Chie Hotta

MHC class I-mediated exogenous antigen presentation by exosomes secreted from immature and mature bone marrow derived dendritic cells

Exosomes are 50-90 nm vesicles with antigen presenting ability carrying major histocompatibility complex (MHC) class I, class II, abundant co-stimulatory molecules and some tetraspan proteins. Although dendritic cells (DCs) are one of the professional antigen presenting cells capable of presenting exogenous antigens in MHC class I-mediated antigen specific manner (cross-presentation), the cross-presentation ability by exosomes from immature or mature DCs are unknown. Here we show that exosomes released from ovalbumin (OVA) protein-pulsed bone marrow derived dendritic cells (BM-DCs) weakly present the peptide determinants to OVA specific MHC class I-restricted CD8(+) T cell hybridomas. The exosomes secreted by OVA(257-264) peptide- or OVA protein-pulsed mature BM-DCs activated OVA specific MHC class I-restricted T cell hybridomas more efficiently than those from immature BM-DCs. Transporters associated with antigen processing (TAP) deficient mice-derived BM-DCs were also used to examine whether functional TAP activity was required for cross-presentation by exosomes. The exosomes obtained from OVA(257-264) peptide-pulsed BM-DCs derived from TAP(-/-) mice showed a significant antigen presenting ability to OVA specific MHC class I-restricted T cell hybridomas. Altogether, our data indicate that BM-DCs secrete exosomes with weak cross-presentation ability.

Shared and Distinct Functions of the Transcription Factors IRF4 and IRF8 in Myeloid Cell Development

Interferon regulatory factor (IRF) 8 and IRF4 are structurally-related, hematopoietic cell-specific transcription factors that cooperatively regulate the differentiation of dendritic cells and B cells. Whilst in myeloid cells IRF8 is known to modulate growth and differentiation, the role of IRF4 is poorly understood. In this study, we show that IRF4 has activities similar to IRF8 in regulating myeloid cell development. The ectopic expression of IRF4 in myeloid progenitor cells in vitro inhibits cell growth, promotes macrophages, but hinders granulocytic cell differentiation. We also show that IRF4 binds to and activates transcription through the IRF-Ets composite sequence (IECS). Furthermore, we demonstrate that Irf8 -/- Irf4 -/- mice exhibit a more severe chronic myeloid leukemia (CML)-like disease than Irf8 -/- mice, involving a disproportionate expansion of granulocytes at the expense of monocytes/macrophages. Irf4 -/- mice, however, display no obvious abnormality in myeloid cell development, presumably because IRF4 is expressed at a much lower level than IRF8 in granulocyte-macrophage progenitors. Our results also suggest that IRF8 and IRF4 have not only common but also specific activities in myeloid cells. Since the expression of both the IRF8 and IRF4 genes is downregulated in CML patients, these results may add to our understanding of CML pathogenesis.

The delivery of an antigen from the endocytic compartment into the cytosol for cross-presentation is restricted to early immature dendritic cells

Dendritic cells (DCs) are the only antigen‐presenting cell population having a cross‐presentation capacity. For cross‐presentation, however, the intracellular antigen‐processing pathway and its regulatory mechanism have not been defined. Here we report the differences in cross‐presentation ability among murine bone marrow‐derived immature DC, early immature day8‐DC and late immature day10‐DC, and fully mature day10 + lipopolysaccharide DC. Day8‐DCs and day10‐DCs show an immature phenotypic profile but are different in morphology. Day8‐DCs can internalize an abundant volume of exogenous soluble ovalbumin (OVA) and result in cross‐presentation. In contrast, day10‐DCs are not able to cross‐present, although they maintain efficient macropinocytosis. Exogenously internalized OVA antigens are stored in the endocytic compartments. The endocytic compartments are temporarily maintained at mildly acidic pH in day8‐DCs and are rapidly acidified in day10‐DCs after uptake of antigens. We show that OVA antigens accumulated in the endocytic compartments move into the cytosol in day8‐DCs but do not in day10‐DCs. NH4Cl‐treatment, which neutralizes the acidic endocytic compartments and/or delays endosomal maturation, restores day10‐DCs for transport the stored OVA antigens from the endocytic compartments into the cytosol. Diphenyleneiodonium chloride‐treatment, which acidifies the endocytic compartments, decreases an amount of transported OVA antigen into the cytosol in day8‐DCs. These data indicate that only the early immature stage of DC interferes with endosomal maturation, even after uptake of exogenous antigens, and then transports the antigens into the cytosol.

Impaired function of dendritic cells in alymphoplasia (aly/aly) mice for expansion of CD25+CD4+ regulatory T cells

Alymphoplasia (aly/aly) mice are from a naturally occurring strain with a mutation in nuclear factor-kappa B inducing kinase (NIK). The NIK mutation causes disruption of the architecture of the thymus and spleen and aly/aly mice show decreased numbers of CD25+CD4+T cells in the spleen. For the expansion of CD25+CD4+T cells, interactions between dendritic cells (DCs) and CD25+CD4+ regulatory T cells are necessary. We investigated the ability of DCs to induce expansion of CD25+CD4+T cells. We found that DCs are reduced in the spleen of aly/aly mice, and showed low expressions of CD80, CD86 and MHC class II molecules on the surface. DCs from aly/aly mice showed decreased ability to present ovalbumin (OVA) to T cells from OVA specific TCR transgenic mice, and a decreased ability for alloantigen presentation. Further, DCs showed a decreased ability to induce expansion of CD25+CD4+T cells in vitro. Our results suggested that the impairment of DCs in aly/aly mice is responsible, at least in part, for the decreased numbers of CD25+CD4+T cells in the periphery of aly/aly mice.

Human invariant V24 natural killer T cells acquire regulatory functions by interacting with IL-10-treated dendritic cells

Glycolipid-reactive Valpha24(+) invariant natural killer T (iNKT) cells have been implicated in regulating a variety of immune responses and in the induction of immunologic tolerance. Activation of iNKT cells requires interaction with professional antigen-presenting cells, such as dendritic cells (DCs). We have investigated the capacity of distinct DC subsets to modulate iNKT cell functions. We demonstrate that tolerogenic DCs (tolDCs), generated by treatment of monocyte-derived DC with interleukin (IL)-10, induced regulatory functions in human iNKT cells. tolDCs, compared with immunogenic DCs, had reduced capacity to induce iNKT-cell proliferation, but these cells produced large amounts of IL-10 and acquired an anergic phenotype. These anergic Valpha24(+) iNKT cells were able to potently inhibit allogeneic CD4(+) T-cell proliferation in vitro. Furthermore, the anergic Valpha24(+) iNKT cells could suppress DC maturation in vitro. We conclude that the interaction of iNKT cells with tolDCs plays an important role in the immune regulatory network, which might be exploited for therapeutic purposes.

The Transcription Factor IRF8 Counteracts BCR-ABL to Rescue Dendritic Cell Development in Chronic Myelogenous Leukemia

BCR-ABL tyrosine kinase inhibitors (TKI) have dramatically improved therapy for chronic myelogenous leukemia (CML). However, several problems leading to TKI resistance still impede a complete cure of this disease. IFN regulatory factor-8 (IRF8) is a transcription factor essential for the development and functions of immune cells, including dendritic cells. Irf8(-/-) mice develop a CML-like disease and IRF8 expression is downregulated in patients with CML, suggesting that IRF8 is involved in the pathogenesis of CML. In this study, by using a murine CML model, we show that BCR-ABL strongly inhibits a generation of dendritic cells from an early stage of their differentiation in vivo, concomitant with suppression of Irf8 expression. Forced expression of IRF8 overrode BCR-ABL (both wild-type and T315I-mutated) to rescue dendritic cell development in vitro, indicating that the suppression of Irf8 causes dendritic cell deficiency. Gene expression profiling revealed that IRF8 restored the expression of a significant portion of BCR-ABL-dysregulated genes and predicted that BCR-ABL has immune-stimulatory potential. Indeed, IRF8-rescued BCR-ABL-expressing dendritic cells were capable of inducing CTLs more efficiently than control dendritic cells. Altogether, our findings suggest that IRF8 is an attractive target in next-generation therapies for CML.

Impaired expression of MHC class I molecules on mouse testicular germ cells is mainly caused by the post-transcriptional mechanism.

Abstract. In this study, we examined expression of the major histocompatibility complex (MHC) class I molecules and of the mRNAs for MHC class I-mediated antigen presentation machinery molecules in highly purified C57BL/6 mouse testicular germ cells. Although H2 class I molecules were not detected on the cell surface, small amounts of the molecules were detected inside the cells by flow cytometric and immunoprecipitation analyses. Interferon (IFN)γ treatment did not affect expression of the molecules on the surface by flow cytometric analysis. In addition, expression of mRNAs for MHC class I-mediated antigen presentation machinery molecules in the germ cells was analyzed by reverse transcription-polymerase chain reaction analysis. The faint constitutive expression of mRNAs for H2-K, β2-microglobulin (β2m), tapasin, and calnexin was observed in the cells. IFNγ treatment up-regulated expression of mRNAs for H2-K, β2m, TAP1 (a peptide transporter), and LMP2 (a proteosomal subunit). These findings indicate that impaired expression of MHC class I molecules on the cell surface of mouse testicular germ cells is mainly caused by the post-transcriptional regulatory mechanism.

Anti-Semaphorin 3A neutralization monoclonal antibody prevents sepsis development in lipopolysaccharide-treated mice

Semaphorin 3A (Sema3A), originally identified as a potent growth cone collapsing factor in developing sensory neurons, is now recognized as a key player in immune, cardiovascular, bone metabolism and neurological systems. Here we established an anti-Sema3A monoclonal antibody that neutralizes the effects of Sema3A both in vitro and in vivo. The anti-Sema3A neutralization chick IgM antibodies were screened by combining an autonomously diversifying library selection system and an in vitro growth cone collapse assay. We further developed function-blocking chick-mouse chimeric and humanized anti-Sema3A antibodies. We found that our anti-Sema3A antibodies were effective for improving the survival rate in lipopolysaccharide-induced sepsis in mice. Our antibody is a potential therapeutic agent that may prevent the onset of or alleviate symptoms of human diseases associated with Sema3A.

Effect of BSA Antigen Sensitization during the Acute Phase of Influenza A Viral Infection on CD11c+ Pulmonary Antigen Presenting Cells

BACKGROUND Influenza A viral infection is concerned with induction of asthma. CD11c+ pulmonary antigen presenting cells (APCs) play a central role in sensitization with inhaled antigens during the acute phase of influenza A viral infection and also reside on bronchial epithelium for the long term after sensitization. To investigate the role of CD11c+ pulmonary APCs in the inhaled antigen sensitization during the acute phase of influenza A viral infection, we analyzed their function. METHODS Mice were infected with influenza A virus and were sensitized intranasally with BSA/alum during the acute phase of influenza A viral infection. Expression of surface antigens on CD11c+ pulmonary APCs was analyzed by FACS. Cytokine production from CD11c+ pulmonary APCs, and interaction between CD11c+ pulmonary APCs and naïve CD4+ T cells was assessed by ELISA. Ability of antigen presentation by CD11c+ pulmonary APCs was measured by proliferation assay. RESULTS BSA antigen sensitization during the acute phase of influenza A viral infection induced eosinophil recruitment into the lungs after BSA antigen challenge and moderately increased expression of MHC class II molecules on CD11c+ pulmonary APCs. The interaction between the CD11c+ pulmonary APCs and naïve CD4+ T cells secreted large amounts of IL-10. CONCLUSIONS BSA antigen sensitization during the acute phase of influenza A viral infection enhanced IL-10 production from naïve CD4+ T cell interaction with CD11c+ pulmonary APCs. The IL-10 secretion evoked Th2 responses in the lungs with downregulation of Th1 responses and was important for the eosinophil recruitment into the lungs after BSA antigen challenge.