Establishment of a human microbiome- and immune system-reconstituted dual-humanized mouse model.

Humanized mice are widely used to study the human immune system in vivo and investigate therapeutic targets for various human diseases. Immunodeficient NOD/Shi-scid-IL2rγnull (NOG) mice transferred with human hematopoietic stem cells are a useful model for studying human immune systems and analyzing engrafted human immune cells. The gut microbiota plays a significant role in the development and function of immune cells and the maintenance of immune homeostasis; however, there is currently no available animal model that has been reconstituted with human gut microbiota and immune systems in vivo. In this study, we established a new model of CD34+ cell-transferred humanized germ-free NOG mice using an aseptic method. Flow cytometric analysis revealed that the germ-free humanized mice exhibited a lower level of human CD3+ T cells than the SPF humanized mice. Additionally, we found that the human CD3+ T cells slightly increased after transplanting human gut microbiota into the germ-free humanized mice, suggesting that the human microbiota supports T cell proliferation or maintenance in humanized mice colonized by the gut microbiota. Consequently, the dual-humanized mice may be useful for investigating the physiological role of the gut microbiota in human immunity in vivo and for application as a new humanized mouse model in cancer immunology.

Vγ5Vδ1 TCR signaling is required to different extents for embryonic versus postnatal development of DETCs.

γδ T cells expressing Vγ5Vδ1 TCR originally develop in the embryonic thymus and migrate to the epidermis, forming dendritic epidermal T cells (DETCs) throughout life. It is thought that a TCR signal is essential for their development; e.g., lack of TCR signal-transducer ZAP70 significantly decreases DETC numbers. On the other hand, lack of ZAP70 does not affect Vγ5Vδ1+ T cells in the embryonic thymus; thus, the involvement of TCR signaling remains elusive. Here, we used SKG mice with attenuated TCR signaling rather than gene-knockout mice. In SKG mice, Vγ5+ T cells showed a marked decrease [10% of wild-type (WT)] in adult epidermis; however, there was just a moderate decrease (50% of WT) in the embryonic thymus. In early postnatal epidermis in SKG mice, substantial numbers of Vγ5+ T cells were observed (50% of WT). Their activation markers including CD122, a component of the IL-15 receptor indispensable for DETC proliferation, were comparable to those of WT. However, the Vγ5+ T cells in SKG mice did not proliferate and form DETCs thereafter. Furthermore, in SKG/+ mice, the number of thymic Vγ5Vδ1+ T cells increased, compared to SKG mice; however, the number of DETCs remained significantly lower than in WT, similar to SKG mice. Our results suggest that signaling via Vγ5Vδ1 TCR is indispensable for DETC development, with distinct contributions to embryonic development and postnatal proliferation.

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-PrkdcscidIl2rgtm1Sug/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.

Creation of an experimental rearing environment for microbiome animal research using an individually ventilated cage system and bioBUBBLE enclosure.

To avoid microbial contamination risk, vinyl film isolators are generally used in animal microbiome experiments involving germ-free (GF) mice and/or gnotobiotic (GB) mice. However, it can take several months to gain expertise in operating the isolator competently. Furthermore, sterilization and sterility testing, which are essential for isolator preparation, can take more than 20 days. Hence, we built an experimental rearing environment that combines an individual ventilation cage system and a bioBUBBLE clean room enclosure to easily set up an experimental animal microbiome environment for animal facilities. In this work, a three-step evaluation was conducted. First, we examined whether GF mice can be maintained in this rearing environment without bacterial contamination. Next, we examined whether GF and GB mice can be maintained without cross-contamination in one individual ventilation cage rack. Finally, we tested whether GF mice can be maintained in a biological safety cabinet controlled by negative pressure. In our series of experiments, no microbial contamination occurred over more than 3 months. These results indicated that our rearing system that combines the individual ventilation cage and bioBUBBLE systems can be used not only for experiments with GF mice but also for Biosafety Level 2 experiments that handle bacteria. Our system can mitigate various disadvantages of using vinyl film isolators. In conclusion, we established an experimental method with improved working time and efficiency compared with those of the previous vinyl isolator method.

Improved engraftment of human peripheral blood mononuclear cells in NOG MHC double knockout mice generated using CRISPR/Cas9.

Humanized mice are widely used to study the human immune system in vivo and develop therapies for various human diseases. Human peripheral blood mononuclear cells (PBMC)-engrafted NOD/Shi-scid IL2rγnull (NOG) mice are useful models for characterization of human T cells. However, the development of graft-versus-host disease (GVHD) limits the use of NOG PBMC models. We previously established a NOG-major histocompatibility complex class I/II double knockout (dKO) mouse model. Although humanized dKO mice do not develop severe GVHD, they have impaired reproductive performance and reduced chimerism of human cells. In this study, we established a novel beta-2 microglobulin (B2m) KO mouse model using CRISPR/Cas9. By crossing B2m KO mice with I-Ab KO mice, we established a modified dKO (dKO-em) mouse model. Reproductivity was slightly improved in dKO-em mice, compared with conventional dKO (dKO-tm) mice. dKO-em mice showed no signs of GVHD after the transfer of human PBMCs; they also exhibited high engraftment efficiency. Engrafted human PBMCs survived significantly longer in the peripheral blood and spleens of dKO-em mice, compared with dKO-tm mice. In conclusion, dKO-em mice might constitute a promising PBMC-based humanized mouse model for the development and preclinical testing of novel therapeutics for human diseases.

Human PBMC-transferred murine MHC class I/II-deficient NOG mice enable long-term evaluation of human immune responses.

Immunodeficient mice engrafted with human peripheral blood cells are promising tools for in vivo analysis of human patient individual immune responses. However, when human peripheral blood mononuclear cells (PBMCs) are transferred into NOG (NOD/Shi-scid, IL-2rgnull) mice, severe graft versus host disease (GVHD) hinders long term detailed analysis. Administration of human PBMCs into newly developed murine MHC class I- and class II-deficient NOG (NOG-dKO; NOG- Iab, B2m-double-knockout) mice showed sufficient engraftment of human immune cells with little sign of GVHD. Immunization with influenza vaccine resulted in an increase in influenza-specific human IgG Ab, indicating induction of antigen-specific B cells in the NOG-dKO mice. Immunization with human dendritic cells pulsed with HLA-A2 restricted cytomegalovirus peptide induced specific cytotoxic T cells, indicating the induction of antigen-specific T cells in the NOG-dKO mice. Adoptive cell therapies (ACTs) using melanoma antigen recognized by T cells (MART-1)-specific TCR-transduced activated T cells showed strong tumor growth inhibition in NOG-dKO mice without any sign of GVHD accompanied by preferential expansion of the transferred MART-1-specific T cells. ACTs using cultured human melanoma infiltrating T cells also showed anti-tumor effects against autologous melanoma cells in NOG-dKO mice, in which changes in human cancer phenotypes by immune intervention, such as increased CD271 expression, could be evaluated. Therefore, NOG-dKO mice are useful tools for more detailed analysis of both the induction and effector phases of T-cell and B-cell responses for a longer period than regular NOG 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-2Rγ null (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.

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-2Rγ(null) (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(-)CD56(high)CD16(+/-) 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.