Chieko Ishifune

Notch2 integrates signaling by the transcription factors RBP-J and CREB1 to promote T cell cytotoxicity

The acquisition of cytotoxic effector function by CD8+ T cells is crucial for the control of intracellular infection and tumor invasion. However, it remains unclear which signaling pathways are required for the differentiation of CD8+ cytotoxic T lymphocytes. We show here that Notch2-deficient T cells had impaired differentiation into cytotoxic T lymphocytes. In addition, dendritic cells with lower expression of the Notch ligand Delta-like 1 induced the differentiation of cytotoxic T lymphocytes less efficiently. We found that the intracellular domain of Notch2 interacted with a phosphorylated form of the transcription factor CREB1, and together these proteins bound the transcriptional coactivator p300 to form a complex on the promoter of the gene encoding granzyme B. Our results suggest that the highly regulated, dynamic control of T cell cytotoxicity depends on the integration of Notch2 and CREB1 signals.

Notch controls the survival of memory CD4+ T cells by regulating glucose uptake

CD4+ T cells differentiate into memory T cells that protect the host from subsequent infection. In contrast, autoreactive memory CD4+ T cells harm the body by persisting in the tissues. The underlying pathways controlling the maintenance of memory CD4+ T cells remain undefined. We show here that memory CD4+ T cell survival is impaired in the absence of the Notch signaling protein known as recombination signal binding protein for immunoglobulin κ J region (Rbpj). Treatment of mice with a Notch inhibitor reduced memory CD4+ T cell numbers and prevented the recurrent induction of experimental autoimmune encephalomyelitis. Rbpj-deficient CD4+ memory T cells exhibit reduced glucose uptake due to impaired AKT phosphorylation, resulting in low Glut1 expression. Treating mice with pyruvic acid, which bypasses glucose uptake and supplies the metabolite downstream of glucose uptake, inhibited the decrease of autoimmune memory CD4+ T cells in the absence of Notch signaling, suggesting memory CD4+ T cell survival relies on glucose metabolism. Together, these data define a central role for Notch signaling in maintaining memory CD4+ T cells through the regulation of glucose uptake.

The lifestyle of the segmented filamentous bacterium: A non-culturable gut-associated immunostimulating microbe inferred by whole-genome sequencing

Numerous microbes inhabit the mammalian intestinal track and strongly impact host physiology; however, our understanding of this ecosystem remains limited owing to the high complexity of the microbial community and the presence of numerous non-culturable microbes. Segmented filamentous bacteria (SFBs), which are clostridia-related Gram-positive bacteria, are among such non-culturable populations and are well known for their unique morphology and tight attachment to intestinal epithelial cells. Recent studies have revealed that SFBs play crucial roles in the post-natal maturation of gut immune function, especially the induction of Th17 lymphocytes. Here, we report the complete genome sequence of mouse SFBs. The genome, which comprises a single circular chromosome of 1 620 005 bp, lacks genes for the biosynthesis of almost all amino acids, vitamins/cofactors and nucleotides, but contains a full set of genes for sporulation/germination and, unexpectedly, for chemotaxis/flagella-based motility. These findings suggest a triphasic lifestyle of the SFB, which comprises two types of vegetative (swimming and epicellular parasitic) phases and a dormant (spore) phase. Furthermore, SFBs encode four types of flagellin, three of which are recognized by Toll-like receptor 5 and could elicit the innate immune response. Our results reveal the non-culturability, lifestyle and immunostimulation mechanisms of SFBs and provide a genetic basis for the future development of the SFB cultivation and gene-manipulation techniques.

Dendritic cell-mediated NK cell activation is controlled by Jagged2–Notch interaction

Natural killer (NK) cells regulate various immune responses by exerting cytotoxic activity or secreting cytokines. The interaction of NK cells with dendritic cells (DC) contributes to NK cell-mediated antitumor or antimicrobial responses. However, the cellular and molecular mechanisms for controlling this interaction are largely unknown. Here, we show an involvement of Jagged2–Notch interaction in augmenting NK cell cytotoxicity mediated by DC. Enforced expression of Jagged2 on A20 cells (Jag2-A20 cells) suppressed their growth in vivo, which was abrogated by depleting NK cells. Moreover, Jag2-A20 cells exerted a suppression on the growth of nonmanipulated A20 cells in SCID mice in an NK-dependent manner. Consistently, coinoculation of A20 cells with DC overexpressing Jagged2 (Jag2-DC) suppressed the growth of A20 cells in mice. Stimulation of NK cells with Jagged2 directly enhanced their cytotoxicity, IFN-γ production, and proliferation. Ligation of Notch2 on NK cells enhanced their cytotoxic activity, and Jag2-DC or CpG-treated DC-mediated NK cell cytotoxicity was suppressed by a γ-secretase inhibitor. These results indicate that the Jagged2–Notch axis plays a crucial role in DC-mediated NK cell cytotoxicity. Furthermore, manipulation of this interaction may provide an approach to induce potent tumor immunity or to inhibit certain autoimmune diseases caused by NK cell activation.

Regulation of monocyte cell fate by blood vessels mediated by Notch signalling

A population of monocytes, known as Ly6Clo monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6Chi monocytes into Ly6Clo monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation.

Differentiation of CD11c+CX3CR1+ cells in the small intestine requires Notch signaling

Significance The gastrointestinal tract directly faces an array of environmental agents, including bacteria and food. Intestine-specific subsets of immune cells maintain gut homeostasis by continuously sampling luminal antigens and maintaining immune tolerance. We here demonstrated that Rbpj, an essential molecule for Notch signaling, is essential for the development of CD11c+CX3CR1+ cells that are crucial for sampling luminal antigens with dendrites projecting through the epithelial cell layer. These findings indicate that Notch signaling is required for maintaining the repertoires of intestinal antigen-presenting cells and suggest new ways to keep immune tolerance by modulating Notch-mediated CD11c+CX3CR1+ cells. The gastrointestinal tract comes into direct contact with environmental agents, including bacteria, viruses, and foods. Intestine-specific subsets of immune cells maintain gut homeostasis by continuously sampling luminal antigens and maintaining immune tolerance. CD11c+CX3CR1+ cells sample luminal antigens in the small intestine and contribute to the trafficking of bacteria to lymph nodes under dysbiotic conditions. The molecular mechanisms crucial for the differentiation of CD11c+CX3CR1+ cells remain unclear. Here we demonstrate that the Notch1– or Notch2–Rbpj axis is essential for the development of CD11c+CX3CR1+ cells. In mice in which Rbpj or Notch1 and Notch2 were deleted from CD11c+ cells, there was a deficit of CD11c+CX3CR1+ cells and an accumulation of CD11clowCX3CR1+ cells. The CD11clowCX3CR1+ cells could not differentiate to CD11c+CX3CR1+ cells, suggesting that CD11clowCX3CR1+ cells represent a lineage distinct from CD11c+CX3CR1+ cells. These data indicate that Notch signaling is essential for lineage fixation of intestinal CD11c+CX3CR1+ cells.

The ARNT–STAT3 axis regulates the differentiation of intestinal intraepithelial TCRαβ + CD8αα + cells

Intestinal intraepithelial T cells contribute to the regulation of inflammatory responses in the intestine; however, the molecular basis for their development and maintenance is unknown. The aryl hydrocarbon receptor complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT) and senses environmental factors, including gut microbiota. Here, we identify ARNT as a critical regulator of the differentiation of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. Mice deficient in either ARNT or aryl hydrocarbon receptor show a greater than- eight-fold reduction in the number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. The number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells is increased by treatment with an aryl hydrocarbon receptor agonist in germ-free mice and is decreased by antibiotic treatment. The Arnt-deficient precursors of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells express low amounts of STAT3 and fail to differentiate towards the TCRαβ(+)CD8αα(+) cell fate after IL-15 stimulation, a deficiency that is overcome by overexpression of Stat3. These data demonstrate that the ARNT-STAT3 axis is a critical regulator of TCRαβ(+)CD8αα(+) intestinal intraepithelial T-cell development and differentiation.

Abrogation of Rbpj Attenuates Experimental Autoimmune Uveoretinitis by Inhibiting IL-22-Producing CD4(+) T Cells

Experimental autoimmune uveoretinitis (EAU) is an organ-specific T cell-mediated disease induced by immunizing mice with interphotoreceptor retinoid binding protein (IRBP). Autoaggressive CD4+ T cells are the major pathogenic population for EAU. We investigated the contribution of Notch signaling in T cells to EAU pathogenesis because Notch signaling regulates various aspects of CD4+ T cell functions. Rbpj is required for Notch signaling, and Rbpj deficiency in T cells inhibited EAU disease severity. The amelioration of EAU in T cell-specific Rbpj-deficient mice correlated with low levels of IL-22 production from CD4+ T cells, although IRBP-specific CD4+ T cell proliferation and Th17 differentiation were unaffected. Administration of recombinant IL-22 during the late phase, but not the early phase, of EAU increased EAU clinical scores in T cell-specific Rbpj-deficient mice. Notch inhibition in mice immunized with IRBP with a γ-secretase inhibitor (GSI) suppressed EAU progression, even when GSI was administered as late as 13 days after IRBP immunization. Our data demonstrate that Rbpj/Notch-mediated IL-22 production in T cells has a key pathological role in the late phase of EAU, and suggest that Notch blockade might be a useful therapeutic approach for treating EAU.

Notch signaling regulates the development of a novel type of Thy1-expressing dendritic cell in the thymus

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.

Notch 1 and Notch 2 synergistically regulate the differentiation and function of invariant NKT cells

Invariant natural killer T cells are a distinct subset of T cells that exert Janus‐like functions. Moreover, Notch signaling is known to have critical roles in the development and functions of T cells. However, it is not known whether Notch signaling contributes to the development or functions of invariant natural killer T cells. Here, we found that CD4‐specific gene ablation of Notch 1 and Notch 2 (N1N2−/−) increased the number of invariant natural killer T cells in the thymus but decreased them in the liver. N1N2−/− mice showed impaired thymic maturation of invariant natural killer T cells from the NK1.1−CD44+ to the NK1.1+CD44+ stage, resulting in accumulation of NK1.1−CD44+ invariant natural killer T cells in the thymus. Upon activation, hepatic invariant natural killer T cells from N1N2−/− mice produced lower cytokine levels and increased apoptosis versus wild‐type invariant natural killer T cells. Furthermore, Notch 1/Notch 2‐deficient, but not wild type, invariant natural killer T cells failed to promote antibody‐induced arthritis in CD1d−/− mice. Unlike N1N2−/− mice, RBP‐jlox/lox CD4‐Cre mice showed similar percentages and numbers of thymic invariant natural killer T cells to wild‐type mice but had defects in their homeostasis, maturation, and cytokine production in the liver. Taken together, our data indicate distinct effects of Notch signaling on invariant natural killer T cells in the thymus and liver, which are at least partly independent of RBP‐j in the thymus.