antibody increased oocyst production (27). CD4⁺ T cells are also an important source of gamma interferon (IFN-γ), and this cytokine plays a key role in the control of infection. Antigen-specific IFN-γ production by restimulated CD4⁺ T cells from humans who recovered from infection was observed, although cells taken during acute infection were not responsive to antigen (6). IFN-γ^−/− mice or mice administered anti-IFN-γ neutralizing antibodies had exacerbated infections compared with control animals (18, 27). IFN-γ activity during C. parvum infection has been associated with a chemokine response by intestinal epithelial cells that attracted both CD4⁺ T cells and macrophages into the lamina propria (10). In addition, IFN-γ has been shown to have a direct effect on parasite growth by activating epithelial cell antimicrobial killing activity (19).Innate immune responses are also able to limit the reproduction of C. parvum. Immunocompromised adult nude mice (lacking T cells) or SCID mice (lacking T and B cells) developed chronic infections that were controlled for a number of weeks but eventually became progressive and fatal (13, 17, 27). IFN-γ was important for the initial resistance of these mice, since administration of anti-IFN-γ neutralizing antibodies to adult or neonatal SCID mice increased susceptibility to infection (14, 28), and repeated antibody treatment resulted in rapid establishment of severe infection (14). In addition, morbidity as a result of parasite reproduction appeared sooner in SCID IFN-γ^−/− mice than in SCID mice (7).NK cells are involved in resistance to intracellular microbial pathogens, including protozoa, and are a major source of IFN-γ in innate immunity (9). NK cells originate mainly in the bone marrow, from where they migrate to other organs (5, 29). Interleukin-15 (IL-15) is essential for differentiation and subsequent survival of NK cells and can also be important in activation of the cells (5, 9). NK cells are activated by ancillary cells, such as dendritic cells (DCs), by direct contact and by proinflammatory cytokines produced by DCs stimulated by antigen (9). Activated NK cells produce IFN-γ and other proinflammatory cytokines and may also become cytotoxic against infected cells.The protective role of NK cells in innate immunity to C. parvum is unclear, but some studies imply that these cells may be involved. Human peripheral blood NK cells treated with IL-15 were shown to have cytolytic activity against human intestinal epithelial cell lines infected with C. parvum (4), and intestinal expression of this cytokine has been detected in humans (20). C. parvum infection was found to be more widespread in SCID mice deficient in NK cell cytotoxicity than in SCID mice with normal NK cell function (17). In addition, in vitro studies demonstrated that splenocytes from SCID mice produced IFN-γ in the presence of cryptosporidial antigens, but if NK cells were depleted, IFN-γ production did not occur (15). However, attempts to show that NK cells were protective in SCID mice infected with C. parvum have not been successful. In separate studies, treatment of these mice with anti-asialo-GM1 antibodies that can deplete NK cells in vivo was shown to have no effect on the course of C. parvum infection (15, 27), and while it has been argued that these antibodies might not have reached the gut in sufficient quantity to be effective, similar antibodies were shown to diminish intestinal NK cell function (30).The aim of the present study was to examine further the role of NK cells and IFN-γ in the innate immune response to C. parvum. The pattern of infection and immune responses were compared in Rag2^−/− mice, which lack T and B cells, and Rag2^−/− γ_c^−/− mice, which, in addition, lack NK cells due to the absence of the γ_c chain component of the IL-15 receptor (5). The results support protective roles for IFN-γ and NK cells in innate immunity to C. parvum but also indicate that IFN-γ from a cell type other than NK cells is important for control of infection.     

HIV-1 immunopathogenesis in humanized mouse models

In recent years, the technology of constructing chimeric mice with humanized immune systems has markedly improved. Multiple lineages of human immune cells develop in immunodeficient mice that have been transplanted with human hematopoietic stem cells. More importantly, these mice mount functional humoral and cellular immune responses upon immunization or microbial infection. Human immunodeficiency virus type I (HIV-1) can establish an infection in humanized mice, resulting in CD4⁺ T-cell depletion and an accompanying nonspecific immune activation, which mimics the immunopathology in HIV-1-infected human patients. This makes humanized mice an optimal model for studying the mechanisms of HIV-1 immunopathogenesis and for developing novel immune-based therapies.     

Humanized mice: novel model for studying mechanisms of human immune-based therapies

The lack of relevant animal models is the major bottleneck for understanding human immunology and immunopathology. In the last few years, a novel model of humanized mouse has been successfully employed to investigate some of the most critical questions in human immunology. We have set up and tested in our laboratory the latest technology for generating mice with a human immune system by reconstituting newborn immunodeficient NOD/SCID-γ ^−/−_c mice with human fetal liver-derived hematopoietic stem cells. These humanized mice have been deemed most competent as human models in a thorough comparative study with other humanized mouse technologies. Lymphocytes in these mice are of human origin while other hematopoietic cells are chimeric, partly of mouse and partly of human origin. We demonstrate that human CD8 T lymphocytes in humanized mice are fully responsive to our novel cell-based secreted heat shock protein gp96^HIV-Ig vaccine. We also show that the gp96^HIV-Ig vaccine induces powerful mucosal immune responses in the rectum and the vagina, which are thought to be required for protection from HIV infection. We posit the hypothesis that vaccine approaches tested in humanized mouse models can generate data rapidly, economically and with great flexibility (genetic manipulations are possible), to be subsequently tested in larger nonhuman primate models and humans.

     

Long‐term human immune system reconstitution in non‐obese diabetic (NOD)‐Rag (–)‐γ chain (–) (NRG) mice is similar but not identical to the original stem cell donor

The murine immune system is not necessarily identical to it human counterpart, which has led to the construction of humanized mice. The current study analysed whether or not a human immune system contained within the non‐obese diabetic (NOD)‐Rag1^null‐γ chain^null (NRG) mouse model was an accurate representation of the original stem cell donor and if multiple mice constructed from the same donor were similar to one another. To that end, lightly irradiated NRG mice were injected intrahepatically on day 1 of life with purified cord blood‐derived CD34⁺ stem and progenitor cells. Multiple mice were constructed from each cord blood donor. Mice were analysed quarterly for changes in the immune system, and followed for periods up to 12 months post‐transplant. Mice from the same donor were compared directly with each other as well as with the original donor. Analyses were performed for immune reconstitution, including flow cytometry, T cell receptor (TCR) and B cell receptor (BCR) spectratyping. It was observed that NRG mice could be ‘humanized’ long‐term using cord blood stem cells, and that animals constructed from the same cord blood donor were nearly identical to one another, but quite different from the original stem cell donor immune system.     

High diversity of the immune repertoire in humanized NOD.SCID.γc−/− mice

The diversity of the human immune repertoire and how it relates to a functional immune response has not yet been studied in detail in humanized NOD.SCID.γc^?/? immunodeficient mice. Here, we used a multiplex PCR on genomic DNA to quantify the combinatorial diversity of all possible V–J rearrangements at the TCR‐β chain and heavy chain Ig locus. We first show that the combinatorial diversity of the TCR‐β chain generated in the thymus was well preserved in the periphery, suggesting that human T cells were not vastly activated in mice, in agreement with phenotypic studies. We then show that the combinatorial diversity in NOD.SCID.γc^?/? mice reached 100% of human reference samples for both the TCR and the heavy chain of Ig. To document the functionality of this repertoire, we show that a detectable but weak HLA‐restricted cellular immune response could be elicited in reconstituted mice after immunization with an adenoviral vector expressing HCV envelope glycoproteins. Altogether, our results suggest that humanized mice express a diversified repertoire and are able to mount antigen‐specific immune responses.     

Preventative role of interleukin-17 producing regulatory T helper type 17 (Treg17) cells in type 1 diabetes in non-obese diabetic mice

T helper type 17 (Th17) cells have been shown to be pathogenic in autoimmune diseases; however, their role in type 1 diabetes (T1D) remains inconclusive. We have found that Th17 differentiation of CD4⁺ T cells from BDC2·5 T cell receptor transgenic non-obese diabetic (NOD) mice can be driven by interleukin (IL)-23 + IL-6 to produce large amounts of IL-22, and these cells induce T1D in young NOD mice upon adoptive transfer. Conversely, polarizing these cells with transforming growth factor (TGF)-β + IL-6 led to non-diabetogenic regulatory Th17 (T_reg17) cells that express high levels of aryl hydrocarbon receptor (AhR) and IL-10 but produced much reduced levels of IL-22. The diabetogenic potential of these Th17 subsets was assessed by adoptive transfer studies in young NOD mice and not NOD.severe combined immunodeficient (SCID) mice to prevent possible transdifferentiation of these cells in vivo. Based upon our results, we suggest that both pathogenic Th17 cells and non-pathogenic regulatory T_reg17 cells can be generated from CD4⁺ T cells under appropriate polarization conditions. This may explain the contradictory role of Th17 cells in T1D. The IL-17 producing T_reg17 cells offer a novel regulatory T cell population for the modulation of autoimmunity.     

Glucagon-reactive islet-infiltrating CD8 T cells in NOD mice

Type 1 diabetes is characterized by T-cell-mediated destruction of the insulin-producing β cells in pancreatic islets. A number of islet antigens recognized by CD8 T cells that contribute to disease pathogenesis in non-obese diabetic (NOD) mice have been identified; however, the antigenic specificities of the majority of the islet-infiltrating cells have yet to be determined. The primary goal of the current study was to identify candidate antigens based on the level and specificity of expression of their genes in mouse islets and in the mouse β cell line MIN6. Peptides derived from the candidates were selected based on their predicted ability to bind H-2K^d and were examined for recognition by islet-infiltrating T cells from NOD mice. Several proteins, including those encoded by Abcc8, Atp2a2, Pcsk2, Peg3 and Scg2, were validated as antigens in this way. Interestingly, islet-infiltrating T cells were also found to recognize peptides derived from proglucagon, whose expression in pancreatic islets is associated with α cells, which are not usually implicated in type 1 diabetes pathogenesis. However, type 1 diabetes patients have been reported to have serum autoantibodies to glucagon, and NOD mouse studies have shown a decrease in α cell mass during disease pathogenesis. Our finding of islet-infiltrating glucagon-specific T cells is consistent with these reports and suggests the possibility of α cell involvement in development and progression of disease.     

Streptozotocin-induced diabetes in mice lacking αβ T cells

Multiple low-dose streptozotocin (MD-STZ) is widely used for the experimental induction of diabetes, but, as non-obese diabetic (NOD)-scid/scid mice have been found to display enhanced susceptibility to MD-STZ, whether or not the model is genuinely autoimmune and T cell-mediated has been unclear. Mice bearing a targeted mutation of the T cell receptor (TCR) α-chain were therefore used to assess whether TCR αβ⁺ cells are involved in the diabetogenic effects of MD-STZ injections. Young NOD mice lacking TCR αβ cells, when given five daily injections of 40 mg/kg STZ, developed diabetes at low frequency (2/12), despite the widespread destruction of pancreatic islet cells. By comparison, most normal control mice became hyperglycaemic (12/23). We conclude that whilst much of the tissue destruction observed in this model is due to the direct toxic effect of STZ, a significant amount is also due to the action of TCR αβ cells tipping the balance between tolerable and clinically damaging action on islet cells.     

Human Hepatocytes and Hematolymphoid Dual Reconstitution in Treosulfan-Conditioned uPA-NOG Mice

Human-specific HIV-1 and hepatitis co-infections significantly affect patient management and call for new therapeutic options. Small xenotransplantation models with human hepatocytes and hematolymphoid tissue should facilitate antiviral/antiretroviral drug trials. However, experience with mouse strains tested for dual reconstitution is limited, with technical difficulties such as risky manipulations with newborns and high mortality rates due to metabolic abnormalities. The best animal strains for hepatocyte transplantation are not optimal for human hematopoietic stem cell (HSC) engraftment, and vice versa. We evaluated a new strain of highly immunodeficient nonobese diabetic/Shi-scid (severe combined immunodeficiency)/IL-2Rγ_c^null (NOG) mice that carry two copies of the mouse albumin promoter-driven urokinase-type plasminogen activator transgene for dual reconstitution with human liver and immune cells. Three approaches for dual reconstitution were evaluated: i) freshly isolated fetal hepatoblasts were injected intrasplenically, followed by transplantation of cryopreserved HSCs obtained from the same tissue samples 1 month later after treosulfan conditioning; ii) treosulfan conditioning is followed by intrasplenic simultaneous transplantation of fetal hepatoblasts and HSCs; and iii) transplantation of mature hepatocytes is followed by mismatched HSCs. The long-term dual reconstitution was achieved on urokinase-type plasminogen activator–NOG mice with mature hepatocytes (not fetal hepatoblasts) and HSCs. Even major histocompatibility complex mismatched transplantation was sustained without any evidence of hepatocyte rejection by the human immune system.In Europe, Australia, and North America at least 25% of HIV-infected persons have a concomitant hepatitis C virus (HCV) infection, and 5% to 10% are co-infected with chronic hepatitis B virus (HBV).¹ Although the incidence of monoinfections (HIV, HBV, and HCV) is declining because of prophylaxis, vaccination, and newly available treatments, co-infections of HIV with HBV/HCV are still problematic, and the medical care of these co-infected patients remains a difficult task.^1,2 These human-specific co-infections require a small animal model to study double infections, such as HIV/HCV or HIV/HBV, and to test new antiviral/antiretroviral therapeutics.A search is ongoing for the best strain of mice for dual reconstitution and available tissue sources. Among multiple mouse models for human hepatocyte transplantation, the most robust and effective for mouse liver cell depletion and human hepatocytes engraftment are tyrosine catabolic enzyme fumarylacetoacetate hydrolase (Fah) mutants³ and transgenic mice with a tandem array of murine urokinase genes under the control of the albumin promoter (Alb-uPA) on a CB-17-scid-bg background.⁴ The advantages (up to 99% of human hepatocyte reconstitution) and the disadvantages (colony maintenance, limited time window for transplantation, and mouse health problems) of these mouse models for liver repopulation with human hepatocytes have been extensively discussed.^5–8 Several models for human hematopoietic stem cell (HSC) transplantation are based on the nonobese diabetic (NOD) and BALB/c background mouse strains, known as NOG (NOD/Shi-scid/IL-2Rγ_c^null),⁹ NSG (NOD/scid/γ_C^−/−/SzJ),¹⁰ and double knockout for Rag2+IL-2Rγ_c.^11,12 A successful co-transplantation of fetal human liver cells and HSCs has been reported in double knockout BALB/c mice with the transgenic expression of the FK506 binding protein–caspase 8 fusion gene driven by the albumin enhancer/promoter.¹³ Despite partial success, the manipulation of newborn animals and the use of fetal tissues create technical and ethical problems.An important issue for any model that is based on human tissue is the source and type of hepatocytes that can be used for transplantation, which must be syngeneic (matched) with HSCs for immune system reconstruction in experimental animals. The fetal liver provides both types of cells for transplantation. A possible choice is fetal liver cells with a progenitor phenotype that expresses epithelial cell adhesion molecule (CD326).¹⁴ The adult hepatocytes can be transplanted with high efficiency and can be sustained long enough⁶; however, the matched sources of HSCs are limited. These cells must be isolated from the same donor bone marrow or peripheral blood. Thus, no existing model is ideal for dual reconstitution.Here, we investigated the utility of a new urokinase-type plasminogen activator (uPA)-NOG strain¹⁵ of mice for dual reconstitution and compared different sources of human cells for transplantation. A homozygous line of uPA-NOG mice carries two copies of the transgene array that stably reinforces transgene expression. Perinatal bleeding, embryonic or neonatal lethality, and severe tissue pathology did not occur in homozygous uPA-NOG mice compared with Alb-uPA/scid mice. The alanine aminotransferase (ALT) levels were persistently elevated along with evidence of modest hepatic injury by 6 weeks of age in the uPA-NOG homozygotes. Compared with Alb-uPA/scid transgenic mice, which have an age-dependent decrease in uPA expression caused by deletion of the integrated transgene, a relatively low frequency of physical loss of the transgene is observed from uPA-NOG mice. The persistence of the hepatic injury marker should facilitate human hepatocyte engraftment and expansion throughout the life of the mouse.¹⁵ This property of uPA-NOG strain allows for the manipulation of adult animals and expands the window for human cell transplantation. The engraftment of HSCs also requires the creation of a niche in mouse bone marrow for human cells. The widely used total body irradiation increases the risks of severe bacteremia and body weight loss. In this study, we used non-myeloablative conditioning with treosulfan as a safe and well-tolerated alternative to total body irradiation for HSC transplantation.^16–20 Human hepatocyte engraftment at the 3% to 5% level is adequate to perform important studies on intrahepatic pathogens, such as those that cause malaria and hepatitis B. Successful HCV infection was achieved with >10% of hepatocytes being of human origin. Efficient hematolymphoid repopulation in combination with partial liver repopulation is sufficient to study HIV co-infection, and uPA-NOG mice offer this possibility.     

Comprehensive Analysis of the Activation and Proliferation Kinetics and Effector Functions of Human Lymphocytes,and Antigen Presentation Capacity of Antigen-Presenting Cells in Xenogeneic Graft-Versus-Host Disease

Xenogeneic graft-versus-host disease (GVHD) models in highly immunodeficient mice are currently being used worldwide to investigate human immune responses against foreign antigens in vivo. However, the individual roles of CD4⁺ and CD8⁺ T cells, and donor/host hematopoietic and nonhematopoietic antigen-presenting cells (APCs) in the induction and development of GVHD have not been fully investigated. In the present study, we comprehensively investigated the immune responses of human T cells and the antigen presentation capacity of donor/host hematopoietic and nonhematopoietic APCs in xenogeneic GVHD models using nonobese diabetic/Shi-scid-IL2rg^null mice. CD4⁺ T cells and, to a lesser extent, CD8⁺ T cells individually mediated potentially lethal GVHD. In addition to inflammatory cytokine production, CD4⁺ T cells also supported the activation and proliferation of CD8⁺ T cells. Using bone marrow chimeras, we demonstrated that host hematopoietic, but not nonhematopoietic, APCs play a critical role in the development of CD4⁺ T cell-mediated GVHD. During early GVHD, we detected 2 distinct populations in memory CD4⁺ T cells. One population was highly activated and proliferated in major histocompatibility complex antigen (MHC)^+/+ mice but not in MHC^?/? mice, indicating alloreactive T cells. The other population showed a less activated and slowly proliferative status regardless of host MHC expression, and was associated with higher susceptibility to apoptosis, indicating nonalloreactive T cells in homeostasis-driven proliferation. These observations are clinically relevant to donor T cell response after allogeneic hematopoietic stem cell transplantation. Our findings provide a better understanding of the immunobiology of humanized mice and support the development of novel options for the prevention and treatment for GVHD.     

Reduction in serum levels of antimitochondrial (M2) antibodies following immunoglobulin therapy in severe combined immunodeficient (SCID) mice reconstituted with lymphocytes from patients with primary biliary cirrhosis (PBC)

The effect of gammaglobulin treatment on autoantibody production was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells (PBMC) obtained from patients with PBC. All reconstituted mice displayed the presence of human antimitochondrial antibodies (αM2Ab) of both IgG and IgM types before treatment with human immunoglobulin. Two weeks after i.p. injection of 20 ×10⁶ PBMC into SCID mice, i.p. treatment with various preparations of human immunoglobulin was initiated. In control animals treated with saline, serum levels of human αM2Ab of the IgG type increased with time, peaking around 4 weeks after reconstitution. In contrast, human IgG autoantibodies rapidly decreased in all animals treated with human IgG. Treatment with a human IgM preparation had no effect on serum levels of αM2Ab of the IgG type. The results may suggest that the pronounced reduction of specific IgG autoantibodies was due to an increased catabolism of human IgG, including the autoantibodies, in the gammaglobulin-treated mice. Although the production of human αM2Ab in reconstituted mice could be easily shown, PBC-specific liver lesions or bile duct destruction were not observed, irrespective of treatment protocol.     

VCAM-1/α4β1 Integrin Interaction Is Crucial for Prompt Recruitment of Immune T Cells into the Brain during the Early Stage of Reactivation of Chronic Infection with Toxoplasma gondii To Prevent Toxoplasmic Encephalitis

Reactivation of chronic infection with Toxoplasma gondii can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals. We examined the role of VCAM-1/α4β1 integrin interaction in T cell recruitment to prevent reactivation of the infection in the brain. SCID mice were infected and treated with sulfadiazine to establish a chronic infection. VCAM-1 and ICAM-1 were the endothelial adhesion molecules detected on cerebral vessels of the infected SCID and wild-type animals. Immune T cells from infected wild-type mice were treated with anti-α4 integrin or control antibodies and transferred into infected SCID or nude mice, and the animals received the same antibody every other day. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Expression of mRNAs for CD3δ, CD4, CD8β, gamma interferon (IFN-γ), and inducible nitric oxide synthase (NOS2) (an effector molecule to inhibit T. gondii growth) and the numbers of CD4⁺ and CD8⁺ T cells in the brain were significantly less in mice treated with anti-α4 integrin antibody than in those treated with control antibody at 3 days after sulfadiazine discontinuation. At 6 days after sulfadiazine discontinuation, cerebral tachyzoite-specific SAG1 mRNA levels and numbers of inflammatory foci associated with tachyzoites were markedly greater in anti-α4 integrin antibody-treated than in control antibody-treated animals, even though IFN-γ and NOS2 mRNA levels were higher in the former than in the latter. These results indicate that VCAM-1/α4β1 integrin interaction is crucial for prompt recruitment of immune T cells and induction of IFN-γ-mediated protective immune responses during the early stage of reactivation of chronic T. gondii infection to control tachyzoite growth.     

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

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

CD34+ Cord Blood Cell-Transplanted Rag2−/− γc−/− Mice as a Model for Epstein-Barr Virus Infection

Recent studies suggest that Epstein-Barr virus (EBV) can infect naïve B cells, driving them to differentiate into resting memory B cells via the germinal center reaction. This hypothesis has been inferred from parallels with the biology of normal B cells but has never been proven experimentally. Rag2^−/− γ_c^−/− mice that were transplanted with human CD34⁺ cord blood cells as newborns were recently shown to develop human B, T, and dendritic cells, constituting lymphoid organs in situ. Here we used this model to better define the strategy of EBV infection of human B cells in vivo and to compare this model system with different conditions of EBV infection in humans. Our results support the model of EBV persistence in vivo in cases that were characterized by follicular hyperplasia and a relatively normal CD4⁺ and CD8⁺ T-cell distribution. Intriguingly, in cases that were characterized by nodular and diffuse proliferation with a preponderance of CD8⁺ T cells, similar to infectious mononucleosis, EBV still infects naïve B cells but also induces clonal expansion and ongoing somatic mutations without germinal center reactions. Our results reveal different strategies of EBV infection in B cells that possibly result from variations in the host immune response. Future experiments might allow understanding of the mechanisms responsible for persistent EBV infection and provide targets for more highly tailored therapeutic interventions.     

Protection of wild-type and severe combined immunodeficiency mice against an intranasal challenge by passive immunization with monoclonal antibodies to the Chlamydia trachomatis mouse pneumonitis major outer membrane protein

Monoclonal antibodies (MAbs) to the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) were characterized for their ability to neutralize the infectivity of this organism in vitro and in vivo. One of the MAbs (MoPn-23) recognizes a nonlinear epitope in the MOMP, MAb MoPn-40 binds to a linear epitope in the variable domain 1 (VD1), and MAb MoPn-32 recognizes the chlamydial lipopolysaccharide. MAb MoPn-23 neutralized 50% of the infectivity of Chlamydia, as measured in vitro by using HAK (FcγIII⁻) and HeLa-229 (FcγIII⁺) cells at a concentration 100 times lower than MAb MoPn-40. MAb MoPn-32 had no neutralizing ability. In comparison to the control normal mouse immunoglobulin G, passive immunization of BALB/c mice with MAb MoPn-23 resulted in a highly significant protection against an intranasal (i.n.) challenge as determined by the change in body weight, the weight of the lungs, and the yield of Chlamydia inclusion-forming units (IFU) from the lungs. Passive immunization with MAb MoPn-40 resulted in a lower degree of protection, and MAb MoPn-32 afforded no protection. MAb MoPn-23 was also tested for its ability to protect wild-type (WT) and severe combined immunodeficient (SCID) C.B-17 mice against an i.n. challenge. Protection based on total body weight, lung weight, and yield of Chlamydia IFU was as effective in SCID as in WT C.B-17 mice. In conclusion, antibodies to MOMP can protect mice against a chlamydial infection in the presence or absence of T and B cells.     

CD11c+ CD103+ cells of Mycobacterium tuberculosis‐infected C57BL/6 but not of BALB/c mice induce a high frequency of interferon‐γ‐ or interleukin‐17‐producing CD4+ cells

The magnitude of the cellular adaptive immune response is critical for the control of Mycobacterium tuberculosis infection in the chronic phase. In addition, the genetic background is equally important for resistance or susceptibility to tuberculosis. In this study, we addressed whether lung populations of dendritic cells, obtained from genetically different hosts, would play a role in the magnitude and function of CD4⁺ populations generated after M. tuberculosis infection. Thirty days post-infection, C57BL/6 mice, which generate a stronger interferon-γ (IFN-γ)-mediated immune response than BALB/c mice, exhibited a higher number and frequency of lung CD11c⁺ CD11b⁻ CD103⁺ cells compared with BALB/c mice, which exhibited a high frequency of lung CD11c⁺ CD11b⁺ CD103⁻ cells. CD11c⁺ CD11b⁻ CD103⁺ cells, purified from lungs of infected C57BL/6 mice, but not from infected BALB/c mice, induced a higher frequency of IFN-γ-producing or interleukin-17 (IL-17)-producing CD4⁺ cells. Moreover, CD4⁺ cells also arrive at the lung of C57BL/6 mice faster than in BALB/c mice. This pattern of immune response seems to be associated with higher gene expression for CCL4, CCL19, CCL20 and CCR5 in the lungs of infected C57BL/6 mice compared with infected BALB/c mice. The results described here show that the magnitude of IFN-γ-producing or IL-17-producing CD4⁺ cells is dependent on CD11c⁺ CD11b⁻ CD103⁺ cells, and this pattern of immune response is directly associated with the host genetic background. Therefore, differences in the genetic background contribute to the identification of immunological biomarkers that can be used to design human assays to predict progression of M. tuberculosis infection.     

Liver Xeno-Repopulation with Human Hepatocytes in Fah?/?Rag2?/? Mice after Pharmacological Immunosuppression

Functional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury–induced donor hepatocyte selection: the uPA/SCID mice and Fah^−/− Rag2^−/−Il2rg^−/− mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GM1 and FK506 in Fah^−/−Rag2^−/− mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fah^−/−Rag2^−/− mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and HBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharmacokinetics and efficacy of potential vaccines.Human hepatocytes xeno-engrafted into the liver of immunodeficient mice could be used as a model to study human hepatitis virus infection in vivo as well as the efficacy of potential vaccines.^1,2,3,4,5,6 Engrafted human hepatocytes can be serially transplanted from primary mice into secondary mice without losing hepatic function.⁷ Mouse recipients of human liver cells must have two capabilities: robust liver repopulation and immune tolerance for human hepatocytes. Liver xeno-repopulation from human hepatocytes was first reported in uPA/Rag2^−/− mice¹ and uPA/SCID mice.^2,3,8 The levels of liver xeno-repopulation varies in several reports, ranging from 10% to as high as 90%.^1,8 Humanized livers in uPA/SCID mice are susceptible to hepatitis B virus (HBV)^1,2 and HCV^3,4 infection. However, uPA mice have several disadvantages: i) neonatal death during colony breeding; ii) transplantation of hepatocytes into newborn mice (within the second week of life) is technically difficult due to a bleeding disorder in the mice; iii) there is uncontrollable selection for donor cells; iv) there is autoreversion of endogenous hepatocytes; and v) kidney damage is induced by the human complement system.^1,2,8,9Recently, robust liver xeno-repopulation from human hepatocytes was found in Fah^−/−Rag2^−/−Il2rg^−/− mice, cross-bred from Fah^−/− mice and Rag2^−/−Il2rg^−/− mice.⁷ Fah^−/−Rag2^−/−Il2rg^−/− mice have advantages over previous immunodeficient uPA models.⁷ First, Rag2^−/−Il2rg^−/− mice lack B, T, and NK cells, rendering more complete immunodeficiency compared with either Rag2^−/− or SCID mice.¹⁰ Second, liver injury in Fah^−/− mice is controllable by switching on and off 2-(2-nitro-4-trifluoro-methyl-benzoyl)-1, 3 cyclohexanedione (NTBC) administration.¹¹ NTBC inhibits accumulation of toxic metabolites in hepatocytes to maintain Fah^−/− mice in a healthy state. When the NTBC is removed, a powerful selection for fumaryl acetoacetate hydrolase (Fah) expressing cells is induced in the liver.¹² However, maintenance of Fah^−/−Rag2^−/−Il2rg^−/− mice during colony breeding, animal growing, and cell transplantation surgery are with high mortality in our experiments. The genotyping of animal offspring is overly elaborate. These concerns have not been discussed in previous publications^7,13 and may present a barrier to larger scale research projects.In comparison, Fah^−/−Rag2^−/− mice were much more tolerant of breeding and surgery procedures. However, Fah^−/−Rag2^−/− mice were thought to have no capacity for liver xeno-repopulation, because their NK cells are intact.⁷ We hypothesized that treatment of Fah^−/− Rag2^−/− mice with anti-asialo GM1 could result in complete depletion of NK cells as seen in Fah^−/−Rag2^−/− Il2rg^−/− mice.^14,15 We further tested the combined treatments of both anti-asialo GM1 and the immunosuppressor tacrolimus (FK506) to Fah^−/−Rag2^−/− mice.^16,17 The results indicated that the combined treatments enabled Fah^−/−Rag2^−/− recipients to have a high level of liver xeno-repopulation by human hepatocytes as seen in Fah^−/−Rag2^−/−Il2rg^−/− mice. Our results revealed a new and easily controlled mouse model with humanized liver. Using the same treatments, liver xeno-repopulation with human fetal liver progenitor cells was also achieved in Fah^−/−Rag2^−/− mice. Finally, for the first time, we were able to prove that human HBV actively replicated in the humanized Fah^−/−Rag2^−/− mice and that viral proteins were released in the serum of humanized Fah^−/−Rag2^−/− mice, which showed no significant difference with previous reports of human HBV infection in humanized uPA/SCID mice.^1,2,5