Development History of Immunodeficient Mice and Their Research Applications
By John Charpentier, Ph.D.
October 25, 2022
Since the characterization of the nude mouse in 1962, genetic mouse models of immunodeficiency have been increasingly developed, diversified, and utilized for both basic discovery research and preclinical studies of therapeutic candidates. Today, highly immunodeficient, genetically-engineered mouse models are invaluable tools for basic biomedical research, especially for the study of cancer, hematopoiesis, infectious diseases, and tissue regeneration. In addition to enabling in vivo studies in the absence of murine immune components, immunodeficient mice are ideal for human immune cell reconstitution and modeling of human immune responses.
In this feature we will briefly review the history of immunodeficient mice, introduce and differentiate Biocytogen’s industry-leading immunodeficient B-NDG model, and describe our latest advances in engineering next-generation strains within the B-NDG family. If you’d prefer to view a video on this material, please see our recent webinar below.
Why Use Immunodeficient Mouse Models?
Highly immunodeficient mouse models enable the study of biological phenomena that are difficult or impossible to evaluate in other systems. Successful xenotransplantation of human cells or tissues, for example, is only feasible in a heterologous graft-tolerant model. This includes the in vivo reconstitution of human immune system components by engraftment of peripheral blood mononuclear cells (PBMCs) or CD34+ hematopoietic stem cells from whole blood donors.
Broad humanization of the immune system in this manner makes tractable the study of human-like immune responses in the mouse and permits the evaluation of therapeutic candidates targeting or modulating them. Additionally, the abrogation of murine immunity enables the study of human-specific pathogens that would not otherwise productively infect rodents. Immunodeficient mice are also a powerful tool to assess the functional potential of embryonic and induced pluripotent stem cells.
History of Immunodeficient Mouse Models
The first genetic mouse model of immunodeficiency, the nude mouse, was discovered in 1962. These mice were characterized by a spontaneous deletion of Foxn1, a master regulator of the thymic epithelial cell lineage. In addition to producing their eponymous hairlessness, deletion of Foxn1 disrupts thymic architecture and inhibits the signal-dependent differentiation of thymocytes. Consequently, nude mice are athymic and lack mature T cells. While they are immunodeficient, nude mice do not efficiently tolerate patient-derived xenografts (PDXs) or cell-derived xenografts (CDXs).
Approximately two decades later, the non-obese diabetic (NOD)/ShiLTJ mouse was discovered. In contrast to nude mice, the NOD/ShiLTJ model is characterized by polymorphic Ctla-4 and Sirpα alleles. The latter encodes an inhibitory receptor that strongly binds human CD47 protein, and the former encodes a variant T cell immune checkpoint receptor with impaired functioning. These mice also exhibit a deficiency of the hemolytic complement component C5.
Disruption of Ctla-4 results in unchecked pancreatic islet infiltration and destruction by lymphocytes, making the NOD mouse an early genetic model of Type I diabetes. The high-affinity binding of human CD47 by polymorphic Sirpα creates a more permissive environment in vivo for human-to-mouse xenografts. NOD mice are therefore more suitable for PDXs and CDX studies than nude mice, but engraftment efficiency remains quite low. Finally, loss of hemolytic complement impairs innate immunity by inhibiting chemotaxis of inflammatory myeloid cells and formation of the membrane attack complex (MAC).
The discovery of Rag1/2 and DNA-PKcs enzymes led to the generation of the severe combined immunodeficiency (SCID) model in 1985. Homozygous mutation of murine genes encoding these proteins severely impairs V(D)J recombination, an essential process for development of lymphocyte immunoreceptors. Consequently, SCID mice, which are congenic with the C.B-17 strain, have non-functional T and B cells and exhibit agammaglobulinemia. The presence of high numbers of murine NK cells, however, diminishes engraftment efficiency in in SCID mice, limiting their research application.
Transferring SCID mutations to the NOD background produced the NOD-SCID model, first characterized in 1990. These mice harbor mutations in both Ctla-4 and Prkdc (encoding a DNA-PKcs enzyme). As the name suggests, NOD-SCID mice exhibit defects in both innate and adaptive immunity seen in the parental strains and are much more suitable for PDX/CDX studies than either parental strain. These mice, however, have an elevated incidence of thymic lymphoma that shortens their average lifespan.
IL2rγnull , NOG, and NSG™ mice
In 1997, the IL-2rγnull mouse model, which carries a null allele of the IL-2 receptor common gamma (cγ) chain, was developed. As the cγ chain is a component of a number of cytokine receptors (i.e., IL-2/-4/-7/-9/-15/-21), these mice show profoundly impaired cytokine signaling and lymphocyte development. A major benefit of this model is the absence of murine NK cells, as IL-15 is required for NK cell ontogeny. The discovery of this model directly led to the further development of the highly immunodeficient models in use today, such as NOG and NSG™ mice, which were developed in the early 2000s. These mice lack Ctla-4, Prkdc, and have disrupted IL-2rγ function, resulting in severe defects in complement, cytokine signaling, and development of T, B, and NK cells.
Biocytogen’s highly immunodeficient B-NDG (NOD.CB17-PrkdcscidIl2rgtm1/Bcgen) mouse was engineered in 2014. Similar to NOG and NSG mice, these mice also lack Ctla-4 and Prkdc, and harbor a null Il2rγ allele. B-NDG mice eliminate some of the limitations of NOD-SCID immunodeficient mice, and do not exhibit variation in phenotypic penetrance, leakiness of disrupted murine genes, reduced longevity, or poor engraftment, proliferation, and functioning of xenotransplanted cells and tissues. B-NDG mice live much longer on average than NOD-SCID mice (18 vs. 8-9 months), likely due to the minimal incidence of thymic lymphoma. Leakage of murine lymphocytes in B-NDG mice is also negligible or absent in B-NDG mice, as detailed below.
B-NDG Mice Lack T, B, and NK Cells
B-NDG mice lack mature T, B, and NK cells. In the figure below, we see two representations of murine T, B, and NK cell populations isolated from 6 week old BALB/c, NOD-SCID, and B-NDG spleens (n = 3 females per group). On the top (A) we see representative flow cytometry plots showing the distribution of T, B, and NK cells among murine CD45+ cells in each model. The bottom panel (B) shows a quantitation of the same data. Note that while T and B cells are absent in both NOD-SCID and B-NDG mice, NK cells are only missing in the latter.
Lymphoid Follicles Are Disrupted in B-NDG Mice
Histological sections of lymphoid follicles from 9 week old, C57BL/6, NOD-SCID, and B-NDG mice show significant structural differences in the spleen and thymus. As shown in the spleen sections below, follicles in C57BL/6 mice are well-defined and exhibit normal morphology while follicles in NOD-SCID mice display white pulp hypoplasia and disordered follicles. B-NDG mice, on the other hand, show a total loss of follicular structure.
Circulating IgG and IgM Are Depleted in B-NDG mice
As expected, B-NDG mice have dramatically reduced numbers of circulating IgG and IgM antibodies. Relative proportions of IgG and IgM in BALB/c and B-NDG mice were measured by ELISA and quantitated in the figure below. IgG and IgM concentrations in B-NDG mice were dramatically reduced compared to the BALB/c positive control and were essentially identical to negative controls. This trend was preserved across IgG subclasses.
Multiple Applications For B-NDG mice
B-NDG mice are ideal for experiments using cell- and patient-derived xenografts (CDX/PDXs), as well as CAR studies. In the experiment summarized below, 5 × 106 Raji-Fluc B cells were transplanted into 8 B-NDG mice, and infused with 2 × 107 (human) CD19-targeting CAR-T cells or negative control 2 weeks later. In vivo bioluminescence imaging (BLI) shows limited expansion of tumor cells over 5 weeks in the post-infusion mice but not untreated control mice, who survived only three weeks following Raji-Fluc B cell transplantation.
B-NDG mice are well-suited for human immune system reconstitution using PBMCs, CD34+ hematopoietic stem cells, or combined fetal bone marrow, liver, and thymus (BLT) transplant. In the experiment below, 5 × 106 human PBMCs were intravenously transplanted into 6 week old, female B-NDG mice (n = 6). Post-transplantation analysis revealed a high percentage of engrafted human CD45+ (hematopoietic) and human CD3+ T cells, as shown below. High levels of human T cell engraftment produces graft-versus-host effects responsible for the reduced body weight and decreased survival seen here.
Next-Generation B-NDG Mouse Models
Biocytogen scientists have further engineered the B-NDG model and developed a family of derived strains for broader research applications. One category of these next-generation models is B-NDG mice expressing transgenes to support human immune cell survival and differentiation.
For example, B-NDG hIL-6 mice show enhanced human B cell and plasma cell differentiation and expansion compared to the parental strain. Similarly, NDG hIL-15 mice support differentiation and proliferation of human lymphoid cells (NK, NKT, CD8+ T cells).
The dually-humanized B-NDG hCSF1/hTHPO model expresses human colony stimulating factor 1 and thrombopoietin to promote the differentiation and survival of human monocytes and macrophages.
Expression of humanized genes on the B-NDG background also permits shaping of the human T cell repertoire in vivo. For example, Biocytogen’s B-NDG HLA-A2.1 model (which also includes hβ2M) not only facilitates enhanced differentiation of human CD8+ T cells, but also enables recognition of HLA-restricted epitopes.
Another category of next-generation B-NDG models are those adding a targeted murine gene knock-out or mutation. B-NDG β2M KO Plus mice lack cell surface expression of MHC-I on dendritic cells. Consequently, engrafted CD8+ T cells cannot mount MHC-I specific T cell responses, extending survival and reducing graft-versus-host responses. This tolerance profile makes it possible to evaluate long-term antitumor effects of therapeutic interventions and/or memory responses in tumor xenograft models bearing reconstituted human immune systems.
Biocytogen also offers B-NDG mice which combine both murine gene knock-out and humanized gene expression. This category is exemplified by our B-NDG β2M KO Plus/hIL-15 mice, which were engineered support human NK cell proliferation and expansion in the absence of murine MHC-I or CD8+ T cells. By selective humanization and targeted knockout of genes on the B-NDG background, many dimensions of human immunobiology can be recapitulated and studied in one high-quality model.
Biocytogen: Your Trusted Source for Immunodeficient Mice
Biocytogen’s B-NDG family of highly-immunodeficient mice constitute a suite of powerful tools to answer research questions that remain intractable in standard models. Currently, 23 of our 30 strains of genetically engineered, highly-immunodeficient mouse models are on the B-NDG background. All animals are bred and shipped from our our 540,000 ft2 SPF facility in Haimen, China. Health reports, including screening for over 80 pathogens, are shared prior to shipment transit, which usually takes 1-2 weeks for most destinations.
To view a complete listing of currently available models in the B-NDG family, please click here.