Analyses of a Mutant Foxp3 Allele Reveal BATF as a Critical Transcription Factor in the Differentiation and Accumulation of Tissue Regulatory T Cells.

Foxp3 controls the development and function of regulatory T (Treg) cells, but it remains elusive how Foxp3 functions in vivo. Here, we established mouse models harboring three unique missense Foxp3 mutations that were identified in patients with the autoimmune disease IPEX. The I363V and R397W mutations were loss-of-function mutations, causing multi-organ inflammation by globally compromising Treg cell physiology. By contrast, the A384T mutation induced a distinctive tissue-restricted inflammation by specifically impairing the ability of Treg cells to compete with pathogenic T cells in certain non-lymphoid tissues. Mechanistically, repressed BATF expression contributed to these A384T effects. At the molecular level, the A384T mutation altered Foxp3 interactions with its specific target genes including Batf by broadening its DNA-binding specificity. Our findings identify BATF as a critical regulator of tissue Treg cells and suggest that sequence-specific perturbations of Foxp3-DNA interactions can influence specific facets of Treg cell physiology and the immunopathologies they regulate.

T-cell receptor repertoire analysis of CD4-positive T cells from blood and an affected organ in an autoimmune mouse model.

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 ß repertoires in blood T cells with the extent of autoimmune signs that varies among individuals. We sequenced TCRα and ß of CD4+ CD44high CD62Llow T cells in the blood and stomachs of mice deficient in autoimmune regulator (Aire) (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.

Plasticity of Foxp3(+) T cells reflects promiscuous Foxp3 expression in conventional T cells but not reprogramming of regulatory T cells.

The emerging notion of environment-induced reprogramming of Foxp3(+) regulatory T (Treg) cells into helper T (Th) cells remains controversial. By genetic fate mapping or adoptive transfers, we have identified a minor population of nonregulatory Foxp3(+) T cells exhibiting promiscuous and transient Foxp3 expression, which gave rise to Foxp3(-) ("exFoxp3") Th cells and selectively accumulated in inflammatory cytokine milieus or in lymphopenic environments including those in early ontogeny. In contrast, Treg cells did not undergo reprogramming under those conditions irrespective of their thymic or peripheral origins. Moreover, although a few Treg cells transiently lose Foxp3 expression, such "latent" Treg cells retained their memory and robustly re-expressed Foxp3 and suppressive function upon activation. This study establishes that Treg cells constitute a stable cell lineage, whose committed state in a changing environment is ensured by DNA demethylation of the Foxp3 locus irrespectively of ongoing Foxp3 expression.

Long-term hindlimb unloading causes a preferential reduction of medullary thymic epithelial cells expressing autoimmune regulator (Aire).

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.

Active demethylation of the Foxp3 locus leads to the generation of stable regulatory T cells within the thymus.

Stable expression of Foxp3 in regulatory T cells (Tregs) depends on DNA demethylation at the Treg-specific demethylated region (TSDR), a conserved, CpG-rich region within the Foxp3 locus. The TSDR is selectively demethylated in ex vivo Tregs purified from secondary lymphoid organs, but it is unclear at which stage of Treg development demethylation takes place. In this study, we show that commitment to a stable lineage occurred during early stages of murine thymic Treg development by engraving of lineage-specific epigenetic marks in parallel with establishment of a Treg-specific gene expression profile. TSDR demethylation was achieved through an active mechanism and involved enzymes of the ten-eleven-translocation family and hydroxylation of methylated cytosines, a modification that is implicated as an initiating step of mitosis-independent DNA demethylation pathways and has not yet been observed at specific loci during immune cell differentiation. Together, our results demonstrate that initiating TSDR demethylation during early stages of thymic Treg development commences stabilization of Foxp3 expression and guarantees full functionality and long-term lineage stability of Tregs.

Mitochondrial protein C15ORF48 is a stress-independent inducer of autophagy that regulates oxidative stress and autoimmunity.

Autophagy is primarily activated by cellular stress, such as starvation or mitochondrial damage. However, stress-independent autophagy is activated by unclear mechanisms in several cell types, such as thymic epithelial cells (TECs). Here we report that the mitochondrial protein, C15ORF48, is a critical inducer of stress-independent autophagy. Mechanistically, C15ORF48 reduces the mitochondrial membrane potential and lowers intracellular ATP levels, thereby activating AMP-activated protein kinase and its downstream Unc-51-like kinase 1. Interestingly, C15ORF48-dependent induction of autophagy upregulates intracellular glutathione levels, promoting cell survival by reducing oxidative stress. Mice deficient in C15orf48 show a reduction in stress-independent autophagy in TECs, but not in typical starvation-induced autophagy in skeletal muscles. Moreover, C15orf48-/- mice develop autoimmunity, which is consistent with the fact that the stress-independent autophagy in TECs is crucial for the thymic self-tolerance. These results suggest that C15ORF48 induces stress-independent autophagy, thereby regulating oxidative stress and self-tolerance.

Sample Preparation and Integrative Data Analysis of a Droplet-based Single-Cell ATAC-sequencing Using Murine Thymic Epithelial Cells.

Accessible chromatin regions modulate gene expression by acting as cis-regulatory elements. Understanding the epigenetic landscape by mapping accessible regions of DNA is therefore imperative to decipher mechanisms of gene regulation under specific biological contexts of interest. The assay for transposase-accessible chromatin sequencing (ATAC-seq) has been widely used to detect accessible chromatin and the recent introduction of single-cell technology has increased resolution to the single-cell level. In a recent study, we used droplet-based, single-cell ATAC-seq technology (scATAC-seq) to reveal the epigenetic profile of the transit-amplifying subset of thymic epithelial cells (TECs), which was identified previously using single-cell RNA-sequencing technology (scRNA-seq). This protocol allows the preparation of nuclei from TECs in order to perform droplet-based scATAC-seq and its integrative analysis with scRNA-seq data obtained from the same cell population. Integrative analysis has the advantage of identifying cell types in scATAC-seq data based on cell cluster annotations in scRNA-seq analysis.

Acute irradiation causes a long-term disturbance in the heterogeneity and gene expression profile of medullary thymic epithelial cells.

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.

Integrative analysis of scRNA-seq and scATAC-seq revealed transit-amplifying thymic epithelial cells expressing autoimmune regulator.

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+) 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+CD80hi). Proliferating Aire+CD80hi mTECs detected using Fucci technology express a minimal number of Aire-dependent TSAs and are converted into quiescent Aire+CD80hi mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit-amplifying Aire+mTEC precursors during the Aire+mTEC differentiation process of the postnatal thymus.

The CCR4-NOT deadenylase complex safeguards thymic positive selection by down-regulating aberrant pro-apoptotic gene expression.

A repertoire of T cells with diverse antigen receptors is selected in the thymus. However, detailed mechanisms underlying this thymic positive selection are not clear. Here we show that the CCR4-NOT complex limits expression of specific genes through deadenylation of mRNA poly(A) tails, enabling positive selection. Specifically, the CCR4-NOT complex is up-regulated in thymocytes before initiation of positive selection, where in turn, it inhibits up-regulation of pro-apoptotic Bbc3 and Dab2ip. Elimination of the CCR4-NOT complex permits up-regulation of Bbc3 during a later stage of positive selection, inducing thymocyte apoptosis. In addition, CCR4-NOT elimination up-regulates Dab2ip at an early stage of positive selection. Thus, CCR4-NOT might control thymocyte survival during two-distinct stages of positive selection by suppressing expression levels of pro-apoptotic molecules. Taken together, we propose a link between CCR4-NOT-mediated mRNA decay and T cell selection in the thymus.

Quantitative analysis reveals reciprocal regulations underlying recovery dynamics of thymocytes and thymic environment in mice.

Thymic crosstalk, a set of reciprocal regulations between thymocytes and the thymic environment, is relevant for orchestrating appropriate thymocyte development as well as thymic recovery from various exogenous insults. In this work, interactions shaping thymic crosstalk and the resultant dynamics of thymocytes and thymic epithelial cells are inferred based on quantitative analysis and modeling of the recovery dynamics induced by irradiation. The analysis identifies regulatory interactions consistent with known molecular evidence and reveals their dynamic roles in the recovery process. Moreover, the analysis also predicts, and a subsequent experiment verifies, a previously unrecognized regulation of CD4+CD8+ double positive thymocytes which temporarily increases their proliferation rate upon the decrease in their population size. Our model establishes a pivotal step towards the dynamic understanding of thymic crosstalk as a regulatory network system.

Dynamic Imprinting of the Treg Cell-Specific Epigenetic Signature in Developing Thymic Regulatory T Cells.

Regulatory T (Treg) cells mainly develop within the thymus and arise from CD25+Foxp3- (CD25+ TregP) or CD25-Foxp3+ (Foxp3+ TregP) Treg cell precursors resulting in Treg cells harboring distinct transcriptomic profiles and complementary T cell receptor repertoires. The stable and long-term expression of Foxp3 in Treg cells and their stable suppressive phenotype are controlled by the demethylation of Treg cell-specific epigenetic signature genes including an evolutionarily conserved CpG-rich element within the Foxp3 locus, the Treg-specific demethylated region (TSDR). Here we analyzed the dynamics of the imprinting of the Treg cell-specific epigenetic signature genes in thymic Treg cells. We could demonstrate that CD25+Foxp3+ Treg cells show a progressive demethylation of most signature genes during maturation within the thymus. Interestingly, a partial demethylation of several Treg cell-specific epigenetic signature genes was already observed in Foxp3+ TregP but not in CD25+ TregP. Furthermore, Foxp3+ TregP were very transient in nature and arose at a more mature developmental stage when compared to CD25+ TregP. When the two Treg cell precursors were cultured in presence of IL-2, a factor known to be critical for thymic Treg cell development, we observed a major impact of IL-2 on the demethylation of the TSDR with a more pronounced effect on Foxp3+ TregP. Together, these results suggest that the establishment of the Treg cell-specific hypomethylation pattern is a continuous process throughout thymic Treg cell development and that the two known Treg cell precursors display distinct dynamics for the imprinting of the Treg cell-specific epigenetic signature genes.

Impact of spaceflight on the murine thymus and mitigation by exposure to artificial gravity during spaceflight.

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 × g 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 × g 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 × g might alleviate the impairment of thymus homeostasis induced by spaceflight.

Down-regulation of GATA1-dependent erythrocyte-related genes in the spleens of mice exposed to a space travel.

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 g by centrifuging in the ISS, to provide information regarding the effect of microgravity and 1 g 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 g 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.

Unique properties of thymic antigen-presenting cells promote epigenetic imprinting of alloantigen-specific regulatory T cells.

Regulatory T cells (Tregs) are potential immunotherapeutic candidates to induce transplantation tolerance. However, stability of Tregs still remains contentious and may potentially restrict their clinical use. Recent work suggested that epigenetic imprinting of Foxp3 and other Treg-specific signature genes is crucial for stabilization of immunosuppressive properties of Foxp3+ Tregs, and that these events are initiated already during early stages of thymic Treg development. However, the mechanisms governing this process remain largely unknown. Here we demonstrate that thymic antigen-presenting cells (APCs), including thymic dendritic cells (t-DCs) and medullary thymic epithelial cells (mTECs), can induce a more pronounced demethylation of Foxp3 and other Treg-specific epigenetic signature genes in developing Tregs when compared to splenic DCs (sp-DCs). Transcriptomic profiling of APCs revealed differential expression of secreted factors and costimulatory molecules, however neither addition of conditioned media nor interference with costimulatory signals affected Foxp3 induction by thymic APCs in vitro. Importantly, when tested in vivo both mTEC- and t-DC-generated alloantigen-specific Tregs displayed significantly higher efficacy in prolonging skin allograft acceptance when compared to Tregs generated by sp-DCs. Our results draw attention to unique properties of thymic APCs in initiating commitment towards stable and functional Tregs, a finding that could be highly beneficial in clinical immunotherapy.

Development of a tightly-regulated tetracycline-dependent transcriptional activator and repressor co-expression system for the strong induction of transgene expression.

The teteracycline (Tc)-dependent and -inducible transcriptional activator (rtTA) system has been used to express regulated transgene expression in vitro and in vivo. However, previous reports have demonstrated that, even in the absence of Tc, the rtTA binds weakly to the tetracycline response element (TRE), leading to a low level of background activity. In order to reduce the leaky gene expression induced by rtTA, we previously established a tightly regulated system (A-IRES-R system) that makes use of both the rtTA (A) and a Tc-dependent repressor (TetR-Kruppel-associated box; KRAB) (R). In addition, others have described a transactivator rtTA2-M2 (M2) that displays higher sensitivity to Dox than rtTA. In this study, to further develop the A-IRES-R system, we generated a derivative Tc system (M2-IRES-R system) that co-expresses both rtTA-M2 and TetR-KRAB from a single vector. We show that compared to the A-IRES-R system, the M2-IRES-R system leads to a greater level of induced TRE-mediated transcription in the presence of doxycycline (Dox) and yet displays a similar level of basal TRE-mediated transcription in the absence of Dox. Furthermore, the M2-IRES-R system also displays less leaky gene expression in the absence of Dox compared to rtTA-M2 and rtTA systems. Taken together, our results suggest that the M2-IRES-R system enables to tightly regulate and highly induce the expression of transgene compared to other systems.

Transgenic up-regulation of alpha-CaMKII in forebrain leads to increased anxiety-like behaviors and aggression.

BACKGROUND: Previous studies have demonstrated essential roles for alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaMKII) in learning, memory and long-term potentiation (LTP). However, previous studies have also shown that alpha-CaMKII (+/-) heterozygous knockout mice display a dramatic decrease in anxiety-like and fearful behaviors, and an increase in defensive aggression. These findings indicated that alpha-CaMKII is important not only for learning and memory but also for emotional behaviors. In this study, to understand the roles of alpha-CaMKII in emotional behavior, we generated transgenic mice overexpressing alpha-CaMKII in the forebrain and analyzed their behavioral phenotypes. RESULTS: We generated transgenic mice overexpressing alpha-CaMKII in the forebrain under the control of the alpha-CaMKII promoter. In contrast to alpha-CaMKII (+/-) heterozygous knockout mice, alpha-CaMKII overexpressing mice display an increase in anxiety-like behaviors in open field, elevated zero maze, light-dark transition and social interaction tests, and a decrease in locomotor activity in their home cages and novel environments; these phenotypes were the opposite to those observed in alpha-CaMKII (+/-) heterozygous knockout mice. In addition, similarly with alpha-CaMKII (+/-) heterozygous knockout mice, alpha-CaMKII overexpressing mice display an increase in aggression. However, in contrast to the increase in defensive aggression observed in alpha-CaMKII (+/-) heterozygous knockout mice, alpha-CaMKII overexpressing mice display an increase in offensive aggression. CONCLUSION: Up-regulation of alpha-CaMKII expression in the forebrain leads to an increase in anxiety-like behaviors and offensive aggression. From the comparisons with previous findings, we suggest that the expression levels of alpha-CaMKII are associated with the state of emotion; the expression level of alpha-CaMKII positively correlates with the anxiety state and strongly affects aggressive behavior.