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We investigated the effects of anti-CD80/86 antibodies in a murine high-risk corneal transplantation rejection model. A mixed lymphocyte reaction (MLR) assay was conducted with anti-CD80/86 antibodies. Inflammatory cytokine levels in the culture supernatant were measured using an enzyme-linked immunosorbent assay. Interferon (IFN)-γ-producing CD4+ T cell frequencies in the MLR were assessed using flow cytometry. In vivo, high-risk corneal allograft survival and IFN-γ-producing CD4+ T cell frequencies in corneal grafts were assessed with intraperitoneal injection of anti-CD80/86 antibodies compared to phosphate-buffered saline (PBS). RNA-sequencing was performed on corneal grafts 2 weeks post-transplantation. Anti-CD80/86 antibodies significantly decreased T-cell proliferation, IFN-γ+-producing CD4+ T cell frequencies, and IFN-γ, interleukin (IL)-1β, IL-2, IL-10, and tumor necrosis factor-α production in the MLR compared to PBS injection. Intraperitoneal injection of anti-CD80/86 antibodies significantly prolonged corneal graft survival and decreased IFN-γ+-producing CD4+ T cell frequencies compared to PBS injection. Gene set enrichment analysis showed that the gene sets mainly enriched in the control group were related to allograft rejection and inflammatory response compared to PBS injection. Anti-CD80/86 antibodies significantly prolonged corneal graft survival by inhibiting T-cell proliferation and inflammatory response.

Emerging evidence supports the theory that tumor cell clusters efficiently metastasize to distant organs. However, the roles of epithelial-to-mesenchymal transition (EMT) in metastasizing tumor cell clusters have not yet been fully elucidated. To investigate this issue, tumor fragments were dissected from 40 colorectal cancer (CRC) patients and implanted subcutaneously into immunodeficient mice. We observed that tumors developed from the tumor fragments obtained from 28 of the 40 CRC patients. The tumors were then dissociated into cell suspensions to be orthotopically injected into secondary mice. The tumors from 13 of the 28 patients progressed. Furthermore, metastases formed spontaneously in the liver and lungs from the tumor fragments obtained from 8 of these 13 patients. Moreover, employing a mathematical analysis, we showed that tumor cell clusters seeded these metastases significantly more often than did single tumor cells. Membrane E-cadherin- and nuclear ZEB1-positive tumor cells indicating the hybrid epithelial/mesenchymal state were also detected in primary tumors of various CRC patients, and in the corresponding patient-derived xenografts (PDXs) and circulating tumor cell clusters in the bloodstreams of mice. In contrast, ZEB1 staining was barely detectable in the patient-matched liver metastases presumably developing through mesenchymal-to-epithelial transition. Inhibition of E-cadherin or ZEB1 expression by shRNA notably prevented the PDX-derived tumor organoids from colonizing the liver, when injected intrasplenically into mice, indicating E-cadherin and ZEB1 expressions to be required for their metastatic colonization. Taken together, these findings suggest that the epithelial/mesenchymal state mediates metastatic seeding of human CRC cell clusters into distant organs.

Background Although the outcome of kidney transplantation (KTx) has improved, various adverse effects of immunosuppressants and chronic rejection aggravate the long-term prognosis of patients. Therefore, the induction of immune tolerance may be an effective therapeutic strategy. Methods A clinical trial aiming at immune tolerance induction was conducted in kidney transplant recipients from HLA mismatched living donors by infusing autologous donor-specific regulatory T cells (Treg). To obtain Treg, recipient's peripheral blood mononuclear cells were cocultured with irradiated donor cells in the presence of anti-CD80/CD86 monoclonal antibody for 2 weeks. For preconditioning, splenectomy + cyclophosphamide (CP) was employed in the first series (group A; n = 9). In group B, splenectomy was substituted by preadministration of rituximab (group B; n = 3). In the latest cases, rituximab + rabbit antithymocyte globulin was administered instead of cyclophosphamide (group C; n = 4). Twelve days after KTx, the cultured cells were intravenously infused, and immunosuppressants were gradually tapered thereafter. Results Although mixed lymphocyte reaction was remarkably suppressed in a donor-specific fashion, 6 out of 9 patients from group A, 1 out of 3 from group B, and 1 out of 4 from group C developed acute rejection within 1 year after KTx. Complete cessation of immunosuppression was not achieved, and a small dose of immunosuppressants was continued. Conclusions The adoptive transfer of autologous ex vivo-expanded Treg is 1 of the options to possibly induce alloimmune hyporesponsiveness. However, in the present study, further regimen optimization is still required and should be the focus of future investigations.

Delta-like (Dll) 1 and Dll4 differently function as Notch ligands in a context-dependent manner. As these ligands share structural properties, the molecular basis for their functional difference is poorly understood. Here, we investigated the superiority of Dll4 over Dll1 with respect to induction of T cell development using a domain-swapping approach in mice. The DOS motif, shared by Notch ligands—except Dll4—contributes to enhancing the activity of Dll for signal transduction. The module at the N-terminus of Notch ligand (MNNL) of Dll4 is inherently advantageous over Dll1. Molecular dynamic simulation revealed that the loop structure in MNNL domain of Dll1 contains unique proline residues with limited range of motion. The Dll4 mutant with Dll1-derived proline residues showed reduced activity. These results suggest that the loop structure—present within the MNNL domain—with a wide range of motion ensures the superiority of Dll4 and uniquely contributes to the triggering of Notch signaling.

Because accumulation of mutations produces unique neoantigen patterns in cancer cells, it has been hypothesized that these patterns are recognized by the patients' immune system. Therefore, the development of cancer vaccines has been challenging owing to the possibility of an anti-cancer effect induced by the immune system against such neoantigens. However, it is difficult to develop effective vaccines because of the variety of mutations induced in cancer cells and human leukocyte antigen (HLA), which is predicted to present the neoantigen-derived peptides. Moreover, the activation of cancer-specific cytotoxic cells is inhibited by cancer cell immunoediting in each patient. Although cellular immunity can be analyzed ex vivo, there are no definite methods to evaluate humoral immunity. A humanized mouse model has been developed and used for evaluating the multipotency of hematopoietic stem cells. Presently, significant improvements in the humanized mouse model approach have partially recapitulated human humoral immunity in vivo, and human antibody production can be induced in the mouse model. These mice can be used to produce new cancer vaccines and to establish polypharmacy protocols in a preclinical model. In this review, we discuss the preclinical evaluation of the cancer vaccines using humanized mice transferred with patient-derived peripheral blood mononuclear cells (PBMCs) to advance a personalized medicine approach.

The in vivo model of pollinosis has been established using rodents, but the model cannot completely mimic human pollinosis. We used Callithrix jacchus, the common marmoset (CM), to establish a pollinosis animal model using intranasal weekly administration of cedar pollen extract with cholera toxin adjuvant. Some of the treated CMs exhibited the symptoms of snitching, excess nasal mucus and/or sneezing, but the period was very short, and the symptoms disappeared after several weeks. The CD4+CD25+ cell ratio in the peripheral blood increased in CMs quickly after the nasal administration of cedar pollen extract, but the timing was not parallel with the symptoms. IL-10 mRNA was enhanced in the peripheral blood mononuclear cells (PBMCs), suggesting CM-induced tolerance for cedar pollen administration. Similarly, Foxp3 mRNA was also detected in the PBMC. Additive sensitization of these CMs with Ascaris egg administration did not enhance chronic inflammation of type 1 allergy to induce the symptoms. These results suggest that the environmental immune cells develop transient allergic symptoms and subsequent immune-tolerance in the intranasally sensitized CMs.

The gut microbiota has a great impact on the host immune systems. Recent evidence suggests that the maternal gut microbiota affects the immune systems of offspring. Metabolites produced by the gut microbiota play crucial roles in the immune system. Previous studies have also revealed that metabolites such as short-chain fatty acids (SCFAs) and the aryl hydrocarbon receptor (AhR) ligands are involved in host health and diseases. Great progress has been made in understanding the roles of diet-derived SCFAs in the offspring's immune system. The findings to date raise the possibility that maternal dietary soluble fiber intake may play a role in the development of the offspring's systemic immune response. In this review, we summarize the present knowledge and discuss future therapeutic possibilities for using dietary soluble fiber intake against inflammatory diseases.

Several recent studies suggest that cancer stem cells (CSCs) are involved in intrinsic resistance to cancer treatment. Maintenance of quiescence is crucial for establishing resistance of CSCs to cancer therapeutics. F-box/WD repeat-containing protein 7 (FBXW7) is a ubiquitin ligase that regulates quiescence by targeting the c-MYC protein for ubiquitination. We previously reported that gefitinib-resistant persisters (GRPs) in EGFR-mutant non-small cell lung cancer (NSCLC) cells highly expressed octamer-binding transcription factor 4 (Oct-4) as well as the lung CSC marker CD133, and they exhibited distinctive features of the CSC phenotype. However, the role of FBXW7 in lung CSCs and their resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors in NSCLC is not fully understood. In this study, we developed GRPs from the two NSCLC cell lines PC9 and HCC827, which express an EGFR exon 19 deletion mutation, by treatment with a high concentration of gefitinib. The GRPs from both PC9 and HCC827 cells expressed high levels of CD133 and FBXW7, but low levels of c-MYC. Cell cycle analysis demonstrated that the majority of GRPs existed in the G0/G1 phase. Knockdown of the FBXW7 gene significantly reduced the cell number of CD133-positive GRPs and reversed the cell population in the G0/G1-phase. We also found that FBXW7 expression in CD133-positive cells was increased and c-MYC expression was decreased in gefitinib-resistant tumors of PC9 cells in mice and in 9 out of 14 tumor specimens from EGFR-mutant NSCLC patients with acquired resistance to gefitinib. These findings suggest that FBXW7 plays a pivotal role in the maintenance of quiescence in gefitinib-resistant lung CSCs in EGFR mutation-positive NSCLC.

Until now, metal allergies have been regarded as a Th1-type immune response. However, because the contribution of a Th2-type immune response has been suggested by clinical findings, we previously examined the Th2-type immune response during the development of metal allergies using a GATA-3 transgenic (GATA-3 Tg) mouse model. As a result, a Th2-type immunization reaction was suggested to be involved in the early phase of metal allergies. Recently, the involvement of NKT cells in metal allergies has been suggested. We examined this possibility using the activation of NKT cells and an NKT cell-deficient mouse model to determine the contribution of NKT cells to nickel allergy in the present study. In NKT cell-deficient mice, ear swelling was remarkably increased, compared with that in control mice. Also, in mice that had been treated with α-galactosylceramide (α-GalCer) to activate NKT cells, the ear swelling response was remarkably inhibited, compared with that in untreated mice. These facts show that NKT cells are involved in the inhibition of nickel allergy-induced ear swelling responses.

Recent advances in genome editing have facilitated the generation of nonhuman primate (NHP) models, with potential to unmask the complex biology of human disease not revealed by rodent models. However, their broader use is hindered by the challenges associated with generation of adult NHP models as well as the cost of their production. Here, we describe the generation of a marmoset model of severe combined immunodeficiency (SCID). This study optimized zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to target interleukin-2 receptor subunit gamma (IL2RG) in pronuclear stage marmoset embryos. Nine of 21 neonates exhibited mutations in the IL2RG gene, concomitant with immunodeficiency, and three neonates have currently survived from 240 days to 1.8 years. Our approach demonstrates highly efficient production of founder NHP with SCID phenotypes, with promises of multiple pre-clinical and translational applications.

UNLABELLED Potent immunosuppressive drugs have significantly improved early patient survival after liver transplantation (LT). However, long-term results remain unsatisfactory because of adverse events that are largely associated with lifelong immunosuppression. To solve this problem, different strategies have been undertaken to induce operational tolerance, for example, maintenance of normal graft function and histology without immunosuppressive therapy, but have achieved limited success. In this pilot study, we aimed to induce tolerance using a novel regulatory T-cell-based cell therapy in living donor LT. Adoptive transfer of an ex vivo-generated regulatory T-cell-enriched cell product was conducted in 10 consecutive adult patients early post-LT. Cells were generated using a 2-week coculture of recipient lymphocytes with irradiated donor cells in the presence of anti-CD80/86 monoclonal antibodies. Immunosuppressive agents were tapered from 6 months, reduced every 3 months, and completely discontinued by 18 months. After the culture, the generated cells displayed cell-number-dependent donor-specific inhibition in the mixed lymphocyte reaction. Infusion of these cells caused no significant adverse events. Currently, all patients are well with normal graft function and histology. Seven patients have completed successful weaning and cessation of immunosuppressive agents. At present, they have been drug free for 16-33 months; 4 patients have been drug free for more than 24 months. The other 3 recipients with autoimmune liver diseases developed mild rejection during weaning and then resumed conventional low-dose immunotherapy. CONCLUSIONS A cell therapy using an ex vivo-generated regulatory T-cell-enriched cell product is safe and effective for drug minimization and operational tolerance induction in living donor liver recipients with nonimmunological liver diseases. (Hepatology 2016;64:632-643).

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited. We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2Rγ(null) (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.

The precise mechanism of how the regulatory T cell population elicits and maintains tolerant state in activated T cells is poorly understood. To address this issue, we established an in vitro coculture system using mouse T cells and showed that tolerant state is serially passed from preinduced-tolerant T cells into new TCR-stimulated T cells across generations in a dendritic cell–independent manner. In this successive induction process of tolerant state, TIGIT was found to play an important role: TIGIT expression on induced-tolerant T cells was promoted in stimulated T cells cocultured with the tolerant cells. In addition, these stimulated T cells in the coculture also expressed high B lymphocyte-induced maturation protein 1 accompanied by IL-2 suppression. Because CD155, a partner of TIGIT, is known to transduce signaling inside by trans-interaction with its ligands, these phenotypical changes in TCR-stimulated naive T cells were reproduced when naive T cells were double cross-linked by CD3 and CD155. These results indicate that TIGIT enhanced on tolerant T cells may function as a ligand of its paired receptor CD155 to transduce signaling into its expressing naive T cells to accelerate new TIGIT expressions as well as IL-2 suppression via B lymphocyte-induced maturation protein 1 enhancement. In consideration of these results, we propose a novel process in which tolerant state in T cell population is maintained by successive generation of new tolerant T cells from naive T cells as one of the regulating mechanisms in immune responses.

The common marmoset (CM; Callithrix jacchus) is a small New World monkey with a high rate of pregnancy and is maintained in closed colonies as an experimental animal species. Although CMs are used for immunological research, such as studies of autoimmune disease and infectious disease, their immunological characteristics are less defined than those of other nonhuman primates. We and others have analyzed antigen recognition-related molecules, the development of hematopoietic stem cells (HSCs), and the molecules involved in the immune response. CMs systemically express Caja-G, a major histocompatibility complex class I molecule, and the ortholog of HLA-G, a suppressive nonclassical HLA class I molecule. HSCs express CD117, while CD34 is not essential for multipotency. CD117+ cells developed into all hematopoietic cell lineages, but compared with human HSCs, B cells did not extensively develop when HSCs were transplanted into an immunodeficient mouse. Although autoimmune models have been successfully established, sensitization of CMs with some bacteria induced a low protective immunity. In CMs, B cells were observed in the periphery, but IgG levels were very low compared with those in humans and mice. This evidence suggests that CM immunity is partially suppressed systemically. Such immune regulation might benefit pregnancy in CMs, which normally deliver dizygotic twins, the placentae of which are fused and the immune cells of which are mixed. In this review, we describe the CM immune system and discuss the possibility of using CMs as a model of human immunity.

The regulation of intestinal homeostasis by the immune system involves the dynamic interplay between gut commensal microbiota and resident immune cells. It is well known that a large and diverse lymphocyte antigen receptor repertoire enables the immune system to recognize and respond to a wide range of invading pathogens. There is also an emerging appreciation for a critical role the T-cell receptor (TCR) repertoire serves in the maintenance of peripheral tolerance by regulatory T cells (Tregs). Nevertheless, how the diversity of the TCR repertoire in Tregs affects intestinal homeostasis remains unknown. To address this question, we studied mice whose T cells express a restricted TCR repertoire. We observed the development of spontaneous colitis, accompanied by the induction of T-helper type 17 cells in the colon that is driven by gut commensal microbiota. We provide further evidence that a restricted TCR repertoire causes a loss of tolerogenicity to microbiota, accompanied by a paucity of peripherally derived, Helios(-) Tregs and hyperactivation of migratory dendritic cells. These results thus reveal a new facet of the TCR repertoire in which Tregs require a diverse TCR repitoire for intestinal homeostasis, suggesting an additional driving force in the evolutional significance of the TCR repertoire.

Delta-like 4 (Dll4)-mediated Notch signaling is critical for specifying T-cell fate, but how Dll4-mediated Notch signaling actually contributes to T-cell development in the thymus remains unclear. To explore this mechanism in the thymic three-dimensional structure, we performed fetal thymus organ culture using Dll4-deficient mice. DN1a/b+DN2mt cells, which had not yet committed to either the αβ T or γδ T/NK cell lineage, did not differentiate into the αβ T-cell lineage in Dll4-deficient thymus despite the lack of cell fate conversion into other lineages. However, DN3 cells efficiently differentiated into a later developmental stage of αβ T cells, the double-positive (DP) stage, although the proliferation was significantly impaired during the differentiation process. These findings suggest that the requirement for Notch signaling differs between the earliest and pre-TCR-bearing precursors and that continued Notch signaling is required for proper differentiation with active proliferation of αβ T lineage cells. Furthermore, we showed that Notch signaling increased the c-Myc expression in DN3 cells in the thymus and that its overexpression rescued the proliferation and differentiation of DN3 cells in the Dll4-null thymus. Therefore, c-Myc plays a central role in the transition from stage DN3 to DP as a downstream target of Notch signaling.

Notch signaling has been shown to contribute to murine pancreatic development at various stages. Delta-like 1 (Dll1) or Jagged1 (Jag1) are the Notch ligands that solely function to trigger this signaling during the pancreatic bud stage (~e9.5) or after birth, respectively. However, it has not been elucidated whether these Notch ligands are required at the later stage (e10.5-18.5) when the particular pancreas structures form. Here, we detected the dual expression of Dll1 and Jag1 in the epithelium after e10.5, which was restricted to the ductal cell lineage, including centroacinar cells expressing Sox9, CD133 and Hes1 but not the ductal cell markers Hnf1β and DBA, at e18.5. To evaluate the significance of the Notch ligands during this period, we established double-floxed mice of Dll1 and Jag1 genes with Ptf1a-Cre knock-in allele and examined the effects on development. The abrogation of both ligands but not a single one led to the loss of centroacinar cells, which was due to the decrease in cell proliferation and the increase in cell death, as well as to the reduction of Sox9. These results suggested that Dll1 and Jag1 function redundantly and are necessary to maintain the centroacinar cells as an environmental niche in the developing pancreas.

Immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMCs) are promising tools to evaluate human immune responses to vaccines. However, these mice usually develop severe graft-versus-host disease (GVHD), which makes estimation of antigen-specific IgG production after antigen immunization difficult. To evaluate antigen-specific IgG responses in PBMC-transplanted immunodeficient mice, we developed a novel NOD/Shi-scid-IL2rγnull (NOG) mouse strain that systemically expresses the human IL-4 gene (NOG-hIL-4-Tg). After human PBMC transplantation, GVHD symptoms were significantly suppressed in NOG-hIL-4-Tg compared to conventional NOG mice. In kinetic analyses of human leukocytes, long-term engraftment of human T cells has been observed in peripheral blood of NOG-hIL-4-Tg, followed by dominant CD4+ T rather than CD8+ T cell proliferation. Furthermore, these CD4+ T cells shifted to type 2 helper (Th2) cells, resulting in long-term suppression of GVHD. Most of the human B cells detected in the transplanted mice had a plasmablast phenotype. Vaccination with HER2 multiple antigen peptide (CH401MAP) or keyhole limpet hemocyanin (KLH) successfully induced antigen-specific IgG production in PBMC-transplanted NOG-hIL-4-Tg. The HLA haplotype of donor PBMCs might not be relevant to the antibody secretion ability after immunization. These results suggest that the human PBMC-transplanted NOG-hIL-4-Tg mouse is an effective tool to evaluate the production of antigen-specific IgG antibodies.

This study highlights the cellular and molecular mechanisms by which stromal fibroblasts enable human breast cancer cells to form tumor cell clusters and acquire highly invasive and metastatic traits., Emerging evidence supports the hypothesis that multicellular tumor clusters invade and seed metastasis. However, whether tumor-associated stroma induces epithelial–mesenchymal plasticity in tumor cell clusters, to promote invasion and metastasis, remains unknown. We demonstrate herein that carcinoma-associated fibroblasts (CAFs) frequently present in tumor stroma drive the formation of tumor cell clusters composed of two distinct cancer cell populations, one in a highly epithelial (E-cadherinhiZEB1lo/neg: Ehi) state and another in a hybrid epithelial/mesenchymal (E-cadherinloZEB1hi: E/M) state. The Ehi cells highly express oncogenic cell–cell adhesion molecules, such as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and CEACAM6 that associate with E-cadherin, resulting in increased tumor cell cluster formation and metastatic seeding. The E/M cells also retain associations with Ehi cells, which follow the E/M cells leading to collective invasion. CAF-produced stromal cell-derived factor 1 and transforming growth factor-β confer the Ehi and E/M states as well as invasive and metastatic traits via Src activation in apposed human breast tumor cells. Taken together, these findings indicate that invasive and metastatic tumor cell clusters are induced by CAFs via epithelial–mesenchymal plasticity.

The common marmoset is a new world monkey, which has become a valuable experimental animal for biomedical research. This study developed cDNA libraries for the common marmoset from five different tissues. A total of 290 426 high-quality EST sequences were obtained, where 251 587 sequences (86.5%) had homology (1E(-100)) with the Refseqs of six different primate species, including human and marmoset. In parallel, 270 673 sequences (93.2%) were aligned to the human genome. When 247 090 sequences were assembled into 17 232 contigs, most of the sequences (218 857 or 15 089 contigs) were located in exonic regions, indicating that these genes are expressed in human and marmoset. The other 5578 sequences (or 808 contigs) mapping to the human genome were not located in exonic regions, suggesting that they are not expressed in human. Furthermore, a different set of 118 potential coding sequences were not similar to any Refseqs in any species, and, thus, may represent unknown genes. The cDNA libraries developed in this study are available through RIKEN Bio Resource Center. A Web server for the marmoset cDNAs is available at http://marmoset.nig.ac.jp/index.html, where each marmoset EST sequence has been annotated by reference to the human genome. These new libraries will be a useful genetic resource to facilitate research in the common marmoset.

After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G1 cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G1-phase extension and represses CD8 expression in a G1 extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G1 extension and CD8 repression. Importantly, forced G1 extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G1 progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G1-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.

Mast cells (MCs) are developed from hematopoietic progenitor cells and play an important role in inflammation. Study of the kinetics of development and accumulation of primate MC in vivo is crucial for the control of human inflammatory diseases, as evolution of the immune system is quite rapid and inflammation including MC response is considered to be different between mouse and human. In the present study, we examined the development of MC from hematopoietic progenitors of Callithrix jacchus (common marmoset), an experimental animal of nonhuman primates. Bone marrow cells were fractionated for the expression of CD34 and CD117 by cell sorting. MCs were developed in vitro or by transplanting the cells to NOD/SCID/IL-2γc knockout (NOG) mice. In vitro culture of CD34(+)CD117(+) (double positive, DP) cells with stem cell factor could generate high-affinity Fc epsilon receptor (FcεR)-expressing CD117(+) cells with typical granules. The developed MC released β-hexosaminidase and produced leukotriene C(4) after the stimulation of FcεRI. Transplantation of DP cells gave rise to a marked expansion of CD34(-)CD45(+)CD117(+)FcεR(+) cells in NOG mice. They expressed transcripts encoding chymase 1 and tryptase β. Differentiation of CD34(-)CD117(+) cells to MCs was relatively limited compared with the DP cells, similarly to human MCs. These results suggest that this marmoset system provides a good model for human MC development.

Peyer's patches (PPs) are potential sites where specific mucosal immune responses and oral tolerance are induced. The unique features of these immune responses are thought to occur in micromilieu and are largely affected by antigen-presenting cells (APCs) such as dendritic cells. In this study, we investigated the cytokine profiles induced by the activation of CD4(+) T cells of PPs. PP cells from TCR transgenic mice secreted greater amounts of IL-5 and IL-6 than spleen cells after antigenic stimulation. IL-5 was mainly produced by PP non-T cells, whereas IL-6 was secreted by PP CD4(+) cells. PPs contained two major populations including naïve and memory/activated CD4(+) cells; both populations secreted IL-6 upon activation. We also found that CD4(+)/CD62L(hi) naïve cells from PPs secreted a greater amount of IL-6 after stimulation than those from the spleen. Furthermore, subtraction and qPCR analyses revealed that PP CD4(+)/CD62L(hi) cells express a greater amount of transcripts of GA-binding protein β subunit 1 than those of the spleen. These results suggest that naïve T cells as well as non-T cells and activated/memory T cells from PPs are distinct from their splenic counterparts and thus cause unique immune responses the in intestine.

Interleukin-10 was originally described as a factor that inhibits cytokine production by murine Th1 clones. Recent studies have since shown that IL-10 can also downregulate Th2 clones and their production of IL-4 and IL-5. Because of its immuno-suppressive properties, IL-10 has been suggested as a potential therapy for allergic inflammation and asthma. However, the pathophysiological role of IL-10 in vivo has not been clearly elucidated. We investigated the effects of IL-10 administration on the production of IgE, cytokine and allergen-induced Th2 cytokine production as well as its effects on eosinophilic inflammation. We established GATA-3/TCR double transgenic (GATA-3/TCR-Tg) mice by crossing GATA-3 transgenic mice with ovalbumin (OVA)-specific TCR transgenic mice; these mice were then sensitized using an intraperitoneal injection of OVA adsorbed to alum and challenged with the intratracheal instillation of an allergen. When GATA-3/TCR-Tg mice sensitized with OVA and alum were injected with C57-IL-10 cells before OVA inhalation, the levels of IL-5, IL-13, and IL-4 decreased by 40-85% and number of eosinophils decreased by 70% (P

Runx1 transcription factor is highly expressed at a CD4/CD8-double-negative (DN) stage of thymocyte development but is down-regulated when cells proceed to the double-positive (DP) stage. In the present study, we examined whether the down-regulation of Runx1 is necessary for thymocyte differentiation from the DN to DP stage. When Runx1 was artificially over-expressed in thymocytes by Lck-driven Cre, the DN3 population was unaffected, as exemplified by proper pre-T-cell receptor expression, whereas the DN4 population was perturbed as shown by the decrease in the CD27(hi) sub-fraction. In parallel, the growth rate of DN4 cells was reduced by half, as measured by bromodeoxyuridine incorporation. These events impaired the transition of DN4 cells to the DP stage, resulting in the drastic reduction of the number of DP thymocytes. The Runx1 gene has two promoters, a proximal and a distal promoter; and, in thymocytes, endogenous Runx1 was mainly transcribed from the distal promoter. Interestingly, only distal, but not proximal, Runx1 over-expression exhibited an inhibitory effect on thymocyte differentiation, suggesting that the distal Runx1 protein may fulfil a unique function. Our collective results indicate that production of the distal Runx1 protein must be adequately down-regulated for thymocytes to transit from the DN to the DP stage, a critical step in the massive expansion of the T-cell lineage.

Objective Common marmosets are considered experimental animals of primates useful for medical research. We developed several monoclonal antibodies (mAbs) directed to CD molecules to gain initial insight into the immune and hematopoietic systems of this organism, and analyzed the basic cellularity and characters of marmoset lymphocytes. Materials and Methods Anti-marmoset CD antigen mAbs were prepared using marmoset antigen-expressing transfectants and used for flow cytometric analyses and cell fractionation. Expression of T-cell−related cytokine gene transcripts was examined in response to T-cell receptor stimulation by reverse transcription polymerase chain reaction analyses. Hematopoietic progenitor activities of marmoset bone marrow cells were examined in fractionated cells by mAbs against CD117 (c-kit) and CD34. Results CD4 and CD8 expression profiles in T-cell subsets of marmoset were essentially similar to those in mouse and human. CD4 + and CD8 + subsets were isolated from marmoset spleens. Detected transcripts after stimulation of T cells included Th1-, Th2-, and Th17-related cytokines in CD4 + cells and cytotoxic proteases in CD8 + cells, respectively. Colony-forming abilities were detected mainly in CD117 (c-kit) + cells, irrespective of CD34 expression. Conclusions Marmoset immune system was basically similar to human and mouse systems.

The common marmoset (Callithrix jacchus) is increasingly attractive for use as a non-human primate animal model in biomedical research. It has a relatively high reproduction rate for a primate, making it potentially suitable for transgenic modification. Although several attempts have been made to produce non-human transgenic primates, transgene expression in the somatic tissues of live infants has not been demonstrated by objective analyses such as polymerase chain reaction with reverse transcription or western blots. Here we show that the injection of a self-inactivating lentiviral vector in sucrose solution into marmoset embryos results in transgenic common marmosets that expressed the transgene in several organs. Notably, we achieved germline transmission of the transgene, and the transgenic offspring developed normally. The successful creation of transgenic marmosets provides a new animal model for human disease that has the great advantage of a close genetic relationship with humans. This model will be valuable to many fields of biomedical research.

As preclinical evaluation in animals does not necessarily portray human responses, liposome-encapsulated hemoglobin (LEH), an artificial oxygen carrier, was tested in immunodeficient mice reconstituted with human hematopoietic stem cells (cord blood-transfused NOD/SCID/IL-2R(gammanull)[CB-NOG] mice). Changes in immunocompetent T-cell and B-cell composition in peripheral blood, spleen, and bone marrow were examined 2 and 7 days after 10 mL/kg of intravenous administration of LEH, empty liposome (EL), or saline using immunohistochemical and flow cytometrical techniques in wild-type mice and CB-NOG mice. Responses to intraperitoneal administration of toxic shock syndrome toxin-1 (TSST-1) under the absence or presence of LEH (10 mL/kg) were also determined 4 h and 3 days later in terms of lymphocyte composition and IL-2 plasma level in wild-type as well as CB-NOG mice. When liposome (LEH or EL) was administered to wild-type or CB-NOG mice, the composition of B-cells and T-cells in the spleen or peripheral blood failed to show any consistent or significant changes. The responses to a bacterial antigen (TSST-1) measured by IL-2 production were comparable regardless of the presence or absence of LEH in wild-type as well as in CB-NOG mice. Cellularity, distribution, and maturation of these human cells in peripheral blood, spleen, and bone marrow were comparable among the groups. The results suggest that simple LEH administration may not change immune cellularity, and LEH presence may not largely affect the early T-cell response to bacterial enterotoxins in murine as well as in reconstituted human immune systems.

Callithrix jacchus, the common marmoset, is a small new world primate that is considered effective as an experimental animal model for various human diseases. In this study, we generated monoclonal antibodies (mAbs) against common marmoset lymphocytes for immunological studies on the common marmoset. We established five hybridoma clones, 6C9, 10D7, 6F10, 7A4 and 5A1, producing anti-marmoset mAbs against cell surface antigens on marmoset T and/or B lymphocytes. We confirmed that 6C9 and 10D7 antibodies recognized CD45 antigen, and 6F10 antibody recognized CD8 antigen by flow cytometry using marmoset cDNA transfectants. We also tested them for application of immunoprecipitation, Western blot analysis and immunohistochemistry. We found that immunohistochemistry using marmoset spleen sections could be applied with all established mAbs but immunoprecipitation and the Western blot analysis could be applied with 6F10 and 10D7 antibodies but not with the other three mAbs. These results show that these monoclonal antibodies are useful for advancing immunological research on the common marmoset.

The Notch ligand Delta-like 1 (Dll1) is critical for the generation of marginal zone (MZ) B cells in the spleen. However, the precise mechanism underlying the differentiation of MZB cells is unclear. To determine whether hematopoietic cells or non-hematopoietic cells provides the Dll1-mediated signals to primitive hematopoietic cells, we transplanted lineage(-)c-kit(+)Sca-1(+) (KSL) bone marrow cells derived from wild-type (Dll1(+/+)) GFP-transgenic mice into lethally irradiated Dll1 conditional knockout (cKO) mice. After transplantation, we examined the kinetics of hematopoietic reconstitution and found that although the frequency of stem/progenitor subsets and of more mature lymphoid, myeloid, and erythroid lineages were normal, the donor-derived hematopoietic cells failed to differentiate into MZB cells. We further demonstrated that while the splenic stromal cells of wild-type mice expressed Dll1 molecule, the splenic stromal cells of recipient Dll1 cKO mice deleted the expression of Dll1. These results suggesting that the expression of Dll1 in splenic non-hematopoietic stromal cells, but not hematopoietic cells, is essential for the development of MZB cells.

The thymic microenvironment is required for T cell development in vivo. However, in vitro studies have shown that when hematopoietic progenitors acquire Notch signaling via Delta-like (Dll)1 or Dll4, they differentiate into the T cell lineage in the absence of a thymic microenvironment. It is not clear, however, whether the thymus supports T cell development specifically by providing Notch signaling. To address this issue, we generated mice with a loxP-flanked allele of Dll4 and induced gene deletion specifically in thymic epithelial cells (TECs). In the thymus of mutant mice, the expression of Dll4 was abrogated on the epithelium, and the proportion of hematopoietic cells bearing the intracellular fragment of Notch1 (ICN1) was markedly decreased. Corresponding to this, CD4 CD8 double-positive or single-positive T cells were not detected in the thymus. Further analysis showed that the double-negative cell fraction was lacking T cell progenitors. The enforced expression of ICN1 in hematopoietic progenitors restored thymic T cell differentiation, even when the TECs were deficient in Dll4. These results indicate that the thymus-specific environment for determining T cell fate indispensably requires Dll4 expression to induce Notch signaling in the thymic immigrant cells.

GATA3 and Notch1 are essential for T cell development at the earliest stage, but their mutual roles in this process remain to be clarified. In this study, we demonstrated that impairment of T lymphopoiesis in hematopoietic progenitor cells (HPC) of GATA3-deficient fetal liver (FL) on day 11.5 of gestation (E11.5) was rescued only by introduction of both GATA3 and the intracellular region of Notch1 but not by either alone. However, the introduction of GATA3 only was sufficient for T cell induction in GATA3-deficient FL cells at the advanced stage, where Notch signaling is well detectable. This indicates that Notch signaling is necessary for GATA3 to function for T cell fate specification but is not sufficient without GATA3. On the other hand, Notch signaling is sufficient for blockage of B cell development without GATA3, suggesting that T cell fate specification at the branching point does not result simply from the developmental arrest of B cell lineage by Notch signaling.

In the evolution of primates, the common marmoset belongs to the new world monkey family and is distinct from the great ape family (which includes humans). In this study, we predicted the amino acid sequences of 30 immunity-related genes from the common marmoset and compared them with those from human and mouse. The domain composition of each orthologous protein was analyzed by the SMART tool and was found to be the same among the three species. A BLAST search revealed that the common marmoset and human proteins were 86% identical on average, whereas the conservation between the common marmoset and mouse or between the human and mouse was only 60%. This indicates that the common marmoset and human proteins are closely related and are similarly divergent from the mouse. We divided the 30 proteins into two categories based on the degree of conservation between the common marmoset and mouse amino acid sequences. One group included 19 proteins and had a relatively high level of conservation (68% identical), whereas the other 11 proteins were less conserved (45% identical). This suggests that these immunity-related genes do not evolve at a uniform rate. Interestingly, however, ligand/receptor pairs such as interleukin-6 and interleukin-6 receptor appear to have evolved simultaneously.

Runx family proteins play indispensable roles in the development of various hematopoietic lineage cells. However, their function in NK cells is still uncertain. We found that NK cells and CD8 T cells dominantly express Runx3 protein, whereas NKT cells and CD4 T cells express Runx1. Reverse transcription-PCR analysis revealed that Runx3 expression is initiated at the NK precursor stage and is maintained along the course of NK cell differentiation. In order to examine their role in the earlier stage of NK cell development, we introduced Runx dominant-negative (Runx dn) form into Lin(-)c-kit(+)Sca-1(+) hematopoietic stem cells, which were applied to NK cell-inducing culture. Post-cultured cells showed a decreased expression of IL-2/IL-15 common receptor beta subunit (CD122), consistent with another finding that Runx binds to promoter region of CD122 gene. To examine the Runx function in the later developmental stage, we used transgenic mouse, in which Runx dn form is expressed in immature and mature NK cells. This mouse showed decreased expressions of NK maturation markers, such as Ly49 family, Mac-1 and CD43, whereas IFN-gamma production was greatly enhanced. These findings suggest that Runx proteins, especially Runx3, play multiple roles in NK cell differentiation.

Mastermind (Mam) is one of the elements of Notch signaling, an ancient system that plays a pivotal role in metazoan development. Genetic analyses in Drosophila and Caenorhabditis elegans have shown Mam to be an essential positive regulator of this signaling pathway in these species. Mam proteins bind to and stabilize the DNA-binding complex of the intracellular domains of Notch and CBF-1, Su(H), Lag-1 (CSL) DNA-binding proteins in the nucleus. Mammals have three Mam proteins, which show remarkable similarities in their functions while having an unusual structural diversity. There have also been recent indications that Mam-1 functionally interacts with other transcription factors including p53 tumor suppressor. We herein describe that Mam-1 deficiency in mice abolishes the development of splenic marginal zone B cells, a subset strictly dependent on Notch2, a CSL protein and Delta1 ligand. Mam-1 deficiency also causes a partially impaired development of early thymocytes, while not affecting the generation of definitive hematopoiesis, processes that are dependent on Notch1. We also demonstrate the transcriptional activation of a target promoter by constitutively active forms of Notch to decrease severalfold in cultured Mam-1-deficient cells. These results indicate that Mam-1 is thus required to some extent for Notch-dependent stages in lymphopoiesis, thus supporting the notion that Mam is an essential component of the canonical Notch pathway in mammals.

We examined the pathogenesis of glomerular damage in Th2 type-dependent GATA-3 transgenic (GATA-3 Tg) mice with IgA nephropathy (IgAN). GATA-3 Tg mice were immunized orally using OVA plus cholera toxin B (CTB), and measurement of the serum IgA antibody level and histopathological examination were performed. Marked increases in the serum levels of OVA-specific IgA antibody, IgA and IgG, C3 deposits analogous to those seen in IgAN, and expansion of the matrix in association with mesangial cell proliferation were observed. Furthermore, glomerular IgA deposits were co-localized with mannan-binding lectin (MBL) deposits, which might actually have been abnormal IgA deposits. In GATA-3/TCR-Tg mice that had been orally sensitized with CTB plus OVA and were re-stimulated with OVA in vitro, cultured Peyer’s patch cells showed the enhanced production of IL-5 and supernatants from cultures of spleen cells showed a reduction of TGF-β production with a simultaneous increase in IL-2 production and the recovery of IFN-γ formation. The amount of TGF-β produced by the spleen cells was found to be correlated with the amount of IFN-γ and IL-IL-2 produced by the cells. Also, the percentage of regulatory T cells (Treg) in the spleens of mice sensitized with OVA plus CTB was lower than that in mice orally sensitized with OVA alone. These results suggest that the increased production of IL-5 from Peyer’s patch cells (PPc) and the restored Th1-type immune response might cause the production of abnormal IgA and might induce the deposition of IgA in glomeruli.

Various immunological strategies for tolerance induction against allogeneic tissue grafts (allografts) have been tested in islet transplant recipients; for example, T cell activating co-stimulatory pathway blockade has been shown to prolong islet allograft survival. However, little is known about whether infiltrating inflammatory cells (e.g., basophils) affect islet allograft fates before antigen-specific immune cell development. Herein, we treated mice with a basophil-specific monoclonal antibody (mAb) and examined whether early acute-phase islet allograft rejection could be prevented in recipients.Pancreatic islets isolated from C57BL/6 (H-2b) or DBA/2 (H-2d) mice were transplanted under the renal capsules of C57BL/6 recipient mice. Recipients receiving allografts were administered the anti-basophil mAb MAR-1 to examine the antibody-mediated effect on graft survival. At days 4 and 7 post-transplantation, graft-bearing recipient kidneys were harvested for immunohistological analysis and stained with anti-insulin antibody to compare the sizes of grafted islets.On day 7 post-transplantation, the transplanted pancreatic islet clusters in allograft-recipient kidneys had rapidly decreased in size, whereas those in syngeneic recipients remained larger in both size and number. However, MAR-1-treated recipients had increased the numbers of larger insulin-positive allograft islet cell clusters.Basophil-specific mAb treatment contributes to enhance and prolong transplanted islet survival in allogeneic recipient mice.