The paradoxical role of an immune receptor, DNAM-1, in tumor development

The 3rd International Symposium on Carcinogenic Spiral & International Symposium on Tumor Biology in Kanazawa, [DATE]: January 24(Thu)-25(Fri),2013, [Place]:Kanazawa Excel Hotel Tpkyu, Kanazawa, Japan, [Organizers]:Infection/Inflammation-Assisted Acceleration of the Carcinogenic Spiral and its Alteration through Vector Conversion of the Host Response to Tumors / Scientific Research on Innovative Areas, a MEXT Grant-in Aid Projec

Development of the novel molecular target therapy for the graft versus host disease (GVHD) using the humanized mice

科学研究費助成事業 研究成果報告書：挑戦的萌芽研究2012-2013 課題番号：2465917

Selective DNAM-1 expression on small peritoneal macrophages contributes to CD4+ T cell costimulation

Mouse peritoneal macrophages consist of two subsets: large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs), defined as CD11bhiF4/80hi and CD11b+F4/80lo cells, respectively. We reveal that SPMs, but not LPMs, have the ability to present antigens to naïve CD4+ T cells. Coculture of SPMs with naïve ovalbumin (OVA) specific CD4+ T cells (OT-II) in the presence of OVA peptide effectively induced CD4+ T cells priming. SPMs, but not LPMs, strongly express DNAM-1, an activating immunoreceptor. Although antigen uptake and processing were comparable between WT and DNAM-1-deficient SPMs, deficiency of DNAM-1 on SPMs or blockade of DNAM-1 and its ligand interaction impaired CD4+ T cells priming by SPMs. Furthermore, T and B cell responses in mediastinal lymph nodes of mice intraperitoneally immunized with trinitrophenyl (TNP)–OVA protein in Alum adjuvant were enhanced by intraperitoneally transferred wild-type, but not DNAM-1-deficient, SPMs. We propose that SPMs are functionally distinct from LPMs, and DNAM-1 plays a costimulatory role in antigen presentation by SPMs

Reciprocal Roles for CCAAT/Enhancer Binding Protein (C/EBP) and PU.1 Transcription Factors in Langerhans Cell Commitment

Myeloid progenitor cells give rise to a variety of progenies including dendritic cells. However, the mechanism controlling the diversification of myeloid progenitors into each progeny is largely unknown. PU.1 and CCAAT/enhancing binding protein (C/EBP) family transcription factors have been characterized as key regulators for the development and function of the myeloid system. However, the roles of C/EBP transcription factors have not been fully identified because of functional redundancy among family members. Using high titer–retroviral infection, we demonstrate that a dominant-negative C/EBP completely blocked the granulocyte–macrophage commitment of human myeloid progenitors. Alternatively, Langerhans cell (LC) commitment was markedly facilitated in the absence of tumor necrosis factor (TNF)α, a strong inducer of LC development, whereas expression of wild-type C/EBP in myeloid progenitors promoted granulocytic differentiation, and completely inhibited TNFα-dependent LC development. On the other hand, expression of wild-type PU.1 in myeloid progenitors triggered LC development in the absence of TNFα, and its instructive effect was canceled by coexpressed C/EBP. Our findings establish reciprocal roles for C/EBP and PU.1 in LC development, and provide new insight into the molecular mechanism of LC development, which has not yet been well characterized

CD226 (DNAM-1) Is Involved in Lymphocyte Function–associated Antigen 1 Costimulatory Signal for Naive T Cell Differentiation and Proliferation

Upon antigen recognition by the T cell receptor, lymphocyte function–associated antigen 1 (LFA-1) physically associates with the leukocyte adhesion molecule CD226 (DNAM-1) and the protein tyrosine kinase Fyn. We show that lentiviral vector-mediated mutant (Y-F322) CD226 transferred into naive CD4+ helper T cells (Ths) inhibited interleukin (IL)-12–independent Th1 development initiated by CD3 and LFA-1 ligations. Moreover, proliferation induced by LFA-1 costimulatory signal was suppressed in mutant (Y-F322) CD226-transduced naive CD4+ and CD8+ T cells in the absence of IL-2. These results suggest that CD226 is involved in LFA-1–mediated costimulatory signals for triggering naive T cell differentiation and proliferation. We also demonstrate that although LFA-1, CD226, and Fyn are polarized at the immunological synapse upon stimulation with anti-CD3 in CD4+ and CD8+ T cells, lipid rafts are polarized in CD4+, but not CD8+, T cells. Moreover, proliferation initiated by LFA-1 costimulatory signal is suppressed by lipid raft disruption in CD4+, but not CD8+, T cells, suggesting that the LFA-1 costimulatory signal is independent of lipid rafts in CD8+ T cells

Accelerated tumor growth in mice deficient in DNAM-1 receptor

Since the identification of ligands for human and mouse DNAM-1, emerging evidence has suggested that DNAM-1 plays an important role in the T cell– and natural killer (NK) cell–mediated recognition and lysis of tumor cells. However, it remains undetermined whether DNAM-1 is involved in tumor immune surveillance in vivo. We addressed this question by using DNAM-1–deficient mice. DNAM-1–deficient cytotoxic T lymphocyte (CTL) and NK cells showed significantly less cytotoxic activity against DNAM-1 ligand-expressing tumors in vitro than wild-type (WT) cells. The methylcholanthrene (MCA)-induced fibrosarcoma cell line Meth A expressed the DNAM-1 ligand CD155, and DNAM-1–deficient mice showed increased tumor development and mortality after transplantation of Meth A cells. Moreover, the DNAM-1–deficient mice developed significantly more DNAM-1 ligand-expressing fibrosarcoma and papilloma cells in response to the chemical carcinogens MCA and 7,12-dimethylbenz[a]anthracene (DMBA), respectively, than did WT mice. These results indicate that DNAM-1 plays an important role in immune surveillance of tumor development

Paired Activating and Inhibitory Immunoglobulin-like Receptors, MAIR-I and MAIR-II, Regulate Mast Cell and Macrophage Activation

Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands. Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other. MAIR-I, expressed on the majority of myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, contains the tyrosine-based sorting motif and the immunoreceptor tyrosine-based inhibitory motif-like sequences in the cytoplasmic domain and mediates endocytosis of the receptor and inhibition of IgE-mediated degranulation from mast cells. On the other hand, MAIR-II, expressed on subsets of peritoneal macrophages and B cells, associates with the immunoreceptor tyrosine-based activation motif-bearing adaptor DAP12 and stimulates proinflammatory cytokines and chemokine secretions from macrophages. Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses

The immunoreceptor adapter protein DAP12 suppresses B lymphocyte–driven adaptive immune responses

Human and mouse B cells lacking functional DAP12 are hyperresponsive, and DAP12 works with MAIR-II (CD300d) to negatively regulate B cell activity

Isolation and characterization of naïve follicular dendritic cells

Follicular dendritic cells (FDC) are specialized antigen-presenting cells to cognate B cells in the follicle of the lymphoid tissues. FDC also support survival and proliferation of the B cells, leading to the germinal center formation. FDC therefore play a central role in humoral immune responses. However, molecular and functional characteristics of FDC are largely unknown, because it is difficult to isolate and analyze FDC due to a very small number of FDC in the lymphoid tissues and the fragility by mechanical and chemical stresses in vitro. In this report, we established a novel method for FDC isolation from the spleen of naïve mice by flow cytometry and analyzed the phenotypical and functional characteristics. The isolated FDC, which accounted for ∼0.2% of the spleen cells of naïve mice, were CD45−, FDC-M2+, and ICAM-1+, and supported the survival and LPS-induced proliferation of B cells. We also showed that a neutralizing antibody against B cell activating factor TNF family (BAFF) suppressed FDC-dependent B cell proliferation in the presence of LPS, but not survival, demonstrating the evidence that FDC-derived BAFF is involved in B cell proliferation

Virulence assessment of six major pathogenic Candida species in the mouse model of invasive candidiasis caused by fungal translocation

Gastrointestinal colonization has been considered as the primary source of candidaemia; however, few established mouse models are available that mimic this infection route. We therefore developed a reproducible mouse model of invasive candidiasis initiated by fungal translocation and compared the virulence of six major pathogenic Candida species. The mice were fed a low-protein diet and then inoculated intragastrically with Candida cells. Oral antibiotics and cyclophosphamide were then administered to facilitate colonization and subsequent dissemination of Candida cells. Mice infected with Candida albicans and Candida tropicalis exhibited higher mortality than mice infected with the other four species. Among the less virulent species, stool titres of Candida glabrata and Candida parapsilosis were higher than those of Candida krusei and Candida guilliermondii. The fungal burdens of C. parapsilosis and C. krusei in the livers and kidneys were significantly greater than those of C. guilliermondii. Histopathologically, C. albicans demonstrated the highest pathogenicity to invade into gut mucosa and liver tissues causing marked necrosis. Overall, this model allowed analysis of the virulence traits of Candida strains in individual mice including colonization in the gut, penetration into intestinal mucosa, invasion into blood vessels, and the subsequent dissemination leading to lethal infections