Tsutomu Kamisako

Highly Sensitive Model for Xenogenic GVHD Using Severe Immunodeficient NOG Mice

Background. Several animal models for xenogenic (xeno) graft versus host disease (GVHD) have been developed in immunodeficient mice, such as C.B-17-scid and nonobese diabetes (NOD)/severe combined immunodeficiency (SCID), by human peripheral blood mononuclear cell (hPBMC) transplantation. However, these models pose problems because they require sublethal total body irradiation of the mice and a large number of hPBMCs to induce GVHD, and the timing of onset of GVHD is also unstable. The aim of this study is to establish improved murine models of xeno-GVHD using novel immunodeficient NOD/Shi-scid IL2rγnull (NOG) mice. Methods. In three strains of immunodeficient mice, NOG, BALB/cA-RAG2null IL2rγnull, and NOD/SCID mice, GVHD was induced by transplantation of hPBMCs with or without total body irradiation, and the GVHD symptoms in these strains were compared. Results. After intravenous transplantation of hPBMCs, NOG mice showed early onset of GVHD symptoms and a small number of hPBMCs (2.5×106) was sufficient to induce GVHD when compared with BALB/cA-RAG2null IL2rγnull and NOD/SCID mice. In addition, total body irradiation was not always necessary in the present model. Conclusions. These results indicate that our model using the NOG mouse is a useful tool to investigate GVHD and to develop effective drugs for GVHD.

Establishment of a Human Allergy Model Using Human IL-3/GM-CSF–Transgenic NOG Mice

The development of animal models that mimic human allergic responses is crucial to study the pathophysiology of disease and to generate new therapeutic methodologies. Humanized mice reconstituted with human immune systems are essential to study human immune reactions in vivo and are expected to be useful for studying human allergies. However, application of this technology to the study of human allergies has been limited, largely because of the poor development of human myeloid cells, especially granulocytes and mast cells, which are responsible for mediating allergic diseases, in conventional humanized mice. In this study, we developed a novel transgenic (Tg) strain, NOD/Shi-scid-IL2rγnull (NOG), bearing human IL-3 and GM-CSF genes (NOG IL-3/GM–Tg). In this strain, a large number of human myeloid cells of various lineages developed after transplantation of human CD34+ hematopoietic stem cells. Notably, mature basophils and mast cells expressing FcεRI were markedly increased. These humanized NOG IL-3/GM–Tg mice developed passive cutaneous anaphylaxis reactions when administered anti–4-hydroxy-3-nitrophenylacetyl IgE Abs and 4-hydroxy-3-nitrophenylacetyl. More importantly, a combination of serum from Japanese cedar pollinosis patients and cedar pollen extract also elicited strong passive cutaneous anaphylaxis responses in mice. Thus, to our knowledge, our NOG IL-3/GM–Tg mice are the first humanized mouse model to enable the study of human allergic responses in vivo and are excellent tools for preclinical studies of allergic diseases.

Predominant development of mature and functional human NK cells in a novel human IL-2-producing transgenic NOG mouse.

We generated a severe immunodeficient NOD/Shi-scid-IL-2Rγnull (NOG) mouse substrain expressing the transgenic human IL-2 gene (NOG–IL-2 Tg). Upon transfer of human cord blood–derived hematopoietic stem cells (HSCs), CD3−CD56highCD16+/− cells developed unexpectedly, predominantly in the NOG–IL-2 Tg (hu-HSC NOG–IL-2 Tg). These cells expressed various NK receptors, including NKp30, NKp44, NKp46, NKG2D, and CD94, as well as a diverse set of killer cell Ig-like receptor molecules at levels comparable to normal human NK cells from the peripheral blood, which is evidence of their maturity. They produced levels of granzyme A as high as in human peripheral blood–derived NK cells, and a considerable amount of perforin protein was detected in the plasma. Human NK cells in hu-HSC NOG–IL-2 Tg produced IFN-γ upon stimulation, and IL-2, IL-15, or IL-12 treatment augmented the in vitro cytotoxicity. Inoculation of K562 leukemia cells into hu-HSC NOG–IL-2 Tg caused complete rejection of the tumor cells, whereas inoculation into hu-HSC NOG fully reconstituted with human B, T, and some NK cells did not. Moreover, when a CCR4+ Hodgkin’s lymphoma cell line was inoculated s.c. into hu-HSC NOG–IL-2 Tg, the tumor growth was significantly suppressed by treatment with a therapeutic humanized anti-CCR4 Ab (mogamulizumab), suggesting that the human NK cells in the mice exerted active Ab-dependent cellular cytotoxicity in vivo. Taken together, these data suggest that the new NOG–IL-2 Tg strain is a unique model that can be used to investigate the biological and pathological functions of human NK cells in vivo.

Efficient Xenoengraftment in Severe Immunodeficient NOD/Shi-scid IL2rγnull Mice Is Attributed to a Lack of CD11c+B220+CD122+ Cells

Xenograft animal models using immunodeficient mice have been widely applied in medical research on various human diseases. NOD/Shi-scid-IL2rγnull (NOG) mice are known to show an extremely high engraftment rate of xenotransplants compared with conventional immunodeficient mice. This high engraftment rate of xenotransplants in NOG mice was substantially suppressed by the transfer of spleen cells from NOD-scid mice that were devoid of NK cells. These results indicate that cell types other than splenic NK cells present in NOD-scid mice but not in NOG mice may be involved in this suppression. To identify the cell types responsible for this effect, we transferred subpopulations of spleen cells from NOD-scid mice into NOG mice and assessed the levels of human cell engraftment after human PBMC (hPBMC) transplantation. These experiments revealed that CD11c+B220+ plasmacytoid dendritic cells (pDCs) from NOD-scid mice markedly inhibited engraftment of human cells. The CD11c+B220+CD122+ cells further fractionated from the pDCs based on the expression of CD122, which is an NK cell marker strongly inhibited during hPBMC engraftment in NOG mice. Moreover, the CD122+ cells in the pDC fraction were morphologically distinguishable from conventional CD122+ NK cells and showed a higher rejection efficiency. The current results suggest that CD11c+B220+CD122+ cells play an important role in xenograft rejection, and their absence in NOG mice may be critical in supporting the successful engraftment of xenotransplants.

A study on cryoprotectant solution suitable for vitrification of rat two-cell stage embryos.

The present study was performed to develop a suitable cryoprotectant solution for cryopreservation of rat two-cell stage embryos. First, we examined the cell permeability of several cryoprotectants; propylene glycol had the fastest permeability compared to dimethyl sulfoxide, ethylene glycol, and glycerol. Embryos were then exposed to a solution containing propylene glycol to evaluate its effects on fetal development. As the development was similar to that of fresh embryos, P10 (10% v/v propylene glycol in PB1) was used as a pretreatment solution. Next, the effects of the vitrification solution components (sucrose, propylene glycol, ethylene glycol, and Percoll) were examined by observing the vitrification status; 10% v/v propylene glycol, 30% v/v ethylene glycol, 0.3 mol sucrose, and 20% v/v Percoll in PB1 (PEPeS) was the minimum essential concentration for effective vitrification without the formation of ice crystals or freeze fractures. A new vitrification method using P10 and PEPeS was tested using rat embryos. The survival rate of vitrified embryos after exposure to P10 for 120, 300, or 600 s ranged from 95.9% to 98.3%. The fetal developmental rate ranged from 57.7% to 65.2%, which was not significantly different from that of fresh embryos. The experimental results indicated that vitrification using a combination of P10 and PEPeS was suitable for cryopreservation of rat early stage embryos.

NOD-Rag2null IL-2Rγnull mice: an alternative to NOG mice for generation of humanized mice.

We have developed NOD-Rag2null IL-2Rγnull (NR2G) mice similar to NOD-scidIL-2Rγnull (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.

Long-term maintenance of peripheral blood derived human NK cells in a novel human IL-15- transgenic NOG mouse

We generated a novel mouse strain expressing transgenic human interleukin-15 (IL-15) using the severe immunodeficient NOD/Shi-scid-IL-2Rγnull (NOG) mouse genetic background (NOG-IL-15 Tg). Human natural killer (NK) cells, purified from the peripheral blood (hu-PB-NK) of normal healthy donors, proliferated when transferred into NOG-IL-15 Tg mice. In addition, the cell number increased, and the hu-PB-NK cells persisted for 3 months without signs of xenogeneic graft versus host diseases (xGVHD). These in vivo-expanded hu-PB-NK cells maintained the original expression patterns of various surface antigens, including NK receptors and killer cell immunoglobulin-like receptor (KIR) molecules. They also contained significant amounts of granzyme A and perforin. Inoculation of K562 leukemia cells into hu-PB-NK-transplanted NOG-IL-15 Tg mice resulted in significant suppression of tumor growth compared with non-transplanted mice. Furthermore, NOG-IL-15 Tg mice allowed for engraftment of in vitro-expanded NK cells prepared for clinical cell therapy. These cells exerted antibody-dependent cell-mediated cytotoxicity (ADCC) on Her2-positive gastric cancer cells in the presence of therapeutic anti-Her2 antibody, and subsequently suppressed tumor growth. Our results collectively suggest that the NOG-IL-15 Tg mice are a useful model for studying human NK biology and evaluating human NK cell-mediated in vivo cytotoxicity.

Strain preservation of experimental animals: Vitrification of two-cell stage embryos for multiple mouse strains

Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains.

Novel reporter and deleter mouse strains generated using VCre/VloxP and SCre/SloxP systems, and their system specificity in mice

DNA site-specific recombination by Cre/loxP is a powerful tool for gene manipulation in experimental animals. VCre/VloxP and SCre/SloxP are novel site-specific recombination systems, consisting of a recombinase and its specific recognition sequences, which function in a manner similar to Cre/loxP. Previous reports using Escherichia coli and Oryzias latipes demonstrated the existence of stringent specificity between each recombinase and its target sites; VCre/VloxP, SCre/SloxP, and Cre/loxP have no cross-reactivity with each other. In this study, we established four novel knock-in (KI) mouse strains in which VloxP-EGFP, SloxP-tdTomato, CAG-VCre, and CAG-SCre genes were inserted into the ROSA26 locus. VloxP-EGFP and SloxP-tdTomato KI mice were reporter mice carrying EGFP or tdTomato genes posterior to the stop codon, which was floxed by VloxP or SloxP fragments, respectively. CAG-VCre and CAG-SCre KI mice carried VCre or SCre genes that were expressed ubiquitously. These two reporter mice were crossed with three different deleter mice, CAG-VCre KI, CAG-SCre KI, and Cre-expressing transgenic mice. Through these matings, we found that VCre/VloxP and SCre/SloxP systems were functional in mice similar to Cre/loxP, and that the recombinases showed tight specificity for their recognition sequences. Our results suggest that these novel recombination systems allow highly sophisticated genome manipulations and will be useful for tracing the fates of multiple cell lineages or elucidating complex spatiotemporal regulations of gene expression.