Runx3 instructs Aire+ mTEC development, TSA gene expression, and central tolerance.

We have identified a novel function for a zinc finger transcription factor in medullary thymic epithelial cell (mTEC) development and central tolerance. Previous studies have shown that the transcription factor has critical functions in immune cell development, however our study is one of the first to describe a role for the transcription factor in the epithelial lineage. Probing publicly available gene expression databases, we found that the transcription factor is highly expressed in mTECs. Recent studies on mTECs have shown considerable cellular heterogeneity within mTECs with at least five mTEC subtypes including a transient amplifying population (TAC-TECs), autoimmune regulator (Aire+) mTECs, Late/Post Aire mTECs, Ccl21a mTECS, and tuft cells. Using a conditional knockout mouse line, we deleted the transcription factor in mTECs which resulted in drastic changes in the cellular composition of mTECs with substantial increase in Ccl21a mTECs and tuft cells and a dramatic decrease in Aire+ mTECs and Late Aire mTECs. Furthermore, the remaining Aire+ mTECs have a significant decrease in tissue-specific antigen gene expression resulting in autoimmunity in mice. To gain mechanistic understanding of the developmental changes we observed, we performed scRNA-seq and scATAC-seq, which showed significant changes in gene expression and the chromatin landscape in mTECs and helps to explain the critical function for this transcription factor in mTEC development.

Mutations in the gene encoding the zinc-finger transcription factor Ikaros (IKZF1) are found in patients with immunodeficiency, leukemia, and autoimmunity. Although Ikaros has a well-established function in modulating gene expression programs important for hematopoietic development, its role in other cell types is less well defined. Here, we uncover functions for Ikaros in thymic epithelial lineage development in mice and show that Ikzf1 expression in medullary thymic epithelial cells (mTECs) is required for both autoimmune regulator-positive (Aire+) mTEC development and tissue-specific antigen (TSA) gene expression. Accordingly, TEC-specific deletion of Ikzf1 in mice results in a profound decrease in Aire+ mTECs, a global loss of TSA gene expression, and the development of autoimmunity. Moreover, Ikaros shapes thymic mimetic cell diversity, and its deletion results in a marked expansion of thymic tuft cells and muscle-like mTECs and a loss of other Aire-dependent mimetic populations. Single-cell analysis reveals that Ikaros modulates core transcriptional programs in TECs that correlate with the observed cellular changes. Our findings highlight a previously undescribed role for Ikaros in regulating epithelial lineage development and function and suggest that failed thymic central tolerance could contribute to the autoimmunity seen in humans with IKZF1 mutations.

Thymic central tolerance is a critical process that prevents autoimmunity but also presents a challenge to the generation of anti-tumor immune responses. Medullary thymic epithelial cells (mTECs) eliminate self-reactive T cells by displaying a diverse repertoire of tissue-specific antigens (TSAs) that are also shared by tumors. Therefore, while protecting against autoimmunity, mTECs simultaneously limit the generation of tumor-specific effector T cells by expressing tumor self-antigens. This ectopic expression of TSAs largely depends on autoimmune regulator (Aire), which is expressed in mature mTECs. Thus, therapies to deplete Aire-expressing mTECs represent an attractive strategy to increase the pool of tumor-specific effector T cells. Recent work has implicated the TNF family members RANK and RANK-Ligand (RANKL) in the development of Aire-expressing mTECs. We show that in vivo RANKL blockade selectively and transiently depletes Aire and TSA expression in the thymus to create a window of defective negative selection. Furthermore, we demonstrate that RANKL blockade can rescue melanoma-specific T cells from thymic deletion and that persistence of these tumor-specific effector T cells promoted increased host survival in response to tumor challenge. These results indicate that modulating central tolerance through RANKL can alter thymic output and potentially provide therapeutic benefit by enhancing anti-tumor immunity.

GENERAL COMMENTARY article Front. Immunol., 19 March 2014Sec. Primary Immunodeficiencies Volume 5 - 2014 | https://doi.org/10.3389/fimmu.2014.00111

Thymic epithelial cells are indispensable for T cell maturation and selection and the induction of central immune tolerance. The self-peptide repertoire expressed by medullary thymic epithelial cells is in part regulated by the transcriptional regulator Aire (Autoimmune regulator) and the transcription factor Fezf2. Due to the high complexity of mTEC maturation stages (i.e., post-Aire, Krt10+ mTECs, and Dclk1+ Tuft mTECs) and the heterogeneity in their gene expression profiles (i.e., mosaic expression patterns), it has been challenging to identify the additional factors complementing the transcriptional regulation. We aimed to identify the transcriptional regulators involved in the regulation of mTEC development and self-peptide expression in an unbiased and genome-wide manner. We used ATAC footprinting analysis as an indirect approach to identify transcription factors involved in the gene expression regulation in mTECs, which we validated by ChIP sequencing. This study identifies Fezf2 as a regulator of the recently described thymic Tuft cells (i.e., Tuft mTECs). Furthermore, we identify that transcriptional regulators of the ELF, ESE, ERF, and PEA3 subfamily of the ETS transcription factor family and members of the Krüppel-like family of transcription factors play a role in the transcriptional regulation of genes involved in late mTEC development and promiscuous gene expression.

Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. Here, we identify a highly conserved noncoding DNA element that is essential for Aire expression. This element shows enrichment of enhancer-associated histone marks in mTECs and also has characteristics of being an NF-κB-responsive element. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance.

T cell tolerance in the thymus is a key step in shaping the developing T cell repertoire. Thymic medullary epithelial cells play multiple roles in this process, including negative selection of autoreactive thymocytes, influencing thymic dendritic cell positioning, and the generation of Foxp3(+) regulatory T cells. Previous studies show that medullary thymic epithelial cell (mTEC) development involves hemopoietic cross-talk, and numerous TNFR superfamily members have been implicated in this process. Whereas CD40 and RANK represent key examples, interplay between these receptors, and the individual cell types providing their ligands at both fetal and adult stages of thymus development, remain unclear. In this study, by analysis of the cellular sources of receptor activator for NF-κB ligand (RANKL) and CD40L during fetal and adult cross-talk in the mouse, we show that the innate immune cell system drives initial fetal mTEC development via expression of RANKL, but not CD40L. In contrast, cross-talk involving the adaptive immune system involves both RANKL and CD40L, with analysis of distinct subsets of intrathymic CD4(+) T cells revealing a differential contribution of CD40L by conventional, but not Foxp3(+) regulatory, T cells. We also provide evidence for a stepwise involvement of TNFRs in mTEC development, with CD40 upregulation induced by initial RANK signaling subsequently controlling proliferation within the mTEC compartment. Collectively, our findings show how multiple hemopoietic cell types regulate mTEC development through differential provision of RANKL/CD40L during ontogeny, revealing molecular differences in fetal and adult hemopoietic cross-talk. They also suggest a stepwise process of mTEC development, in which RANK is a master player in controlling the availability of other TNFR family members.

The crucial capability of T cells for discrimination between self and non-self peptides is based on negative selection of developing thymocytes by medullary thymic epithelial cells (mTECs). The mTECs purge autoreactive T cells by expression of cell-type specific genes referred to as tissue-restricted antigens (TRAs). Although the autoimmune regulator (AIRE) protein is known to promote the expression of a subset of TRAs, its mechanism of action is still not fully understood. The expression of TRAs that are not under the control of AIRE also needs further characterization. Furthermore, expression patterns of TRA genes have been suggested to change over the course of mTEC development. Herein we have used single-cell RNA-sequencing to resolve patterns of TRA expression during mTEC development. Our data indicated that mTEC development consists of three distinct stages, correlating with previously described jTEC, mTEChi and mTEClo phenotypes. For each subpopulation, we have identified marker genes useful in future studies. Aire-induced TRAs were switched on during jTEC-mTEC transition and were expressed in genomic clusters, while otherwise the subsets expressed largely overlapping sets of TRAs. Moreover, population-level analysis of TRA expression frequencies suggested that such differences might not be necessary to achieve efficient thymocyte selection.

Tolerance in developing T cells is dependent on medullary thymic epithelial cells (mTEC) and mTEC development in turns requires signals from mature SP thymocytes. Several TNF family receptor-ligand pairs have been shown to be important players in the thymic crosstalk required to foster mTEC development including RANK-RANKL, LTαβ-LTβR and CD40L-CD40. In addition to confirming the important roles of TNF family members in thymic crosstalk, we have recently identified CD28-CD80/86 interactions as an additional pathway important for normal thymic medullary compartment development. CD80 and CD86, similar to LTβR, CD40 and RANK, are expressed by mTEC as well as by bone marrow (BM) derived cells in the thymus. To address the question of which cell type(s) need to express CD80/86, LTβR, CD40 and RANK in order to support mTEC development, we have generated and analyzed BM chimeras or conditional knockout mice in which either non-BM-derived cells (including thymic epithelium) or BM-derived cells express the molecule in question. For LTβR and RANK, we find that expression on radiation-resistant non-BM-derived cells, is required. Interestingly, however, we find that for CD80/86 and CD40, expression on either non-BM-derived or BM-derived cells is sufficient to promote mTEC development. Our findings suggest that models of thymic crosstalk must consider not only communication between TEC and thymocytes but with other bone marrow derived cell types as well.

ABSTRACTWe previously established three mouse cell lines (Aire+TEC1, Aire+TEC2 and Aire+DC) from the medullary thymic epithelial cells (mTECs) and dendritic cells (mDCs). These cells constitutively expressed “autoimmune regulator (Aire) gene” and they exhibited various features of self antigen-presenting cells (self-APCs) present in the thymic medullary region.Here, we confirmed our previous observation that Aire+ thymic epithelial cells adhere to fresh thymocytes and kill them by inducing apoptosis, thus potentially reproducing in vitro some aspects of the negative selection of T cells in vivo. In this system, a single Aire+ cell appeared able to kill ∼30 thymocytes within 24 hrs. Moreover, we observed that ectopic expression of peripheral tissue-specific antigens (TSAs), and expression of several surface markers involved in mTEC development, increased as Aire+ cell density increases toward confluency. Thus, these Aire+ cells appear to behave like differentiating mTECs as if they pass through the developmental stages from intermediate state toward mature state. Surprisingly, an in vitro co-culture system consisting of Aire+ cells and fractionated sub-populations of fresh thymocytes implied the possible existence of two distinct subtypes of thymocytes (named as CD4+ killer and CD4− rescuer) that may determine the fate (dead or alive) of the differentiating Aire+mTECs. Thus, our in vitro co-culture system appears to mimic a part of “in vivo thymic crosstalk”.

Negative selection induces central tolerance in which self-reactive T cells are deleted by medullary thymic epithelial cells (mTECs) to prevent autoimmunity. The transcriptional factor, autoimmune regulator (Aire), controls the expression of tissue-specific antigens (TSAs) by mTECs for negative selection. The mechanisms by which Aire targets loci which encode TSAs are unknown in detail; recently, however, the ATF7ip-MBD1 complex was identified as an Aire-interacting transcriptional protein complex required for its targeting the loci. Lineage tracing of Aire(+) mTECs identified that mTECs have a post-Aire stage during the development, where they lost maturation markers but maintained intermediate TSA expression, and Aire is required for the terminal differentiation of mTEC's. Extrathymic Aire-expressing cells (eTACs) are identified in murine and human secondary lymphoid organs. eTACs express major histocompatibility complex class II(hi), CD80(lo), CD86(lo), epithelial cell adhesion molecule(hi), CD45(lo) bone marrow-derived peripheral antigen-presenting cell population, which is distinct from mTECs and dendritic cells. They can induce activation-induced cell death of self-reactive CD8(+) T cells and unresponsiveness of self-reactive CD4(+) T cells through a mechanism that does not require regulatory T cells, suggesting that peripheral Aire plays a complementary role for central tolerance.

Down syndrome (DS), a chromosomal condition caused by the trisomy of chromosome 21, is associated with various immunological impairments, including a high incidence of autoimmune diseases (e.g., hypothyroidism, type 1 diabetes) and anatomical changes in the thymus. The autoimmune regulator (AIRE) is a transcription factor whose gene maps in 21q22.3 and controls the ectopic expression of a large set of peripheral tissue antigen genes (PTA) in medullary thymic epithelial cells (mTECs). This phenomenon, termed promiscuous gene expression (pGE), plays a key role in central tolerance as demonstrated by the inactivation of AIRE resulting in the rare recessive autoimmune polyendocrinophathy-candidiasis-ectodermal dystrophy syndrome (APECED). DS patients carry three copies of AIRE which may result in its overexpression or, more rarely, its underexpression. To investigate the possibility that DS associated autoimmunity is favored by the impairment of pGE as a consequence of changes in AIRE expression and/or anatomical disorganisation of the thymus, we investigated AIRE, PTA and cell marker gene expression in thymi of DS individuals vs controls. Gene expression was assessed on cDNA from 19 DS and 21 controls total thymus samples by qPCR using TaqMan probes. Interestingly, AIRE gene expression was significantly reduced in thymus from DS vs controls (p = 0.0003). 7 PTA genes (CHRNA1, GAD1, PLP1, KLK3, SAG, TG and TSHR) were also reduced, more markedly for KLK3 and SAG (P = 0.002 and 0.0004 respectively). This changes seems unlikely to result from a reduction in the number of epithelial thymic cell since the levels of keratin expression were not reduced. Allele-specific quantification of three alleles of the AIRE gene demonstrated that, in spite of its overall reduction, the three copies of AIRE genes are expressed in the thymus of DS patients, thus indicating that reduction of AIRE expression is not caused by allele silencing (imprinting). Conclusion, autoimmunity associated to DS is probably in part caused by a reduction of AIRE expression that results in an impaired pGE rather that by a reduction of thymic epithelial cells as part of anatomical disorganization.

The thymic medulla represents a key site for the induction of T cell tolerance. In particular, autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) provide a spectrum of tissue-restricted Ags that, through both direct presentation and cross-presentation by dendritic cells, purge the developing T cell repertoire of autoimmune specificities. Despite this role, the mechanisms of Aire(+) mTEC development remain unclear, particularly those stages that occur post-Aire expression and represent mTEC terminal differentiation. In this study, in mouse thymus, we analyze late-stage mTEC development in relation to the timing and requirements for Aire and involucrin expression, the latter a marker of terminally differentiated epithelium including Hassall's corpuscles. We show that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events that are controlled by distinct mechanisms. We find that whereas mature thymocytes are not essential for Aire(+) mTEC development, use of an inducible ZAP70 transgenic mouse line--in which positive selection can be temporally controlled--demonstrates that the emergence of involucrin(+) mTECs critically depends upon the presence of mature single positive thymocytes. Finally, although initial formation of Aire(+) mTECs depends upon RANK signaling, continued mTEC development to the involucrin(+) stage maps to activation of the LTα-LTβR axis by mature thymocytes. Collectively, our results reveal further complexity in the mechanisms regulating thymus medulla development and highlight the role of distinct TNFRs in initial and terminal differentiation stages in mTECs.

The essential role of tissue specific antigen (TSA) expression in medullary thymic epithelial cells (mTECs) in mediating central tolerance has been well-established. We have previously shown that mice with mTEC-specific insulin deletion develop autoimmune diabetes within 3 weeks postnatal. To further understand the mechanisms of mTEC-mediated self-tolerance, we generated the Aire-YFP mice by crossing R26R-YFP reporters with Aire-Cre transgenic mice. Three subsets of mTECs were isolated from these mice: the YFPLOWCD80LOWClass IILOW pre-Aire cells; the YFPHICD80HIClass IIHI Aire+ cells; and the YFPHICD80INTClass IIINT post-Aire cells. Diverse TSA transcripts were detected in Aire+ mTECs, but not, or more restricted in pre- and post-Aire subsets, respectively. To investigate the temporal differentiation of mTECs, we generated the Aire-Cre:R26R-DTR (Aire-DTR) mice, which marked both Aire+ and post-Aire mTECs with diphtheria toxin (DT) receptors. Consecutive administration of DT resulted in ablation of both subsets, whereas pre-Aire mTECs remained intact. Upon DT withdrawal, transcripts of Aire and TSAs remained absent at day 3, but became detectable at day 5. Interestingly, no autoimmunity was observed in Aire-DTR mice treated weekly with a single dose of DT, suggesting that the newly generated Aire+ mTECs could reestablish self-tolerance in a timely manner. Our data provide new insights into the temporal regeneration of mTECs and its importance in maintaining central tolerance.

Thymic crosstalk between medullary thymic epithelial cells (mTEC) and mature SP thymocytes is critical for tolerance in developing T cells as well as for mTEC development. Several TNF family receptor-ligand pairs, including RANK-RANKL, LTαβ-LTβR and CD40L-CD40, have been shown to be important in thymic crosstalk, in part via RelB mediated non-canonical NFκB pathway. Recently, we identified CD28-CD80/86 as an additional pathway important for mTEC development. CD80/86, LTβR, CD40, RANK, as well as MHCII (also shown to be important for mTEC development) are expressed by both mTEC and by bone marrow (BM) derived cells in the thymus. Thus, it is important to ask which cell types need to express these molecules in order to support mTEC development. To address the question, we have generated and analyzed BM chimeras or conditional knockout mice in which either non-BM-derived cells (including thymic epithelium) or BM-derived cells express the molecule in question. Interestingly, for CD40 and CD80/86, we find that expression on either non-BM or BM-derived cells is sufficient to promote mTEC development. For LTβR, and RANK, we find that expression on radiation-resistant non-BM derived cells is required. The ability of MHCII expression on specific cell populations to drive mTEC development is currently being examined. Our findings suggest that models of thymic crosstalk must consider not only communication between TEC and thymocytes but with other BM derived cell types as well.