堀江 健太

The thymus has the ability to regenerate from acute injury caused by radiation, infection, and stressors. In addition to thymocytes, thymic epithelial cells in the medulla (mTECs), which are crucial for T cell self-tolerance by ectopically expressing and presenting thousands of tissue-specific antigens (TSAs), are damaged by these insults and recover thereafter. However, given recent discoveries on the high heterogeneity of mTECs, it remains to be determined whether the frequency and properties of mTEC subsets are restored during thymic recovery from radiation damage. Here we demonstrate that acute total body irradiation with a sublethal dose induces aftereffects on heterogeneity and gene expression of mTECs. Single-cell RNA-sequencing (scRNA-seq) analysis showed that irradiation reduces the frequency of mTECs expressing AIRE, which is a critical regulator of TSA expression, 15 days after irradiation. In contrast, transit-amplifying mTECs (TA-mTECs), which are progenitors of AIRE-expressing mTECs, and Ccl21a-expressing mTECs, were less affected. Interestingly, a detailed analysis of scRNA-seq data suggested that the proportion of a unique mTEC cluster expressing Ccl25 and a high level of TSAs was severely decreased by irradiation. In sum, we propose that the effects of acute irradiation disrupt the heterogeneity and properties of mTECs over an extended period, which potentially leads to an impairment of thymic T cell selection.

<jats:title>Abstract</jats:title><jats:p>One hallmark of some autoimmune diseases is the variability of symptoms among individuals. Organs affected by the disease differ between patients, posing a challenge in diagnosing the affected organs. Although numerous studies have investigated the correlation between T cell antigen receptor (TCR) repertoires and the development of infectious and immune diseases, the correlation between TCR repertoires and variations in disease symptoms among individuals remains unclear. This study aimed to investigate the correlation of TCRα and β repertoires in blood T cells with the extent of autoimmune signs that varies among individuals. We sequenced TCRα and β of CD4<jats:sup>+</jats:sup>CD44<jats:sup>high</jats:sup>CD62L<jats:sup>low</jats:sup> T cells in the blood and stomachs of mice deficient in autoimmune regulator (<jats:italic>Aire</jats:italic>) (AIRE KO), a mouse model of human autoimmune polyendocrinopathy‐candidiasis‐ectodermal dystrophy. Data analysis revealed that the degree of similarity in TCR sequences between the blood and stomach varied among individual AIRE KO mice and reflected the extent of T cell infiltration in the stomach. We identified a set of TCR sequences whose frequencies in blood might correlate with extent of the stomach manifestations. Our results propose a potential of using TCR repertoires not only for diagnosing disease development but also for diagnosing affected organs in autoimmune diseases.</jats:p>

<jats:title>Abstract</jats:title><jats:p>Sphingosine 1-phosphate receptor 1 (S1PR1), a G protein-coupled receptor, is required for lymphocyte trafficking, and is a promising therapeutic target in inflammatory diseases. To find potent S1PR1 antagonists, identification of the structural basis for drug efficacy is important. Here, we synthesized a novel antagonist, KSI-6666, that persistently inhibits S1PR1 activity and effectively suppresses pathogenic inflammation. Metadynamics simulation suggested that the interaction of a benzene ring moiety in KSI-6666 with a methionine residue in the ligand-binding pocket of S1PR1 inhibits the dissociation of KSI-6666 from S1PR1, generating a metastable binding state. Consistently,<jats:italic>in vitro</jats:italic>functional and mutational analyses revealed that KSI-6666 causes pseudoirreversible inhibition of S1PR1, dependent on the methionine residue of the protein and substituents on the distal benzene ring of KSI-6666. Moreover,<jats:italic>in vivo</jats:italic>study suggested that this pseudoirreversible inhibition is responsible for the persistent activity of KSI-6666. These findings will contribute to the rational design of potent S1PR1 antagonists for the treatment of inflammatory disorders.</jats:p>

<jats:p>Medullary thymic epithelial cells (mTECs) are critical for self-tolerance induction in T cells via promiscuous expression of tissue-specific antigens (TSAs), which are controlled by the transcriptional regulator, AIRE. Whereas AIRE-expressing (Aire<jats:sup>+</jats:sup>) mTECs undergo constant turnover in the adult thymus, mechanisms underlying differentiation of postnatal mTECs remain to be discovered. Integrative analysis of single-cell assays for transposase-accessible chromatin (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) suggested the presence of proliferating mTECs with a specific chromatin structure, which express high levels of Aire and co-stimulatory molecules, CD80 (Aire<jats:sup>+</jats:sup>CD80<jats:sup>hi</jats:sup>). Proliferating Aire<jats:sup>+</jats:sup>CD80<jats:sup>hi</jats:sup> mTECs detected using Fucci technology express a minimal number of Aire-dependent TSAs and are converted into quiescent Aire<jats:sup>+</jats:sup>CD80<jats:sup>hi</jats:sup> mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit-amplifying Aire<jats:sup>+</jats:sup>mTEC precursors during the Aire<jats:sup>+</jats:sup>mTEC differentiation process of the postnatal thymus.</jats:p>

<jats:title>Abstract</jats:title><jats:p>The impact of spaceflight on the immune system has been investigated extensively during spaceflight missions and in model experiments conducted on Earth. Data suggest that the spaceflight environment may affect the development of acquired immunity, and immune responses. Herein we summarize and discuss the influence of the spaceflight environment on acquired immunity. Bone marrow and the thymus, two major primary lymphoid organs, are evidently affected by gravitational change during spaceflight. Changes in the microenvironments of these organs impair lymphopoiesis, and thereby may indirectly impinge on acquired immunity. Acquired immune responses may also be disturbed by gravitational fluctuation, stressors, and space radiation both directly and in a stress hormone-dependent manner. These changes may affect acquired immune responses to pathogens, allergens, and tumors.</jats:p>

<jats:title>Abstract</jats:title><jats:p>The environment experienced during spaceflight may impact the immune system and the thymus appears to undergo atrophy during spaceflight. However, molecular aspects of this thymic atrophy remain to be elucidated. In this study, we analysed the thymi of mice on board the international space station (ISS) for approximately 1 month. Thymic size was significantly reduced after spaceflight. Notably, exposure of mice to 1 × <jats:italic>g</jats:italic> using centrifugation cages in the ISS significantly mitigated the reduction in thymic size. Although spaceflight caused thymic atrophy, the global thymic structure was not largely changed. However, RNA sequencing analysis of the thymus showed significantly reduced expression of cell cycle-regulating genes in two independent spaceflight samples. These reductions were partially countered by 1 × <jats:italic>g</jats:italic> exposure during the space flights. Thus, our data suggest that spaceflight leads to reduced proliferation of thymic cells, thereby reducing the size of the thymus, and exposure to 1 × <jats:italic>g</jats:italic> might alleviate the impairment of thymus homeostasis induced by spaceflight.</jats:p>

<jats:title>Abstract</jats:title><jats:p>Secondary lymphoid organs are critical for regulating acquired immune responses. The aim of this study was to characterize the impact of spaceflight on secondary lymphoid organs at the molecular level. We analysed the spleens and lymph nodes from mice flown aboard the International Space Station (ISS) in orbit for 35 days, as part of a Japan Aerospace Exploration Agency mission. During flight, half of the mice were exposed to 1 <jats:italic>g</jats:italic> by centrifuging in the ISS, to provide information regarding the effect of microgravity and 1<jats:italic> g</jats:italic> exposure during spaceflight. Whole-transcript cDNA sequencing (RNA-Seq) analysis of the spleen suggested that erythrocyte-related genes regulated by the transcription factor GATA1 were significantly down-regulated in ISS-flown vs. ground control mice. GATA1 and Tal1 (regulators of erythropoiesis) mRNA expression was consistently reduced by approximately half. These reductions were not completely alleviated by 1 <jats:italic>g</jats:italic> exposure in the ISS, suggesting that the combined effect of space environments aside from microgravity could down-regulate gene expression in the spleen. Additionally, plasma immunoglobulin concentrations were slightly altered in ISS-flown mice. Overall, our data suggest that spaceflight might disturb the homeostatic gene expression of the spleen through a combination of microgravity and other environmental changes.</jats:p>

Hindlimb unloading (HU) of rodents has been used as a ground-based model of spaceflight. In this study, we investigated the detailed impact of 14-day HU on the murine thymus. Thymic mass and cell number were significantly reduced after 14 days of hindlimb unloading, which was accompanied by an increment of plasma corticosterone. Although corticosterone reportedly causes selective apoptosis of CD4+CD8+ thymocytes (CD4+CD8+DPs) in mice treated with short-term HU, the reduction of thymocyte cellularity after the 14-day HU was not selective for CD4+CD8+DPs. In addition to the thymocyte reduction, the cellularity of thymic epithelial cells (TECs) was also reduced by the 14-day HU. Flow cytometric and RNA-sequencing analysis suggested that medullary TECs (mTECs) were preferentially reduced after HU. Moreover, immunohistochemical staining suggested that the 14-day HU caused a reduction of the mTECs expressing autoimmune regulator (Aire). Our data suggested that HU impacts both thymocytes and TECs. Consequently, these data imply that thymic T cell repertoire formation could be disturbed during spaceflight-like stress.