Not published

In the complex field of epilepsy, glial cells are emerging as key players. Here, I present our findings on the role of microglia and astrocytes in specific epileptic conditions, exploring their mechanisms of action and potential therapeutic implications.　First, we examined auditory dysfunction reported in patients with temporal lobe epilepsy (TLE). Although increased neuronal activity has been observed in auditory pathways, including the primary auditory cortex and medial geniculate body (MGB), the cellular underpinnings remain elusive. Using a mouse model that mimics TLE after status epilepticus we identified the role of microglia in the disinhibition of these pathways. Over an 8-week period after status epilepticus, microglia were found to reduce inhibitory synapses on MGB relay neurons, catalyzing hyperactivity in auditory pathways. Interestingly, local removal of microglia from the MGB reduced this hyperactivity and restored auditory discrimination. This sheds light on the central role of thalamic microglia in epilepsy-induced auditory dysfunction.　On another front, we investigated the role of astrocytes in mesial temporal lobe epilepsy (MTLE), a challenging form of epilepsy characterized by hippocampal seizures. Using transcriptome analysis in an MTLE mouse model, we identified a significant increase in insulin-like growth factor binding protein 2 (Igfbp2) levels within gliosis-associated astrocytes. Preliminary results suggest that astrocytic Igfbp2 may be instrumental in driving epileptogenesis, providing a promising therapeutic avenue for the treatment of MTLE.　Taken together, these findings underscore the critical influence of glial cells in epilepsy and open avenues for novel therapeutic interventions targeting these cell types.

Microglia are tissue macrophages of the brain and play essential roles beyond the immune cell framework from brain development to homeostasis. Recent studies in microglia revealed the cellular origin of microglia as primitive macrophages that arise in the yolk sac, and this discovery prompted the development of the differential protocol of microglia from stem cells. The stem cell-derived primitive macrophages/microglia are then cocultured with stem cell-derived neurons and neurospheres to produce a further appropriate brain model which may contribute to providing new insights into the mechanism of physiological function of microglia. We also developed human 2D-and 3D brain organoid-model containing microglia to examine the roles of microglia in neurodegenerative diseases, especially Alzheimer’s disease, using amyloid-β oligomers, and found the neuroprotective function of microglia against amyloid-β neurotoxicity. This presentation will outline recent trends in vitro culture models of human brains. We will further discuss its significance, limitations so far, and future prospects as a microphysiological system that is expected to provide a new approach to elucidate the pathogenesis of neurodegenerative diseases and develop therapeutic strategies.

In the central nervous system (CNS), microglia are resident immune cells and play an important role in maintaining homeostasis via inflammatory regulation and engulfment of cell debris or beta-amyloid. Thus, functional abnormalities in microglia are involved in the onset and progression of various CNS diseases. Recently, it has been reported that cellular senescence in microglia progresses more remarkably with aging than in other cells in the brain. In addition, senescent cells present senescent-associated secretory phenotype (SASP) which secretes inflammatory cytokines and chemokines. Therefore, the progression of cellular senescence in microglia might be a key factor in the pathogenesis of age-related neurodegenerative diseases. However, in vitro systems, primary cultured microglia isolated from rodent embryos or neonates have generally been used, but the details of age-related changes in microglial function have not been adequately analyzed in these cell systems. In this symposium, I will introduce our recently established model system of cellular senescence microglia and discuss the possibility that microglial dysfunction by cellular senescence is a potential drug target for age-related neurodegenerative diseases.

Glaucoma is leading cause of blindness worldwide, which is characterized by progressive optic neuropathy and degeneration of retinal ganglion cells (RGCs), one of the retinal neurons transmits visual information to the brain. Because RGC degeneration is one of the hall marks of the disease, many studies have paid attention to only neurons. In this symposium, we report that impaired glial function triggers pathogenesis of normal-tension glaucoma. We have recently discovered that astrocyte dysfunction by lacking ATP-binding cassette transporter A1 (ABCA1) causes glaucoma-like phenotypes. The astrocyte-specific ABCA1KO (Astro-KO) mice showed RGC degeneration and visual impairment at 12 months old without changes in an intraocular pressure, a conventional glaucoma risk factor which can cause damages in RGCs. To further clarifying the molecular mechanisms, we performed bulk and single-cell RNA-sequence of retina. We found that RGCs and retinal astrocytes up-regulate neuroinflammatory pathways including CXCR4 and CCR5 signaling. CXCL12 and CCL5 were up-regulated in astrocytes in vitro and in vivo. Because CXCR4 and CCR5 were highly expressed in RGCs, the astrocyte-derived chemokines might affect RGC functions. We identified a novel RGC subclass enriched in a unique set of NMDA subunit genes, implicating higher sensitivity to excitotoxicity. Supporting this, intravitreal NMDA injection caused exacerbated RGC damages in Astro-KO mice. Thus, our data demonstrate that astrocytic dysfunction causes non-cell-autonomous optic neuropathy.

In addition to microglia, lymphocytes are involved in the pathology of brain diseases. Lymphocytes in the brain are discriminated into brain-resident lymphocytes and peripheral lymphocytes infiltrating into the brain. Recently, the function of the meningeal lymphatic vessels was revealed as an infiltrating route for lymphocytes into the brain. The presenter is clarifying the pathological role of peripheral lymphocytes infiltrating into the brain in the disease by surgical blocking of the meningeal lymphatic route and by pharmacological intervention. Here, our recent findings from such study on the function of peripheral lymphocytes in the brain disease are introduced.

We identified Nelfinavir (NFV), an HIV-1 protease inhibitor, as a potential therapeutic agent through drug repositioning during the COVID-19 pandemic (Ohashi et al., iScience, 2021). At that time, there were no antiviral drugs available for novel coronaviruses, and international research groups were conducting clinical trials on various therapeutic candidates, yet no effective treatments had been identified. Beyond the challenge of finding candidates with strong antiviral efficacy, we contemplated whether the design of clinical trials itself could be optimized. To address this, we gathered extensive clinical data and employed a mathematical model to analyze virus dynamics in infected patients. Comprehensive simulations unveiled considerable individual variations in infection dynamics, demonstrating that the effectiveness of treatment heavily relied on the timing of drug administration (Kim et al., PLOS Biol., 2021). Moreover, incorporating these insights, we devised a quantitative simulator that emulated a randomized controlled trial, thereby refining the optimal clinical trial design (Iwanami et al., PLOS Med., 2021). The outcomes indicated that by focusing solely on cases within a few days of disease onset, the number of participants in the trial could be substantially reduced. Additionally, we fine-tuned the dosage and regimen of the therapeutic drug along with the primary endpoint, maximizing their alignment within practical constraints (Hosogaya et al., Trial, 2021). Harnessing digital twin technology, we remarkably initiated the clinical trial within a mere "4 months" after the commencement of drug repositioning (jRCT2071200023). Despite the termination of numerous clinical trials worldwide, we successfully concluded this trial (Miyazaki et al., Microbiol Spectr., 2023). In this presentation, we will elucidate our digital twin research, offering insights into drug discovery from a multifaceted perspective encompassing fundamental research, clinical exploration, and societal implementation.

Psychiatric disorders are diagnosed based on the clinical interviews by psychiatrists and there has been no useful biological markers in the present clinical settings. Human brain magnetic resonance imaging (MRI) studies for schizophrenia have indicated structural alterations in the cortical and subcortical area. In addition, these structural alterations change progressively according to the onset of first episode psychotic symptoms. However, recent cohort studies focused on adolescent development have shown the decrease in most structural features, and the patterns of the alteration are non-linear and diverse across the features. These results suggest that we need to know whether the structural alterations found in clinical investigations would rely on the emergence of pathological characteristics in psychiatric disorders or the course of typical adolescent brain development. Here, we want to talk about the human brain MRI findings for schizophrenia from the clinical multi-site studies and for general adolescents from Tokyo TEEN Cohort (TTC) study project, and discuss about the brain change in schizophrenia along with adolescent development.

The Pre-Disease Database, conceived under the aegis of Moonshot Goal 2's Aihara Projects, aims to revolutionize research data management. This lecture will provide a comprehensive examination of the database's intrinsic functionalities, real-world applications, and future trajectory. The database operates on two distinct yet complementary platforms: GakuNin RDM for data management, and JAIRO Cloud for data dissemination. GakuNin RDM serves as the managerial platform, equipped with advanced data analysis features. It permits seamless integration with various computational resources, including supercomputers, High-Performance Computing (HPC), and mdx platforms. Additionally, its provenance management assures accurate traceability of the data lineage. Concurrently, JAIRO Cloud functions as the public platform, offering an unmatched level of flexibility for user interface (UI) and metadata customization. It also collaborates with international scholarly communication networks. This lecture, in the context of Moonshot Goal 2, will showcase how the Pre-Disease Database fosters efficient data sharing and utilization among multiple projects. The lecture aims to engage physicians, pharmaceutical researchers, and healthcare organizations in a substantive dialogue regarding the database's potential utility, strategic implementation, and prospective developments, while also addressing its anticipated societal and academic impacts.

There is a gap of about 10 years between Japan's average life expectancy and healthy life expectancy. In recent years, to shorten this period, "mibyou" and its improvement have been attracting attention. There are various approaches to improve "mibyou", including dietary habits and exercise. In the Aihara Project of JST Moonshot Research and Development Project Goal 2, research is being conducted to mathematically define the pre-symptomatic conditions before the transition from a healthy state to a diseased state, and to realize ultra-early disease prediction and prevention. This is a major challenge that has the potential to change the concepts of "health" and "disease." Therefore, it is also necessary to consider the impact on society if this becomes a reality. In addition, research involves the collection of a large amount of data from patients, therefore it is necessary to gain the public's understanding of the necessity of research and to provide sufficient and appropriate explanations regarding the research to patients. For this reason, the project has established an ELSI support team that examines legal, ethical, and social issues (ELSI) and supports researchers. In this presentation, I will show the results of the questionnaire survey that we conducted in 2022 and discuss ELSI in pre-symptomatic research, taking into consideration issues that are often pointed out regarding the realization of genomic medicine.

It is becoming increasingly difficult to explore novel small-molecule compounds or antibody drug candidates. Therefore, it is essential to develop new technologies different from conventional methods to acquire lead compounds or lead antibodies.

We are engaged in research aimed at producing new natural products from fungi and creating candidate monoclonal antibodies for antibody drugs using synthetic genome technology. The first involves a technique called the TAQing system, which utilizes conditionally-activated restriction enzymes within living cells, cutting the genomic DNA at multiple sites, promoting large-scale genome rearrangements, and inducing phenotypic changes. We have used this technology to alter the traits of yeast, plants, and filamentous fungi. Applying this system to filamentous fungi enabled induced expression of dormant biosynthesis gene clusters for natural products.

The second approach involves the Human ADLib system using a chicken B-cell line with human antibody genes introduced into the chromosomes. This system enabled production of human monoclonal antibodies against the target antigen rapidly, followed by seamless affinity maturation to levels close to the antibody medicines. Through these two examples, we also want to consider the possibilities of synthetic genome technologies in recent drug discovery.

We recently developed a cell-free system for propagating circular DNAs by reconstituting the whole replication cycle of the Escherichia coli chromosome using 26 purified proteins. The propagation proceeds exponentially, even from a single DNA molecule, with extremely high fidelity. We further combined a multiple-DNA fragment assembly technique to generate large circular DNA, which provides a powerful tool for the cell-free synthesis of genome-length DNA without biological cloning. In this symposium, I’ll talk about our cell-free genome synthesis technology as a tool for drug discovery and deployment.

Cell poration technologies offer opportunities not only to understand the activities of biological molecules but also to investigate genetic manipulation possibilities. Unfortunately, transferring large molecules that can carry huge genomic information is challenging. In this presentation, I will introduce electromechanical poration using a core–shell-structured microbubble generator, consisting of a fine microelectrode covered with a dielectric material. By introducing a microcavity at its tip, we could concentrate the electrical field with the application of electric pulses and generate microbubbles for electromechanical stimulation of cells. Specifically, the technology enables transfection with molecules that are thousands of kDa even into osteoblasts and Chlamydomonas, which are generally considered to be difficult to inject. Notably, we found that the transfection efficiency can be enhanced by adjusting the viscosity of the cell suspension, which was presumably achieved by remodeling of the membrane cytoskeleton. The applicability of the approach to a variety of cell types opens up numerous emerging gene engineering applications.

RNA and DNA delivery technology using lipid nanoparticles (LNPs) has reached the practical application stage, including its use in mRNA vaccines. To date, 21 bp siRNA (Onpattro) and 4300 nucleotides mRNA vaccines for COVID-19 have been commercialized; however, the decrease in transfection efficiency with the increase in the size of the delivered RNA and DNA is a major challenge. In particular, compared with RNA transfection, plasmid DNA (pDNA) transfection requires multiple steps, including cellular uptake, endosomal escape, nuclear translocation, transcription, and translation. The low transfection efficiency of large pDNA is a critical limitation in the development of artificial cells and functionalization of cells. In this study, we report polymer-lipid hybrid nanoparticles for large-sized pDNA transfection. We demonstrated that positively charged pDNA-polycation core nanoparticle-loaded LNPs showed a four-fold higher transfection efficiency of 15 kbp pDNA than that of the conventional LNPs. Based on the assessment of the size and internal structure of polymer-lipid nanoparticles, hemolysis assay, and cellular uptake, we propose a strategy to enhance large-sized pDNA transfection using LNPs. This strategy will accelerate the delivery of large-sized pDNA in vivo and the development of artificial cells.

For precision cancer medicine, we have been developing an Integrated Cancer Clinical Database System(ICDB) with AI, which automatically collects and stores structured labeled data, such as clinical data in text format, pathological data, genome and liquid biopsy data, from the Electronic Health Record(EHR). The ICDB is an independent and vendor-neutral database system which can connect to any EHR. Oncologists can use AI powered ICDB to recommend and share the most suitable cancer treatment for each patient. During shared decision-making process AI communication system supports patients and physicians for precise approach and communication, regarding patient personality and level of understanding. An AI communication robot supports us during cancer pharmaceutical treatments as an electric patient reported outcome. Cancer information is growing at an accelerated rate, which exceeds the human ability to organize, memorize, and appropriately use. Therefore, AI assistance in Cancer Medical Development is essential. Positive and active application of AI will enable compensation for uneven distribution of experts and labor shortages of healthcare professionals due to an aging society. We are consistently developing future cancer precision treatment with AI assistance.

Osaka University Hospital (hereinafter referred to as "Osaka University Hospital") has set forth "Futurability: a hospital that all people can look forward to, a hospital that looks to the possibilities of the future and continues to solve new problems in new ways" as its vision for the hospital. While progress in medical care is advancing, there are also negative aspects of medical care that have become increasingly specialized and complex. The use of AI in the medical field is important to overcome this situation. When one thinks of AI in the medical field, programmed medical devices for image diagnosis and diagnostic support come to mind, but AI is not limited to this, but is also being incorporated into operational support and patient services. In this presentation, we will introduce some examples of AI applications in the medical field. While he is convinced that AI will be incorporated into medical care and operations in a natural way in the near future, he also believes that medical problems will not be solved only from a technological perspective, and aims to create a place where medical professionals who can use AI can maximize their abilities.

In recent years, artificial intelligence (AI)-based medical and diagnostic assistance programs have been developed all over the world. On the other hand, AI diagnostic support technologies that can be used for adults cannot be applied directly to children or pregnant women, and it is necessary to develop AI technologies suited to the field of pediatric and perinatal medicine that can be applied to children and pregnant women. We, the National Center for Child Health and Development, promotes medical treatment (child health care) and research related to diseases that occur during the life cycle, starting from fertilization and pregnancy, through the fetal, neonatal, infant, school-age, and adolescent stages, and ending in adulthood, when the next generation is being raised. Program (SIP), "Research Project on Advanced Diagnosis and Treatment System by AI (Artificial Intelligence) Hospital," and has been conducting empirical research on medical AI technology from the perspective of pediatric and perinatal care, based on the necessity and challenges of AI technology in the medical field. This presentation will introduce the efforts of the AI Hospital Project at the National Center for Child Health and Development, and outline the present and future of the AI Hospital Project.

The medical dictionary that can be used by AI systems, which was the challenge of the AI Hospital Project, was constructed under the guidance of Dr. Aramaki of Nara Institute of Science and Technology. This dictionary is characterized by its format as a network-type dictionary. Broadly speaking, it consists of two structures: a “relation table based on weighting and association between terms” and a ”term table listing a variety of medical terms”. The total number of medical terms is 380,000. Automatic voice input system makes it possible to generate " natural conversational sentences" by collecting corpus. However, since conversations in medical settings involve differences, special phrasing such as dialects may exist depending on the environment in which the term appears. Therefore, translation using a dictionary is required. The relationship table, which is a major feature of this dictionary, is a mechanism that allows you to visualize the frequency of co-occurrence between terms by structuring the relationships between terms, such as drugs and diagnosis names, symptoms and diagnosis names, etc. is being built. The terminology table consists of "disease name/symptoms", "medicine", etc., and also supports pronunciation, linking with ICD codes, and English translation.These tables are intended for use in "AI diagnosis candidate support systems," "automatic voice input support systems," and "hospitals, clinics, etc."

In actual verification research, it has been confirmed that IBM's automatic voice input system and diagnostic support system have improved performance. We hope that we can contribute to "``medical care that meets the needs of our patients."

Patient-centric medical care is necessary to improve patients' quality of life. However, to realize patient-centric medical care, it is necessary to increase the time spent facing patients by reducing the burden of routine tasks and paperwork performed by healthcare professionals, including physicians, nurses, and pharmacists. Since 2018, Hitachi, Ltd. has been conducting research and development of smart communication technology that coordinates people with IT, robots, and other technologies in collaboration with several medical institutions, under the "Innovative Artificial Intelligence (AI) Hospital Program", one of twelve in the 2nd term of Cross-ministerial Strategic Innovation Promotion Program (SIP) led by Cabinet Office.

In this presentation, we will mainly describe AI interview system for a side effect of outpatients undergoing drug therapy for cancer and eConsent, which digitizes the process of obtaining consent from patients required in various situations such as examination, treatment, and hospitalization at medical institutions.

By using these digital technologies, we would like to contribute to realization of the digital transformation of Healthcare and Medical care, which will contribute to reforming the work styles of healthcare professionals and improving patient QoL.

Metabolism in different tissues/organs is considered to be systemically regulated in a coordinated manner. We have identified several neuronal networks as being involved in inter-organ metabolic communication. A broad range of metabolic information is sent from peripheral organs/tissues and transmitted by neuronal relays consisting of afferent and efferent nerves, resulting in cooperative metabolic regulation of functions, such as energy expenditure, pancreatic β cell mass, lipid metabolism and adaptive thermogenesis. These mechanisms are considered to function in maintaining metabolic homeostasis at the whole-body level, but ironically, they contribute to major clinical features of the metabolic syndrome in states of continuous excessive energy intake.

We further identified anatomical and molecular mechanisms underlying selective β cell proliferation induced by neuronal signals. Furthermore, we have succeeded in enhancing both glucose-stimulated insulin secretion and pancreatic β cell proliferation by optogenetic stimulation of vagal nerve innervating the pancreas. Thus, by taking advantage of these features of the neuronal system, metabolic homeostasis can be properly maintained at the whole-body level. The inter-organ system is a potential target of developing therapeutic strategies for metabolic disorders including diabetes.

Spinal cord injury (SCI) causes continuous disability in the motor, sensory, and higher neuronal functions, which depends on insufficient neuronal regeneration spontaneously in the adult. The neuronal network regeneration is inhibited by the molecular expressions from the cells in the central nervous system (CNS). CNS contains many types of cells that control neuronal regeneration, and recently Type A-pericyte has been identified as the cell that forms a scar around the lesion after injury. Pericyte proliferation in response to injury is an essential process to create the inhibitory environment in the CNS, however, the molecular mechanism that regulates pericyte proliferation has not been elucidated. Previously, we focused on the role of the circulating factor that controls CNS regeneration, because circulating factors leak into the CNS around the lesion after that injury disrupts the vascular barrier. In this talk, I would like to introduce our recent findings that circulating factor promotes pericyte scar formation in mice after SCI.

The interaction between the nervous system and the immune system has received much attention. Using a mouse model of cerebral infarction, we researched about the importance of immune responses during the acute and chronic phases after stroke. We have found that innate immune-mediated inflammation plays a major role in the acute phase, whereas a large number of T cells infiltrate the brain in the chronic phase. In particular, we found that regulatory T cells (Treg) acquire brain-specific properties and contribute to the recovery of neurological symptoms by regulating excessive activation of microglia and astrocytes.

Tregs are also elevated in the peripheral blood during the chronic phase of stroke, and when stroke recurs, Tregs are elevated and inflammation is suppressed, resulting in reduced infarct volume in the brain. Tregs with the same T cell receptor were more likely to infiltrate during initial and recurrent infarction, suggesting that once Tregs recognize the antigen, they can quickly reactivate and respond. We also identified oxytocin as another inhibitory factor that is elevated in the brain and peripheral blood during the chronic phase of stroke. Oxytocin reduced brain infarction, and oxytocin receptor antagonists delayed recovery of neurological symptoms during the chronic phase of stroke, suggesting that oxytocin may be important in suppressing inflammation.

The kidneys are highly developed organs and have a variety of functions. Currently, angiotensin II receptor blockers (ARBs) and sodium glucose cotransporter 2 (Sglt2) inhibitors are used to treat chronic kidney disease, but there are no fundamental treatments for the disease and new treatments are urgently needed. The autonomic nervous system, consisting of sympathetic and parasympathetic nervous systems, plays an important role in maintaining homeostasis in the body. Some immune cells have receptors for the neurotransmitters and respond to inflammation, such as infection, eliciting an immune response via the peripheral and central nervous system. The kidneys are very richly innervated by sympathetic nerves, while little parasympathetic innervation has been identified. Despite this, we have previously shown that vagus nerve stimulation exerts a very strong renoprotective effect. Using optogenetics and single-cell RNA-seq technology, we have also found that vagal afferent stimulation exerts tubular protective effects via medullary C1 neurons, sympathetic nerves, and spleen. Most recently, we have shown that acetylcholine-stimulated macrophages exert anti-inflammatory and renal protective effects by affecting other macrophages. In this symposium, I would like to introduce the anti-inflammatory and renoprotective mechanisms mediated by the autonomic nervous and immune systems that I have revealed and discuss for further research development.

Drug discovery research with protein-targeted small molecules is becoming difficult because proteins as potential drug targets are already exhausted. For this reason, RNA, which is responsible for protein synthesis, is attracting attention from pharmaceutical companies as new target for drug discovery. Since RNA is essential for the modulation of genetic information and gene regulation in human cells and for maintaining biological functions, it has been shown to be directly involved in many genetic diseases. However, unlike proteins, RNA molecules contain structured regions and are inherently flexible, and their conformational changes have been reported to play an important role in maintaining biological functions. In order to validate RNAs with these characteristics as new drug target to replace proteins, drug discovery research should be conducted from the perspective of chemistry, molecular biology and pharmacology. In this section, we will outline the examples of RNA-targeted small molecules under clinical development and characteristics of them, as well as novel approach in Reborna Biosciences, Inc.

Targeted Protein Degradation (TPD) is an emerging technology in the pharmaceutical industry. Heterobifunctional degraders, known as proteolysis-targeting chimeras (PROTACs), offer an attractive and novel modality. These degraders consist of a Protein of Interest (POI) binder and a binder that recruits E3 ubiquitin ligase, which are joined by a linker. They effectively hijack the ubiquitin-proteasome system, leading to proteolysis of POI.

This novel approach of targeted proteolysis holds significant promise as a therapeutic strategy to regulate protein levels in previously “undruggable” targets, including proteins lacking enzymatic activity or proteins having scaffold function.

In this session, we will introduce a novel therapeutic strategy for the cancer-immunotherapy by combining IRAK-M (interleukin 1 receptor associated kinase 3) silent binder and targeted protein degradation technology that shifts the balance between tolerance and immunity by releasing immunosuppressive activity, leading to a more favorable tumor microenvironment to enhance host immunity.

Nucleic acid medicines represented by antisense oligonucleotides (ASO) and siRNA have been attracting attention as a new modality to treat genetic disorders and intractable diseases. To date, 17 drugs in this modality have been approved.  A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties such as increased resistance to nuclease and improved target binding affinity. The toxicity associated with chemical modification and efficient delivery to target tissues are the major hurdles remaining for further application of nucleic acid medicines to a wider range of disorders.

　Heteroduplex oligonucleotides (HDO) is a platform technology which could be applied to solve these issues. HDO has the features of DNA/RNA or DNA/DNA double-stranded oligonucleotide consisting of the ASO strand and a complementary sequence to the ASO as carrier strand which is conjugated with specific ligands. Ligand-molecules can work to deliver HDO to the target tissues. Now, research and development based on HDO is actively underway as a new modality that improves overall drug discovery potentials. Toxicity reduction and efficient delivery are the critical elements to be realized in the HDO format by utilizing the characteristics of double stranded structure and introducing suitable ligands.

　In this presentation, current status and issues for nucleic acid medicines are summarized and expecting future approaches are discussed with referring to the HDO technology.

Cell therapy to regenerate organ function lost in the body is becoming real in the world. Based on their pluripotent and proliferative characteristics, induced pluripotent stem cells (iPSCs) can provide great opportunities to save patients suffering from diseases without effective treatments, and several iPSC-derived differentiated cells have already progressed into clinical trials. However, multiple challenges still exist in the development of iPSC-derived cell therapy product with respect to the demonstration of safety, efficacy and quality with scalable manufacturing process tolerable to the implantation in human. In safety perspective, absence of undifferentiated iPSCs and malignant transformed cells after differentiation needs to be proven with long term tumorigenicity study. In efficacy studies, translatability of immunocompromised animals used as recipients should be considered regarding immune responses and dosage. For quality control, precise, stable and reliable parameters are required to realize the ideal cell product although differentiated cells are often heterogeneous. In this presentation, the potential of cell therapy and challenges in the product development are introduced by showing some actual cases of iPSC-derived pancreatic islet cells (iPIC), a novel cell therapy product candidate for type 1 diabetes.

A critical role of serotonergic neurons has been shown in reward and aversive information processing. Rewarding stimuli activate serotoninergic neurons in the dorsal raphe nucleus (DRN), while optogenetic stimulation of DRN serotoninergic neurons elicits reward-like effects. However, pharmacological enhancement of serotonin neurotransmission produces neither rewarding nor aversive effects. These findings suggest the existence of another serotonergic neuron that plays an opposite role to DRN in processing reward and aversion information. Previous reports have suggested that the median raphe nucleus (MRN) is involved in processing negative emotional stimuli. To clarify the role of MRN serotoninergic neurons in these processing, we recorded changes in serotonergic activity in mice in response to rewarding and aversive stimuli. We also used optogenetic manipulation to test whether these changes were sufficient to induce rewarding and aversive behaviors. The GCaMP fluorescence from MRN serotonergic neurons decreased in response to rewarding stimuli and increased after aversive stimuli. Optogenetic inhibition of MRN serotonergic neurons induced reward-related behavior, while optogenetic stimulation elicited aversion-related behavior. Moreover, we found that the projection pathway of MRN serotonergic neurons to the interpeduncular nucleus was important for these information processes. These results suggest that MRN serotonergic neurons play a key role in processing reward and aversive information and have an opposite function to the DRN.

Central serotonergic system has multiple roles in animal physiology and behavior, including sleep-wake regulation. However, its role in the regulation of brain energy metabolism accompanying the sleep-wake state changes of animals has remained unclear. We demonstrated that in vivo optogenetic activation of raphe serotonergic neurons increased cortical neuronal intracellular levels of adenosine triphosphate (ATP), an indispensable cellular energy molecule. The serotonergic neuronal activation-induced increase in neuronal ATP level was suppressed by inhibiting neuronal uptake of lactate derived from astrocytes, a type of glial cells. The serotonergic neuronal activation increased cortical astrocytic Ca²⁺ and cAMP levels and extracellular lactate concentrations, suggesting the facilitation of lactate release from astrocytes. Furthermore, chemogenetic inhibition of raphe serotonergic neurons partly attenuated the increase in cortical neuronal intracellular ATP levels as arousal increased in mice. In conclusion, raphe serotonergic neuronal activation increased in cortical neuronal intracellular ATP levels, partly mediated by the facilitation of astrocyte-neuron lactate shuttle, which contributes to state-dependent regulation of neuronal intracellular energy levels.

Serotonin (5-HT) is the neurotransmitter which has been strongly implicated in aggression in species ranging from invertebrates to humans. However, little is known about how the 5-HT system is modulated when animals engage in species-typical (adaptive) or escalated aggressive behavior. Previously, we have shown that glutamatergic input in the dorsal raphe nucleus (DRN) is increased when male mice are engaging in an elevated level of aggressive behavior after social provocation. We found that glutamatergic projections from the lateral habenula (LHb) to the DRN were activated by the social provocation, and optogenetic activation of the LHb-DRN projecting neurons increased aggressive behavior of male mice. Anatomical analysis showed that DRN neurons that receive input from the LHb were mainly non-serotonergic and they project to the ventral tegmental area (VTA). Indeed, optogenetic activation of the DRN to VTA projecting neurons increased inter-male aggression. On the other hand, we found that optogenetic activation of 5-HT neurons blocked social instigation-heightened aggression without affecting species-typical level of aggression. These results indicate that the DRN contains both aggression-accelerating neurons and aggression-decelerate neurons in the situation of social provocation.

We have demonstrated that the activation of serotonin neurons in the median raphe nucleus (MRN) facilitated anxiety- and fear-related behavior while that in the dorsal raphe nucleus (DRN) exerted antidepressant-like effects. Nonetheless, drawing a direct causal link between the neuronal activity of serotonin neurons and negative/positive emotions remains to be determined because the results of traditional behavioral tests have large variances and are difficult to interpret. A facial expression analysis might be a solution. It would be a more direct assessment of emotional responses. We recorded alterations in facial expressions using a high-resolution camera and employed image analysis based on Histogram of Oriented Gradients (HOG) features to assess changes in facial expression before and after the activation of serotonin neurons. I found that the activation of MRN serotonin neurons alone resulted in significant alterations in facial expressions, even without any external environmental stimuli. Remarkably, the facial expressions induced by MRN activation bore similar to those elicited by aversive stimuli. Furthermore, preliminary results showed that the activation of DRN serotonin neurons could induce positive emotion-related facial expressions. These results collectively suggest that serotonin neurons in the MRN are involved in negative emotions, while those in the DRN are involved in positive emotions.

[Background] The number of patients with depression is increasing, but the cause of the onset remains unclear. We found that exposure of social defeat stress to Shati/Nat8l increased its expression only in the striatum. Shati/Nat8l has N-acetyltransferase activity and synthesizes N-acetylaspartic acid (NAA) from L-aspartic acid and acetyl-CoA. One of the causes of depression is the BDNF hypothesis, and there are reports that the expression level of BDNF is epigenetically regulated. Since Shati/Nat8 uses acetyl-CoA, which is necessary for acetylation, as a substrate, we focused on the regulation of acetylation of BDNF and performed this study to clarify the relationship with Shati/Nat8l.

[Methods/Results] BDNF levels in the striatum of mice exposed to social defeat stress for 10 days were measured, and mRNA levels increased. Furthermore, BDNF histone acetylation was also measured by chromatin immunoprecipitation, and an increase was observed. Shati/Nat8l striatal local KO mice were found to be resistant to social defeat stress in various behavioral experiments.

[Discussion] We found that the reduction of Shati/Nat8l in the mouse striatum plays an important role in the development of resistance to depression-like behavior. Part of the mechanism is thought to be through regulation of BDNF acetylation. There are few reports on the relationship between the striatum and the onset of depression, and the results of this study indicate the possibility of Shati/Nat8l as a therapeutic target for depression in the future.

The number of patients with mood disorders including depression has increased by 700,000 over the past decade. But, the rate of consultations with psychiatrists is still low. It is thought that depression patients manifest not only psychiatric symptoms but also various other symptoms such as insomnia, fatigue, anorexia, and headache. On the other hand, chronic diseases such as cancer, chronic pain, cardiac disease, diabetes, dementia, and epilepsy tend to cause depression, and side effects of drugs such as interferon (IFN) and steroids can also cause depression. Early detection and treatment are effective in depression. It is important that general physicians detect depression in patients at an early stage and provide early intervention in collaboration (liaison) with psychiatrists. However, the diagnosis of depression is mainly by a psychiatrist interview but not by objective criterion such as blood tests. Thus, the depression biomarkers to determine the onset and severity are needed. In this symposium, we will introduce the development of depression biomarkers based on the findings from basic research using animal models of depression, and their application to clinical research in the field of psychiatric liaison.

This presentation will explore the current diagnosis and treatment for depression, utilizing insights from a recently published, comprehensive review.

Depression is diagnosed based on established diagnostic criteria. According to these criteria, individuals must exhibit five or more characteristic depressive symptoms, including depressed mood and diminished interest, lasting at least two weeks. Severity is assessed using rating scales that evaluate specific depressive symptoms. While there might be some concerns regarding these diagnostic criteria and rating scales, they are essential for accurately assessing patients.

Preventative measures range from universal to selective and indicated interventions. Treatment options encompass psychotherapy and antidepressant treatments for initial depressive episodes, and for recurrent or persistent depression, approaches include augmentation, combination treatments, and neuromodulation. Guided self-help has shown potential to be as effective as standard care. The term "difficult-to-treat depression" has recently been introduced, describing cases where depression continues to be burdensome despite standard treatment efforts. There is an increasing focus on emerging treatments, including neuromodulation, glutamatergic drugs, and mindfulness-based cognitive behavioral therapy.

Depression has been the most common cause of disability, and affects as many as one in ten individuals at any given time｣ｬboth in worldwide and China. The COVID-19 pandemic in past 3 years increases nearly 30% of the patients with MDD, especially among younger people (COVID-19 Mental Disorders Collaborators, 2021). Depression presents a global health crisis, and there is an urgent need to reduce its burden. Antidepressant treatment, mainly working as to regulate monoamine neurotransmitters, has been the present treatment mainstream in China. While, there still is approximately one-third of treated patients with MDD remaining non-remission. Traditional Chinese medicine (TCM) has a long history of understanding and treating depression, and has been used widely in treating patients with depression in China. More recent clinical trials have been carried out to assess the efficacy and safety of TCM, and to explore the mechanisms of action in relation to the treatment of depression. In this session, we would like to present the effectiveness and application of TCM in the treatment of depression in China, consider their possible mechanisms, and characterize the relationships between their efficacy and mechanisms.

Epigenetic regulation mechanisms, in which the expression state of a gene is altered by changes in intracellular conditions or external stimuli, independent of DNA sequence, have attracted much attention since they have been found to be "key" in various acquired diseases, such as cancer and lifestyle-related diseases. We hypothesized that DNA sequence-independent epigenetic regulation of expression is involved in the development of cataracts, and initiated our research based on the fact that there are patients who show differences in the progression of cataracts between right and left eye despite having the same organs. 

In this study, we screened by galactose-induced cataract model using rat lenses and found that histone acetyltransferase inhibitors could resolve the opacity once formed. In order to clarify the mechanism by which the opacity was resolved, we prepared lens sections and observed that new lens fiber cells were formed in the arch region and pushed the vacuoles in the cortex into the interior. In addition, microarray analysis of cataract model animals (ICR rats) and human samples identified genes whose expression levels fluctuate with the progression of cataracts. 

Currently, research into the development of a new treatment for retinopathy based on the findings from the development of a cataract treatment has also been initiated, and we would also like to discuss the latest findings of this study.

Approximately 28,000 long noncoding RNAs (lncRNAs) are transcribed from the human genome, with some localized in the nucleus. These lncRNAs have been revealed to play vital and diverse roles in nuclear functions, including transcriptional regulation, DNA damage repair, and maintenance of nuclear compartments. Importantly, dysregulation of these lncRNAs has been linked to many human diseases, especially cancer. Here we show that TUG1, a highly expressed lncRNA in many types of cancers, plays a crucial role in resolving pathogenic R-loops, a source of DNA damage and genome instability. Depleting TUG1 by antisense oligonucleotides (ASO) resulted in an overabundant R-loops and DNA damage, leading to significant inhibition of cell proliferation. In a glioblastoma xenograft mouse model, treatment with TUG1 ASO, coupled with a tumor-specific drug delivery system, substantially inhibited tumor growth. Our study unveils a novel role of lncRNA in maintaining genome stability in cancer cells and provides a strong rationale for targeting TUG1 as a potent therapeutic approach for cancer treatment.

Histone post-translational modification is one of important epigenetic mechanisms to regulate chromatin structures and gene expression. Many kinds of histone-modifying enzymes are reported, and regulation strategies for such protein complexes have been actively studied. In the field of chemical epigenetics, it is highly demanded to develop novel molecules that can modulate activity of target histone-modifying enzymes. However, it is still difficult to identify specific modulators due to the structural complexity of these types of proteins.

We aimed to develop novel molecules that modulate the levels of histone H3 lysine 27 (H3K27) methyl marks by targeting polycomb repressive complex 2 (PRC2). PRC2 contains four subunits (EZH2, EED, SUZ12 and RbAp46/48), and catalyzes sequential methylation reactions at H3K27. Both the gain-of-function and loss-of-function mutations of PRC2 have been reported in several types of cancers. Therefore, development of PRC2 inhibitors, and also activators can be effective for some types of cancers. 

In the presentation, our recent results will be shown to discuss strategies for modulation of H3K27 methylation with molecules.

Chromatin modifications, which involve alterations in DNA methylation and histone modifications, have an epigenetic influence on biological processes such as cellular differentiation and developmental transitions. Among the various post-translational histone modifications, lysine methylation has been extensively studied. The methylation of histone lysine residues is a reversible process, managed by enzymes known as histone lysine methyltransferases and demethylases. One specific demethylase, lysine-specific demethylase 1 (LSD1), removes methyl groups from mono- and di-methylated lysine 4 of histone H3 (H3K4me1/2) through an enzymatic oxidation reliant on flavin adenine dinucleotide. In addition, LSD1 has been highlighted as a potential target for lung cancer, neuroblastoma, and leukemia. Therefore, we have performed a drug discovery study on LSD1 and identified small molecules and peptides as LSD1 inhibitors. In this presentation, we introduced and discussed some peptide-based LSD1 inhibitors and proteolysis targeting chimeras (PROTACs) designed based on SNAIL1, which is a member of the SNAIL/SCRATCH family of transcription factors and interacts with LSD1.

Liquid-liquid phase separation (LLPS) is a phenomenon in which different proteins spontaneously assemble within a cellular compartment that recognized as a membraneless organelle. LLPS plays important roles in neurons, particularly in the structural organization of postsynaptic densities.

Calmodulin-dependent protein kinase II (CaMKII) is a major synaptic protein kinase and a key regulator of synaptic plasticity. CaMKII forms calcium-gated condensates with the glutamate receptor subunit GluN2B via LLPS. Since the interaction with GluN2B maintains CaMKII in an active conformation, we hypothesize that the LLPS of CaMKII serves as a molecular machine to maintain biochemical activity as a memory. Given that a protein in a liquid condensate can be replaced by an external protein, a protein cannot persist in the condensate for long time, but the condensate itself can persist for a long time. This unique property reasonably explains how the condensate of CaMKII can be a memory unit that lasts longer than the lifetime of the protein.

In this symposium, I will present our recent progress on how CaMKII maintains its activity at the synapse using the in vitro reconstituted LLPS assay and nanobody-assisted synaptic translocation of CaMKII into the synapse combined with the FRET sensor of CaMKII.

The ubiquitin-proteasome system (UPS) plays an essential role in maintaining proteostasis by selective degradation of aberrant proteins. Ubiquitin inclusion is a hallmark of almost all neurodegenerative diseases, thereby suggesting that dysfunction of the UPS leads to neurodegenerative diseases. Recently, ubiquitin-dependent phase separation and transition have been implicated in disease pathogenesis, but little is known about the molecular mechanisms involved. We previously found that the nuclear proteasomes undergo liquid-liquid phase separation (LLPS) with ubiquitylated substrates upon hyperosmotic stress, forming proteolytic droplets. Furthermore, we have found that various stresses induce the formation of distinct co-condensates of ubiquitin and proteasomes, suggesting that their co-condensation is the universal cellular response machinery. Unexpectedly, some of the co-condensates are unlikely proteolytic droplets. They exhibit the hydrogel-like properties and potentially non-proteolytic functions. Here, we present and discuss the current perspectives on the co-condensation of ubiquitin and proteasome.

Autophagy is an intracellular degradation system that contributes not only to securing nutrient source during starvation, but also to intracellular clearance under normal conditions. Upon starvation, a new lipid membrane called the isolation membrane is generated from the pre-autophagosome structure (PAS), which is the starting point of autophagy, and forms a double membrane autophagosome by wrapping around a portion of the cytoplasm. We were the first to identify that PAS is a droplet formed by the liquid-liquid phase separation of the Atg protein. However, it is still unclear what kind of reactions occur in PAS using the droplet characteristics. We performed an in vitro analysis of the introduction of downstream Atg proteins into the initial PAS droplets formed by purified Atg1 complex and found that the efficiency of introduction into the droplets varied among Atg proteins. Interestingly, the Atg12-Atg5-Atg16 complex, the E3 enzyme of the lipid-binding reaction system of the autophagy core factor Atg8, was significantly enriched in the droplet, whereas Atg4, the delipidase of Atg8, was not, thus promoting the formation of lipidated Atg8 in the droplet. One of the reasons why PAS is a droplet may be that it provides an efficient site for enzymatic reactions essential for autophagy to occur.

The mechanism underling dysfunction of cellular proteostasis on α-synuclein (αSyn) leading to pathogenesis of synucleinopathy remains unclear. Recently, we reported that the binding of an RNA secondary structure G-quadruplex (G4RNA) to a prion-like protein causes its sol-gel phase transition, leading to neurodegeneration in a hereditary neurodegenerative disease (Sci Adv. 2021). Herein, we demonstrate that G4RNA assembly forms scaffolds for αSyn aggregation, contributing to neurodegeneration. Purified αSyn binds to G4RNA specifically, but not other structures. G4RNA undergo Ca²⁺-induced phase separation and assembly, accelerating αSyn sol-gel phase transition. In mouse primary neurons, G4RNA aggregation was immediately observed under cellular stress conditions, thereafter co-aggregation of αSyn with G4RNA was occurred. Moreover, RIP-seq revealed endogenous G4RNA associated with αSyn aggregation. We also demonstrated that artificial assembly of G4RNA using an optogenetic approach initiated αSyn aggregation, thereby elicits neuronal dysfunction. Finally, G4 ligand can ameliorate G4RNA-elicited αSyn phase transition and neurodegeneration. These results suggest that G4RNA assembly evoked by various cellular stress triggers to develop aggregation of αSyn, which may be a cellular mechanism underlying onset of sporadic synucleinopathy.

Since the pandemic of COVID-19, various strains of the virus have appeared causing stringency on medical resources each time. Predicting the short- and long-term prognosis of severity of COVID-19 patients will not only provide indicators for prevention or treatment, but it is also extremely useful in building systems for securing medical supplies, resources, and hospital beds.

We have developed AI models that predict long- and short-term changes in disease severity (increasing severity, recovery, and death) using individuals' conditions and medical histories for the patients infected with COVID-19 and hospitalized in ICU, during the period from 2020 to 2022, so-called 1^st to 6^th pandemic wave. We adopted Deep Insight method which converts numerical data into an image format to build a classifier using deep learning framework, in which the accuracy and kappa scores are as high as 0.9 and 0.8, respectively towards the validation data of the specific period identical to the training data.

However, in reality, building and optimization of AI model with the latest data requires a huge amount of procedures to ensure the security of personal information for use of medical data for analysis outside the hospital, and a lot of interactions and resources in and between multiple organizations including data cleansing, AI model optimization, data and model management.

Here we propose and demonstrate a framework for Medical MLOps, using the above COVID-19 severity prediction model as an example, towards the goal of end-to-end automatic orchestration of data flow, model tuning and model deployment at clinical site for sustainable operation for medical AI models.

We have developed a SaMD to implement dCBT for insomnia with the aim of promoting cognitive behavioral therapy recommended by guidelines in the treatment of insomnia. After consulting with the PMDA, we have conducted a clinical trial under the GCP ordinance. The clinical trial was conducted at several medical institutions in Japan from May 2021 for patients with insomnia according to ICSD-3 diagnostic criteria. Patients were randomized to a therapeutic app group or a sham app group, with an 8-week treatment intervention followed by a 2-week follow-up. The primary endpoint, change (mean ± SD) in the Athens Insomnia Scale (AIS), was -6.7 ± 4.4 (95% CI -7.6 to -5.8) for the treatment app versus -3.3 ± 4.0 (95% CI -4.1 to -2.5), p < 0.001, indicating a significant treatment effect. Furthermore, at 10 weeks after the intervention, the AIS values were 5.9 ± 4.0 (95% CI, 5.1 to 6.7) for the therapeutic app and 9.7 ± 4.9 (95% CI, 8.7 to 10.6) for the sham app, p < 0.001. The results of the clinical trial confirmed that the treatment effect was maintained without the side effect of rebound insomnia, a withdrawal symptom that is an issue with sleeping pills. Based on the results of the clinical trial in Japan, the product was approved as a SaMD for insomnia on February 15, 2023.

In this presentation, I will introduce the research, development, and social implementation of "sheet-type bio-electrical potential measurement systems," utilizing flexible, and stretchable electronic devices realized by highly integrating functional organic materials. Specifically, I will introduce the electrical characteristics of lightweight electronics that can be attached to human skin to accurately measure bio-electrical potentials and present our efforts to develop the world's first medical-use patch-type electroencephalograph (EEG) using these technologies. We are currently working with several medical institutions to develop algorithms for measuring dementia, menopausal disorders, developmental disorders, and epilepsy and their visualization (EEG AI analysis tool). Furthermore, we are working to develop the Brain-Tech market, which contributes to our life, not only in medicine, but also in healthcare.

Part of this research was supported by JST Moonshot Goal 1 (JPMJMS2012).

Shionogi's strategic plan, STS2030, represents a pivotal shift from a conventional research-focused pharmaceutical entity to a provider of "Healthcare as a Service (HaaS)," primarily focused on addressing customer healthcare needs.

We are pursuing several key initiatives. These include investments in digital health solutions, such as mobile apps, wearable devices, and data analytics platforms for real-time patient health monitoring. They are also fostering collaborations with digital health startups and technology companies to fuse pharmaceutical expertise with advanced tech for innovative healthcare solutions.

Furthermore, a strong commitment to patient-centricity is evident, with efforts to enhance patient engagement via digital channels, providing easy access to information and virtual care platforms for remote consultations and monitoring.

In summary, Shionogi's strategy centers on harnessing digital advancements in the pharmaceutical sector. The STS2030 plan signifies our transformation into a HaaS provider, emphasizing digital health, collaborations, process enhancement, and patient-centric approaches. Our goal is to remain at the forefront of innovation in an ever-evolving healthcare landscape.

Single-cell RNA-sequencing (scRNA-seq) can obtain comprehensive gene expression information on tens of thousands of single cells and accelerate pathophysiological insights at the single cell level. However, there are many technical challenges in terms of sensitivity and accuracy. We previously developed TAS-Seq, a sensitive and accurate scRNA-seq method, but it only applicable to nanowell-based system, which limits its utility. To solve these issues, we have developed a cDNA amplification method, TAS-Seq2, which is highly sensitive and broadly applicable to various scRNA-seq platforms, including 10X Chromium and BD Rhapsody. TAS-Seq2 exerted 1.5-2 times higher gene-detection sensitivity and better cell subset separation than the original methods.

Next, we analyzed silica-induced lung fibrosis using TAS-Seq, and identified C1q as a novel specific marker for interstitial macrophages (IMs). By using Ccr2^-/- mice, BrdU, and parabiosis, C1q⁺ Lyve1^- CD163^- IMs increased depending on local proliferation and CCR2-independent monocytes. In addition, C1q itself induced lung fibrosis, fibroblasts/epithelial cell activation with gene-expression changes associated with fibrosis. Furthermore, in human fibrotic lungs, C1Q⁺ SELENOP⁺ IMs were found as a distinct population from SPP1⁺ macrophages, and C1Q⁺ IMs were increased in fibrotic area. These results suggest that C1Q⁺ IMs might exacerbate lung fibrosis by inducing fibroblast and epithelial cell activation through the production of C1q.

Post-intensive care syndrome (PICS), characterized by physical and mental/cognitive symptoms occurs in some intensive care unit (ICU) survivors.  We developed a mouse model of PICS by combining acute lung injury (ALI) with lower limb immobilization in both young and aged mice. Clinically, these animals exhibited characteristics of PICS including disuse muscle atrophy, signs compatible with depression, and pulmonary and systemic inflammation. Single cell transcriptomic analysis in brain demonstrated the effect of ageing in several cell types, specially microglia and endothelial cells and analysis of treatment effect showed that aged mice is more susceptible to changes in gene expression by the induction of ALI. Shared upregulated genes in aged and young treated mice were identified. Our data indicates that the combination of ALI and immobilization induces gene programs in brain associated with depression or neurodegenerative disorders and differentially affects young and aged individuals.

Human T cell receptors (TCRs) develop an extremely diverse repertoire, among which the pathogen-specific T cells have high diversity and low frequency in homeostatic conditions. Single-cell–based TCR- and RNA-sequencing analyses enable us to acquire paired TCR sequences, together with the gene expression signature of these individual T cells; however, the throughput is limited. To identify clonotypes associated with SARS-CoV-2 defense, we filtered T cells by recall antigen stimulation followed by sorting of responded T cells, in addition to other layers of analyses, such as deep bulk TCR sequencing and public TCR sequence databases. From convalescent COVID-19 patients, we identified TCRs of public circulating follicular helper (cTfh) clonotypes that were significantly expanded in patients that have recovered from mild COVID-19 but not in severely affected patients. In SARS-CoV-2 vaccinees, we found that highly responsive cTfh cells were more abundant in donors with sustained anti-S antibody titer (sustainers) than those who had declined antibody titer (decliners) after the 2nd dose of vaccination. These T-cell clonotypes tend to recognize conserved epitopes, some of which could be presented on various HLAs and activate T cells in multiple donors. These high-resolution platforms could provide invaluable information for future vaccine development.