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Bacteroides is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of Bacteroides with epithelial function, the gut immune system and systemic metabolism in the host. The mariner family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the mariner family transposon is expected to identify key molecules for human-Bacteroides symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (ermF/ITR), which harbors the erythromycin resistant marker (ermF) and the inverted repeat sequences (ITRs) recognized by Himar1 transposase, to Bacteroides via electrotransformation. pMI07 successfully delivered ermF/ITR to the Bacteroides genomes and generated thousands of insertion mutants/μg of pMI07 in B. thetaiotaomicron, B. fragilis, B. ovatus, and also, although to a lesser extent, B. vulgatus. Analyses of the ermF/ITR insertion sites in B. thetaiotaomicron and B. vulgatus revealed that the cassette targeted the dinucleotide TA and integrated into the genomes in an unbiased manner. The data reported here will provide useful information for transposon mutagenesis in Bacteroides species, which will enable identification of the genes responsible for their unique phenotypes.

IL-23 plays a critical role in the expansion of highly proinflammatory Th17 cells secreting IL-17 and IL-22. Recently, we demonstrated that Notch signaling drives IL-22 secretion through the aryl hydrocarbon receptor (AHR) and plays a protective role in Con A-induced hepatitis. In this study, we investigated the role of IL-23 in hepatitis using IL-23p19- and IL-17-deficient mice. In WT mice, the injection of Con A induced the upregulation of various cytokines, which included IL-23, IL-22, IL-17, IFN-γ and TNF-α. In IL-23p19-deficient mice, exacerbated hepatitis was observed and serum IL-22 and IL-17 levels were greatly reduced, whereas in IL-17-deficient mice, ameliorated hepatitis was observed. The injection of exogenous IL-22 protected p19-deficient mice from hepatitis, whereas the injection of exogenous IL-23 significantly increased the serum levels of not only IL-22 but also IL-17, and less effectively protected against hepatitis in IL-17-dependent and -independent manners. Finally, it was revealed that STAT3, STAT4 and Notch contributed to the production of both the cytokines, and that the AHR was important only for IL-22 production in response to Con A and IL-23 in liver mononuclear cells. These results suggest that IL-23 plays a protective role in hepatitis through IL-22 production and also a pathological role via IL-17-dependent and -independent mechanisms.

Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8α(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs.

CD4+ T cells are the major effector T cells against blood-stage Plasmodium yoelii infection. On the other hand, the lethal strain of P. yoelii (PyL) has acquired an escape mechanism from host T cell immunity by activating CD4+CD25+ regulatory T cells (Treg). Although the activation of Treg during PyL infection precludes the clearance of PyL from mice, it remains unclear whether activation of Treg is attributable to a specific response against PyL infection. Thus, we examined here whether Treg proliferate in an antigen-dependent manner during PyL infection. We also investigated the effector and regulatory mechanisms of Treg. Infection with PyL increased the number of CD4+CD25+ T cells, in which expression of Foxp3 mRNA is up-regulated. The Treg that were transferred into mice infected with PyL, but not with a non-lethal strain of P. yoelii (PyNL), proliferated during the initial 5 days following infection. The Treg from PyL-infected mice showed strong suppression compared with those from naive or PyNL-infected mice, and could suppress T cell activation by recognizing PyL- but not PyNL-derived antigens. Furthermore, the suppressive function of Treg activated in PyL-infected but not in naive mice could not be inhibited by treatment with an anti-glucocorticoid-induced TNFR family-related protein (GITR) mAb. These findings indicate that PyL infection specifically activates Treg that are specific for PyL-derived antigens. The infection also induces resistance for Treg to GITR signaling, and this eventually contributes to the escape of parasites from host T cell immunity.

Objective To determine whether eyedrop administration of an anti-CD4 monoclonal antibody (mAb) is effective in the treatment of Sjogren's syndrome (SS) using a mouse model of the disease. Methods The anti-CD4 mAb was administered daily into the eyes of mice with SS from ages 4 to 8 weeks or ages 10 to 12 weeks. During treatment, tear volume was monitored and after final treatment, histologic features of the lacrimal and salivary glands, the phenotypes and function of T cells, and serum titers of anti–α-fodrin antibody were examined. Results Eyedrop administration of anti-CD4 mAb before the onset of SS prevented the autoimmune pathology seen in the lacrimal glands but not that in the salivary glands. Furthermore, eyedrop administration of anti-CD4 mAb after the development of SS inhibited mononuclear cell infiltration and the destruction of parenchyma only in the lacrimal glands. Eyedrop administration of anti-CD4 mAb suppressed the local activation of CD4+ T cells rather than deleting CD4+ T cells, which reduced the expansion of pathologic CD4+ T cells against α-fodrin. Conclusion These results demonstrate the remarkable efficacy of anti-CD4 mAb eyedrops in the treatment of SS eye symptoms, which illustrates a new antibody-based therapeutic strategy for patients with eye problems caused by SS as well as other diseases.

Familial hemophagocytic lymphohistiocytosis (FHL) is a disorder of immune homeostasis characterized by fever, cytopenias, hepatosplenomegaly, and coagulopathy. We studied the outcomes of 13 FHL patients who underwent the first unrelated cord blood transplantation (UCBT) after non-myeloablative conditionings. The major regimen consisted of fludarabine (FLU; n = 12)+melphalan (MEL; n = 11)± low-dose total body irradiation (TBI 2-4 Gy; n = 6). The median age at presentation and period to UCBT were 6 and 5 months, respectively. Central nervous system (CNS) disease developed in one infant at diagnosis, and in two others until UCBT. HLH activity was controlled in all but one at the time of UCBT. Ten patients had early engraftment on median day 21 with no grade >2 treatment-related toxicity and two controllable grade >2 acute GVHD. Two patients with early rejection successfully underwent subsequent UCBT after myeloablative conditioning. Two others had late graft failure following mixed donor chimerism. Two deaths occurred from HLH; early liver failure and late CNS disease. Of 11 FLU+MEL-conditioned patients, the frequency of disease-free complete engraftment was higher for MEL (≥120 mg/m(2) )+TBI, or high-dose MEL (180 mg/m(2) ) than for others (83% vs. 25%, p = 0.036). The FLU+MEL-based non-myeloablative regimen was acceptable for FHL infants undergoing UCBT, although further studies will be needed for confirmation.

We reported that LEC rats are genetically deficient in the development of thymic CD4+8- cells and that this defect is caused by bone marrow (BM)-derived stem cells. To determine which BM-derived cells are responsible for the arrest of T-cell development in LEC rats, fetal thymuses of LEC rats, or LEA rats which bear the same major histocompatibility complex (MHC) as LEC rats but are immunologically normal, were engrafted under the kidney capsule of severe combined immunodeficiency (SCID) mice (LEC-TG and LEA-TG mice, respectively). We than examined the differentiation of T cells and their immunological functions in the SCID mice. A large number of rat-derived CD4+ T cells appeared in the peripheral blood, lymph nodes (LN) and spleens in LEC-TG mice. Furthermore, the peripheral LN cells in LEC-TG mice appeared to be functional. These cells produced IL-2 upon Con A stimulation, whereas LN cells from LEC rats produced no IL-2 in the same conditions. Thymopoiesis was observed at 3 weeks in LEC-TG as well as LEA-TG mice. The distribution of thymocyte subsets with respect to CD4 and CD8 expression in LEC-TG mice closely resembled that of LEA rat thymus and that in LEA-TG mice, suggesting that normal T-cell differentiation occurred in LEC-TG mice. The results indicated that BM-derived progenitor T cells of LEC rats could differentiate to functional CD4+ T cells.