Takeshi Takahashi

Current advances in humanized mouse models

Humanized mouse models that have received human cells or tissue transplants are extremely useful in basic and applied human disease research. Highly immunodeficient mice, which do not reject xenografts and support cell and tissue differentiation and growth, are indispensable for generating additional appropriate models. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγnull gene (e.g., NOD/SCID/γcnull and Rag2nullγcnull mice). These strains show not only a high rate of human cell engraftment, but also generate well-differentiated multilineage human hematopoietic cells after human hematopoietic stem cell (HSC) transplantation. These humanized mice facilitate the analysis of human hematology and immunology in vivo. However, human hematopoietic cells developed from HSCs are not always phenotypically and functionally identical to those in humans. More recently, a new series of immunodeficient mice compensates for these disadvantages. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γcnull and Rag2nullγcnull mice. In this review, we describe the current knowledge of human hematopoietic cells developed in these mice. Various human disease mouse models using these humanized mice are summarized.

IL-2 induces in vivo suppression by CD4+CD25+Foxp3+ regulatory T cells

Interleukin‐2 (IL‐2) treatment is currently used to enhance T cell‐mediated immune responses against tumors or in viral infections. At the same time, IL‐2 is essential for the peripheral homeostasis of CD4+CD25+Foxp3+ regulatory T cells (Treg). In our study, we show that IL‐2 is also an important activator of Treg suppressive activity in vivo. IL‐2 treatment induces Treg expansion as well as IL‐10 production and increases their suppressive potential in vitro. Importantly, in vivo application of IL‐2 via gene‐gun vaccination using IL‐2 encoding DNA plasmids (pIL‐2) inhibited naive antigen‐specific T cell proliferation as well as a Th1‐induced delayed type hypersensitivity response. The suppressive effect can be transferred onto naive animals by Treg from IL‐2‐treated mice and the suppression depends on the synergistic action of IL‐10 and TGF‐β. These data highlight that during therapeutic treatment with IL‐2 the concomitant activation of Treg may indeed counteract the intended activation of cellular immunity.

OX40–OX40 Ligand Interaction in T-Cell-Mediated Immunity and Immunopathology

T-cell activation is mediated not only by antigen stimulation through T-cell receptors but also by costimulatory signals through costimulatory molecules. Among several costimulatory molecules, the tumor necrosis factor (TNF) receptor family member OX40 plays a key role in the survival and homeostasis of effector and memory T cells. According to the conventional understanding of OX40 costimulation, an interaction between OX40 and OX40 ligand (OX40L) occurs when activated T cells bind to professional antigen-presenting cells (APCs). The T-cell functions, including cytokine production, expansion, and survival, are then enhanced by the OX40 costimulatory signals. Over the last half-decade, evidence has accumulated that OX40 signals are critical for controlling the function and differentiation of Foxp3(+) regulatory T cells, indicating a new aspect of OX40-mediated autoimmunity. Furthermore, the expression of OX40L by mast cells was shown to be important for controlling inflammation through regulatory T-cell function. Besides the essential role played by OX40 signaling in generating memory CD4 T cells, recent reports show that it also has a unique role in generating memory CD8 T cells. In addition, recent genome-wide association studies have identified single-nucleotide polymorphisms of the OX40L and OX40 genes that are related to cardiovascular diseases and SLE, providing direct evidence for the involvement of the OX40-OX40L interaction in human diseases. Here, we review recent progress on how the OX40-OX40L interaction regulates T-cell tolerance, peripheral T-cell homeostasis, and T-cell-mediated inflammatory diseases.

Phase II Study of Irinotecan and Cisplatin as First-Line Chemotherapy in Advanced or Recurrent Cervical Cancer

Irinotecan (CPT-11) and cisplatin are singly active against cervical cancer. We evaluated the efficacy and toxicity of CPT-11 plus cisplatin as first-line chemotherapy in patients with advanced or recurrent cervical cancer. Twenty-nine chemotherapy-naive patients with advanced or recurrent cervical cancer were treated with CPT-11 (60 mg/m2) on days 1, 8, and 15 by intravenous infusion over 90 min, followed by cisplatin (60 mg/m2 i.v.) on day 1 over 90 min. The patients’ median age was 57 years (range 35–75). Nineteen patients (66%) had advanced primary disease. Six patients with recurrent disease (21%) had been treated with prior radiotherapy. The remaining 4 patients (14%) had residual or recurrent disease after radical surgery. The histologic diagnoses were squamous cell carcinoma in 25 patients (87%), adenocarcinoma in 3, and adenosquamous cell carcinoma in 1. All eligible patients were included in the toxicity and response analysis based on the intent to treat. Two patients (7%) achieved a complete response and 15 (52%) a partial response (overall response rate: 59%, 95% confidence interval; 41–74%). Stable disease was recorded in 6 patients (21%) and progressive disease in 3 patients (10%). In 3 patients, image-guided evaluation of response was judged to be unfeasible at the time of independent extramural review (10%). The median time to response was 32 days (range 16–62 days). The median survival was 27.7+ months (range, 6.4–52.8+ months). Two dose-limiting side effects were observed: grade 3 (28%) or 4 (45%) neutropenia and grade 3 (7%) or 4 (7%) diarrhea. Other severe toxicities included anemia (45%), thrombocytopenia (3%), nausea/vomiting (31%), and alopecia (7%). The combination of CPT-11 with cisplatin is an active regimen for treatment of advanced or recurrent cervical cancer albeit with a significant degree of myelosuppression.

The Behavior of Endometrial Hyperplasia: A Prospective Study

Objective: To clarify the behavior of endometrial hyperplasia in a prospective study.

Induction of human humoral immune responses in a novel HLA-DR-expressing transgenic NOD/Shi-scid/γcnull mouse

Mounting evidence has demonstrated that NOD-Shi/scid/γc(null) (NOG) mice are one of the most suitable mouse strains for humanized mouse technologies, in which various human cells or tissues can be engrafted without rejection and autonomously maintained. We have characterized and analyzed various features of the human immune system reconstituted in NOG mice by transplanting human hematopoietic stem cells (hu-HSC). One of the problems of the quasi-immune system in these hu-HSC NOG mice is that the quality of immune responses is not always sufficient, as demonstrated by the lack of IgG production in response to antigen challenge. In this study, we established a novel transgenic NOG sub-strain of mice bearing the HLA-DRA and HLA-DRB1:0405 genes, which specifically expresses HLA-DR4 molecules in MHC II-positive cells. This mouse strain enabled us to match the haplotype of HLA-DR between the recipient mice and human donor HSC. We demonstrated that T-cell homeostasis was differentially regulated in HLA-matched hu-HSC NOG mice compared with HLA-mismatched control mice, and antibody class switching was induced after immunization with exogenous antigens in HLA-matched mice. This novel mouse strain improves the reconstituted human immune systems that develop in humanized mice and will contribute to future studies of human humoral immune responses.

OX40 Costimulatory Signals Potentiate the Memory Commitment of Effector CD8+ T Cells

A T cell costimulatory molecule, OX40, contributes to T cell expansion, survival, and cytokine production. Although several roles for OX40 in CD8+ T cell responses to tumors and viral infection have been shown, the precise function of these signals in the generation of memory CD8+ T cells remains to be elucidated. To address this, we examined the generation and maintenance of memory CD8+ T cells during infection with Listeria monocytogenes in the presence and absence of OX40 signaling. We used the expression of killer cell lectin-like receptor G1 (KLRG1), a recently reported marker, to distinguish between short-lived effector and memory precursor effector T cells (MPECs). Although OX40 was dispensable for the generation of effector T cells in general, the lack of OX40 signals significantly reduced the number and proportion of KLRG1low MPECs, and, subsequently, markedly impaired the generation of memory CD8+ T cells. Moreover, memory T cells that were generated in the absence of OX40 signals in a host animal did not show self-renewal in a second host, suggesting that OX40 is important for the maintenance of memory T cells. Additional experiments making use of an inhibitory mAb against the OX40 ligand demonstrated that OX40 signals are essential during priming, not only for the survival of KLRG1low MPECs, but also for their self-renewing ability, both of which contribute to the homeostasis of memory CD8+ T cells.

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.

Homeostatic Proliferation of Naive CD4+ T Cells in Mesenteric Lymph Nodes Generates Gut-Tropic Th17 Cells

Homeostatic proliferation of naive T cells in the spleen and cutaneous lymph nodes supplies memory–phenotype T cells. The “systemic” proliferative responses divide distinctly into fast or slow cell division rates. The fast proliferation is critical for generation of effector memory T cells. Because effector memory T cells are abundant in the lamina propria of the intestinal tissue, “gut-specific” homeostatic proliferation of naive T cells may be important for generation of intestinal effector memory T cells. However, such organ-specific homeostatic proliferation of naive T cells has not yet been addressed. In this study, we examined the gut-specific homeostatic proliferation by transferring CFSE-labeled naive CD4+ T cells into sublethally irradiated mice and separately evaluating donor cell division and differentiation in the intestine, mesenteric lymph nodes (MLNs), and other lymphoid organs. We found that the fast-proliferating cell population in the intestine and MLNs had a gut-tropic α4β7+ Th17 phenotype and that their production was dependent on the presence of commensal bacteria and OX40 costimulation. Mesenteric lymphadenectomy significantly reduced the Th17 cell population in the host intestine. Furthermore, FTY720 treatment induced the accumulation of α4β7+IL-17A+ fast-dividing cells in MLNs and eliminated donor cells in the intestine, suggesting that MLNs rather than intestinal tissues are essential for generating intestinal Th17 cells. These results reveal that MLNs play a central role in inducing gut-tropic Th17 cells and in maintaining CD4+ T cell homeostasis in the small intestine.

Development of a Multi-Step Leukemogenesis Model of MLL-Rearranged Leukemia Using Humanized Mice

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ−/− (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia.