Exp. Immunol., Immunol. Frontier Res. Ctr., Osaka …pharmacology.main.jp/jps93/pdf/pdf/SL.pdf2-PL Pharmacological control of regulatory T cells in immunological diseases Shimon Sakaguchi

2-PLPharmacological control of regulatory T cells in immunological diseases

Shimon Sakaguchi

Exp. Immunol., Immunol. Frontier Res. Ctr., Osaka Univ.

Regulatory T (Treg) cells, which are expressing the transcription factor Foxp3, are actively engaged in themaintenance of immunological self-tolerance and homeostasis. Depletion or functional impairment of Treg cells isable to enhance cancer and microbial immunity, while their numerical expansion or functional augmentation isinstrumental in treating autoimmune disease and establishing graft tolerance. How to achieve these aims by targetingTreg cells with biologicals (such as monoclonal antibodies) or chemicals has been an issue of intense investigation.We have recently shown that certain tyrosine kinase inhibitors that blocked T-cell receptor-proximal signaling in Tcells were able to specifically deplete mature Treg cells, thereby enhancing tumor immunity in humans. On the otherhand, inhibitors of a serine threonine kinase involved in a T-cell signaling pathway evoked Foxp3 expression inconventional T cells and converted them to functionally competent Treg-like cells, which effectively suppressedautoimmune disease and allergy in animal models. It will be discussed how Treg cells be pharmacologically targeted tocontrol a variety of physiological and pathological immune responses.

正常個体中に存在する制御性T細胞 (Regulatory T cells, Treg)は、免疫自己寛容の維持、様々な免疫応答の抑制的制御に枢要である。内在性Tregの大部分は胸腺で、機能的に成熟した形で産生される。転写因子Foxp3は、Tregに特異的に発現し、Tregの発生、機能発現を制御するマスター制御遺伝子である。Foxp3+Tregの量的・質的異常は、様々な自己免疫／炎症性疾患の直接的原因となる。逆に、正常T細胞にFoxp3を発現させると、機能、表現型の点で内在性Tregと同等のTreg様T細胞に転換できる。一方、抗体、小分子を用いてTregの量的減少、抑制活性の減弱を図れば、がん免疫、微生物免疫を亢進できる。本講演では、Tregによる免疫抑制の分子機構、およびその機能、細胞系譜の維持機構について述べる。さらに、如何にTregを増やし、あるいは通常T細胞をTregに転換できるか、またTregを用いた細胞療法による自己免疫病、炎症性腸炎などの予防・治療が可能か、について議論する。

Plenary Lecture

Copyright © The Japanese Pharmacological Society

1-SL01Toward the Mysteries of Sleep

Masashi Yanagisawa

WPI-IIIS, Univ. Tsukuba

Despite the fact that the executive neurocircuitry and neurochemistry for sleep/wake switching has been increasinglyrevealed in recent years, the mechanism for homeostatic regulation of sleep, as well as the neural substrate for"sleepiness" (sleep need), remains unknown. To crack open this black box, we have initiated a large-scale forwardgenetic screen of sleep/wake phenotype in mice based on true somnographic (EEG/EMG) measurements. We haveso far screened >8,000 heterozygous ENU-mutagenized founders and established a number of pedigrees exhibitingheritable and specific sleep/wake abnormalities. By combining linkage analysis and the next-generation whole exomesequencing, we have molecularly identified and verified the causal mutation in several of these pedigrees. Biochemicaland neurophysiological analyses of these mutations are underway. Since these dominant mutations cause strongphenotypic traits, we expect that the mutated genes will provide new insights into the elusive pathway regulatingsleep/wakefulness. Indeed, through a systematic cross-comparison of the Sleepy mutants and sleep-deprived mice, wehave recently found that the cumulative phosphorylation state of a specific set of mostly synaptic proteins may be themolecular substrate of sleep need.

睡眠覚醒調節の根本的な原理、つまり「眠気」（睡眠圧）の脳内での実体とはいったい何なのか、またそもそもなぜ睡眠が必要なのか等、睡眠学の基本課題は全く明らかになっていない。私たちはこのブラックボックスの本質に迫るべく、ランダムな突然変異を誘発したマウスを8,000匹以上作成し、脳波測定により睡眠覚醒異常を示す少数のマウスを選別して原因遺伝子変異を同定するという探索的な研究を行なってきた。このフォワード・ジェネティクス研究の進展により、睡眠覚醒制御メカニズムの中核を担うと考えられる複数の遺伝子の同定に成功し、現在その機能解析を進めている。最近、フォワード・ジェネティクスによって同定されたSleepy変異マウスと断眠マウスの解析から、シナプス蛋白質の累積的リン酸化状態が睡眠圧の本態の一部である可能性が提示された。本講演では、筑波大学WPI-IIISの私どものラボにおける睡眠覚醒の謎への探索的アプローチを紹介する。

Special Lecture1


1-SL02Role of transporter sciences in new drug discovery and development.

Yuichi Sugiyama

Sugiyama Lab. RIKEN

Recently, many studies on genetic polymorphisms (PGx) in drug transporters and transporter-mediated drug-druginteractions(DDI) have been published, and these are part of mechanisms of interindividual difference in drugresponse. It is important to predict such interindividual difference at early stage of drug development. With a PBPKmodel, the effect of changes in transporter function on the Pharmacokinetics (PK),and, ultimately, thePharmacodynamics (PD) and/or Toxicodynamics (TD) will be predicted. I will focus on the following subjects.Recent reports provided quantitative predictions for OATP1Bs-mediated DDIs between statins and cyclosporine A(CsA)/rifampicin(RIF) based on PBPK models. In the process of the analyses, the in vitro–in vivo discrepancies inthe Ki values for OATPs were suggested. I will share with you our approach to make the bottom-up predictionpossible. Finally, it should be also challenging to reproduce the inter-individual variabilities by considering the meanvalue and deviation of each PK parameter in a certain population with PBPK model (so-called "virtual clinical study").

生体内では多様な輸送担体（トランスポーター;TP）が生体に必要な物質を積極的に取り込み、異物を積極的に排泄する機能を担っている。TPの分子認識に基づいた創薬を行うことにより、薬効を維持したまま副作用に関連する組織・細胞への移行を抑える理想的な動態特性を持つ薬の開発も可能になると期待され、医薬品開発の面からもTP研究への関心が高まっている。TPは、薬物代謝酵素と同様に、分子多様性、遺伝子多型、臓器特異性、広範な基質認識性という特性を持つことがわかってきた。医薬品開発においては、in vitroからin vivoへの外挿法などを確立していくことで、医薬品開発過程での遺伝子発現系の利用、薬物間相互作用の評価、遺伝子多型による個人間変動の解析などTP研究を実用段階へと進めていく必要がある。当日はその実例を示したいと思う。近年、米国FDAが中心となり、生理学的薬物速度論モデル（PBPK model）による薬物動態予測を、臨床試験の必要性の判断、投与量の設定に活かそうという動きがある。この実例についても触れながらの講演にしたい。

Special Lecture2


1-SL03The activation mechanism and drug discovery of G protein-coupled receptor

Shunguang Yuan

The Research Center for Computer-aided Drug Discovery, Shenzhen Institutes of Advanced Technology, ChineseAcademy of Science

More than 800 human GPCRs allow the selective detection of extracellular signals as diverse as photons, odorants,flavors, nucleotides, hormones, neurotransmitters – revealing GPCRs fundamental role in signal transduction. As theyregulate many central physiological processes and are thus implicated in many diseases, GPCRs are among the mostimportant targets for modern medicines. In spite of this medical importance and the recent progress in elucidating the3D structures of various GPCRs, central questions how these receptors recognize extracellular chemical signals andtransfer them across the cellular membrane to finally evoke an intracellular response are largely unresolved at amolecular level, mainly because the different steps during signal transmission are not directly accessible byexperiments. In this context we are concentrating on central questions of GPCR mediated cellular signalling usingcomputer based molecular dynamics simulations. Our work revealed for the first time, in atomic detail, the entireprocess of transmembrane signalling of various GPCRs: we found that ligand binding induces a series ofconformational changes within a GPCR which opened a gate inside the receptor for water molecules entering theinternal region of the receptor and subsequently driving conformational switches within the receptor which finally ledto the activation of a G protein on the intracellular side of the receptor. We have applied our findings successfully tovarious GPCR targeted drug discovery projects.

Special Lecture3


1-SL04Biological roles of ATP receptors and microglia: Pharmacologists areessential for drug discovery

Kazuhide Inoue

Kyushu Univ.

Microglia are thought to work as immunocompetent cells in central nervous system. Microglia are also thought to bederived from primitive macrophages in the yolk sac. In normal conditions, microglia are ubiquitously distributed inthe spinal cord and brain, and have small cell bodies bearing branched and motile processes, which seem to monitorthe local environment. In the pathophysiological condition, microglia are activated, change morphologically, increasein cell number, and alter the expression of genes, including neurotransmitter receptors, such as P2 purinergicreceptors. Activated microglia express several subtypes of ionotropic (P2XRs) and metabotropic P2 receptors(P2YRs). Extracellular ATP stimulates microglial P2 receptors to evoke various cellular responses. For example,P2X4Rs play a key role in evoking and maintaining neuropathic pain. P2Y12Rs and P2Y6Rs play very important rolefor microglial trafficking and phagocytosis, respectively. These findings will open new strategy for drug development.Indeed, the inhibitor of P2X4R has a high potential for being a first in class medicine against neuropathic pain. I thinkthat Pharmacologists are essential for drug discovery.

ミクログリアは中枢神経系での免疫担当細胞、起源として卵黄嚢の未分化マクロファージが源流であるとされている。正常時、ミクログリアは脊髄や脳に均等に存在し、小さな細胞体と長く可動性の突起をもち、それらを頻繁に動かして周辺の環境を監視しているように見える。病態時には、ミクログリアは活性化され、形態を変化させ、分裂増殖し、遺伝子発現の状況に変化を与え、例えばP2プリナージック受容体のような様々な神経伝達物質受容体の遺伝子発現に影響を与える。活性化ミクログリアはいくつかのATP 受容体サブタイプを発現する。外液のATPはこれらの受容体を刺激し、ミクログリアに様々な生理反応を引き起こす。その結果、例えば、P2X4受容体は神経障害性疼痛発症に重要な役目を果たす。また、P2Y12受容体およびP2Y6受容体は、それぞれミクログリアの遊走と貪食に重要な役割を持つ。この様な発見は、新たな医薬品創製の道を切り開くものである。実際に、P2X4受容体阻害剤は優れたFirst in classの神経障害性疼痛治療薬として有望視されている。医薬品開発には薬理学者が欠かせないと私は実感している。

Special Lecture4


2-SL05Harnessing gut commensals to combat disease

Kenya Honda

Dep. Microbiol. & Immunol., Keio Univ. Sch. Med.

The mammalian alimentary tract harbors hundreds of species of commensal microbes that critically influence amultitude of host physiological functions. Unfavorable alterations of the gut microbiota composition often correlatewith several negative health outcomes. Thus, the amelioration of microbiota dysfunction is a promising route forfuture therapeutics for several diseases. We have been aiming to understand the features and functions, particularlyimmunological attributes, of the microbiota, and trying to identify responsible bacterial species and factors forshaping the immune system. We have succeeded in isolation of human and mouse gut-associated commensal bacterialstrains that specifically affect the development and function of Th17 cells, Treg cells, Th1 cells or CD8 T cells. Inaddition, we have identified trypsin-degrading bacterial species. Our findings would allow for designing bacterialconsortia that activate or suppress specific adaptive immune programs, potentially resulting in development of bettertherapeutics for numerous diseases involving the immune system, including infectious disease, autoimmunity, allergy,and cancer.

ほ乳類の腸管には数百の腸内細菌（マイクロバイオータ）が存在し、宿主の生理機能に深く影響を及ぼしている。従ってマイクロバイオータに人為的に介入することが出来れば、複数の疾患に対する新たな治療戦略となり得る。我々は、消化管の恒常性維持機構を理解すると共に、個々の腸内細菌種が免疫システムにどのように影響を与えているかを還元化して把握して行く独自の研究手法を確立してきた。この方法によってこれまでに、制御性T細胞、Th17細胞、Th1細胞、CD8 T細胞を特異的に誘導する腸内細菌種の同定に成功した。これらの成果は、免疫系が関わる様々な疾患（感染症・アレルギー・自己免疫・がんなど）に対する新しい治療法開発に繋がる可能性がある。

Special Lecture5


2-SL06CryoEM of GPCRs: from molecular mechanism to drug discovery

Georgios Skiniotis

Stanford University, USA

G protein coupled receptors (GPCRs) comprise the main conduit for information transfer between cells and theirenvironment and participate in most physiological processes through innumerable signaling networks. Accordingly,GPCRs are the largest family of pharmaceutical targets in therapeutic areas ranging from cardiovascular andmetabolic diseases to neuropsychiatric and behavioral disorders. Here, I will describe our results from the structuraland functional characterization of GPCRs in complex with a range of ligands and signal transducers, providinggeneral mechanisms of transmembrane signal instigation from different receptor classes in this family.

Special Lecture6


2-SL07Spying on neuromodulation by constructing new genetically encodedfluorescent sensors

Yulong Li

Peking University School of Life Sciences, PKU-IDG/McGovern Institute for Brain Research, Peking-TsinghuaCenter for Life Sciences, Beijing, P.R.China

Monoamine transmitters, e.g. dopamine (DA), norepinephrine (NE) and serotonin (5-HT), are playing importantroles in a plethora of physiological processes, including reward, attention and mood regulation. Malfunction of themonoaminergic signaling is implicated in diseases such as addiction, stress and depression. Take the DA as anexample, a longstanding yet largely unmet goal is to measure DA changes reliably and specifically with highspatiotemporal precision, particularly in animals executing complex behaviors. We developed the new geneticallyencoded GPCR-activation-based-DA (GRABDA) sensors that enable these measurements. GRABDA sensors canresolve a single-electrical-stimulus-evoked DA release in mouse brain slices and detect endogenous DA release inliving flies, fish, and mice. We have further expanded the repertoire of GRAB sensors for detecting monoamines,purinergic transmitters (e.g. adenosine, ADP and ATP) and neuropeptides (e.g. cholecystokinin, somatostatin andvasoactive intestinal peptide). In sum, these newly developed GRAB sensors provide powerful tools for understandingthe regulation and malfunction of monoaminergic and purinergic systems and neuropeptides in both physiologicaland pathological processes.

Special Lecture7


2-SL08Synaptic Plasticity: from bench to bedside

Takuya Takahashi

Dept. Physiol., Grad. Sch. Med., Yokohama City Univ.

Glutamatergic synapses play central roles in almost all of neuronal functions such as learning, motor and sensoryfunctions. Among glutamate receptors, AMPARs are the "actual mediator" at glutamatergic synapses. Despite theaccumulation of knowledge of physiological roles of AMPARs, its clinical translation is limited. Main reason for this isthat we are not currently able to visualize AMPARs in living human brain. We developed novel PET probe forAMPARs, named [11C]K-2. We detected [11C]K-2 signals reflecting specific binding to AMPARs in rat, non-humanprimate and human. We detected significant positive correlation between [11C]K-2-signals and protein amount ofAMPARs with surgically removed tissue from epileptic patients. Thus, our PET probe for AMPARs specificallydetects AMPARs and the first PET probe to visualize AMPAR in living human brain. We are currently imagingpatients with neuropsychiatric disorders. Further, we have recently identified CRMP2-binding compound, edonerpicmaleate, facilitates synaptic AMPAR delivery and results in the acceleration of motor function recovery after braindamage in rodent and non-human primate.

シナプス可塑性の基礎研究については多くの知見が蓄積されている一方で、その臨床応用は非常に乏しいというのが現状である。グルタミン酸シナプスは脳内での情報処理において非常に重要な役割を果たしている。脳に可塑的な変化がおこる際、グルタミン酸受容体であるAMPA受容体がシナプスへ移行することは当研究室の研究も含めた多くの研究が明らかにしている。

当教室ではAMPA受容体シナプス移行を促進する化合物を特定しており、脳卒中後のリハビリテーション効果促進薬としての可能性を模索している。現在げっ歯類においては劇的な効果が証明されており、霊長類においても同様の効果が見られている。今年度から臨床治験の開始を予定している。

現在の精神神経疾患の診断治療は基礎研究の根拠に乏しい。本講演ではAMPA受容体を認識するPET Probeの開発について当教室の最新の知見を紹介する。AMPA受容体をヒトで可視化するPET Probeは未だ存在しない。この技術を用いて、「シナプス機能分子による精神神経疾患の再分類」が可能になり、今後の「基礎研究の根拠」に基づいた新規診断が可能になる。

Special Lecture8


3-SL09Resolvins & Pro-Resolving Mediators with Novel Mechanisms in Infectious-Inflammation

Charles N. Serhan

Brigham & Women’s Hospital - Harvard Medical School

Inflammation is an integral component of many diseases e.g. arthritis, periodontal disease, asthma, cardiovasculardiseases and neurodegenerative diseases. Using a systems approach with self-limited inflammatory infectious exudatesto map tissue events, cell traffic and identification of protein and chemical mediators, we uncovered three structurallydistinct families of potent omega-3 fatty acid-derived (EPA, DPA, DHA) novel endogenous mediators, namedresolvins, protectins and maresins coined specialized pro-resolving mediators (SPM) and recently the biosynthesis ofpeptidio-SPM within these families that are involved in tissues regeneration (denoted , cys-SPM). Completestructural elucidation and total organic synthesis of each new molecule and pathway intermediates confirmed theirroles in vivo in the resolution of inflammation in animal models. Each member of this super-family is structurallydistinct and is a pro-resolving mediator controlling the duration and magnitude of acute inflammatory responses withactions in pico-nanogram range in animal disease models. Mapping of these resolution circuits provides new avenuesto probe the molecular basis of many widely occurring diseases (CN Serhan, Nature 2014, Molecular Aspects ofMedicine 2017, Serhan, Levy JCI 2018). This special presentation will focus on our recent advances in thebiosynthesis and functions of SPM and the role of the vagus nerve in controlling infectious inflammation. We'veoperationalized LC-MS-MS based targeted metabololipidomics to profile SPM and recently cross validated thisapproach with other laboratories using coded samples from human endotoxin challenges establishing SPMbiosynthesis in humans and function (Norris et al 2019). Our recent evidence indicates a new role for the vagus nerveand vagotomy in regulation of lipid mediators. Specifically, vagotomy reduces pro-resolving mediators such aslipoxins, resolvins, protectins and maresins delaying resolution in mouse peritonitis and E. coli infections. The vagusregulates peritoneal Group 3 innate lymphoid cell (ILC3) number and peritoneal macrophage responses with lipidmediator profile signatures showing elevated pro-inflammatory mediators and reduced resolvins, including the novelprotective immunoresolvent agonist protectin conjugate in tissue regeneration 1 (PCTR1; Dalli et al., Immunity).Results obtained with human vagus ex vivo indicate that vagus produces both proinflammatory lipid mediators (i.e.,prostaglandins and leukotrienes) as well as SPM obtained using targeted LC-MS-MS profiling. Electrical stimulationof human vagus ex vivo reduces both prostaglandins and leukotrienes and increases resolvins and other SPMs. Theseresults elucidate a host-protective mechanism mediated by vagus stimulation. Moreover, they define a new pro-resolution of inflammation reflex operative in mice and isolated human tissue that involves a vagus-SPM circuit.Together these results indicate that endogenous resolution pathways may underlie prevalent diseases associated withuncontrolled inflammation and open the potential for resolution-based physiology, resolution pharmacology andresolution-bioelectric medicine.

Special Lecture9


3-SL10A Chemical Approach to Controlling Cell Fate

Sheng Ding

University of California, San Francisco, USA

Recent advances in stem cell biology may make possible new approaches for the treatment of a number of diseases. Abetter understanding of molecular mechanisms that control stem cell fate as well as an improved ability to manipulatethem are required. Toward these goals, we have developed and implemented high throughput cell-based screenings ofchemical libraries, and identified and further characterized small molecules that can control stem cell fate in varioussystems. This talk will provide latest examples of discovery efforts in my lab that have advanced our ability andunderstanding toward controlling stem cell fate, including self-renewal, survival, differentiation and reprogrammingof stem cells.

Special Lecture10


2-JALThe structural basis of peptide GPCR activation and signalling

Denise Wootten

Monash Institute of Pharmaceutical Sciences, Monash University, Australia

G protein-coupled receptors (GPCRs) are the largest family of cell surface drug targets. Consequently, there is highinterest in understanding the structure of members of this receptor superfamily and molecular detail of how ligandsand transducer proteins interact with them. Our laboratory has been applying single particle cryo-EM todetermination of active GPCR structures, using minimally modified receptors. Our work has been principally focusedon the class B GPCR subfamily that bind large peptide hormones and are well established clinical targets for thetreatment of major disease, including migraine, irritable bowel syndrome, diabetes, obesity and neurodegeneration.We have now solved structures of multiple different receptors, providing wide structural coverage of the majorsubfamilies of class B GPCRs. Included within this are structures of the same receptor bound to native peptideagonists, biased peptide agonists and non-peptide agonists and receptors in complex with accessory proteins thatallosterically modulate receptor function. In combination with molecular pharmacology and molecular dynamicssimulations, we are gaining substantial insights into diverse modes of ligand binding, receptor activation andmodulation that lead to G protein coupling and downstream signalling.

JPS-ASCEPT Lecture


3-SL11Biology of LipoQuality: Omega-3 fatty acid cascade that controlsinflammation and tissue homeostasis

Makoto Arita1,2,3

1Div. of Physiol. Chem.& Metab., Grad. Sch. Pharmaceut. Sci., Keio Univ., 2Lab. for Metabolomics, RIKEN IMS,3Grad. Sch. of Med Life Sci,. Yokohama City Univ.

Polyunsaturated fatty acids (PUFAs) exhibit a wide range of biological effects, many of which are mediated throughthe formation and actions of their bioactive metabolites. It is well appreciated that different PUFA balance affectsinflammation and related diseases, and recent advances in mass spectrometry-based lipidomics technologies haverevealed potential links between PUFA metabolism and biological phenotypes. Omega-3 PUFAs such as EPA andDHA are widely held to be beneficial in maintaining tissue homeostasis. To date, we've developed LC-MS/MS-basedlipidomics system to monitor fatty acid metabolites comprehensively, and uncovered the roles of omega-3 PUFAmetabolites in controlling inflammatory responses. I'll describe recent advances on omega-3 PUFA–derivedmediators, mainly focusing on omega-3 oxygenation pathway that may hold a key for the beneficial effects of omega-3PUFAs by structure-specific mechanisms in controlling inflammation and tissue homeostasis. Also I'll introduce ourrecent technology development, namely non-targeted lipidomics, that has a strong potential to search for lipids ofinterest globally, and to identify unknown lipid species in a non-biased fashion.

生体内には多様な脂質分子種が存在し、その質（リポクオリティ）の違いや代謝バランスの変化が,さまざまな炎症・代謝性疾患の背後に潜む重要な要素であることが示唆されている。一方で近年の質量分析技術の進歩は、質の異なる脂肪酸分子種の代謝、分布、動態を詳細に捉えることを可能にした。我々は、生体内の脂肪酸やリン脂質の代謝を網羅的かつ定量的に把握するためのリピドミクス解析システムを構築し、炎症・代謝性疾患の制御において脂肪酸代謝バランスが重要であることを示してきた。中でも、EPAやDHAなどω３脂肪酸が脂肪酸オキシゲナーゼにより活性代謝物に変換され、積極的に抗炎症作用や組織保護作用を発揮することを見出してきた。本講演では、生体内のリポクオリティが炎症制御や組織恒常性に及ぼす影響およびその分子機序について紹介する。さらに、生体内のリポクオリティの変化をノンバイアスかつ広範囲に捉えるための新技術、ノンターゲットリピドミクスの有用性と今後の発展性について議論したい。


Exp. Immunol., Immunol. Frontier Res. Ctr., Osaka …pharmacology.main.jp/jps93/pdf/pdf/SL.pdf2-PL Pharmacological control of regulatory T cells in immunological diseases Shimon Sakaguchi

Documents