Bonnie L. Lyons

Human Lymphoid and Myeloid Cell Development in NOD/LtSz-scid IL2Rγnull Mice Engrafted with Mobilized Human Hemopoietic Stem Cells

Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC). To overcome this limitation, small animal models of human HSC engraftment have been used. We report the development and characterization of a new genetic stock of IL-2R common γ-chain deficient NOD/LtSz-scid (NOD-scid IL2Rγnull) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. NOD-scid IL2Rγnull mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. Engraftment of NOD-scid IL2Rγnull mice with human HSC generate 6-fold higher percentages of human CD45+ cells in host bone marrow than with similarly treated NOD-scid mice. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Spleens from engrafted NOD-scid IL2Rγnull mice contain human Ig+ B cells and lower numbers of human CD3+ T cells. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4+CD8+ thymocytes as well human CD4+CD8− and CD4−CD8+ peripheral blood and splenic T cells. De novo human T cell development in NOD-scid IL2Rγnull mice was validated by 1) high levels of TCR excision circles, 2) complex TCRβ repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Thus, NOD-scid IL2Rγnull mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment.

Chemotherapy-resistant human AML stem cells home to and engraft within the bone-marrow endosteal region

Acute myelogenous leukemia (AML) is the most common adult leukemia, characterized by the clonal expansion of immature myeloblasts initiating from rare leukemic stem (LS) cells. To understand the functional properties of human LS cells, we developed a primary human AML xenotransplantation model using newborn nonobese diabetic/severe combined immunodeficient/interleukin (NOD/SCID/IL)2rγnull mice carrying a complete null mutation of the cytokine γc upon the SCID background. Using this model, we demonstrated that LS cells exclusively recapitulate AML and retain self-renewal capacity in vivo. They home to and engraft within the osteoblast-rich area of the bone marrow, where AML cells are protected from chemotherapy-induced apoptosis. Quiescence of human LS cells may be a mechanism underlying resistance to cell cycle–dependent cytotoxic therapy. Global transcriptional profiling identified LS cell–specific transcripts that are stable through serial transplantation. These results indicate the potential utility of this AML xenograft model in the development of novel therapeutic strategies targeted at LS cells.

NOD/LtSz-Rag1null Mice: An Immunodeficient and Radioresistant Model for Engraftment of Human Hematolymphoid Cells, HIV Infection, and Adoptive Transfer of NOD Mouse Diabetogenic T Cells

Development of a small animal model for the in vivo study of human immunity and infectious disease remains an important goal, particularly for investigations of HIV vaccine development. NOD/Lt mice homozygous for the severe combined immunodeficiency (Prkdcscid) mutation readily support engraftment with high levels of human hematolymphoid cells. However, NOD/LtSz-scid mice are highly radiosensitive, have short life spans, and a small number develop functional lymphocytes with age. To overcome these limitations, we have backcrossed the null allele of the recombination-activating gene (Rag1) for 10 generations onto the NOD/LtSz strain background. Mice deficient in RAG1 activity are unable to initiate V(D)J recombination in Ig and TCR genes and lack functional T and B lymphocytes. NOD/LtSz-Rag1null mice have an increased mean life span compared with NOD/LtSz-scid mice due to a later onset of lymphoma development, are radioresistant, and lack serum Ig throughout life. NOD/LtSz-Rag1null mice were devoid of mature T or B cells. Cytotoxic assays demonstrated low NK cell activity. NOD/LtSz-Rag1null mice supported high levels of engraftment with human lymphoid cells and human hemopoietic stem cells. The engrafted human T cells were readily infected with HIV. Finally, NOD/LtSz-Rag1null recipients of adoptively transferred spleen cells from diabetic NOD/Lt+/+ mice rapidly developed diabetes. These data demonstrate the advantages of NOD/LtSz-Rag1null mice as a radiation and lymphoma-resistant model for long-term analyses of engrafted human hematolymphoid cells or diabetogenic NOD lymphoid cells.

Non-obese diabetic-recombination activating gene-1 (NOD-Rag1 null) interleukin (IL)-2 receptor common gamma chain (IL2r gamma null) null mice: a radioresistant model for human lymphohaematopoietic engraftment

Immunodeficient hosts engrafted with human lymphohaematopoietic cells hold great promise as a preclinical bridge for understanding human haematopoiesis and immunity. We now describe a new immunodeficient radioresistant non‐obese diabetic mice (NOD) stock based on targeted mutations in the recombination activating gene‐1 (Rag1null) and interleukin (IL)‐2 receptor common gamma chain (IL2rγnull), and compare its ability to support lymphohaematopoietic cell engraftment with that achieved in radiosensitive NOD.CB17–Prkdcscid (NOD–Prkdcscid) IL2rγnull mice. We observed that immunodeficient NOD–Rag1null IL2rγnull mice tolerated much higher levels of irradiation conditioning than did NOD–Prkdcscid IL2rγnull mice. High levels of human cord blood stem cell engraftment were observed in both stocks of irradiation‐conditioned adult mice, leading to multi‐lineage haematopoietic cell populations and a complete repertoire of human immune cells, including human T cells. Human peripheral blood mononuclear cells also engrafted at high levels in unconditioned adult mice of each stock. These data document that Rag1null and scid stocks of immunodeficient NOD mice harbouring the IL2rγnull mutation support similar levels of human lymphohaematopoietic cell engraftment. NOD–Rag1null IL2rγnull mice will be an important new model for human lymphohaematopoietic cell engraftment studies that require radioresistant hosts.

Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion

To obtain insights into the cardiomyogenic potential of hematopoietic tissue, we intravenously (i.v.) injected purified hematopoietic stem/progenitor cells into newborn recipients that may fully potentiate the developmental plasticity of stem cells. Transplantation of mouse bone marrow (BM) lineage antigen‐negative (Lin−) cells resulted in the generation of the cells that displayed cardiomyocyte‐specific antigenic profiles and contractile function when transplanted into syngeneic newborn recipients. To clarify the mechanism underlying the cardiomyogenic potential, green fluorescent protein (GFP)‐labeled BM Lin−ScaI+ hematopoietic progenitors were transplanted into neonatal mice constitutively expressing cyan fluorescence protein (CFP). Lambda image acquisition and linear unmixing analysis using confocal microscopy successfully separated GFP and CFP, and revealed that donor GFP+ cardiomyocytes coexpressed host‐derived CFP. We further reconstituted human hemopoietic‐ and immune systems in mice by injecting human cord blood (CB)‐derived Lin−CD34+CD38− hematopoietic stem cells (HSCs) into neonatal T cell−B cell−NK cell− immune‐deficient NOD/SCID/IL2rγnull mice. Fluoroescence in situ hybridization analysis of recipient cardiac tissues demonstrated that human and murine chromosomes were colocalized in the same cardiomyocytes, indicating that cell fusion occurred between human hematopoietic progeny and mouse cardiomyocytes. These syngeneic‐ and xenogeneic neonatal transplantations provide compelling evidence that hematopoietic stem/progenitor cells contribute to the postnatal generation of cardiomyocytes through cell fusion, not through transdifferentiation.— Ishikawa, F., Shimazu, H., Shultz, L. D., Fukata, M., Nakamura, R., Lyons, B., Shimoda, K., Shimoda, S., Kanemaru, T., Nakamura, K‐i., Ito, H., Kaji, Y., Perry, A. C. F., Harada, M. Purified human hematopoietic stem cells contribute to the generation of cardiomyocytes through cell fusion. FASEB J. 20, E11–E17 (2006)

Human antibodies induce arthritis in mice deficient in the low-affinity inhibitory IgG receptor FcγRIIB

Rheumatoid arthritis (RA) is a complex autoimmune disease with a poorly understood pathogenesis. The disease is associated with polyclonal B cell activation and the production of autoantibodies (autoAbs), but there is a longstanding controversy as to whether such Abs contribute to, or are secondary to, the pathogenesis of RA. To address the potential pathogenicity of human RA–associated Abs, we developed a passive transfer model involving mice deficient in the low-affinity inhibitory Fc receptor, FcγRIIB. We report that plasma or serum from patients with active RA can induce inflammation and histological lesions in FcγRIIB−/− mice consistent with arthritis, and that this pathogenic activity is caused by the immunoglobulin G–rich fraction. Our results suggest that humoral autoimmunity can contribute directly to autoimmune arthritis, and that FcγRIIB−/− mice are a promising model to evaluate the arthritogenic potential of human autoAbs.

Treatment of BXSB-Yaa mice with IL-21R-Fc fusion protein minimally attenuates systemic lupus erythematosus.

Abstract:  Interleukin‐21 (IL‐21) is a pleiotropic cytokine whose function is only now being unraveled. Abundant evidence indicates that activated CD4 T cells are the primary, if not the only, source of IL‐21. While it is clear that IL‐21 is actively transcribed by naïve activated T cells, recent studies have shown that IL‐21 potentially promotes a developmental shift of naïve T cells toward the Th2 phenotype. BXSB‐Yaa mice develop an autoimmune syndrome similar to systemic lupus erythematosus (SLE), affecting males earlier than females on account of the presence of the Yaa (Y‐linked autoimmune acceleration) locus. Previous results indicate the elevation of IL‐21 expression by BXSB‐Yaa mice at an age when the early characteristics of autoimmune processes first become evident. We set out to determine whether IL‐21 was necessary for disease progression in BXSB‐Yaa mice. Mice were treated for 24 weeks with soluble IL‐21R‐Fc in order to therapeutically neutralize the IL‐21 present. The results overall suggest a biphasic effect of IL‐21, negatively influencing survival early on and positively influencing survival at later stages. We propose that IL‐21 exerts a pleiotropic effect in which it promotes the protective effects of CD8+ suppressor cells in the early disease phase and then promotes the humoral components of SLE in the later disease stages. This experiment provides preliminary evidence for a role of IL‐21 in modulating the severity of SLE in BXSB‐Yaa mice.

NOD/LtSz-Rag1nullPfpnull mice: a new model system with increased levels of human peripheral leukocyte and hematopoietic stem-cell engraftment.

Background. A critical need exists for effective small-animal models that accept engraftment of human hematopoietic progenitor cells and mature lymphocytes. The purpose of this study was to determine the phenotypic effects of perforin (Pfp) deficiency on nonobese diabetic (NOD)-Rag1null mice and to evaluate the ability of NOD/LtSz-Rag1nullPfpnull recipients to support engraftment with human hematolymphoid cells. Methods. A new genetic stock of NOD mice doubly homozygous for targeted mutations at the recombination activating gene (Rag)-1 and Pfp genes was developed. NOD/LtSz-Rag1nullPfpnull mice were studied for immunopathologic and hematologic abnormalities. The ability of these mice to support engraftment with human peripheral blood mononuclear cells (PBMC) and umbilical-cord blood hematopoietic progenitor cells was assessed. Results. NOD/LtSz-Rag1nullPfpnull mice lacked mature B cells, T cells, natural killer (NK) cell cytotoxic activity and were devoid of serum immunoglobulin (Ig) throughout a 37-week lifespan. These mice supported heightened engraftment with human PBMC as compared with NOD/LtSz-Rag1null controls as evidenced by a 4- to 5-fold increase in percentages of human lymphocytes and a 7- to 13-fold increase in percentages of CD4+ T cells in the peripheral blood and spleen. Total numbers of human CD4+ T cells were increased approximately 20-fold in the spleens of NOD/LtSz-Rag1nullPfpnull mice. These mice also showed approximately 12-fold higher levels of engraftment with human umbilical-cord blood cells compared with NOD/LtSz-Rag1null mice. Conclusions. NOD/LtSz-Rag1nullPfpnull mice are devoid of mature B cell, T cell, and NK cell cytotoxic activity, engraft at high levels with human PBMC, and hematopoietic progenitor cells and provide a new NK cell-deficient model for human hematolymphoid cell engraftment.

Humanized NOD/LtSz‐scid IL2 Receptor Common Gamma Chain Knockout Mice in Diabetes Research

Abstract:  There are many rodent models of autoimmune diabetes that have been used to study the pathogenesis of human type 1 diabetes (T1D), including the non‐obese diabetic (NOD) mouse, the biobreeding (BB) rat, and the transgenic mouse models. However, mice and rats are not humans, and these rodent models do not completely recapitulate the autoimmune pathogenesis of the human disease. In addition, many of the reagents, tools, and therapeutics proposed for use in humans may be species specific and cannot be investigated in rodents. Researchers have used nonhuman primates to more closely mimic the human immune system and, to study species‐specific therapeutics, but these studies are associated with additional ethical and economic constraints and, to date, no model of autoimmune diabetes in this species has been described. New animal models are needed that will permit the in vivo investigation of human immune systems and analyses of the pathogenesis of human T1D without putting individuals at risk. To fill this need, we are developing humanized mouse models for the in vivo study of T1D. These models are based on our newly generated stock of NOD‐scid IL2rγnull mice, which engraft at higher levels with human hematolymphoid cells and exhibit enhanced function of the engrafted human immune systems compared with previous humanized mouse models. Overall, development of these new generations of humanized mice should facilitate in vivo studies of the human immune system as well as permit the investigation of the pathogenesis and effector phases of human T1D.

Dystrophin and dysferlin double mutant mice: a novel model for rhabdomyosarcoma.

Although researchers have yet to establish a link between muscular dystrophy (MD) and sarcomas in human patients, literature suggests that the MD genes dystrophin and dysferlin act as tumor suppressor genes in mouse models of MD. For instance, dystrophin-deficient mdx and dysferlin-deficient A/J mice, models of human Duchenne MD and limb-girdle MD type 2B, respectively, develop mixed sarcomas with variable penetrance and latency. To further establish the correlation between MD and sarcoma development, and to test whether a combined deletion of dystrophin and dysferlin exacerbates MD and augments the incidence of sarcomas, we generated dystrophin and dysferlin double mutant mice (STOCK-Dysf(prmd)Dmd(mdx-5Cv)). Not surprisingly, the double mutant mice develop severe MD symptoms and, moreover, develop rhabdomyosarcoma (RMS) at an average age of 12 months, with an incidence of >90%. Histological and immunohistochemical analyses, using a panel of antibodies against skeletal muscle cell proteins, electron microscopy, cytogenetics, and molecular analysis reveal that the double mutant mice develop RMS. The present finding bolsters the correlation between MD and sarcomas, and provides a model not only to examine the cellular origins but also to identify mechanisms and signal transduction pathways triggering development of RMS.