腸管のリンパ組織における免疫応答に関わる細胞の機能についての研究

学位の種別: 課程博士審査委員会委員 : （主査）東京大学准教授 八村 敏志, 東京大学教授 田之倉 優, 東京大学教授 高橋 直樹, 東京大学教授 佐藤 隆一郎, 東京大学准教授 戸塚 護University of Tokyo(東京大学

Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBVencoded RNA.We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-a, and arginase 1, suggesting the immune regulatory role of BART miRNAs.The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma.These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV1 B-cell lymphoma. (Blood. 2018;131(23):2552-2567)

Imbalanced expression of polycistronic miRNA in acute myeloid leukemia

miR-1 and miR-133 are clustered on the same chromosomal loci and are transcribed together as a single transcript that is positively regulated by ecotropic virus integration site-1 (EVI1). Previously, we described how miR-133 has anti-tumorigenic potential through repression of EVI1 expression. It has also been reported that miR-1 is oncogenic in the case of acute myeloid leukemia (AML). Here, we show that expression of miR-1 and miR-133, which have distinct functions, is differentially regulated between AML cell lines. Interestingly, the expression of miR-1 and EVI1, which binds to the promoter of the miR-1/miR-133 cluster, is correlative. The expression levels of TDP-43, an RNA-binding protein that has been reported to increase the expression, but inhibits the activity, of miR-1, were not correlated with expression levels of miR-1 in AML cells. Taken together, our observations raise the possibility that the balance of polycistronic miRNAs is regulated post-transcriptionally in a hierarchical manner possibly involving EVI1,suggesting that the deregulation of this balance may play some role in AML cells with high EVI1 expression

Humoral immunity for durable control of SARS-CoV-2 and its variants

Abstract The coronavirus disease 2019 (COVID-19) pandemic is ongoing because of the repeated emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, highlighting the importance of developing vaccines for variants that may continue to emerge. In the present review, we discuss humoral immune responses against SARS-CoV-2 with a focus on the antibody breadth to the variants. Recent studies have revealed that the temporal maturation of humoral immunity improves the antibody potency and breadth to the variants after infection or vaccination. Repeated vaccination or infection further accelerates the expansion of the antibody breadth. Memory B cells play a central role in this phenomenon, as the reactivity of the B-cell antigen receptor (BCR) on memory B cells is a key determinant of the antibody potency and breadth recalled upon vaccination or infection. The evolution of memory B cells remarkably improves the reactivity of BCR to antigenically distinct Omicron variants, to which the host has never been exposed. Thus, the evolution of memory B cells toward the variants constitutes an immunological basis for the durable and broad control of SARS-CoV-2 variants

miRNAs in Normal and Malignant Hematopoiesis

Lineage specification is primarily regulated at the transcriptional level and lineage-specific transcription factors determine cell fates. MicroRNAs (miRNAs) are 18–24 nucleotide-long non-coding RNAs that post-transcriptionally decrease the translation of target mRNAs and are essential for many cellular functions. miRNAs also regulate lineage specification during hematopoiesis. This review highlights the roles of miRNAs in B-cell development and malignancies, and discusses how miRNA expression profiles correlate with disease prognoses and phenotypes. We also discuss the potential for miRNAs as therapeutic targets and diagnostic tools for B-cell malignancies

T396I Mutation of Mouse <i>Sufu</i> Reduces the Stability and Activity of Gli3 Repressor

<div><p>Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3^FL) to an N-terminal repressor (Gli3^REP). Recent studies using a <i>Sufu</i> knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, <i>Sufu^T396I</i>, showed that Thr³⁹⁶residue of Sufu played a key role in regulation of Gli3 activity. <i>Sufu^T396I/T396I</i> embryos exhibited severe polydactyly, which is indicative of compromised <i>Gli3</i> activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3^FL) and processed Gli3 (Gli3^REP) were observed <i>in vivo</i> as well as <i>in vitro</i>. Genetic experiments showed that patterning defects in the limb buds of <i>Sufu^T396I/T396I</i> were rescued by a constitutive Gli3^REPallele (<i>Gli3^∆699</i>), strongly suggesting that Sufu^T396I reduced the truncated Gli3 repressor. In contrast, Sufu^T396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr³⁹⁶residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.</p></div

Sufu^T396I can regulate Gli activator function.

<p>(<b>A</b>) Western blotting of Gli2 from embryo lysates at E9.5. Genotypes and antibodies are indicated at the top and left, respectively. The image presented in the Fig. is representative of independent triplicated experiments. The broad gel images are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.s005" target="_blank">S5A Fig</a>. and relative expression of Gli2 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.s005" target="_blank">S5B Fig</a>. (<b>B, C</b>) Western blotting of immunoprecipitates or lysates from 293T cells transfected with expression constructs as indicated at the top. Antibodies used for immunoprecipitation and western blotting are indicated to the left. The complete gel images are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.s005" target="_blank">S5C and D Fig</a>. Gli1 protein appears to show multiple bands; the reason for this is unknown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.ref042" target="_blank">42</a>] [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.ref046" target="_blank">46</a>]. Relative band intensities of lane 5 and 6 (C) are shown. It is note worthy that the expression levels of Gli2 were proportional to the amount of Sufu irrespective of either wild-type or T396I substitutions. (<b>D, E</b>) Luciferase reporter assay in 3T3 transfected with expression constructs as indicated in figures. Error bars indicate standard deviations. In our experimental settings, we observed approximately only 4-fold induction of luciferase reporter activity by Gli1 expression, as had been observed previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.ref046" target="_blank">46</a>].</p

<i>Sufu</i>^{<i>T396I/T396I</i>} shows reduction of Gli3 activity but not Gli2 activity.

<p>(<b>A–H</b>) Immunofluorescence images of transverse sections at thoracic level. Sections from wild-type (A and E), <i>Sufu</i>^{<i>T396I/T396I</i>} (B and F), and <i>Smo</i>^{<i>G457X/G457X</i>}; <i>Sufu</i>^{<i>T396I/T396I</i>} (C and G) embryos at E10.5 and <i>Smo</i>^{<i>G457X/G457X</i>} embryos (D and H) at E9.5 were immunostained with anti-Olig2 (A–D, magenta), anti-Nkx2.2 (A–D, green), and anti-FoxA2 (E–H, green) antibodies. Dashed lines outline the neural tubes. Scale bar, 100 μm. Images with nuclear staining are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.s007" target="_blank">S7 Fig</a>.</p

Identification of novel <i>Sufu</i> mutants.

<p>(<b>A</b>) Schematic diagrams of the mouse Sufu protein. The numbers refer to amino acid residues. Filled boxes indicate binding domains with GSK3β [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.ref021" target="_blank">21</a>] and the C- and N-terminal regions of Gli1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.ref026" target="_blank">26</a>]. An asterisk marks the position of a 1-base substitution that results in a change of Thr³⁹⁶ to Ile (T396I). <i>Sufu</i>^<i>R146X</i> is a 1-base pair deletion in <i>Sufu</i>, leading to premature termination of the protein product after addition of an aberrant 33 amino acid stretch at the C terminal when translated. (<b>B, C</b>) Wild-type and <i>Sufu</i>^{<i>T396I/T396I</i>} embryos at E13.5. Scale bar, 4 mm. (<b>D</b>) Limb phenotype of <i>Sufu</i>^{<i>T396I/T396I</i>} at E15. Scale bar, 2 mm. (<b>E, F</b>) Lungs of wild-type and <i>Sufu</i>^{<i>T396I/T396I</i>} at E15.5. Scale bars, 2 mm. (<b>G, H</b>) Wild-type and <i>Sufu</i>^{<i>R146X/R146X</i>} embryos at E9.5. Scale bars, 1 mm. Homozygous embryos died at approximately E9.5 and exhibited an open brain and failure to undergo embryonic turning, characteristics identical to those reported in <i>Sufu</i> knockout embryos [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.ref022" target="_blank">22</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119455#pone.0119455.ref051" target="_blank">51</a>].</p