Publications by authors named "Takuya Ohtani"

11 Publications

  • Page 1 of 1

Exploration of T-Cell Diversity Using Mass Cytometry.

Methods Mol Biol 2020 ;2111:1-20

Department of Systems Pharmacology and Translational Therapeutics, Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, USA.

T-cell diversity is multifactorial and includes variability in antigen specificity, differentiation, function, and cell-trafficking potential. Spectral overlap limits the ability of traditional flow cytometry to fully capture the diversity of T-cell subsets and function. The development of mass cytometry permits deep immunoprofiling of T-cell subsets, activation state, and function simultaneously from even small volumes of blood. This chapter describes our methods for mass cytometry and high-throughput data analysis of T cells in patient cohorts. We provide a pipeline that includes practical considerations when customizing a panel for mass cytometry. We also provide protocols for the conjugation and titration of metal-labeled antibodies (including two T-cell panels) and a staining procedure. Finally, with the aim to support translational science, we provide R scripts that contain a detailed workflow for initial evaluation of high-dimensional data generated from cohorts of patients.
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http://dx.doi.org/10.1007/978-1-0716-0266-9_1DOI Listing
January 2021

Common variable immunodeficiency-associated endotoxemia promotes early commitment to the T follicular lineage.

J Allergy Clin Immunol 2019 12 22;144(6):1660-1673. Epub 2019 Aug 22.

Division of Immunology and Allergy, Children's Hospital of Philadelphia, Philadelphia, Pa; Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pa; Institute for Immunology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pa. Electronic address:

Background: Although chiefly a B-lymphocyte disorder, several research groups have identified common variable immunodeficiency (CVID) subjects with numeric and/or functional T cell alterations. The causes, interrelationships, and consequences of CVID-associated CD4 T-cell derangements to hypogammaglobulinemia, autoantibody production, or both remain unclear.

Objective: We sought to determine how circulating CD4 T cells are altered in CVID subjects with autoimmune cytopenias (AICs; CVID+AIC) and the causes of these derangements.

Methods: Using hypothesis-generating, high-dimensional single-cell analyses, we created comprehensive phenotypic maps of circulating CD4 T cells. Differences between subject groups were confirmed in a large and genetically diverse cohort of CVID subjects (n = 69) by using flow cytometry, transcriptional profiling, multiplex cytokine/chemokine detection, and a suite of in vitro functional assays measuring naive T-cell differentiation, B-cell/T-cell cocultures, and regulatory T-cell suppression.

Results: Although CD4 T cell profiles from healthy donors and CVID subjects without AICs were virtually indistinguishable, T cells from CVID+AIC subjects exhibited follicular features as early as thymic egress. Follicular skewing correlated with IgA deficiency-associated endotoxemia and endotoxin-induced expression of activin A and inducible T-cell costimulator ligand. The resulting enlarged circulating follicular helper T-cell population from CVID+AIC subjects provided efficient help to receptive healthy donor B cells but not unresponsive CVID B cells. Despite this, circulating follicular helper T cells from CVID+AIC subjects exhibited aberrant transcriptional profiles and altered chemokine/cytokine receptor expression patterns that interfered with regulatory T-cell suppression assays and were associated with autoantibody production.

Conclusions: Endotoxemia is associated with early commitment to the follicular T-cell lineage in IgA-deficient CVID subjects, particularly those with AICs.
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http://dx.doi.org/10.1016/j.jaci.2019.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900457PMC
December 2019

PRMT5 Associates With the FOXP3 Homomer and When Disabled Enhances Targeted p185 Tumor Immunotherapy.

Front Immunol 2019 8;10:174. Epub 2019 Feb 8.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.

Regulatory T cells (Tregs) are a subpopulation of T cells that are specialized in suppressing immune responses. Here we show that the arginine methyl transferase protein PRMT5 can complex with FOXP3 transcription factors in Tregs. Mice with conditional knock out (cKO) of PRMT5 expression in Tregs develop severe scurfy-like autoimmunity. In these PRMT5 cKO mice, the spleen has reduced numbers of Tregs, but normal numbers of Tregs are found in the peripheral lymph nodes. These peripheral Tregs that lack PRMT5, however, display a limited suppressive function. Mass spectrometric analysis showed that FOXP3 can be di-methylated at positions R27, R51, and R146. A point mutation of Arginine (R) 51 to Lysine (K) led to defective suppressive functions in human CD4 T cells. Pharmacological inhibition of PRMT5 by DS-437 also reduced human Treg functions and inhibited the methylation of FOXP3. In addition, DS-437 significantly enhanced the anti-tumor effects of anti-erbB2/neu monoclonal antibody targeted therapy in Balb/c mice bearing CT26Her2 tumors by inhibiting Treg function and induction of tumor immunity. Controlling PRMT5 activity is a promising strategy for cancer therapy in situations where host immunity against tumors is attenuated in a FOXP3 dependent manner.
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http://dx.doi.org/10.3389/fimmu.2019.00174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375878PMC
February 2020

Epigenomic-Guided Mass Cytometry Profiling Reveals Disease-Specific Features of Exhausted CD8 T Cells.

Immunity 2018 05;48(5):1029-1045.e5

Department of Microbiology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA, USA; Institute for Immunology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA, USA; Parker Institute for Cancer Immunotherapy, University of Pennsylvania Perelman School Medicine, Philadelphia, PA, USA. Electronic address:

Exhausted CD8 T (Tex) cells are immunotherapy targets in chronic infection and cancer, but a comprehensive assessment of Tex cell diversity in human disease is lacking. Here, we developed a transcriptomic- and epigenetic-guided mass cytometry approach to define core exhaustion-specific genes and disease-induced changes in Tex cells in HIV and human cancer. Single-cell proteomic profiling identified 9 distinct Tex cell clusters using phenotypic, functional, transcription factor, and inhibitory receptor co-expression patterns. An exhaustion severity metric was developed and integrated with high-dimensional phenotypes to define Tex cell clusters that were present in healthy subjects, common across chronic infection and cancer or enriched in either disease, linked to disease severity, and changed with HIV therapy. Combinatorial patterns of immunotherapy targets on different Tex cell clusters were also defined. This approach and associated datasets present a resource for investigating human Tex cell biology, with implications for immune monitoring and immunomodulation in chronic infections, autoimmunity, and cancer.
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http://dx.doi.org/10.1016/j.immuni.2018.04.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010198PMC
May 2018

Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression patterns.

J Immunol Methods 2018 02 19;453:3-10. Epub 2017 Mar 19.

Department of Microbiology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania Perelman School Medicine, Philadelphia, PA 19104, USA. Electronic address:

The elimination of infected or tumor cells by direct lysis is a key T and NK cell effector function. T and NK cells can kill target cells by coordinated secretion of cytotoxic granules containing one or both pore-forming proteins, perforin and granulysin and combinations of granzyme (Gzm) family effector proteases (in humans: Gzm A, B, K, M and H). Understanding the pattern of expression of cytotoxic molecules and the relationship to different states of T and NK cells may have direct relevance for immune responses in autoimmunity, infectious disease and cancer. Approaches capable of simultaneously evaluating expression of multiple cytotoxic molecules with detailed information on T and NK differentiation state, however, remain limited. Here, we established a high dimensional mass cytometry approach to comprehensively interrogate single cell proteomic expression of cytotoxic programs and lymphocyte differentiation. This assay identified a coordinated expression pattern of cytotoxic molecules linked to CD8 T cell differentiation stages. Coordinated high expression of perforin, granulysin, Gzm A, Gzm B and Gzm M was associated with markers of late effector memory differentiation and expression of chemokine receptor CX3CR1. However, classical gating and dimensionality reduction approaches also identified other discordant patterns of cytotoxic molecule expression in CD8 T cells, including reduced perforin, but high Gzm A, Gzm K and Gzm M expression. When applied to non-CD8 T cells, this assay identified different patterns of cytotoxic molecule co-expression by CD56 versus CD56 defined NK cell developmental stages; in CD4 T cells, low expression of cytotoxic molecules was found mainly in TH1 phenotype cells, but not in Tregs or T follicular helper cells (TFH). Thus, this comprehensive, single cell, proteomic assessment of cytotoxic protein co-expression patterns demonstrates specialized cytotoxic programs in T cells and NK cells linked to their differentiation stages. Such comprehensive cytotoxic profiling may identify distinct patterns of cytotoxic potential relevant for specific infections, autoimmunity or tumor settings.
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http://dx.doi.org/10.1016/j.jim.2017.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605401PMC
February 2018

Trib2 Suppresses Tumor Initiation in Notch-Driven T-ALL.

PLoS One 2016 18;11(5):e0155408. Epub 2016 May 18.

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Institute of Medicine and Engineering, Institute for Immunology, Center for Personalized Diagnostics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States of America.

Trib2 is highly expressed in human T cell acute lymphoblastic leukemia (T-ALL) and is a direct transcriptional target of the oncogenic drivers Notch and TAL1. In human TAL1-driven T-ALL cell lines, Trib2 is proposed to function as an important survival factor, but there is limited information about the role of Trib2 in primary T-ALL. In this study, we investigated the role of Trib2 in the initiation and maintenance of Notch-dependent T-ALL. Trib2 had no effect on the growth and survival of murine T-ALL cell lines in vitro when expression was blocked by shRNAs. To test the function of Trib2 on leukemogenesis in vivo, we generated Trib2 knockout mice. Mice were born at the expected Mendelian frequencies without gross developmental anomalies. Adult mice did not develop pathology or shortened survival, and hematopoiesis, including T cell development, was unperturbed. Using a retroviral model of Notch-induced T-ALL, deletion of Trib2 unexpectedly decreased the latency and increased the penetrance of T-ALL development in vivo. Immunoblotting of primary murine T-ALL cells showed that the absence of Trib2 increased C/EBPα expression, a known regulator of cell proliferation, and did not alter AKT or ERK phosphorylation. Although Trib2 was suggested to be highly expressed in T-ALL, transcriptomic analysis of two independent T-ALL cohorts showed that low Trib2 expression correlated with the TLX1-expressing cortical mature T-ALL subtype, whereas high Trib2 expression correlated with the LYL1-expressing early immature T-ALL subtype. These data indicate that Trib2 has a complex role in the pathogenesis of Notch-driven T-ALL, which may vary between different T-ALL subtypes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155408PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871414PMC
July 2017

Pim-2 Kinase Influences Regulatory T Cell Function and Stability by Mediating Foxp3 Protein N-terminal Phosphorylation.

J Biol Chem 2015 Aug 18;290(33):20211-20. Epub 2015 May 18.

From the Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,

Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg cell development and function. Foxp3 function itself is directly regulated by multiple posttranslational modifications that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macromolecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here we identified a mechanism regulating Foxp3 level and activity that operates through discrete phosphorylation. The Pim-2 kinase can phosphorylate Foxp3, leading to decreased suppressive functions of Treg cells. The amino-terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to altered expression of proteins related to Treg cell functions and increased Treg cell lineage stability. Treg cell suppressive function can be up-regulated by either pharmacologically inhibiting Pim-2 kinase activity or by genetically knocking out Pim-2 in rodent Treg cells. Deficiency of Pim-2 activity increases murine host resistance to dextran sodium sulfate-induced colitis in vivo, and a Pim-2 small molecule kinase inhibitor also modified Treg cell functions. Our studies define a pathway for limiting the regulation of Foxp3 function because the Pim-2 kinase represents a potential therapeutic target for modulating the Treg cell suppressive activities in controlling immune responses.
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http://dx.doi.org/10.1074/jbc.M115.638221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536430PMC
August 2015

Dynamic interactions between TIP60 and p300 regulate FOXP3 function through a structural switch defined by a single lysine on TIP60.

Cell Rep 2014 Jun 15;7(5):1471-1480. Epub 2014 May 15.

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The human FOXP3 molecule is an oligomeric transcriptional factor able to mediate activities that characterize T regulatory cells, a class of lymphocytes central to the regulation of immune responses. The activity of FOXP3 is regulated at the posttranslational level, in part by two histone acetyltransferases (HATs): TIP60 and p300. TIP60 and p300 work cooperatively to regulate FOXP3 activity. Initially, p300 and TIP60 interactions lead to the activation of TIP60 and facilitate acetylation of K327 of TIP60, which functions as a molecular switch to allow TIP60 to change binding partners. Subsequently, p300 is released from this complex, and TIP60 interacts with and acetylates FOXP3. Maximal induction of FOXP3 activities is observed when both p300 and TIP60 are able to undergo cooperative interactions. Conditional knockout of TIP60 in Treg cells significantly decreases the Treg population in the peripheral immune organs, leading to a scurfy-like fatal autoimmune disease.
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http://dx.doi.org/10.1016/j.celrep.2014.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4064594PMC
June 2014

Notch regulation of early thymocyte development.

Semin Immunol 2010 Oct 13;22(5):261-9. Epub 2010 Jul 13.

The Department of Pathology & Laboratory Medicine and the Abramson Family Cancer Research Institute at the University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

Notch signaling plays multiple roles in T cell development. Following thymic entry, Notch signals are required to specify the T cell fate from a multipotent hematopoietic progenitor. At subsequent steps in early T cell development, Notch provides important differentiation, survival, proliferation and metabolic signals. This review focuses on the multiple functions of Notch in early T cell development, from T cell specification in the thymus through beta selection.
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http://dx.doi.org/10.1016/j.smim.2010.04.015DOI Listing
October 2010

Pre-TCR signaling inactivates Notch1 transcription by antagonizing E2A.

Genes Dev 2009 Jul;23(14):1665-76

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Precise control of the timing and magnitude of Notch signaling is essential for the normal development of many tissues, but the feedback loops that regulate Notch are poorly understood. Developing T cells provide an excellent context to address this issue. Notch1 signals initiate T-cell development and increase in intensity during maturation of early T-cell progenitors (ETP) to the DN3 stage. As DN3 cells undergo beta-selection, during which cells expressing functionally rearranged TCRbeta proliferate and differentiate into CD4(+)CD8(+) progeny, Notch1 signaling is abruptly down-regulated. In this report, we investigate the mechanisms that control Notch1 expression during thymopoiesis. We show that Notch1 and E2A directly regulate Notch1 transcription in pre-beta-selected thymocytes. Following successful beta-selection, pre-TCR signaling rapidly inhibits Notch1 transcription via signals that up-regulate Id3, an E2A inhibitor. Consistent with a regulatory role for Id3 in Notch1 down-regulation, post-beta-selected Id3-deficient thymocytes maintain Notch1 transcription, whereas enforced Id3 expression decreases Notch1 expression and abrogates Notch1-dependent T-cell survival. These data provide new insights into Notch1 regulation in T-cell progenitors and reveal a direct link between pre-TCR signaling and Notch1 expression during thymocyte development. Our findings also suggest new strategies for inhibiting Notch1 signaling in pathologic conditions.
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http://dx.doi.org/10.1101/gad.1793709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2714710PMC
July 2009

A point mutation of Tyr-759 in interleukin 6 family cytokine receptor subunit gp130 causes autoimmune arthritis.

J Exp Med 2002 Oct;196(7):979-90

Department of Molecular Oncology (C7), Graduate School of Medicine, Osaka University, Suita, Japan.

We generated a mouse line in which the src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-2 binding site of gp130, tyrosine 759, was mutated to phenylalanine (gp130(F759/F759)). The gp130(F759/F759) mice developed rheumatoid arthritis (RA)-like joint disease. The disease was accompanied by autoantibody production and accumulated memory/activated T cells and myeloid cells. Before the disease onset, the T cells were hyperresponsive and thymic selection and peripheral clonal deletion were impaired. The inhibitory effect of IL-6 on Fas ligand expression during activation-induced cell death (AICD) was augmented in gp130(F759/F759) T cells in a manner dependent on the tyrosine residues of gp130 required for signal transducer and activator of transcription 3 activation. Finally, we showed that disease development was dependent on lymphocytes. These results provide evidence that a point mutation of a cytokine receptor has the potential to induce autoimmune disease.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2194024PMC
http://dx.doi.org/10.1084/jem.20020619DOI Listing
October 2002