Tissue-restricted Antigens Research Articles

Transcript splicing optimizes the thymic self-antigen repertoire to suppress autoimmunity.

Immunological self-tolerance is established in the thymus by the expression of virtually all self-antigens, including tissue-restricted antigens (TRAs) and cell-type-restricted antigens (CRAs). Despite a wealth of knowledge about the transcriptional regulation of TRA genes, posttranscriptional regulation remains poorly understood. Here, we show that protein arginine methylation plays an essential role in central immune tolerance by maximizing the self-antigen repertoire in medullary thymic epithelial cells (mTECs). Protein arginine methyltransferase-5 (Prmt5) was required for pre-mRNA splicing of certain key genes in tolerance induction, including Aire as well as various genes encoding TRAs. Mice lacking Prmt5 specifically in thymic epithelial cells exhibited an altered thymic T cell selection, leading to the breakdown of immune tolerance accompanied by both autoimmune responses and enhanced antitumor immunity. Thus, arginine methylation and transcript splicing are essential for establishing immune tolerance and may serve as a therapeutic target in autoimmune diseases as well as cancer immunotherapy.

PRMT5: splicing up tolerance.

Expression of tissue-restricted antigens (TRAs) within the thymus is critical for the establishment of self-tolerance; however, exact mechanisms regulating the expression of TRAs has proven more complex than previously appreciated. In this issue of the JCI, Muro et al. identify a central role for protein arginine methyltransferase 5 (PRMT5) in posttranscriptional regulation of TRAs and thereby central tolerance. Using conditional KO mice, the authors showed that thymic deficiency of Prmt5 predisposed mice to developing autoimmune diseases while enhancing antitumor efficacy. These studies provide insight into the role of PRMT5 in shaping the T cell repertoire with implications for the development of therapies.

Skewed epithelial cell differentiation and premature aging of the thymus in the absence of vitamin D signaling.

Central tolerance of thymocytes to self-antigen depends on the medullary thymic epithelial cell (mTEC) transcription factor autoimmune regulator (Aire), which drives tissue-restricted antigen (TRA) gene expression. Vitamin D signaling regulates Aire and TRA expression in mTECs, providing a basis for links between vitamin D deficiency and autoimmunity. We find that mice lacking Cyp27b1, which cannot produce hormonally active vitamin D, display profoundly reduced thymic cellularity, with a reduced proportion of Aire+ mTECs, attenuated TRA expression, and poorly defined cortical-medullary boundaries. Markers of T cell negative selection are diminished, and organ-specific autoantibodies are present in knockout (KO) mice. Single-cell RNA sequencing revealed that loss of Cyp27b1 skews mTEC differentiation toward Ccl21+ intertypical TECs and generates a gene expression profile consistent with premature aging. KO thymi display accelerated involution and reduced expression of thymic longevity factors. Thus, loss of thymic vitamin D signaling disrupts normal mTEC differentiation and function and accelerates thymic aging.

Abstract 4778: Development of self-amplifying RNA vaccines targeting prostate tissue-restricted and tumor-associated antigens

Abstract Prostate cancer is the most frequent cancer type in men, with increasing incidence at advanced age. As this cancer is frequently slow growing, opportunities for cancer interception exist, particularly among men with low-grade tumors who frequently undergo active surveillance. A well-tolerated vaccine intervention would be ideal for this population. Self-amplifying RNA (saRNA) vaccines built on a replication-deficient alphavirus backbone such as VEEV, offer several advantages for vaccine development. Sequences for relatively large antigens can be encoded and efficiently expressed, at low vaccine doses. Practical and efficient delivery of the saRNA is possible with advanced nanoparticles such as cationic inorganic lipid nanoparticles (LION). Among candidate antigens for a prostate cancer interception vaccine, we selected prostein (SLC45A3), a protein whose natural expression is restricted to the prostate gland and prostate cancer and ERG, a member of the ETS family of transcription factors frequently involved in chromosomal translocations in prostate cancer. We generated saRNA constructs with several design strategies and demonstrated that upon in vitro transfection, both antigens are expressed as assessed by mass spectrometry and Western blot analysis. This was achieved using commercial transfection reagents and LION. Prostein expression was particularly notable in that this eleven-transmembrane protein could be detected in HEK-293 cells using flow-cytometry and cell-surface staining. Nearly 50% of the cell population stained positively. Immunogenicity of the vaccines was tested in mice. Following formulation with LION particles i.m. injection and boosting, saRNAs coding for prostein elicited strong cellular immunity as demonstrated by IFN-γ ELISPOT analysis. Detailed analysis of intracellular cytokine expression indicated a strong CD8 response. saRNAs coding for ERG also showed antigen-specific ELISPOTs. Using commercially available recombinant ERG protein, an ELISA was developed and a strong humoral response to the ERG saRNA vaccine was demonstrated. The saRNA vaccines will be further credentialled with additional preclinical studies including cancer preventive efficacy studies in genetically engineered mouse models of prostate cancer. Citation Format: Robert H. Shoemaker, Jesse H. Erasmus, Peter Berglund, Jason D. Marshall, Yuri Koboziev, Bradley M. Beckman, Brian D. Cholewa, Steven G. Reed. Development of self-amplifying RNA vaccines targeting prostate tissue-restricted and tumor-associated antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4778.

FEZF2 and AIRE1: An Evolutionary Trade-off in the Elimination of Auto-reactive T Cells in the Thymus.

Autoimmune Regulator 1 (AIRE1) and Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2) play pivotal roles in orchestrating the expression of tissue-restricted antigens (TRA) to facilitate the elimination of autoreactive T cells. AIRE1's presence in the gonads of various vertebrates has raised questions about its potential involvement in gene expression control for germline cell selection. Nevertheless, the evolutionary history of these genes has remained enigmatic, as has the rationale behind their apparent redundancy in vertebrates. Furthermore, the origin of the elimination process itself has remained elusive. To shed light on these mysteries, we conducted a comprehensive evolutionary analysis employing a range of tools, including multiple sequence alignment, phylogenetic tree construction, ancestral sequence reconstruction, and positive selection assessment. Our investigations revealed intriguing insights. AIRE1 homologs emerged during the divergence of T cells in higher vertebrates, signifying its role in this context. Conversely, FEZF2 exhibited multiple homologs spanning invertebrates, lampreys, and higher vertebrates. Ancestral sequence reconstruction demonstrated distinct origins for AIRE1 and FEZF2, underscoring that their roles in regulating TRA have evolved through disparate pathways. Furthermore, it became evident that both FEZF2 and AIRE1 govern a diverse repertoire of genes, encompassing ancient and more recently diverged targets. Notably, FEZF2 demonstrates expression in both vertebrate and invertebrate embryos and germlines, accentuating its widespread role. Intriguingly, FEZF2 harbors motifs associated with autophagy, such as DKFPHP, SYSELWKSSL, and SYSEL, a process integral to cell selection in invertebrates. Our findings suggest that FEZF2 initially emerged to regulate self-elimination in the gonads of invertebrates. As organisms evolved toward greater complexity, AIRE1 likely emerged to complement FEZF2's role, participating in the regulation of cell selection for elimination in both gonads and the thymus. This dynamic interplay between AIRE1 and FEZF2 underscores their multifaceted contributions to TRA expression regulation across diverse evolutionary contexts.

Extrathymic AIRE-Expressing Cells: A Historical Perspective.

Since its discovery, Aire has been the topic of numerous studies in its role as a transcriptional regulator in the thymus where it promotes the "promiscuous" expression of a large repertoire of tissue-restricted antigens (TRAs) that are normally expressed only in the immune periphery. This process occurs in specialized medullary thymic epithelial cells (mTECs) and mediates the elimination of self-reactive T cells or promotestheir conversion to the Foxp3+ regulatory T cell lineage, both of which are required for the prevention of autoimmunity. In recent years, there has been increasing interest in the role of extrathymic Aire expression in peripheral organs. The focus has primarily been on the identification of the cellular source(s) and mechanism(s) by which extrathymic AIRE affects tolerance-related or other physiological processes. A cadre of OMICs tools including single cell RNA sequencingand novel transgenic models to trace Aire expression to perform lineage tracing experiments haveshed light on a phenomenon that is more complex than previously thought. In this chapter, we provide a deeper analysis of how extrathymic Aire research hasdeveloped and progressed, how cellular sources were identified, and how the function of AIRE was determined. Current data suggests that extrathymic AIRE fulfills a function that differs from what has been observed in the thymus and strongly argues that its main purpose is to regulate transcriptional programs in a cell content-dependent manner. Surprisingly, there is data that also suggests a non-transcriptional role of extrathymic AIRE in the cytoplasm. We have arrived at a potential turning point that will take the field from the classical understanding of AIRE as a transcription factor in control of TRA expression to its role in immunological and non-immunological processes in the periphery.

Insm1 regulates mTEC development and immune tolerance.

The expression of self-antigens in medullary thymic epithelial cells (mTECs) is essential for the establishment of immune tolerance, but the regulatory network that controls the generation and maintenance of the multitude of cell populations expressing self-antigens is poorly understood. Here, we show that Insm1, a zinc finger protein with known functions in neuroendocrine and neuronal cells, is broadly coexpressed with an autoimmune regulator (Aire) in mTECs. Insm1 expression is undetectable in most mimetic cell populations derived from mTECs but persists in neuroendocrine mimetic cells. Mutation of Insm1 in mice downregulated Aire expression, dysregulated the gene expression program of mTECs, and altered mTEC subpopulations and the expression of tissue-restricted antigens. Consistent with these findings, loss of Insm1 resulted in autoimmune responses in multiple peripheral tissues. We found that Insm1 regulates gene expression in mTECs by binding to chromatin. Interestingly, the majority of the Insm1 binding sites are co-occupied by Aire and enriched in superenhancer regions. Together, our data demonstrate the important role of Insm1 in the regulation of the repertoire of self-antigens needed to establish immune tolerance.

Establishment of CD8+ T Cell Thymic Central Tolerance to Tissue-Restricted Antigen Requires PD-1.

Highly self-reactive T cells are censored from the repertoire by both central and peripheral tolerance mechanisms upon receipt of high-affinity TCR signals. Clonal deletion is considered a major driver of central tolerance; however, other mechanisms such as induction of regulatory T cells and functional impairment have been described. An understanding of the interplay between these different central tolerance mechanisms is still lacking. We previously showed that impaired clonal deletion to a model tissue-restricted Ag did not compromise tolerance. In this study, we determined that murine T cells that failed clonal deletion were rendered functionally impaired in the thymus. Programmed cell death protein 1 (PD-1) was induced in the thymus and was required to establish cell-intrinsic tolerance to tissue-restricted Ag in CD8+ thymocytes independently of clonal deletion. In bone marrow chimeras, tolerance was not observed in PD-L1-deficient recipients, but tolerance was largely maintained following adoptive transfer of tolerant thymocytes or T cells to PD-L1-deficient recipients. However, CRISPR-mediated ablation of PD-1 in tolerant T cells resulted in broken tolerance, suggesting different PD-1 signaling requirements for establishing versus maintaining tolerance. Finally, we showed that chronic exposure to high-affinity Ag supported the long-term maintenance of tolerance. Taken together, our study identifies a critical role for PD-1 in establishing central tolerance in autoreactive T cells that escape clonal deletion. It also sheds light on potential mechanisms of action of anti-PD-1 pathway immune checkpoint blockade and the development of immune-related adverse events.

Aire drives steroid hormone biosynthesis by medullary thymic epithelial cells.

Thymic epithelial cells (TECs) produce glucocorticoids, which antagonize negative selection of autoreactive thymocytes and promote a competent T cell antigen-specific repertoire. To characterize their source, we generated a knock-in reporter mouse in which endogenous Cyp11b1, the final enzyme in de novo production of active glucocorticoids, was fluorescently tagged with mScarlet. Here, we find that Cyp11b1 is expressed in medullary TECs (mTECs) but not cortical TECs or other cells in the thymus. A distinct characteristic of mTECs is the presence of Aire, a transcription factor that drives expression of tissue-restricted antigens (TRAs) important for establishing immune tolerance. Cyp11b1 expression was highest in Aire+ mTECs, lower in post-Aire mTECs, and absent in mTECs of Aire-deficient mice. Transcriptomic analyses found that multiple enzymatic biosynthetic pathways are expressed specifically in mTECs and are also Aire dependent. In particular, we found that the thymus expresses messenger RNA for enzymes that catalyze production of many bioactive steroids and that glucocorticoids and sex steroids were secreted by cultured thymi. Expression of the transcripts for these genes and production of their final steroid products were markedly reduced in the absence of Aire. Thus, in addition to its well-established role in inducing TRAs that promote negative selection, Aire has an additional and contrary function of inducing glucocorticoids that antagonize negative selection, which together may expand and enhance the TCR repertoire. Furthermore, because Aire drives expression of multiple enzymes responsible for production of other non-gene-encoded bioactive molecules, it might have yet other roles in thymus development and function.

Revisiting Aire and tissue-restricted antigens at single-cell resolution.

The thymus is a highly specialized organ that plays an indispensable role in the establishment of self-tolerance, a process characterized by the "education" of developing T-cells. To provide competent T-cells tolerant to self-antigens, medullary thymic epithelial cells (mTECs) orchestrate negative selection by ectopically expressing a wide range of genes, including various tissue-restricted antigens (TRAs). Notably, recent advancements in the high-throughput single-cell analysis have revealed remarkable heterogeneity in mTECs, giving us important clues for dissecting the mechanisms underlying TRA expression. We overview how recent single-cell studies have furthered our understanding of mTECs, with a focus on the role of Aire in inducing mTEC heterogeneity to encompass TRAs.

AIRE in Male Fertility: A New Hypothesis.

Male infertility affects approximately 14% of all European men, of which ~44% are characterized as idiopathic. There is an urgency to identify the factors that affect male fertility. One such factor, Autoimmune Regulator (AIRE), a protein found in the thymus, has been studied in the context of central tolerance functioning as a nuclear transcription modulator, responsible for the expression of tissue-restricted antigens in specialized thymic cells that prevent autoimmunity. While its expression in the testes remains enigmatic, we recently observed that sterility in mice correlates with the absence of Aire in the testes, regardless of the deficient expression in medullary thymic epithelial cells or cells of the hematopoietic system. By assessing the Aire transcript levels, we discovered that Sertoli cells are the exclusive source of Aire in the testes, where it most likely plays a non-immune role, suggesting an unknown mechanism by which testicular Aire regulates fertility. Here, we discuss these results in the context of previous reports which have suggested that infertility observed in Aire deficient mice is of an autoimmune aetiology. We present an alternative point of view for the role of Aire in testes in respect to fertility altering the perspective of how Aire’s function in the testes is currently perceived.

Trappc1 deficiency impairs thymic epithelial cell development by breaking endoplasmic reticulum homeostasis.

Thymic epithelial cells (TECs) are important for T cell development and immune tolerance establishment. Although comprehensive molecular regulation of TEC development has been studied, the role of transport protein particle complexes (Trappcs) in TECs is not clear. Using TEC-specific homozygous or heterozygous Trappc1 deleted mice model, we find that Trappc1 deficiency cause severe thymus atrophy with decreased cell number and blocked maturation of TECs. Mice with a TEC-specific Trappc1 deletion show poor thymic T cell output and have a greater percentage of activated/memory T cells, suffered from spontaneous autoimmune disorders. Our RNA-seq and molecular studies indicated that the decreased endoplasmic reticulum (ER) and Golgi apparatus, enhanced unfolded protein response (UPR) and subsequent Atf4-CHOP-mediated apoptosis, and reactive oxygen species (ROS)-mediated ferroptosis coordinately contributed to the reduction of Trappc1-deleted TECs. Additionally, reduced Aire+ mTECs accompanied by the decreased expression of Irf4, Irf8, and Tbx21 in Trappc1 deficiency mTECs, may further coordinately block the tissue-restricted antigen expression. In this study, we reveal that Trappc1 plays an indispensable role in TEC development and maturation and provide evidence for the importance of inter-organelle traffic and ER homeostasis in TEC development.

Cytosolic Nuclear Sensor Dhx9 Controls Medullary Thymic Epithelial Cell Differentiation by p53-Mediated Pathways.

Thymic epithelial cells (TECs) critically participate in T cell maturation and selection for the establishment of immunity to foreign antigens and immune tolerance to self-antigens of T cells. It is well known that many intracellular and extracellular molecules elegantly have mastered the development of medullary TECs (mTECs) and cortical TECs (cTECs). However, the role played by NTP-dependent helicase proteins in TEC development is currently unclear. Herein, we created mice with a TEC-specific DExD/H-box helicase 9 (Dhx9) deletion (Dhx9 cKO) to study the involvement of Dhx9 in TEC differentiation and function. We found that a Dhx9 deficiency in TECs caused a significant decreased cell number of TECs, including mTECs and thymic tuft cells, accompanied by accelerated mTEC maturation but no detectable effect on cTECs. Dhx9-deleted mTECs transcriptionally expressed poor tissue-restricted antigen profiles compared with WT mTECs. Importantly, Dhx9 cKO mice displayed an impaired thymopoiesis, poor thymic T cell output, and they suffered from spontaneous autoimmune disorders. RNA-seq analysis showed that the Dhx9 deficiency caused an upregulated DNA damage response pathway and Gadd45, Cdkn1a, Cdc25, Wee1, and Myt1 expression to induce cell cycle arrest in mTECs. In contrast, the p53-dependent upregulated RANK-NF-κB pathway axis accelerated the maturation of mTECs. Our results collectively indicated that Dhx9, a cytosolic nuclear sensor recognizing viral DNA or RNA, played an important role in mTEC development and function in mice.

MiR-155 exerts posttranscriptional control of autoimmune regulator (Aire) and tissue-restricted antigen genes in medullary thymic epithelial cells

BackgroundThe autoimmune regulator (Aire) gene is critical for the appropriate establishment of central immune tolerance. As one of the main controllers of promiscuous gene expression in the thymus, Aire promotes the expression of thousands of downstream tissue-restricted antigen (TRA) genes, cell adhesion genes and transcription factor genes in medullary thymic epithelial cells (mTECs). Despite the increasing knowledge about the role of Aire as an upstream transcriptional controller, little is known about the mechanisms by which this gene could be regulated.ResultsHere, we assessed the posttranscriptional control of Aire by miRNAs. The in silico miRNA-mRNA interaction analysis predicted thermodynamically stable hybridization between the 3’UTR of Aire mRNA and miR-155, which was confirmed to occur within the cellular milieu through a luciferase reporter assay. This finding enabled us to hypothesize that miR-155 might play a role as an intracellular posttranscriptional regulator of Aire mRNA. To test this hypothesis, we transfected a murine mTEC cell line with a miR-155 mimic in vitro, which reduced the mRNA and protein levels of Aire. Moreover, large-scale transcriptome analysis showed the modulation of 311 downstream mRNAs, which included 58 TRA mRNAs. Moreover, miR-155 mimic-transfected cells exhibited a decrease in their chemotaxis property compared with control thymocytes.ConclusionOverall, the results indicate that miR-155 may posttranscriptionally control Aire mRNA, reducing the respective Aire protein levels; consequently, the levels of mRNAs encode tissue-restricted antigens were affected. In addition, miR-155 regulated a crucial process by which mTECs allow thymocytes’ migration through chemotaxis.

T Cells Engineered to Express Immunoreceptors Targeting the Frequently Expressed Medullary Thyroid Cancer Antigens Calcitonin, CEA, and RET M918T.

Background: Medullary thyroid cancer (MTC) is a rare malignancy originating from the calcitonin-producing C cells of the thyroid. Despite recent therapeutic advances, metastatic MTC remains incurable. Adoptive cell therapy (ACT) using genetically engineered T cells targeting either tissue-restricted tumor-associated antigens or mutated neoantigens has led to durable remissions in other metastatic solid tumors. The majority of MTC express the tumor-associated antigens calcitonin and carcinoembryonic antigen (CEA), and ∼40% of MTC harbor the RET M918T oncogenic driver mutation. Methods: We developed and characterized three immunoreceptors that recognize extracellular CEA, a calcitonin epitope presented by HLA-A*24:02, or an RET M918T neoepitope restricted by HLA-DPB1*04:01/02. The chimeric antigen receptor (CAR) targeting CEA was synthetically designed, while the T cell receptors (TCRs) targeting calcitonin and RET M918T were isolated from a transgenic mouse and patient with MTC, respectively. These immunoreceptors were genetically engineered into peripheral blood T cells and tested for antigen specificity and antitumor activity. Results: T cells expressing the anti-CEA CAR or the calcitonin-reactive TCR produced effector cytokines and displayed cytotoxicity against cell lines expressing their cognate antigen in vitro. In immunodeficient mice harboring a human MTC cell line, the adoptive transfer of T cells engineered to express the anti-CEA CAR or calcitonin-reactive TCR led to complete tumor regression. T cells expressing the HLA-DPB1*04:01/02-restricted TCR targeting RET M918T, which was cloned from peripheral blood CD4+ T cells of a patient with MTC, demonstrated specific reactivity against cells pulsed with the mutated peptide and MTC tumor cells that expressed HLA-DPB1*04:01 and RET M918T. Conclusion: The preclinical data presented herein demonstrate the potential of using genetically engineered T cells targeting CEA, calcitonin, and/or RET M918T to treat metastatic MTC.

Central tolerance is impaired in the middle-aged thymic environment.

One of the earliest hallmarks of immune aging is thymus involution, which not only reduces the number of newly generated and exported T cells, but also alters the composition and organization of the thymus microenvironment. Thymic T‐cell export continues into adulthood, yet the impact of thymus involution on the quality of newly generated T‐cell clones is not well established. Notably, the number and proportion of medullary thymic epithelial cells (mTECs) and expression of tissue‐restricted antigens (TRAs) decline with age, suggesting the involuting thymus may not promote efficient central tolerance. Here, we demonstrate that the middle‐aged thymic environment does not support rapid motility of medullary thymocytes, potentially diminishing their ability to scan antigen presenting cells (APCs) that display the diverse self‐antigens that induce central tolerance. Consistent with this possibility, thymic slice assays reveal that the middle‐aged thymic environment does not support efficient negative selection or regulatory T‐cell (Treg) induction of thymocytes responsive to either TRAs or ubiquitous self‐antigens. This decline in central tolerance is not universal, but instead impacts lower‐avidity self‐antigens that are either less abundant or bind to TCRs with moderate affinities. Additionally, the decline in thymic tolerance by middle age is accompanied by both a reduction in mTECs and hematopoietic APC subsets that cooperate to drive central tolerance. Thus, age‐associated changes in the thymic environment result in impaired central tolerance against moderate‐avidity self‐antigens, potentially resulting in export of increasingly autoreactive naive T cells, with a deficit of Treg counterparts by middle age.

Disruption of thymic central tolerance by infection with murine roseolovirus induces autoimmune gastritis.

Infections with herpesviruses, including human roseoloviruses, have been proposed to cause autoimmune disease, but defining a causal relationship and mechanism has been difficult due to the ubiquitous nature of infection and development of autoimmunity long after acute infection. Murine roseolovirus (MRV) is highly related to human roseoloviruses. Herein we show that neonatal MRV infection induced autoimmune gastritis (AIG) in adult mice in the absence of ongoing infection. MRV-induced AIG was dependent on replication during the neonatal period and was CD4+ T cell and IL-17 dependent. Moreover, neonatal MRV infection was associated with development of a wide array of autoantibodies in adult mice. Finally, neonatal MRV infection reduced medullary thymic epithelial cell numbers, thymic dendritic cell numbers, and thymic expression of AIRE and tissue-restricted antigens, in addition to increasing thymocyte apoptosis at the stage of negative selection. These findings strongly suggest that infection with a roseolovirus early in life results in disruption of central tolerance and development of autoimmune disease.

The Lineage Differentiation and Dynamic Heterogeneity of Thymic Epithelial Cells During Thymus Organogenesis.

Although much progress has been made recently in revealing the heterogeneity of the thymic stromal components, the molecular programs of cell lineage divergency and temporal dynamics of thymic epithelial cell (TEC) development are largely elusive. Here, we constructed a single-cell transcriptional landscape of non-hematopoietic cells from mouse thymus spanning embryonic to adult stages, producing transcriptomes of 30,959 TECs. We resolved the transcriptional heterogeneity of developing TECs and highlighted the molecular nature of early TEC lineage determination and cortico-medullary thymic epithelial cell lineage divergency. We further characterized the differentiation dynamics of TECs by clarification of molecularly distinct cell states in the thymus developing trajectory. We also identified a population of Bpifa1+ Plet1+ mTECs that was preserved during thymus organogenesis and highly expressed tissue-resident adult stem cell markers. Finally, we highlighted the expression of Aire-dependent tissue-restricted antigens mainly in Aire+ Csn2+ mTECs and Spink5+ Dmkn+ mTECs in postnatal thymus. Overall, our data provided a comprehensive characterization of cell lineage differentiation, maturation, and temporal dynamics of thymic epithelial cells during thymus organogenesis.

Redox regulation of age-associated defects in generation and maintenance of Tcell self-tolerance and immunity to foreign antigens.

SUMMARYThymic atrophy reduces naive T cell production and contributes to increased susceptibility to viral infection with age. Expression of tissue-restricted antigen (TRA) genes also declines with age and has been thought to increase autoimmune disease susceptibility. We find that diminished expression of a model TRA gene in aged thymic stromal cells correlates with impaired clonal deletion of cognate T cells recognizing an autoantigen involved in atherosclerosis. Clonal deletion in the polyclonal thymocyte population is also perturbed. Distinct age-associated defects in the generation of antigen-specific T cells include a conspicuous decline in generation of T cells recognizing an immunodominant influenza epitope. Increased catalase activity delays thymic atrophy, and here, we show that it mitigates declining production of influenza-specific T cells and their frequency in lung after infection, but does not reverse declines in TRA expression or efficient negative selection. These results reveal important considerations for strategies to restore thymic function.

The transcription factor Sox4 is required for thymic tuft cell development.

Medullary thymic epithelial cells (mTECs) help shape the thymic microenvironment for T-cell development by expressing a variety of peripheral tissue-restricted antigens (TRAs). The self-tolerance of T cells is established by negative selection of autoreactive T cells that bind to TRAs. To increase the diversity of TRAs, a fraction of mTECs terminally differentiates into distinct subsets resembling atypical types of epithelial cells in specific peripheral tissues. As such, thymic tuft cells that express peripheral tuft cell genes have recently emerged. Here, we show that the transcription factor SRY-box transcription factor 4 (Sox4) is highly expressed in mTECs and is essential for the development of thymic tuft cells. Mice lacking Sox4 specifically in TECs had a significantly reduced number of thymic tuft cells with no effect on the differentiation of other mTEC subsets, including autoimmune regulator (Aire)+ and Ccl21a+ mTECs. Furthermore, Sox4 expression was diminished in mice deficient in TEC-specific lymphotoxin β receptor (LTβR), indicating a role for the LTβR-Sox4 axis in the differentiation of thymic tuft cells. Given that Sox4 promotes differentiation of peripheral tuft cells, our findings suggest that mTECs employ the same transcriptional program as peripheral epithelial cells. This mechanism may explain how mTECs diversify peripheral antigen expression to project an immunological self within the thymic medulla.