Language：English   Publishing type：Research paper (scientific journal)  

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

DOI： 10.1021/acs.jproteome.0c00303

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Journal of Cancer and Chemotherapy Publishers Inc.  

The cancer immunotherapies based on adoptive T cell therapy (ACT) has been receiving increased attention by improvement of the curative effect. T cells for ACT are harvested from the patient, then activated and expanded in vitro. However, in vitro activated T cells frequently show dysfunction after adoptive transfer, such as the exhaustion and the senescence. The exhausted/senescent T cells reduces the effector functions and fails to eliminate tumor cells. Therefore, the development of the culture method avoiding a T cell exhaustion and senescence. Recent findings reveal the dramatic changes of the metabolic status in T cells during T-cell receptor (TCR)-mediated activation. We recently reported that the activation status of glutami-nolysis during TCR-stimulation determines the activated CD8T cell fate. We considered that the therapeutic effect of ACT will be improved by the modulation of glutaminolysis. We demonstrated that the CD8 T cell exhaustion and/or senescence is prevented and the antitumor activity of adoptively transferred CD8T cells is reinforced by the glutamine restriction during in vitro culture. The adoptively transferred CD8 T cells cultured under glutamine-restricted conditions shows higher infiltration in the tumor sites than that of CD8 T cells cultured under normal conditions. The expression of inhibitory receptors, such as PD-1 is decreased in tumor-infiltrating CD8 T cells cultured under glutamine-restricted conditions. Furthermore, the restriction of glutamine during CD8T cell activation in vitro drives memory T cell development after adoptive transfer. The effect of glutamine restriction is antagonized by α-ketoglutarate, a metabolite of glutaminolysis. Thus, our recent findings suggest that the glutamine-restricted culture of CD8 T cells in vitro will improve the efficacy of CD8 T cell-based ACT.

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DOI： 10.1038/s41598-019-53856-1

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DOI： 10.4049/jimmunol.1801083

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DOI： 10.1111/cas.13827

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Language：English   Publishing type：Research paper (scientific journal)  

While menin plays an important role in preventing T-cell dysfunction, such as senescence and exhaustion, the regulatory mechanisms remain unclear. We found that menin prevents the induction of dysfunction in activated CD8 T cells by restricting the cellular metabolism. mTOR complex 1 (mTORC1) signaling, glycolysis, and glutaminolysis are augmented by menin deficiency. Rapamycin treatment prevents CD8 T-cell dysfunction in menin-deficient CD8 T cells. Limited glutamine availability also prevents CD8 T-cell dysfunction induced by menin deficiency, and its inhibitory effect is antagonized by α-ketoglutarate (α-KG), an intermediate metabolite of glutaminolysis. α-KG-dependent histone H3K27 demethylation seems to be involved in the dysfunction in menin-deficient CD8 T cells. We also found that α-KG activates mTORC1-dependent central carbon metabolism. These findings suggest that menin maintains the T-cell functions by limiting mTORC 1 activity and subsequent cellular metabolism.

DOI： 10.1038/s41467-018-05854-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

Menin, a tumor suppressor protein, is encoded by the MEN1 gene in humans. Certain germinal mutations of MEN1 induce an autosomal-dominant syndrome that is characterized by concurrent parathyroid adenomas and several other tumor types. Although menin is also expressed in hematopoietic lineages, its role in CD8(+) T cells remains unclear. We generated Menin(flox/flox) CD4-Cre (Menin-KO) mice by crossing Menin(flox/flox) mice with CD4-Cre transgenic (Tg) mice to determine the role of menin in CD8(+) T cells. Wild-type (WT) and Menin-KO mice were infected with Listeria monocytogenes expressing OVA to analyze the immune response of Ag-specific CD8(+) T cells. Menin deficiency resulted in an impaired primary immune response by CD8+ T cells. On day 7, there were fewer Menin-KO OVA-specific CD8(+) T cells compared with WT cells. Next, we adoptively transferred WT and Menin-KO OT-1 Tg CD8(+) T cells into congenic recipient mice and infected them with L. monocytogenes expressing OVA to determine the CD8(+) T cell-intrinsic effect. Menin-KO OT-1 Tg CD8(+) T cells were outcompeted by the WT cells upon infection. Increased expression of Blimp-1 and T-bet, cell cycle inhibitors, and proapoptotic genes was observed in the Menin-KO OT-1 Tg CD8(+) T cells upon infection. These data suggest that menin inhibits differentiation into terminal effectors and positively controls proliferation and survival of Ag-specific CD8(+) T cells that are activated upon infection. Collectively, our study uncovered an important role for menin in the immune response of CD8(+) T cells to infection.

DOI： 10.4049/jimmunol.1502295

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Although Bach2 has an important role in regulating the Th2-type immune response, the underlying molecular mechanisms remain unclear. We herein demonstrate that Bach2 associates with Batf and binds to the regulatory regions of the Th2 cytokine gene loci. The Bach2-Batf complex antagonizes the recruitment of the Batf-Irf4 complex to AP-1 motifs and suppresses Th2 cytokine production. Furthermore, we find that Bach2 regulates the Batf and Batf3 expressions via two distinct pathways. First, Bach2 suppresses the maintenance of the Batf and Batf3 expression through the inhibition of IL-4 production. Second, the Bach2-Batf complex directly binds to the Batf and Batf3 gene loci and reduces transcription by interfering with the Batf-Irf4 complex. These findings suggest that IL-4 and Batf form a positive feedback amplification loop to induce Th2 cell differentiation and the subsequent Th2-type immune response, and Bach2-Batf interactions are required to prevent an excessive Th2 response.

DOI： 10.1038/ncomms12596

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Gfi1 plays an important role in the development and maintenance of many hematopoietic linage cells. However, the impact of Gfi1-deficiency on the iNKT cell differentiation remains unclear. We herein demonstrate a critical role of Gfi1 in regulating the development of iNKT cell subsets. In the thymus of T cell-specific Gfi1-deficient mice, iNKT cells normally developed up to stage 2, while the number of stage 3 NK1.1(pos) iNKT cells was significantly reduced. Furthermore, CD4(pos) iNKT cells were selectively reduced in the peripheral organs of T cell-specific Gfi1-deficient mice. The alpha-GalCer-dependent production of IFN-gamma and Th2 cytokines, but not IL-17A, was severely reduced in T cell-specific Gfi1-deficient mice. In addition, a reduction of the alpha-GalCer-induced anti-tumor activity was observed in Gfi1-deficient mice. These findings demonstrate the important role of Gfi1 in regulating the development and function of NKT1- and NKT2-type iNKT cell subsets.

DOI： 10.1371/journal.pone.0157395

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

A transcriptional repressor Gfi1 promotes T helper type 2 (Th2) cell development and inhibits Th17 and inducible regulatory T-cell differentiation. However, the role of Gfi1 in regulating Th1 cell differentiation and the Th1-type immune response remains to be investigated. We herein demonstrate that Gfi1 inhibits the induction of the Th1 programme in activated CD4 T cells. The activated Gfi1-deficient CD4 T cells spontaneously develop into Th1 cells in an interleukin-12-and interferon-gamma-independent manner. The increase of Th1-type immune responses was confirmed in vivo in Gfi1-deficient mice using a murine model of nickel allergy and delayed-type hypersensitivity (DTH). The expression levels of Th1-related transcription factors were found to increase in Gfi1-deficient activated CD4 T cells. Tbx21, Eomes and Runx2 were identified as possible direct targets of Gfi1. Gfi1 binds to the Tbx21, Eomes and Runx2 gene loci and reduces the histone H3K4 methylation levels in part by modulating Lsd1 recruitment. Together, these findings demonstrate a novel regulatory role of Gfi1 in the regulation of the Th1-type immune response.

DOI： 10.1111/imm.12580

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The human artificial chromosome (HAC) vector is a promising tool to improve the problematic suppression and position effects of transgene expression frequently seen in transgenic cells and animals produced by conventional plasmid or viral vectors. We generated transgenic mice maintaining a single HAC vector carrying two genomic bacterial artificial chromosomes (BACs) from human HLA-DR loci (DRA and DRB1). Both transgenes on the HAC in transgenic mice exhibited tissue-specific expression in kidney, liver, lung, spleen, lymph node, bone marrow, and thymus cells in RT-PCR analysis. Stable functional expression of a cell surface HLA-DR marker from both transgenes, DRA and DRB1 on the HAC, was detected by flow cytometric analysis of splenocytes and maintained through at least eight filial generations. These results indicate that the de novo HAC system can allow us to manipulate multiple BAC transgenes with coordinated expression as a surface antigen through the generation of transgenic animals.

DOI： 10.1007/s00412-014-0488-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Stat3 signaling is essential for the induction of ROR gamma t and subsequent Th17 cell differentiation. However, the downstream targets of Stat3 for ROR gamma t expression remain largely unknown. We show here that a novel isoform of Sox5, named Sox5t, is induced in Th17 cells in a Stat3-dependent manner. In vivo, T cell-specific Sox5-deficient mice exhibit impaired Th17 cell differentiation and are resistant to experimental autoimmune encephalomyelitis and delayed-type hypersensitivity. Retrovirus-mediated induction of Sox5 together with c-Maf induces Th17 cell differentiation even in Stat3-deficient CD4(+) T cells but not in ROR gamma t-deficient CD4(+) T cells, indicating that Sox5 and c-Maf induce Th17 cell differentiation as downstream effectors of Stat3 and as upstream inducers of ROR gamma t. Moreover, Sox5 physically associates with c-Maf via the HMG domain of Sox5 and DNA-binding domain of c-Maf, and Sox5 together with c-Maf directly activates the promoter of ROR gamma t in CD4(+) T cells. Collectively, our results suggest that Sox5 and c-Maf cooperatively induce Th17 cell differentiation via the induction of ROR gamma t as downstream targets of Stat3.

DOI： 10.1084/jem.20130791

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Although CD4 T-cell senescence plays an important role in immunosenescence, the mechanism behind this process remains unclear. Here we show that T cell-specific Menin deficiency results in the premature senescence of CD4 T cells, which is accompanied by the senescence-associated secretory phenotype after antigenic stimulation and dysregulated cytokine production. Menin is required for the expansion and survival of antigen-stimulated CD4 T cells in vivo and acts by targeting Bach2, which is known to regulate immune homeostasis and cytokine production. Menin binds to the Bach2 locus and controls its expression through maintenance of histone acetylation. Menin binding at the Bach2 locus and the Bach2 expression are decreased in the senescent CD4 T cells. These findings reveal a critical role of the Menin-Bach2 pathway in regulating CD4 T-cell senescence and cytokine homeostasis, thus indicating the involvement of this pathway in the inhibition of immunosenescence.

DOI： 10.1038/ncomms4555

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Language：English   Publishing type：Research paper (scientific journal)  

IL-5 is a key cytokine that plays an important role in the development of pathological conditions in allergic inflammation. Identifying strategies to inhibit IL-5 production is important in order to establish new therapies for treating allergic inflammation. We found that SH-2251, a novel thioamide-related small compound, selectively inhibits the differentiation of IL-5-producing Th2 cells. SH-2251 inhibited the induction of active histone marks at the Il5 gene locus during Th2 cell differentiation. The recruitment of RNA polymerase II, and following expression of the Th2 cell-specific intergenic transcripts around the Il5 gene locus was also inhibited. Furthermore, Th2 cell-dependent airway inflammation in mice was suppressed by the oral administration of SH-2251. Gfi1, a transcriptional repressor, was identified as a downstream target molecule of SH-2251 using a DNA microarray analysis. The Gfi1 expression dramatically decreased in SH-2251-treated Th2 cells, and the SH-2251-mediated inhibition of IL-5-producing Th2 cell differentiation was restored by transduction of Gfi1. Therefore, our study unearthed SH-2251 as a novel therapeutic candidate for allergic inflammation that selectively inhibits active histone marks at the Il5 gene locus. © 2013 Suzuki et al.

DOI： 10.1371/journal.pone.0061785

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

IL-4 plays an important role in the induction of Th2 and Th9 cells, as well as in the inhibition of Th1 cell generation. We show that a combination of IL-4 and TGF-beta augments the development of Th1 cells that express CD103 (CD103(+) Th1 cells) if IFN-gamma is present. The T-box-containing transcription factor eomesodermin (Eomes) is preferentially expressed in CD103(+) Th1 cells and is involved in IFN-gamma production. The induction of T-bet during early T cell activation is essential for the formation of the active chromatin at both the Eomes and IFN-gamma gene loci. TGF-beta is required for the induction of Eomes and CD103, as well as the inhibition of Th2 cytokine expression. In addition, IL-4 induces Eomes transcription through activation of the Stat6-signaling pathway. IFN-gamma-producing CD103(+) Th1 cells are detected in the intraepithelial lymphocytes of normal mice, and their numbers significantly decrease in Tbet- and Stat6-deficient mice. To our knowledge, these results represent the first molecular mechanism of IL-4/TGF-beta-dependent augmentation of Th1 cell generation and raise the possibility that IL-4 and TGF-beta simultaneously enhance the Th1 cell-mediated immune responses under certain cytokine conditions. The Journal of Immunology, 2012, 188: 4846-4857.

DOI： 10.4049/jimmunol.1103799

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