Publications by authors named "Hyota Takamatsu"

57 Publications

IL-33 Induces Sema4A Expression in Dendritic Cells and Exerts Antitumor Immunity.

J Immunol 2021 09 11;207(5):1456-1467. Epub 2021 Aug 11.

Laboratory of Immunopathology, Immunology Frontier Research Center, World Premier International Research Center, Osaka University, Suita, Osaka, Japan;

Cancer immunotherapy has shown great promise as a new standard therapeutic strategy against cancer. However, the response rate and survival benefit remain unsatisfactory because most current approaches, such as the use of immune checkpoint inhibitors, depend on spontaneous antitumor immune responses. One possibility for improving the efficacy of immunotherapy is to promote antitumor immunity using adjuvants or specific cytokines actively. IL-33 has been a candidate for such cytokine therapies, but it remains unclear how and in which situations IL-33 exerts antitumor immune effects. In this study, we demonstrate the potent antitumor effects of IL-33 using syngeneic mouse models, which included marked inhibition of tumor growth and upregulation of IFN-γ production by tumor-infiltrating CD8 T cells. Of note, IL-33 induced dendritic cells to express semaphorin 4A (Sema4A), and the absence of Sema4A abolished the antitumor activity of IL-33, indicating that Sema4A is intrinsically required for the antitumor effects of IL-33 in mice. Collectively, these results not only present IL-33 and Sema4A as potential therapeutic targets but also shed light on the potential use of Sema4A as a biomarker for dendritic cell activation status, which has great value in various fields of cancer research, including vaccine development.
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http://dx.doi.org/10.4049/jimmunol.2100076DOI Listing
September 2021

A design and optimization of a high throughput valve based microfluidic device for single cell compartmentalization and analysis.

Sci Rep 2021 Jun 21;11(1):12995. Epub 2021 Jun 21.

Graduate School of Engineering, Osaka University, Suita, Osaka, 565-0871, Japan.

The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. However, this technique is throughput limited by the number of compartments in the array. Thus, one big challenge lies in increasing the number of microvalves to several thousand that can be actuated in the microfluidic device to confine enzymes and substrates in picoliter volumes. This work explores the design and optimizations done on a microfluidic platform to achieve high-throughput single cell compartmentalization as applied to single-cell enzymatic assay for protein expression quantification. Design modeling through COMSOL Multiphysics was utilized to determine the circular microvalve's optimized parameters, which can close thousands of microchambers in an array at lower sealing pressure. Multiphysical modeling results demonstrated the relationships of geometry, valve dimensions, and sealing pressure, which were applied in the fabrication of a microfluidic device comprising of up to 5000 hydrodynamic traps and corresponding microvalves. Comparing the effects of geometry, actuation media and fabrication technique, a sealing pressure as low as 0.04 MPa was achieved. Applying to single cell enzymatic assay, variations in granzyme B activity in Jurkat and human PBMC cells were observed. Improvement in the microfluidic chip's throughput is significant in single cell analysis applications, especially in drug discovery and treatment personalization.
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http://dx.doi.org/10.1038/s41598-021-92472-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217553PMC
June 2021

The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II.

Nat Commun 2021 06 7;12(1):3333. Epub 2021 Jun 7.

Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

Lysosomes are involved in nutrient sensing via the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 is tethered to lysosomes by the Ragulator complex, a heteropentamer in which Lamtor1 wraps around Lamtor2-5. Although the Ragulator complex is required for cell migration, the mechanisms by which it participates in cell motility remain unknown. Here, we show that lysosomes move to the uropod in motile cells, providing the platform where Lamtor1 interacts with the myosin phosphatase Rho-interacting protein (MPRIP) independently of mTORC1 and interferes with the interaction between MPRIP and MYPT1, a subunit of myosin light chain phosphatase (MLCP), thereby increasing myosin II-mediated actomyosin contraction. Additionally, formation of the complete Ragulator complex is required for leukocyte migration and pathophysiological immune responses. Together, our findings demonstrate that the lysosomal Ragulator complex plays an essential role in leukocyte migration by activating myosin II through interacting with MPRIP.
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http://dx.doi.org/10.1038/s41467-021-23654-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184920PMC
June 2021

PlexinA1 deficiency in BALB/cAJ mice leads to excessive self-grooming and reduced prepulse inhibition.

IBRO Rep 2020 Dec 22;9:276-289. Epub 2020 Oct 22.

Department of Physiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan.

PlexinA1 (PlxnA1) is a transmembrane receptor for semaphorins, a large family of proteins that act as axonal guidance cues during nervous system development. However, there are limited studies on PlxnA1 function in neurobehavior. The present study examined if PlxnA1 deficiency leads to behavioral abnormalities in BALB/cAJ mice. PlxnA1 knockout (KO) mice were generated by homologous recombination and compared to wild type (WT) littermates on a comprehensive battery of behavioral tests, including open field assessment of spontaneous ambulation, state anxiety, and grooming, home cage grooming, the wire hang test of muscle strength, motor coordination on the rotarod task, working memory on the Y maze alternation task, cued and contextual fear conditioning, anxiety on the elevated plus maze, sociability to intruders, and sensory processing as measured by prepulse inhibition (PPI). Measures of motor performance, working memory, fear memory, and sociability did not differ significantly between genotypes, while PlxnA1 KO mice displayed excessive self-grooming, impaired PPI, and slightly lower anxiety. These results suggest a crucial role for PlxnA1 in the development and function of brain regions controlling self-grooming and sensory gating. PlxnA1 KO mice may be a valuable model to investigate the repetitive behaviors and information processing deficits characteristic of many neurodevelopmental and psychiatric disorders.
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http://dx.doi.org/10.1016/j.ibror.2020.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607060PMC
December 2020

The future of microfluidics in immune checkpoint blockade.

Cancer Gene Ther 2021 Sep 27;28(9):895-910. Epub 2020 Oct 27.

Graduate School of Engineering, Osaka University, Suita, Osaka, 565-0871, Japan.

Recent advances in microfluidic techniques have enabled researchers to study sensitivities to immune checkpoint therapy, to determine patients' response to particular antibody treatment. Utilization of this technology is helpful in antibody discovery and in the design of personalized medicine. A variety of microfluidic approaches can provide several functions in processes such as immunologic, genomic, and/or transcriptomic analysis with the aim of improving the efficacy and coverage of immunotherapy, particularly immune checkpoint blockade (ICB). To achieve this requires researchers to overcome the challenges in the current state of the technology. This review looks into the advancements in microfluidic technologies applied to researches on immune checkpoint blockade treatment and its potential shift from proof-of-principle stage to clinical application.
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http://dx.doi.org/10.1038/s41417-020-00248-7DOI Listing
September 2021

Recovery from prolonged thrombocytopenia in patients with TAFRO syndrome: case series and literature review.

Mod Rheumatol Case Rep 2020 07 3;4(2):302-309. Epub 2020 Feb 3.

Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Osaka, Japan.

TAFRO syndrome is a newly proposed disease that is characterised by thrombocytopenia, anasarca, fever, reticulin fibrosis (or renal dysfunction), and organomegaly. Generally, high doses of corticosteroids are recommended for the initial treatment of TAFRO syndrome; however, some patients experience prolonged refractory thrombocytopenia after initiating such therapies. If corticosteroid treatment alone is ineffective, additional immunosuppressive therapies such as cyclosporine A are recommended. Since long-term use of immunosuppressive therapies with TAFRO syndrome sometimes causes serious infection, it is important to recognise the time to recovery from thrombocytopenia. In this study, we investigated how long it took to recover from thrombocytopenia, to aid clinicians in decision-making regarding the need to strengthen treatment for prolonged thrombocytopenia. Here, we describe three of our patients with TAFRO syndrome exhibiting prolonged thrombocytopenia. We also investigated the median period to recovery from this complication (defined as the time to increase the platelet count above 50,000/µL) after the initiation of high-dose corticosteroid treatment in our 3 cases and 38 peer-reviewed cases. We found that it took our patients 61 days to recover from thrombocytopenia; in comparison, our investigation of the 38 peer-reviewed case reports revealed a median recovery time of 47.5 days among previously reported patients. We showed the time to recovery from thrombocytopenia in patients with TAFRO syndrome for the first time. Our findings ought to be useful for decision-making among clinicians regarding the administration of other immunosuppressive treatments in addition to corticosteroid.
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http://dx.doi.org/10.1080/24725625.2020.1717747DOI Listing
July 2020

A case of SAPHO syndrome with the lesions limited to the skull.

Rheumatol Adv Pract 2020 14;4(2):rkaa034. Epub 2020 Jul 14.

Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine.

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http://dx.doi.org/10.1093/rap/rkaa034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494080PMC
July 2020

Pathological and therapeutic implications of eosinophil-derived semaphorin 4D in eosinophilic chronic rhinosinusitis.

J Allergy Clin Immunol 2020 03 5;145(3):843-854.e4. Epub 2020 Feb 5.

Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita City, Osaka, Japan; Laboratory of Immunopathology, World Premier International Immunology Frontier Research Center, Suita City, Osaka, Japan; Institute for Open and Transdisciplinary Research Initiatives, Suita City, Osaka, Japan. Electronic address:

Background: Eosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis. Clinical markers for ECRS disease activity and treatment strategies have not been sufficiently established. Although semaphorins are originally identified as neuronal guidance factors, it is becoming clear that they play key roles in immune regulation and inflammatory diseases.

Objective: We sought to investigate the pathological functions and therapeutic potential of semaphorin 4D (SEMA4D) in ECRS.

Methods: Serum soluble SEMA4D levels in patients with paranasal sinus diseases were measured by ELISA. The expression of SEMA4D in blood cells and nasal polyp tissues was assessed by flow cytometry and immunohistochemistry, respectively. Generation of soluble SEMA4D was evaluated in matrix metalloproteinase-treated eosinophils. Endothelial cells were stimulated with recombinant SEMA4D, followed by eosinophil transendothelial migration assays. Allergic chronic rhinosinusitis was induced in mice using Aspergillus protease with ovalbumin. The efficacy of treatment with anti-SEMA4D antibody was evaluated histologically and by nasal lavage fluid analysis.

Results: Serum soluble SEMA4D levels were elevated in patients with ECRS and positively correlated with disease severity. Tissue-infiltrated eosinophils in nasal polyps from patients with ECRS stained strongly with anti-SEMA4D antibody. Cell surface expression of SEMA4D on eosinophils from patients with ECRS was reduced, which was due to matrix metalloproteinase-9-mediated cleavage of membrane SEMA4D. Soluble SEMA4D induced eosinophil transendothelial migration. Treatment with anti-SEMA4D antibody ameliorated eosinophilic infiltration in sinus tissues and nasal lavage fluid in the ECRS animal model.

Conclusions: Eosinophil-derived SEMA4D aggravates ECRS. Levels of serum SEMA4D reflect disease severity, and anti-SEMA4D antibody has therapeutic potential as a treatment for ECRS.
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http://dx.doi.org/10.1016/j.jaci.2019.12.893DOI Listing
March 2020

Real-Time Monitoring and Detection of Single-Cell Level Cytokine Secretion Using LSPR Technology.

Micromachines (Basel) 2020 Jan 19;11(1). Epub 2020 Jan 19.

Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

Cytokine secretion researches have been a main focus of studies among the scientists in the recent decades for its outstanding contribution to clinical diagnostics. Localized surface plasmon resonance (LSPR) technology is one of the conventional methods utilized to analyze these issues, as it could provide fast, label-free and real-time monitoring of biomolecule binding events. However, numerous LSPR-based biosensors in the past are usually utilized to monitor the average performance of cell groups rather than single cells. Meanwhile, the complicated sensor structures will lead to the fabrication and economic budget problems. Thus, in this paper, we report a simple synergistic integration of the cell trapping of microwell chip and gold-capped nanopillar-structured cyclo-olefin-polymer (COP) film for single cell level Interleukin 6 (IL-6) detection. Here, in-situ cytokine secreted from the trapped cell can be directly observed and analyzed through the peak red-shift in the transmittance spectrum. The fabricated device also shows the potential to conduct the real-time monitoring which would greatly help us identify the viability and biological variation of the tested single cell.
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http://dx.doi.org/10.3390/mi11010107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019717PMC
January 2020

Single Cell Analysis of Neutrophils NETs by Microscopic LSPR Imaging System.

Micromachines (Basel) 2019 Dec 31;11(1). Epub 2019 Dec 31.

Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan.

A simple microengraving cell monitoring method for neutrophil extracellular traps (NETs) released from single neutrophils has been realized using a polydimethylsiloxane (PDMS) microwell array (MWA) sheet on a plasmon chip platform. An imbalance between NETs formation and the succeeding degradation (NETosis) are considered associated with autoimmune disease and its pathogenesis. Thus, an alternative platform that can conduct monitoring of this activity on single cell level at minimum cost but with great sensitivity is greatly desired. The developed MWA plasmon chips allow single cell isolation of neutrophils from 150 µL suspension (6.0 × 10 cells/mL) with an efficiency of 36.3%; 105 microwells with single cell condition. To demonstrate the utility of the chip, trapped cells were incubated between 2 to 4 h after introducing with 100 nM phorbol 12-myristate 13-acetate (PMA) before measurement. Under observation using a hyperspectral imaging system that allows high-throughput screening, the neutrophils stimulated by PMA solution show a significant release of fibrils and NETs after 4 h, with observed maximum areas between 314-758 µm. An average absorption peak wavelength shows a redshift of Δλ = 1.5 nm as neutrophils release NETs.
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http://dx.doi.org/10.3390/mi11010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019790PMC
December 2019

A Microfluidic Platform for Single Cell Fluorometric Granzyme B Profiling.

Theranostics 2020 1;10(1):123-132. Epub 2020 Jan 1.

Graduate School of Engineering, Osaka University, Suita, Osaka, 565-0871, JAPAN.

Granzyme B (GrB) is an essential cytotoxic effector in cancer immunotherapy as it can be a potential biomarker to predict the efficacy of immunotherapies including checkpoint inhibitors. Monitoring the Granzyme B activity in cells would help determine a patient's clinical response to treatment and lead to better treatment strategies by preventing administration of ineffective therapies and avoid adverse events resulting in a delay in subsequent treatment. : A microfluidic device with hydrodynamic traps and pneumatic valving system was fabricated using photo and soft lithography. Single cell Granzyme B (GrB) activity was detected and measured fluorometrically using a commercial assay kit with a peptide substrate containing GrB recognition sequence (Ac-IEPD-AFC) and AFC (7-Amino-4-trifluoromethylcoumarin) label. Fluorescence was observed and measured using a confocal microscope with CSU-W1 scanner unit and CCD camera as well as an inverted microscope with photodetector. Model cells (NK-92, GrB-transduced Jurkat, and THP1 cells) and human PBMCs from healthy donor and lung cancer patients including an anti-PD-1 antibody treated patient were profiled of its GrB activity as proof of concept. : GrB expression from the model cells was found to be markedly different. NK-92 cells were found to have higher GrB activity than the GrB-transduced Jurkat cells. THP-1 was found to have relatively no significant activity. A marked increase in GrB expression was also observed in anti-PD-1 treated lung cancer patient sample in comparison to PBMC from a healthy donor. TCR+ Ig-G4+ PBMC cells were found to have high activity which signifies a clear response to PD-1 blockade. : As proof of concept, we have shown the capability of a microfluidic platform to measure GrB production through a single cell enzymatic activity assay. Our platform might be a promising tool for evaluating the sensitivity of immunotherapies and identifying specific T cell subset responsible for the anti-tumor response.
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http://dx.doi.org/10.7150/thno.37728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929635PMC
April 2021

PlexinA1 is crucial for the midline crossing of callosal axons during corpus callosum development in BALB/cAJ mice.

PLoS One 2019 20;14(8):e0221440. Epub 2019 Aug 20.

Department of Physiology, Faculty of Pharmacy, Meijo University, Nagoya, Japan.

The corpus callosum (CC) is the biggest commissure that links cerebral hemispheres. Guidepost structures develop in the cortical midline during CC development and express axon guidance molecules that instruct neurons regarding the proper direction of axonal elongation toward and across the cortical midline. Neuropilin-1 (Npn1), a high affinity receptor for class 3 semaphorins (Sema3s) localized on cingulate pioneering axons, plays a crucial role in axon guidance to the midline through interactions with Sema3s. However, it remains unclear which type of Plexin is a component of Sema3 holoreceptors with Npn1 during the guidance of cingulate pioneering axons. To address the role of PlexinA1 in CC development, we examined with immunohistochemistry the localization of PlexinA1, Npn1, and Sema3s using embryonic brains from wild-type (WT) and PlexinA1-deficient (PlexinA1 knock-out (KO)) mice with a BALB/cAJ background. The immunohistochemistry confirmed the expression of PlexinA1 in callosal axons derived from the cingulate and neocortex of the WT mice on embryonic day 17.5 (E17.5) but not in the PlexinA1 KO mice. To examine the role of PlexinA1 in the navigation of callosal axons, the extension of callosal axons toward and across the midline was traced in brains of WT and PlexinA1 KO mice at E17.5. As a result, callosal axons in the PlexinA1 KO brains had a significantly lower incidence of midline crossing at E17.5 compared with the WT brains. To further examine the role of PlexinA1 in CC development, the CC phenotype was examined in PlexinA1 KO mice at postnatal day 0.5 (P0.5). Most of the PlexinA1 KO mice at P0.5 showed agenesis of the CC. These results indicate the crucial involvement of PlexinA1 in the midline crossing of callosal axons during CC development in BALB/cAJ mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221440PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701775PMC
March 2020

Response to: 'Blood plasma versus serum: which is right for sampling circulating membrane microvesicles in human subjects?' by Liu .

Ann Rheum Dis 2020 06 16;79(6):e74. Epub 2019 May 16.

Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita City, Japan.

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http://dx.doi.org/10.1136/annrheumdis-2019-215540DOI Listing
June 2020

Semaphorin 7A promotes EGFR-TKI resistance in EGFR mutant lung adenocarcinoma cells.

JCI Insight 2018 12 20;3(24). Epub 2018 Dec 20.

Department of Thoracic Oncology and.

Although responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) are initially positive, 30%-40% of patients with EGFR-mutant tumors do not respond well to EGFR-TKIs, and most lung cancer patients harboring EGFR mutations experience relapse with resistance. Therefore, it is necessary to identify not only the mechanisms underlying EGFR-TKI resistance, but also potentially novel therapeutic targets and/or predictive biomarkers for EGFR-mutant lung adenocarcinoma. We found that the GPI-anchored protein semaphorin 7A (SEMA7A) is highly induced by the EGFR pathway, via mTOR signaling, and that expression levels of SEMA7A in human lung adenocarcinoma specimens were correlated with mTOR activation. Investigations using cell culture and animal models demonstrated that loss or overexpression of SEMA7A made cells less or more resistant to EGFR-TKIs, respectively. The resistance was due to the inhibition of apoptosis by aberrant activation of ERK. The ERK signal was suppressed by knockdown of integrin β1 (ITGB1). Furthermore, in patients with EGFR mutant tumors, higher SEMA7A expression in clinical samples predicted poorer response to EGFR-TKI treatment. Collectively, these data show that the SEMA7A-ITGB1 axis plays pivotal roles in EGFR-TKI resistance mediated by ERK activation and apoptosis inhibition. Moreover, our results reveal the potential utility of SEMA7A not only as a predictive biomarker, but also as a potentially novel therapeutic target in EGFR-mutant lung adenocarcinoma.
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http://dx.doi.org/10.1172/jci.insight.123093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338389PMC
December 2018

Response to: 'Some concerns from Turkey' by Bilgin .

Ann Rheum Dis 2020 02 8;79(2):e16. Epub 2018 Dec 8.

Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan

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http://dx.doi.org/10.1136/annrheumdis-2018-214776DOI Listing
February 2020

Response to: 'Tocilizumab in patients with adult-onset Still's disease refractory to glucocorticoid treatment' by Lee.

Ann Rheum Dis 2019 12 10;78(12):e134. Epub 2018 Nov 10.

Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan.

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http://dx.doi.org/10.1136/annrheumdis-2018-214653DOI Listing
December 2019

SEMA4A promotes eosinophil survival and contributes to eosinophil-mediated allergic diseases.

Allergol Int 2019 Apr 19;68(2):274-276. Epub 2018 Oct 19.

Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Osaka University Graduate School of Medicine, Osaka, Japan; Laboratory of Immunopathology, World Premier International Immunology Frontier Research Center, Osaka, Japan; Integrated Frontier Research for Medical Science Division, The Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka, Japan.

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http://dx.doi.org/10.1016/j.alit.2018.10.001DOI Listing
April 2019

Clinical implications of monitoring nivolumab immunokinetics in non-small cell lung cancer patients.

JCI Insight 2018 10 4;3(19). Epub 2018 Oct 4.

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.

Background: The PD-1-blocking antibody nivolumab persists in patients several weeks after the last infusion. However, no study has systematically evaluated the maximum duration that the antibody persists on T cells or the association between this duration and residual therapeutic efficacy or potential adverse events.

Methods: To define the duration of binding and residual efficacy of nivolumab after discontinuation, we developed a simplified strategy for T cell monitoring and used it to analyze T cells from peripheral blood from 11 non-small cell lung cancer patients previously treated with nivolumab. To determine the suitability of our method for other applications, we compared transcriptome profiles between nivolumab-bound and nivolumab-unbound CD8 T cells. We also applied T cell monitoring in 2 nivolumab-treated patients who developed progressive lung tumors during long-term follow-up.

Results: Prolonged nivolumab binding was detected more than 20 weeks after the last infusion, regardless of the total number of nivolumab infusions (2-15 doses) or type of subsequent treatment, in 9 of the 11 cases in which long-term monitoring was possible. Ki-67 positivity, a proliferation marker, in T cells decreased in patients with progressive disease. Transcriptome profiling identified the signals regulating activation of nivolumab-bound T cells, which may contribute to nivolumab resistance. In 2 patients who restarted nivolumab, T cell proliferation markers exhibited the opposite trend and correlated with clinical response.

Conclusions: Although only a few samples were analyzed, our strategy of monitoring both nivolumab binding and Ki-67 in T cells might help determine residual efficacy under various types of concurrent or subsequent treatment.

Trial Registration: University Hospital Medical Information Network Clinical Trials Registry, UMIN000024623.

Funding: This work was supported by Japan Society for the Promotion of Science KAKENHI (JP17K16045, JP18H05282, and JP15K09220), Japan Agency for Medical Research and Development (JP17cm0106310, JP18cm0106335 and JP18cm059042), and Core Research for Evolutional Science and Technology (JPMJCR16G2).
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http://dx.doi.org/10.1172/jci.insight.59125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237460PMC
October 2018

Tocilizumab in patients with adult-onset still's disease refractory to glucocorticoid treatment: a randomised, double-blind, placebo-controlled phase III trial.

Ann Rheum Dis 2018 12 2;77(12):1720-1729. Epub 2018 Oct 2.

Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan

Objective: To evaluate the efficacy and safety of tocilizumab, an interleukin-6 receptor antibody, in patients with adult-onset Still's disease.

Methods: In this double-blind, randomised, placebo-controlled phase III trial, 27 patients with adult-onset Still's disease refractory to glucocorticoids were randomised to tocilizumab at a dose of 8 mg/kg or placebo given intravenously every 2 weeks during the 12-week, double-blind phase. Patients received open-label tocilizumab for 40 weeks subsequently. The primary outcome was American College of Rheumatology (ACR) 50 response at week 4. The secondary outcomes included ACR 20/50/70, systemic feature score, glucocorticoid dose and adverse events at each point.

Results: In the full analysis set, ACR50 response at week 4 was achieved in 61.5% (95% CI 31.6 to 86.1) in the tocilizumab group and 30.8% (95% CI 9.1 to 61.4) in the placebo group (p=0.24). The least squares means for change in systemic feature score at week 12 were -4.1 in the tocilizumab group and -2.3 in the placebo group (p=0.003). The dose of glucocorticoids at week 12 decreased by 46.2% in the tocilizumab group and 21.0% in the placebo group (p=0.017). At week 52, the rates of ACR20, ACR50 and ACR70 were 84.6%, 84.6% and 61.5%, respectively, in both groups. Serious adverse events in all participants who received one dose of tocilizumab were infections, aseptic necrosis in the hips, exacerbation of adult-onset Still's disease, drug eruption and anaphylactic shock.

Conclusion: The study suggests that tocilizumab is effective in adult-onset Still's disease, although the primary endpoint was not met and solid conclusion was not drawn.
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http://dx.doi.org/10.1136/annrheumdis-2018-213920DOI Listing
December 2018

Eosinophil-derived neurotoxin enhances airway remodeling in eosinophilic chronic rhinosinusitis and correlates with disease severity.

Int Immunol 2019 02;31(1):33-40

Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Yamadaoka, Suita, Osaka, Japan.

Eosinophilic chronic rhinosinusitis (ECRS) is a subtype of chronic rhinosinusitis (CRS) that is characterized by intractable nasal polyp formation. Eosinophil-derived neurotoxin (EDN) is an eosinophil granule protein that is closely related to allergic inflammation, but the pathological implications of EDN in ECRS remain unknown. In this study, we evaluated the function of EDN in ECRS pathogenesis and assessed its potential as a disease activity marker. Serum EDN levels were significantly higher in patients with ECRS than in those with other nasal and paranasal diseases, and were positively correlated with clinical disease activity. Production of EDN from isolated human eosinophils was induced by stimulation with IL-5 in vitro. Human nasal epithelial cells were stimulated with EDN, and the resultant changes in gene expression were detected by RNA sequencing. Pathway analysis revealed that the major canonical pathway affected by EDN stimulation was 'regulation of the epithelial-mesenchymal transition pathway'; the only gene in this pathway to be up-regulated was matrix metalloproteinase 9 (MMP-9). Consistent with this, immunostaining analysis revealed intense staining of both EDN and MMP-9 in nasal polyps from patients with ECRS. In conclusion, our data demonstrate that serum EDN level is a useful marker for the evaluation of ECRS severity. Furthermore, EDN induces production of MMP-9 from the nasal epithelium, which may be involved in the pathogenesis of ECRS.
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http://dx.doi.org/10.1093/intimm/dxy061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6364622PMC
February 2019

Apoptosis-derived membrane vesicles drive the cGAS-STING pathway and enhance type I IFN production in systemic lupus erythematosus.

Ann Rheum Dis 2018 10 26;77(10):1507-1515. Epub 2018 Jun 26.

Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Osaka, Japan.

Objective: Despite the importance of type I interferon (IFN-I) in systemic lupus erythematosus (SLE) pathogenesis, the mechanisms of IFN-I production have not been fully elucidated. Recognition of nucleic acids by DNA sensors induces IFN-I and interferon-stimulated genes (ISGs), but the involvement of cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) and stimulator of interferon genes (STING) in SLE remains unclear. We studied the role of the cGAS-STING pathway in the IFN-I-producing cascade driven by SLE serum.

Methods: We collected sera from patients with SLE (n=64), patients with other autoimmune diseases (n=31) and healthy controls (n=35), and assayed them using a cell-based reporter system that enables highly sensitive detection of IFN-I and ISG-inducing activity. We used Toll-like receptor-specific reporter cells and reporter cells harbouring knockouts of cGAS, STING and IFNAR2 to evaluate signalling pathway-dependent ISG induction.

Results: IFN-I bioactivity and ISG-inducing activities of serum were higher in patients with SLE than in patients with other autoimmune diseases or healthy controls. ISG-inducing activity of SLE sera was significantly reduced in STING-knockout reporter cells, and STING-dependent ISG-inducing activity correlated with disease activity. Double-stranded DNA levels were elevated in SLE. Apoptosis-derived membrane vesicles (AdMVs) from SLE sera had high ISG-inducing activity, which was diminished in cGAS-knockout or STING-knockout reporter cells.

Conclusions: AdMVs in SLE serum induce IFN-I production through activation of the cGAS-STING pathway. Thus, blockade of the cGAS-STING axis represents a promising therapeutic target for SLE. Moreover, our cell-based reporter system may be useful for stratifying patients with SLE with high ISG-inducing activity.
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http://dx.doi.org/10.1136/annrheumdis-2018-212988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6161667PMC
October 2018

Semaphorin 6D reverse signaling controls macrophage lipid metabolism and anti-inflammatory polarization.

Nat Immunol 2018 06 18;19(6):561-570. Epub 2018 May 18.

Department of Immunopathology, Immunology Frontier Research Center, Osaka University, Suita City, Osaka, Japan.

Polarization of macrophages into pro-inflammatory or anti-inflammatory states has distinct metabolic requirements, with mechanistic target of rapamycin (mTOR) kinase signaling playing a critical role. However, it remains unclear how mTOR regulates metabolic status to promote polarization of these cells. Here we show that an mTOR-Semaphorin 6D (Sema6D)-Peroxisome proliferator receptor γ (PPARγ) axis plays critical roles in macrophage polarization. Inhibition of mTOR or loss of Sema6D blocked anti-inflammatory macrophage polarization, concomitant with severe impairments in PPARγ expression, uptake of fatty acids, and lipid metabolic reprogramming. Macrophage expression of the receptor Plexin-A4 is responsible for Sema6D-mediated anti-inflammatory polarization. We found that a tyrosine kinase, c-Abl, which associates with the cytoplasmic region of Sema6D, is required for PPARγ expression. Furthermore, Sema6D is important for generation of intestinal resident CX3CR1 macrophages and prevents development of colitis. Collectively, these findings highlight crucial roles for Sema6D reverse signaling in macrophage polarization, coupling immunity, and metabolism via PPARγ.
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http://dx.doi.org/10.1038/s41590-018-0108-0DOI Listing
June 2018

Lysosomal Protein Lamtor1 Controls Innate Immune Responses via Nuclear Translocation of Transcription Factor EB.

J Immunol 2018 06 23;200(11):3790-3800. Epub 2018 Apr 23.

Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan;

Amino acid metabolism plays important roles in innate immune cells, including macrophages. Recently, we reported that a lysosomal adaptor protein, Lamtor1, which serves as the scaffold for amino acid-activated mechanistic target of rapamycin complex 1 (mTORC1), is critical for the polarization of M2 macrophages. However, little is known about how Lamtor1 affects the inflammatory responses that are triggered by the stimuli for TLRs. In this article, we show that Lamtor1 controls innate immune responses by regulating the phosphorylation and nuclear translocation of transcription factor EB (TFEB), which has been known as the master regulator for lysosome and autophagosome biogenesis. Furthermore, we show that nuclear translocation of TFEB occurs in alveolar macrophages of myeloid-specific Lamtor1 conditional knockout mice and that these mice are hypersensitive to intratracheal administration of LPS and bleomycin. Our observation clarified that the amino acid-sensing pathway consisting of Lamtor1, mTORC1, and TFEB is involved in the regulation of innate immune responses.
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http://dx.doi.org/10.4049/jimmunol.1701283DOI Listing
June 2018

Lamtor1 Is Critically Required for CD4 T Cell Proliferation and Regulatory T Cell Suppressive Function.

J Immunol 2017 09 2;199(6):2008-2019. Epub 2017 Aug 2.

Department of Immunopathology, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan;

Mechanistic target of rapamycin complex (mTORC)1 integrates intracellular sufficiency of nutrients and regulates various cellular functions. Previous studies using mice with conditional knockout of mTORC1 component proteins (i.e., mTOR, Raptor, and Rheb) gave conflicting results on the roles of mTORC1 in CD4 T cells. Lamtor1 is the protein that is required for amino acid sensing and activation of mTORC1; however, the roles of Lamtor1 in T cells have not been investigated. In this article, we show that Lamtor1-deficient CD4 T cells exhibited marked reductions in proliferation, IL-2 production, mTORC1 activity, and expression of purine- and lipid-synthesis genes. Polarization of Th17 cells, but not Th1 and Th2 cells, diminished following the loss of Lamtor1. Accordingly, -driven Lamtor1-knockout mice exhibited reduced numbers of CD4 and CD8 T cells at rest, and they were completely resistant to experimental autoimmune encephalomyelitis. In contrast, genetic ablation of Lamtor1 in Foxp3 T cells resulted in severe autoimmunity and premature death. Lamtor1-deficient regulatory T cells survived ex vivo as long as wild-type regulatory T cells; however, they exhibited a marked loss of suppressive function and expression of signature molecules, such as CTLA-4. These results indicate that Lamtor1 plays essential roles in CD4 T cells. Our data suggest that Lamtor1 should be considered a novel therapeutic target in immune systems.
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http://dx.doi.org/10.4049/jimmunol.1700157DOI Listing
September 2017

Semaphorin 4D inhibits neutrophil activation and is involved in the pathogenesis of neutrophil-mediated autoimmune vasculitis.

Ann Rheum Dis 2017 Aug 17;76(8):1440-1448. Epub 2017 Apr 17.

Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita City, Osaka, Japan.

Objectives: Inappropriate activation of neutrophils plays a pathological role in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The aim of this study was to investigate the functions of semaphorin 4D (SEMA4D) in regulation of neutrophil activation, and its involvement in AAV pathogenesis.

Methods: Serum levels of soluble SEMA4D were evaluated by ELISA. Blood cell-surface expression of membrane SEMA4D was evaluated by flow cytometry. To determine the functional interactions between neutrophil membrane SEMA4D and endothelial plexin B2, wild-type and mice neutrophils were cultured with an endothelial cell line (MS1) stained with SYTOX green, and subjected to neutrophil extracellular trap (NET) formation assays. The efficacy of treating human neutrophils with recombinant plexin B2 was assessed by measuring the kinetic oxidative burst and NET formation assays.

Results: Serum levels of soluble SEMA4D were elevated in patients with AAV and correlated with disease activity scores. Cell-surface expression of SEMA4D was downregulated in neutrophils from patients with AAV, a consequence of proteolytic cleavage of membrane SEMA4D. Soluble SEMA4D exerted pro-inflammatory effects on endothelial cells. Membranous SEMA4D on neutrophils bound to plexin B2 on endothelial cells, and this interaction decreased NET formation. Recombinant plexin B2 suppressed neutrophil Rac1 activation through SEMA4D's intracellular domain, and inhibited pathogen-induced or ANCA-induced oxidative burst and NET formation.

Conclusions: Neutrophil surface SEMA4D functions as a negative regulator of neutrophil activation. Proteolytic cleavage of SEMA4D as observed in patients with AAV may amplify neutrophil-mediated inflammatory responses. SEMA4D is a promising biomarker and potential therapeutic target for AAV.
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http://dx.doi.org/10.1136/annrheumdis-2016-210706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738596PMC
August 2017

Classification of idiopathic interstitial pneumonias using anti-myxovirus resistance-protein 1 autoantibody.

Sci Rep 2017 02 23;7:43201. Epub 2017 Feb 23.

Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita City, Osaka 565-0871, Japan.

Chronic fibrosing idiopathic interstitial pneumonia (IIP) can be divided into two main types: idiopathic pulmonary fibrosis (IPF), a steroid-resistant and progressive disease with a median survival of 2-3 years, and idiopathic non-specific interstitial pneumonia (INSIP), a steroid-sensitive and non-progressive autoimmune disease. Although the clinical courses of these two diseases differ, they may be difficult to distinguish at diagnosis. We performed a comprehensive analysis of serum autoantibodies from patients definitively diagnosed with IPF, INSIP, autoimmune pulmonary alveolar proteinosis, and sarcoidosis. We identified disease-specific autoantibodies and enriched KEGG pathways unique to each disease, and demonstrated that IPF and INSIP are serologically distinct. Furthermore, we discovered a new INSIP-specific autoantibody, anti-myxovirus resistance-1 (MX1) autoantibody. Patients positive for anti-MX1 autoantibody constituted 17.5% of all cases of chronic fibrosing IIPs. Notably, patients rarely simultaneously carried the anti-MX1 autoantibody and the anti-aminoacyl-transfer RNA synthetase autoantibody, which is common in chronic fibrosing IIPs. Because MX1 is one of the most important interferon-inducible anti-viral genes, we have not only identified a new diagnostic autoantibody of INSIP but also obtained new insight into the pathology of INSIP, which may be associated with viral infection and autoimmunity.
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http://dx.doi.org/10.1038/srep43201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322336PMC
February 2017

Polarization of M2 macrophages requires Lamtor1 that integrates cytokine and amino-acid signals.

Nat Commun 2016 10 12;7:13130. Epub 2016 Oct 12.

Department of Immunopathology, World Premier International Immunology Frontier Research Center, Osaka University, Yamadaoka 2-2, Osaka 565-0871 Japan.

Macrophages play crucial roles in host defence and tissue homoeostasis, processes in which both environmental stimuli and intracellularly generated metabolites influence activation of macrophages. Activated macrophages are classified into M1 and M2 macrophages. It remains unclear how intracellular nutrition sufficiency, especially for amino acid, influences on macrophage activation. Here we show that a lysosomal adaptor protein Lamtor1, which forms an amino-acid sensing complex with lysosomal vacuolar-type H-ATPase (v-ATPase), and is the scaffold for amino acid-activated mTORC1 (mechanistic target of rapamycin complex 1), is critically required for M2 polarization. Lamtor1 deficiency, amino-acid starvation, or inhibition of v-ATPase and mTOR result in defective M2 polarization and enhanced M1 polarization. Furthermore, we identified liver X receptor (LXR) as the downstream target of Lamtor1 and mTORC1. Production of 25-hydroxycholesterol is dependent on Lamtor1 and mTORC1. Our findings demonstrate that Lamtor1 plays an essential role in M2 polarization, coupling immunity and metabolism.
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http://dx.doi.org/10.1038/ncomms13130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064021PMC
October 2016

LRRK1 is critical in the regulation of B-cell responses and CARMA1-dependent NF-κB activation.

Sci Rep 2016 05 11;6:25738. Epub 2016 May 11.

Department of Immunopathology, World Premier International (WPI) Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.

B-cell receptor (BCR) signaling plays a critical role in B-cell activation and humoral immunity. In this study, we discovered a critical function of leucine-rich repeat kinase 1 (LRRK1) in BCR-mediated immune responses. Lrrk1(-/-) mice exhibited altered B1a-cell development and basal immunoglobulin production. In addition, these mice failed to produce IgG3 antibody in response to T cell-independent type 2 antigen due to defects in IgG3 class-switch recombination. Concomitantly, B cells lacking LRRK1 exhibited a profound defect in proliferation and survival upon BCR stimulation, which correlated with impaired BCR-mediated NF-κB activation and reduced expression of NF-κB target genes including Bcl-xL, cyclin D2, and NFATc1/αA. Furthermore, LRRK1 physically interacted and potently synergized with CARMA1 to enhance NF-κB activation. Our results reveal a critical role of LRRK1 in NF-κB signaling in B cells and the humoral immune response.
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http://dx.doi.org/10.1038/srep25738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863158PMC
May 2016

mTOR Complex Signaling through the SEMA4A-Plexin B2 Axis Is Required for Optimal Activation and Differentiation of CD8+ T Cells.

J Immunol 2015 Aug 26;195(3):934-43. Epub 2015 Jun 26.

Department of Immunopathology, World Premier International Research Center, Immunology Frontier Research Center, Osaka University, Suita City, Osaka 565-0871, Japan; Department of Respiratory Medicine, Allergy, and Rheumatic Disease, Osaka University Graduate School of Medicine, Suita City, Osaka 565-0871, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Suita City, Osaka 565-0871, Japan;

Mammalian target of rapamycin (mTOR) plays crucial roles in activation and differentiation of diverse types of immune cells. Although several lines of evidence have demonstrated the importance of mTOR-mediated signals in CD4(+) T cell responses, the involvement of mTOR in CD8(+) T cell responses is not fully understood. In this study, we show that a class IV semaphorin, SEMA4A, regulates CD8(+) T cell activation and differentiation through activation of mTOR complex (mTORC) 1. SEMA4A(-/-) CD8(+) T cells exhibited impairments in production of IFN-γ and TNF-α and induction of the effector molecules granzyme B, perforin, and FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8(+) T cell responses were significantly impaired in SEMA4A(-/-) mice. Furthermore, SEMA4A(-/-) CD8(+) T cells exhibited reduced mTORC1 activity and elevated mTORC2 activity, suggesting that SEMA4A is required for optimal activation of mTORC1 in CD8(+) T cells. IFN-γ production and mTORC1 activity in SEMA4A(-/-) CD8(+) T cells were restored by administration of recombinant Sema4A protein. In addition, we show that plexin B2 is a functional receptor of SEMA4A in CD8(+) T cells. Collectively, these results not only demonstrate the role of SEMA4A in CD8(+) T cells, but also reveal a novel link between a semaphorin and mTOR signaling.
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http://dx.doi.org/10.4049/jimmunol.1403038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505953PMC
August 2015
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