Doctor of Medicine, Kyoto University (1999)
Doctor of Philosophy, Kyoto University (2009)
Irving Weissman, Postdoctoral Faculty Sponsor
Mobilization of the T-cell response against cancer has the potential to achieve long-lasting cures. However, it is not known how to harness antigen-presenting cells optimally to achieve an effective antitumor T-cell response. In this study, we show that anti-CD47 antibody-mediated phagocytosis of cancer by macrophages can initiate an antitumor T-cell immune response. Using the ovalbumin model antigen system, anti-CD47 antibody-mediated phagocytosis of cancer cells by macrophages resulted in increased priming of OT-I T cells [cluster of differentiation 8-positive (CD8(+))] but decreased priming of OT-II T cells (CD4(+)). The CD4(+) T-cell response was characterized by a reduction in forkhead box P3-positive (Foxp3(+)) regulatory T cells. Macrophages following anti-CD47-mediated phagocytosis primed CD8(+) T cells to exhibit cytotoxic function in vivo. This response protected animals from tumor challenge. We conclude that anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer but also can initiate an antitumor cytotoxic T-cell immune response.
View details for DOI 10.1073/pnas.1305569110
View details for Web of Science ID 000321978000057
View details for PubMedID 23690610
Phagocytes, including macrophages, recognize phosphatidylserine exposed on apoptotic cells as an "eat me" signal. Milk Fat Globule EGF Factor VIII (MFG-E8) is secreted by one subset of macrophages, whereas Tim4, a type I membrane protein, is expressed by another. These proteins bind tightly to phosphatidylserine on apoptotic cells and enhance their engulfment by macrophages. To study the contribution of these proteins to the engulfment of apoptotic cells, we established a mouse line that was deficient in the genes encoding MFG-E8 and Tim4. The null mutation of Tim4 impaired the ability of resident peritoneal macrophages, but not thioglycollate-elicited macrophages, to engulf apoptotic cells. Mice deficient in either MFG-E8 or Tim4 on the C57BL/6 background developed hardly any autoantibodies, but aged female mice deficient in both MFG-E8 and Tim4 developed autoantibodies in an age-dependent manner. Tumour necrosis factor (TNF) α is known to protect against systemic lupus erythematosus-type autoimmunity, whereas type I IFN accelerates the disease. Indeed, the administration of an anti-TNFα antibody or a reagent that stimulates the IFN-α production [2,6,10,14-tetramethylpentadecane (TMPD; also known as pristane)] enhanced the production of autoantibodies in the MFG-E8- and Tim4-double-deficient mice. These results suggest that the double deficiency of Tim4 and MFG-E8, phosphatidylserine-binding proteins, can trigger autoimmunity and that TNFα and type I IFN regulate reciprocally the development of autoimmune disease.
View details for DOI 10.1093/intimm/dxs064
View details for Web of Science ID 000308016000002
View details for PubMedID 22723547
In programmed cell death, a large number of cells undergo apoptosis, and are engulfed by macrophages to avoid the release of noxious materials from the dying cells. In definitive erythropoiesis, nuclei are expelled from erythroid precursor cells and are engulfed by macrophages. Phosphatidylserine is exposed on the surface of apoptotic cells and on the nuclei expelled from erythroid precursor cells; it works as an 'eat me' signal for phagocytes. Phosphatidylserine is also expressed on the surface of exosomes involved in intercellular signalling. Here we established a library of hamster monoclonal antibodies against mouse peritoneal macrophages, and found an antibody that strongly inhibited the phosphatidylserine-dependent engulfment of apoptotic cells. The antigen recognized by the antibody was identified by expression cloning as a type I transmembrane protein called Tim4 (T-cell immunoglobulin- and mucin-domain-containing molecule; also known as Timd4). Tim4 was expressed in Mac1+ cells in various mouse tissues, including spleen, lymph nodes and fetal liver. Tim4 bound apoptotic cells by recognizing phosphatidylserine via its immunoglobulin domain. The expression of Tim4 in fibroblasts enhanced their ability to engulf apoptotic cells. When the anti-Tim4 monoclonal antibody was administered into mice, the engulfment of apoptotic cells by thymic macrophages was significantly blocked, and the mice developed autoantibodies. Among the other Tim family members, Tim1, but neither Tim2 nor Tim3, specifically bound phosphatidylserine. Tim1- or Tim4-expressing Ba/F3 B cells were bound by exosomes via phosphatidylserine, and exosomes stimulated the interaction between Tim1 and Tim4. These results indicate that Tim4 and Tim1 are phosphatidylserine receptors for the engulfment of apoptotic cells, and may also be involved in intercellular signalling in which exosomes are involved.
View details for DOI 10.1038/nature06307
View details for Web of Science ID 000250918600058
View details for PubMedID 17960135
Neuropilin-1 (NRP-1) is a receptor for both semaphorin and vascular endothelial growth factor and is up-regulated in a variety of human cancers. While there are some reports of NRP-1 expression in ovarian neoplasm, those results differ in pattern of its expression and its role in ovarian cancer is still unclear. We sought to investigate the expression pattern and role of NRP-1 in ovarian cancer.NRP-1 expression was analyzed with eighty-seven ovarian tissue samples by immunohistochemistry and four ovarian cell lines by quantitative RT-PCR and Western blotting. To detect its molecular role in ovarian cancer, WST-1 assay, invasion assay and soft agar assay were performed with or without NRP-1 suppression by the introduction of short hairpin RNAs.NRP-1 expression was found to be enhanced in ovarian cancer compared with ovarian surface epithelium (OSE), benign adenoma and tumors of low malignant potential. In vitro, NRP-1 expression was augmented threefold during malignant transformation of OSE cells with oncogene ras, suggesting an association between NRP-1 and oncogenesis. Suppression of NRP-1 reduced cell proliferation in a dense state, indicating that persistently high expression of NRP-1 in ovarian cancer enhances proliferation through evasion of contact inhibition. Suppression of NRP-1 also decreased cell growth in soft agar and invasion to the extracellular matrix in vitro.These results suggest that NRP-1 is not only associated with oncogenesis, but also with ovarian cancer malignancy, and this molecule is a targeting candidate for the treatment of ovarian malignancies.
View details for DOI 10.1016/j.ygyno.2007.02.005
View details for Web of Science ID 000246815700024
View details for PubMedID 17376520
Malignant transformation is caused by multi-step genetic mutations, and growth factors are believed to play important roles in developing and maintaining malignant phenotype. However, there is no direct evidence that a specific growth factor contributes to malignant transformation of phenotypically normal cells. In order to assess the function of Acrogranin (also known as granulin epithelial precursor; GEP) in ovarian carcinogenesis, ovarian surface epithelial (OSE) cells, which are supposed to be the origin of primary ovarian epithelial cancer, were transfected with combined genes of hTERT, SV40 LT, and Acrogranin. Introduction of hTERT and SV40 LT was sufficient for immortalizing OSE cells but not enough for tumor formation in nude mice. In contrast, transfection and overexpression of Acrogranin in immortalized OSE cells showed augmented clonogenicity in soft agar and obvious tumorigenicity in nude mice. This is the first study showing evidence that a specific growth factor plays a direct role in malignant transformation in ovarian cancer development.
View details for Web of Science ID 000243611400009
View details for PubMedID 17203169
To identify potential oncogenes that contribute to the development of uterine leiomyosarcoma, we conducted a cDNA microarray analysis between normal uterine smooth muscle and uterine leiomyosarcoma. We found that acrogranin (also named PCDGF or progranulin) is overexpressed in uterine leiomyosarcoma. With immunohistochemical staining of 12 leiomyosarcoma cases, we verified acrogranin expression in tumor cells. Furthermore, the intensity of acrogranin expression correlated with high histologic grade and poor prognosis. To directly analyze the oncogenic properties of acrogranin, we established an immortalized uterine smooth muscle cell line by transfection of human telomerase reverse transcriptase into primary culture. This cell line retained the original characteristics of uterine smooth muscle cells, including spindle-shaped extension as well as expression of vimentin, estrogen receptor alpha, progesterone receptor, and alpha smooth muscle actin. Transfection of acrogranin into the immortalized uterine smooth muscle cells resulted in colony formation in soft agar, but the diameter of the colonies did not exceed 100 mum. Transfection of both acrogranin and SV40 early region (SV40ER) into the immortalized uterine smooth muscle cells resulted in an increased number of colonies and increased colony size in soft agar versus transfection of SV40ER alone. We show that only immortalized uterine smooth muscle cells expressing both acrogranin and SV40ER are capable of tumor formation in nude mice. Thus, acrogranin is overexpressed in uterine leiomyosarcoma cells, particularly in high-grade cases, and forced expression of acrogranin in immortalized uterine smooth muscle cells contributes to malignant transformation, which suggest that acrogranin plays an important role in the pathogenesis of uterine leiomyosarcoma.
View details for DOI 10.1158/1078-0432.CCR-05-2003
View details for Web of Science ID 000235988000003
View details for PubMedID 16533762