mTEC damage risks immune recovery

Whether autologous hematopoietic stem cell transplantation is free from graft-versus-host disease is controversial. Alawam et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20211239) now demonstrate that prolonged damage in thymic medullary epithelial cells causes the failure in self-tolerance in newly generated T cells and provokes post-transplant autoimmunity.

“You don’t know what you’ve got till it’s gone.” —Joni Mitchell, “Big Yellow Taxi” Hematopoietic stem cell transplantation (HSCT) is an important treatment of cancer and other diseases (Copelan, 2006; Saad et al., 2020). Complications associated with HSCT include graft-versus-host disease (GVHD), which has been detected even when the combination between grafts and hosts is autologous (Kline et al., 2008). However, whether and how autologous HSCT triggers the breakdown of self-tolerance in the immune system is controversial (Otegbeye et al., 2014). The new study by Alawam et al. published in this issue of Journal of Experimental Medicine demonstrates that in a mouse model of syngeneic HSCT, the recovery of medullary thymic epithelial cells (mTECs) in the thymus was noticeably inefficient and slow compared with cortical TECs (cTECs; Alawam et al., 2021). Importantly, as mTECs have an essential role for the establishment of self-tolerance in newly generated T cells (Abramson and Anderson, 2017), Alawam et al. report that the sustained failure in mTEC recovery during syngeneic HSCT is accompanied by reduced number in thymic dendritic cells (DCs), delayed development of Foxp3⁺ regulatory T (Treg) cells, and defective T cell self-tolerance, which triggers autoimmunity in mice (Alawam et al., 2021). The prolonged damage in mTECs may explain the onset of GVHD-like complications during autologous HSCT.Insights from Yousuke Takahama.In the new study, Alawam et al. examined how syngeneic bone marrow cell transplantation in irradiated mice affects TECs (Alawam et al., 2021). They noticed that the number of mTECs was significantly reduced, while the number of cTECs was not altered, during the process of syngeneic bone marrow cell transplantation. The reduced mTECs included the Aire⁺ MHC II^high CD80^high subpopulation, which has an essential role in inducing self-tolerance in newly generated T cells, by supporting the promiscuous expression of virtually all genes to display genome-wide self-components in the thymic medullary microenvironment (Kyewski and Klein, 2006). Other mTEC subpopulations, including CCL21⁺ cells and thymic tuft cells (Kadouri et al., 2020), were also reduced. CCL21⁺ mTECs have an essential role in inducing self-tolerance in T cells, by supporting the migration of positively selected cortical thymocytes into the thymic medulla (Kozai et al., 2017). Interestingly, the number of Aire⁺ mTECs, which was significantly reduced during the first 3 wk after transplantation, recovered afterward, suggesting that the post-HSCT damage in mTECs is transient and the recovery of mTECs is slower than other cells. Along with the prolonged decrease in mTECs, the number of thymic DCs was also reduced after the bone marrow cell transplantation and their recovery was inefficient and delayed.cTECs are responsible for the production and positive selection of T cells in the thymic cortex (Takahama et al., 2017). Alawam et al. showed that no significant reduction in cTECs during HSCT coincided with the rapid progression of thymocyte development after the bone marrow cell transplantation. In contrast, mTECs and DCs in the thymic medulla are cooperatively responsible for the establishment of self-tolerance in T cells by inducing negative selection of self-reactive T cells and by promoting the generation of Treg cells (Abramson and Anderson, 2017). Importantly, Alawam et al. showed the reduction in caspase 3–cleaved apoptotic TCRβ^high CD5^high thymocytes and the escape of self-mammary tumor virus superantigen-reactive thymocytes from thymic deletion. They also showed a prolonged reduction and delayed recovery in the number of Foxp3⁺ Treg cells during the bone marrow cell transplantation. These results indicate that during syngeneic bone marrow cell transplantation, the thymic cortex keeps its functional capability to produce T cells, but thymic medulla function is substantially impaired, resulting in a failure to establish self-tolerance in T cells newly produced in the cortex. Alawam et al. further showed that Treg cell–depleted CD4⁺CD8⁻ mature thymocytes generated after transplantation readily cause autoimmunity in mice by detecting lymphocyte infiltration in the liver and autoantibodies against various tissues upon the transfer in athymic nude mice. Collectively, Alawam et al. report that during syngeneic bone marrow cell transplantation, the thymus can support production of TCRαβ⁺ T cells rapidly and efficiently, despite that the thymic function is not fully operational as the thymic medulla remains damaged and insufficient to cause defective self-tolerance in T cells.In a normal thymus, hematopoietic stem cell (HSC)–derived precursors enter the thymic cortex to become T-lineage cells and to generate functionally capable repertoire of T cell specificities. Newly generated T-lineage cells migrate to the thymic medulla to acquire self-tolerance with the help of mTECs and DCs. Stepwise supports by thymic cortex and medulla establish a pool of functional and self-tolerant T cells (left). Alawam et al. (2021) demonstrate that after HSCT, the thymic cortex functions undisturbed but the thymic medulla is damaged. Selective damage in the thymic medulla causes the failure in self-tolerance in newly generated T cells and leads to the generation of functional but autoreactive T cell pool (right).By revealing the uncoupling of T cell–producing function by the thymic cortex and self-tolerance–inducing function by the thymic medulla, the study by Alawam et al. suggests a scenario that prolonged failure in thymic medulla is responsible for the loss of self-tolerance in T cells during autologous HSCT. Such a failure in thymic medulla and subsequent autoimmunity during immune reconstitution may explain GVHD-like tissue damages and associated symptoms detected in patients who received autologous HSCT (Abudayyeh et al., 2015; El-Jurdi et al., 2017; Hammami et al., 2018; Hierlmeier et al., 2018).Total body irradiation before HSCT is likely responsible for the damage in mTECs. However, mechanisms for the selective and sustained loss in mTECs (when cTECs seem to remain undisturbed) and the prolonged failure in thymic medulla function during HSCT are interesting issues. Further studies would lead toward a better understanding of the mechanism for the generation and regeneration of heterogenous TEC subpopulations, including cTECs, Aire⁺ mTECs, and CCL21⁺ mTECs. How long the defects persist is another interesting question. Alawam et al. reports that total mTEC number stayed reduced even after 8 wk after transplantation, although Aire⁺ mTECs and Foxp3⁺ Treg cells recovered by then. It is possible that thymic medulla failures in initial stages of immune reconstitution are accompanied by autoimmunity but are later tempered by the availability of Treg cells. Seeking a synchronized regeneration of multiple TEC subpopulations appears an important challenge toward further successful hematopoietic cell reconstitution without complications with the risk of autoimmunity.     

RANK signals from CD4(+)3(-) inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla

Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4(+)3(-) inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4(+)3(-) cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire(+) mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80(-)Aire(-) mTEC progenitors into CD80(+)Aire(+) mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire(+) mTECs and highlight a previously unrecognized role for CD4(+)3(-)RANKL(+) inducer cells in intrathymic self-tolerance.     

Aire controls the differentiation program of thymic epithelial cells in the medulla for the establishment of self-tolerance

The roles of autoimmune regulator (Aire) in the expression of the diverse arrays of tissue-restricted antigen (TRA) genes from thymic epithelial cells in the medulla (medullary thymic epithelial cells [mTECs]) and in organization of the thymic microenvironment are enigmatic. We approached this issue by creating a mouse strain in which the coding sequence of green fluorescent protein (GFP) was inserted into the Aire locus in a manner allowing concomitant disruption of functional Aire protein expression. We found that Aire⁺ (i.e., GFP⁺) mTECs were the major cell types responsible for the expression of Aire-dependent TRA genes such as insulin 2 and salivary protein 1, whereas Aire-independent TRA genes such as C-reactive protein and glutamate decarboxylase 67 were expressed from both Aire⁺ and Aire⁻ mTECs. Remarkably, absence of Aire from mTECs caused morphological changes together with altered distribution of mTECs committed to Aire expression. Furthermore, we found that the numbers of mTECs that express involucrin, a marker for terminal epidermal differentiation, were reduced in Aire-deficient mouse thymus, which was associated with nearly an absence of Hassall's corpuscle-like structures in the medulla. Our results suggest that Aire controls the differentiation program of mTECs, thereby organizing the global mTEC integrity that enables TRA expression from terminally differentiated mTECs in the thymic microenvironment.     

Failures in thymus medulla regeneration during immune recovery cause tolerance loss and prime recipients for auto-GVHD

Bone marrow transplantation (BMT) is a widely used therapy for blood cancers and primary immunodeficiency. Following transplant, the thymus plays a key role in immune reconstitution by generating a naive αβT cell pool from transplant-derived progenitors. While donor-derived thymopoiesis during the early post-transplant period is well studied, the ability of the thymus to synchronize T cell development with essential tolerance mechanisms is poorly understood. Using a syngeneic mouse transplant model, we analyzed T cell recovery alongside the regeneration and function of intrathymic microenvironments. We report a specific and prolonged failure in the post-transplant recovery of medullary thymic epithelial cells (mTECs). This manifests as loss of medulla-dependent tolerance mechanisms, including failures in Foxp3⁺ regulatory T cell development and formation of the intrathymic dendritic cell pool. In addition, defective negative selection enables escape of self-reactive conventional αβT cells that promote autoimmunity. Collectively, we show that post-transplant T cell recovery involves an uncoupling of thymopoiesis from thymic tolerance, which results in autoimmune reconstitution caused by failures in thymic medulla regeneration.     

Enhancement of an anti-tumor immune response by transient blockade of central T cell tolerance

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.Medullary thymic epithelial cells (mTECs) contribute to self-tolerance through the ectopic expression of tissue-specific antigens (TSAs) in the thymus (Derbinski et al., 2001; Anderson et al., 2002; Metzger and Anderson, 2011). This TSA expression in mTECs is largely dependent on autoimmune regulator (Aire), which is expressed in mature mTECs (Gäbler et al., 2007; Gray et al., 2007; Metzger and Anderson, 2011). Through the recognition of TSAs, developing autoreactive T cells are either negatively selected from the pool of developing thymocytes or recruited into the regulatory T (T reg) cell lineage (Liston et al., 2003; Anderson et al., 2005; DeVoss et al., 2006; Shum et al., 2009; Taniguchi et al., 2012; Malchow et al., 2013). The overall importance of this process is underscored by the development of a multi-organ autoimmune syndrome in patients or mice with defective AIRE expression (Consortium, 1997; Nagamine et al., 1997; Anderson et al., 2002).Although central tolerance provides protection against autoimmunity, this process also represents a challenge for anti-tumor immunity (Kyewski and Klein, 2006; Malchow et al., 2013). Because many of the TSAs expressed in the thymus are also expressed in tumors, high-affinity effector T cells capable of recognizing tumor self-antigens may normally be deleted in the thymus (Bos et al., 2005; Cloosen et al., 2007; Träger et al., 2012; Zhu et al., 2013). Transiently suppressing central tolerance by depleting mTECs or modulating Aire expression may provide a therapeutic window for the generation of T cells capable of recognizing tumor self-antigens. Many current cancer immune therapies rely on activating relatively weak tumor-specific T cell responses through modulating peripheral tolerance (Swann and Smyth, 2007; Chen and Mellman, 2013). In contrast, manipulation of central tolerance has the potential to increase the pool and affinity of effector T cells that can recognize and contribute to effective anti-tumor responses. Furthermore, such high-affinity, self-reactive T cells may be more resistant to peripheral tolerance mechanisms that typically restrain an anti-tumor response (Swann and Smyth, 2007). Thus, the development of methods that selectively and transiently deplete Aire-expressing mTECs may be an attractive method to enhance tumor-specific immune responses.Previous work has identified agents that can inhibit the growth and development of TECs such as corticosteroids, cyclosporine, and some inflammatory cytokines (Anz et al., 2009; Fletcher et al., 2009). Despite their clear inhibitory effects on TECs, however, these agents do not appear to have selectivity for blocking mTEC development. Interestingly, recent studies have demonstrated a role for TNF family member pairs RANK–RANKL and CD40-CD40L in the embryological development of Aire⁺ mTECs (Rossi et al., 2007; Akiyama et al., 2008; Hikosaka et al., 2008; Roberts et al., 2012). Recent work has also demonstrated that mTECs in particular have a relatively fast turnover in adult mice with an estimated half-life of ∼2 wk (Gäbler et al., 2007; Gray et al., 2007). Given these findings, we speculated that in vivo blockade of RANKL in adult hosts could both selectively and transiently inhibit the development and turnover of mTECs with potential to alter central T cell tolerance. To this end, we performed in vivo RANKL blockade in adult mice and investigated its effects on both TECs and developing thymocytes. We show that anti-RANKL treatment not only depleted mTECs but could also be used therapeutically to break central tolerance and, as a result, increase the generation of tumor-specific T cells.     

The thymic medulla: a unique microenvironment for intercellular self-antigen transfer

Central tolerance is shaped by the array of self-antigens expressed and presented by various types of thymic antigen-presenting cells (APCs). Depending on the overall signal quality and/or quantity delivered in these interactions, self-reactive thymocytes either apoptose or commit to the T regulatory cell lineage. The cellular and molecular complexity underlying these events has only recently been appreciated. We analyzed the ex vivo presentation of ubiquitous or tissue-restricted self-antigens by medullary thymic epithelial cells (mTECs) and thymic dendritic cells (DCs), the two major APC types present in the medulla. We found that the ubiquitously expressed nuclear neo–self-antigen ovalbumin (OVA) was efficiently presented via major histocompatibility complex class II by mTECs and thymic DCs. However, presentation by DCs was highly dependent on antigen expression by TECs, and hemopoietic cells did not substitute for this antigen source. Accordingly, efficient deletion of OVA-specific T cells correlated with OVA expression by TECs. Notably, OVA was only presented by thymic but not peripheral DCs. We further demonstrate that thymic DCs are constitutively provided in situ with cytosolic as well as membrane-bound mTEC-derived proteins. The subset of DCs displaying transferred proteins was enriched in activated DCs, with these cells being most efficient in presenting TEC-derived antigens. These data provide evidence for a unique, constitutive, and unidirectional transfer of self-antigens within the thymic microenvironment, thus broadening the cellular base for tolerance induction toward promiscuously expressed tissue antigens.The scope of central tolerance is determined by the recognition of self-antigens presented by various APCs in the thymus. The thymic medulla is the major but not exclusive site of central tolerance induction (McCaughtry et al., 2008). The complexity of this process with regard to the composition and role of distinct APC types, and the sets of self-antigens displayed has only recently become apparent. Self-antigens can be supplied by the proteome of residential epithelial cells (i.e., cortical and medullary thymic epithelial cells [mTECs]) and BM-derived APCs (i.e., DCs, macrophages [MΦ], and B cells). In addition, self-antigens from the periphery can reach the thymus medulla via the blood stream or be imported via immigrating antigen-laden DCs (Klein et al., 2001; Bonasio et al., 2006; Li et al., 2009). Among these different self-antigen pools, the array of promiscuously expressed tissue-restricted self-antigens by mTECs is of particular interest, because it preempts the peripheral self. Qualitative or quantitative alterations of this antigen pool predispose to organ-specific autoimmunity. How central tolerance to these antigens is achieved is closely connected to the questions of which APCs present these antigens and which processing pathways are involved (Klein and Kyewski, 2000).Previous studies addressing these issues have been limited to broadly expressed self-antigens (i.e., Eα; Humblet et al., 1994), or neo–self-antigens driven by promoters resulting in expression in a major subset of mTECs (i.e., the Aire promoter; Aschenbrenner et al., 2007) or in more restricted expression, e.g., the rat insulin promoter (Zhang et al., 2003; Gallegos and Bevan, 2004; Zehn and Bevan, 2006). In case of the latter transgenes, it remained however unclear whether the frequency of antigen-positive mTECs and the expression levels per cell faithfully reflected the pattern of promiscuously expressed tissue-restricted antigens (TRAs). Both parameters are critical for T cell (TC) selection.In this report, we analyzed ex vivo presentation of the two native self-antigens, P1A and proteolipid protein (PLP). The tumor rejection antigen P1A is a prototypic cytosolic TRA whose expression is tightly regulated and only detectable in male germ cells and rare mTECs (Derbinski et al., 2001). PLP, an oligodendrocyte-specific myelin protein, is strongly expressed in mTECs and weakly expressed in thymic DCs (Derbinski et al., 2001). Moreover, we wanted to delineate the relative contribution of thymic APC types to tolerance when antigen expression is not confined to a particular cell type. To this end, we chose the neo–self-antigen OVA, expressed ubiquitously and targeted specifically to the nucleus (Kawahata et al., 2002).We found both thymic DCs and mTECs to present these self-antigens ex vivo and in vivo. Efficient presentation by DCs, however, was contingent on antigen provision by TECs irrespective of their subcellular localization or expression pattern. This efficient self-antigen transfer, which apparently is unique to the thymus, broadens the cellular base of self-tolerance and thus enhances its efficacy.     

Constitutive ablation of dendritic cells breaks self-tolerance of CD4 T cells and results in spontaneous fatal autoimmunity

Lack of immunological tolerance against self-antigens results in autoimmune disorders. During onset of autoimmunity, dendritic cells (DCs) are thought to be critical for priming of self-reactive T cells that have escaped tolerance induction. However, because DCs can also induce T cell tolerance, it remains unclear whether DCs are required under steady-state conditions to prevent autoimmunity. To address this question, we crossed CD11c-Cre mice with mice that express diphtheria toxin A (DTA) under the control of a loxP-flanked neomycin resistance (neo^R) cassette from the ROSA26 locus. Cre-mediated removal of the neo^R cassette leads to DTA expression and constitutive loss of conventional DCs, plasmacytoid DCs, and Langerhans cells. These DC-depleted (ΔDC) mice showed increased frequencies of CD4 single-positive thymocytes and infiltration of CD4 T cells into peripheral tissues. They developed spontaneous autoimmunity characterized by reduced body weight, splenomegaly, autoantibody formation, neutrophilia, high numbers of Th1 and Th17 cells, and inflammatory bowel disease. Pathology could be induced by reconstitution of wild-type (WT) mice with bone marrow (BM) from ΔDC mice, whereas mixed BM chimeras that received BM from ΔDC and WT mice remained healthy. This demonstrates that DCs play an essential role to protect against fatal autoimmunity under steady-state conditions.The adaptive immune system can respond to a huge variety of pathogens as a result of a broad repertoire of antigen receptors on T and B cells generated by genomic recombination during development of these cells. To avoid autoimmune reactions, self-reactive lymphocytes have to be deleted or rendered tolerant. Normal polyclonal and self-tolerant T cell repertoires depend on positive and negative selection of developing T cells in the thymus. Positive selection is mediated by thymic cortical epithelial cells, whereas negative selection can occur in the cortex or in the medulla and is induced by both BM–derived cells and medullary thymic epithelial cells (1–5). It has been demonstrated that thymic DCs are very efficient in mediating negative selection of developing thymocytes (5–9). Furthermore, peripheral DCs can migrate to the thymus and contribute to negative selection (9, 10). However, because B cells (11), and perhaps other cells of hematopoietic origin, could also be involved in negative selection, it remains unclear whether a selective lack of DCs would result in impaired clonal deletion and release of self-reactive T cells into the periphery. Self-reactive T cells that escaped clonal deletion in the thymus need to be further controlled by peripheral tolerance mechanisms to prevent tissue damage (12). Under steady-state conditions, DCs are thought to play an important role in peripheral tolerance induction by various mechanisms, including production of soluble factors like IL-10, TGF-β or indoleamine 2,3-dioxygenase (13–15), induction of T reg cells (16–18), and initiation of abortive T cell proliferation resulting in clonal deletion of autoreactive T cells (19, 20). However, it remains unclear whether DCs are required to protect from spontaneous onset of autoimmunity. To address this important question, we generated constitutively DC-depleted mice. These mice rapidly developed spontaneous autoimmunity, which demonstrates for the first time that DCs are essential to maintain a self-tolerant immune system.     

The thymic medulla is required for Foxp3+ regulatory but not conventional CD4+ thymocyte development

A key role of the thymic medulla is to negatively select autoreactive CD4⁺ and CD8⁺ thymocytes, a process important for T cell tolerance induction. However, the involvement of the thymic medulla in other aspects of αβ T cell development, including the generation of Foxp3⁺ natural regulatory T cells (nT_reg cells) and the continued maturation of positively selected conventional αβ T cells, is unclear. We show that newly generated conventional CD69⁺Qa2⁻ CD4 single-positive thymocytes mature to the late CD69⁻Qa2⁺ stage in the absence of RelB-dependent medullary thymic epithelial cells (mTECs). Furthermore, an increasing ability to continue maturation extrathymically is observed within the CD69⁺CCR7^−/loCCR9⁺ subset of conventional SP4 thymocytes, providing evidence for an independence from medullary support by the earliest stages after positive selection. In contrast, Foxp3⁺ nT_reg cell development is medullary dependent, with mTECs fostering the generation of Foxp3⁻CD25⁺ nT_reg cell precursors at the CD69⁺CCR7⁺CCR9⁻ stage. Our results demonstrate a differential requirement for the thymic medulla in relation to CD4 conventional and Foxp3⁺ thymocyte lineages, in which an intact mTEC compartment is a prerequisite for Foxp3⁺ nT_reg cell development through the generation of Foxp3⁻CD25⁺ nT_reg cell precursors.In the thymus, positive selection of CD4⁺8⁺ thymocytes recognizing self-peptide/MHC on cortical thymic epithelial cells (TECs) triggers the entry of CD4/CD8 single-positive (SP) T cells into the thymic medulla, a process essential for tolerance induction (Kurobe et al., 2006). Additionally, the medulla is also considered a key site of differentiation that supports thymocyte maturation after positive selection, including stages defined by loss of CD24/CD69 and acquisition of CD62L/Qa2 (McCaughtry et al., 2007; Li et al., 2007).Although the medulla also contains SP4 Foxp3⁺ natural regulatory T cells (nT_reg cells; Liston et al., 2008), its role in nT_reg cell generation remains unclear, with both medullary TECs (mTECs) and DCs being implicated (Aschenbrenner et al., 2007; Proietto et al., 2008; Spence and Green, 2008; Wirnsberger et al., 2009; Hinterberger et al., 2010). Importantly, nT_reg cell development is a multistage process, with TCR–MHC (Lio and Hsieh, 2008) and CD28–CD80/86 interactions (Lio et al., 2010; Vang et al., 2010; Hinterberger et al., 2011) driving the generation of Foxp3⁻CD25⁺ nT_reg cell precursors that give rise to Foxp3⁺CD25⁺ nT_reg cells (Lio and Hsieh, 2008). However, the role of mTECs during Foxp3⁻CD25⁺ nT_reg cell precursor generation is unknown.Here, we define steps in both conventional and nT_reg SP4 thymocyte maturation, mapping their requirements for a RelB-dependent mTEC compartment (Burkly et al., 1995; Weih et al., 1995; Heino et al., 2000). We show that conventional SP4 thymocytes can complete their maturation in the absence of RelB-dependent mTECs, with evidence of thymic independence occurring by the CD69⁺CCR7^−/loCCR9⁺ SP4 thymocyte stage. In contrast, Foxp3⁺ nT_reg cells require an intact thymic medulla, with a requirement for RelB-dependent mTEC mapping to the generation of Foxp3⁻CD25⁺ nT_reg cell precursors at the CD69⁺CCR7⁺CCR9⁻ stage. Collectively, our data reveal the differential importance of the thymic medulla during SP4 thymocyte development and highlight a specific role for mTECs in Foxp3⁻CD25⁺ precursor generation.     

Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development

Dendritic cells (DCs) in the thymus (tDCs) are predominantly accumulated in the medulla and contribute to the establishment of self-tolerance. However, how the medullary accumulation of tDCs is regulated and involved in self-tolerance is unclear. We show that the chemokine receptor XCR1 is expressed by tDCs, whereas medullary thymic epithelial cells (mTECs) express the ligand XCL1. XCL1-deficient mice are defective in the medullary accumulation of tDCs and the thymic generation of naturally occurring regulatory T cells (nT reg cells). Thymocytes from XCL1-deficient mice elicit dacryoadenitis in nude mice. mTEC expression of XCL1, tDC medullary accumulation, and nT reg cell generation are diminished in Aire-deficient mice. These results indicate that the XCL1-mediated medullary accumulation of tDCs contributes to nT reg cell development and is regulated by Aire.     

Identification of a novel cis-regulatory element essential for immune tolerance

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.The establishment and maintenance of immune tolerance requires the successful detection and control of self-reactive T cells in the thymus and the periphery. In the thymus, medullary thymic epithelial cells (mTECs) play a crucial role in this process, presenting a varied repertoire of antigens and thereby eliminating self-reactive T cells or driving them to a regulatory T cell fate (Derbinski et al., 2001; Malchow et al., 2013). Along with ubiquitous self-antigens, mTECs present thousands of tissue-specific antigens (TSAs), and expression of many of these TSAs depends on the Autoimmune regulator (Aire) gene (Anderson et al., 2002). Humans and mice with mutations in Aire develop an organ-specific autoimmune syndrome, underscoring the critical role of Aire in immune tolerance (Aaltonen et al., 1997; Nagamine et al., 1997; Anderson et al., 2002).Interestingly, Aire expression is highly tissue restricted. It is expressed broadly early in embryogenesis but then restricted to a subset of mature mTECs and rare extrathymic Aire-expressing cells (eTACs), a DC-lineage–derived population present in secondary lymphoid tissues (Gardner et al., 2008, 2013). Previous research has demonstrated the importance of a few TNF receptor superfamily members, particularly Tnfrsf11a (RANK), in the development and maintenance of Aire-expressing mTECs (Rossi et al., 2007; Akiyama et al., 2008; Hikosaka et al., 2008; Khan et al., 2014). Likewise, noncanonical NF-κB family members, which can be activated via RANK signaling, are also essential for the development of Aire-expressing mTECs (Heino et al., 2000; Zhu et al., 2006). Despite this knowledge of factors that help promote expression of Aire, the DNA regulatory elements that help coordinate this process have not been clearly delineated. We sought to identify cis-regulatory elements (CREs) important for regulating Aire expression.In vertebrates, gene regulation involves both proximal CREs—the promoter—and distal CREs, including enhancers and silencers. The sequences of such elements are often conserved (Noonan and McCallion, 2010). In addition, several epigenetic markers, including p300 binding and the histone modification acetylated lysine 27 of histone 3 (H3K27ac), often mark active enhancers (Visel et al., 2009; Creyghton et al., 2010). Here, we searched for candidate Aire-regulating elements through the use of both sequence conservation and chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq). This focused our investigation on a highly conserved region ∼3 kb upstream of Aire that is an NF-κB-responsive element and is essential for Aire expression and immune tolerance.     

Distinct roles for PTEN in prevention of T cell lymphoma and autoimmunity in mice

Mutations in the tumor-suppressor gene phosphatase and tensin homolog deleted on chromosome 10 (Pten) are associated with multiple cancers in humans, including T cell malignancies. Targeted deletion of Pten in T cells induces both a disseminated “mature phenotype” lymphoma and a lymphoproliferative autoimmune syndrome in mice. Here, we have shown that these two diseases are separable and mediated by T lineage cells of distinct developmental stages. Loss of PTEN was found to be a powerful driver of lymphomagenesis within the thymus characterized by overexpression of the c-myc oncogene. In an otherwise normal thymic environment, PTEN-deficient T cell lymphomas invariably harbored RAG-dependent reciprocal t(14:15) chromosomal translocations involving the T cell receptor alpha/delta locus and c-myc, and their survival and growth was TCR dependent, but Notch independent. However, lymphomas occurred even if TCR recombination was prevented, although these lymphomas were less mature, arose later in life, and, importantly, were dependent upon Notch pathways to upregulate c-myc expression. In contrast, using the complementary methods of early thymectomy and adoptive transfers, we found that PTEN-deficient mature T cells were unable to undergo malignant transformation but were sufficient for the development of autoimmunity. These data suggest multiple and distinct regulatory roles for PTEN in the molecular pathogenesis of lymphoma and autoimmunity.     

CVID-associated TACI mutations affect autoreactive B cell selection and activation

Common variable immune deficiency (CVID) is an assorted group of primary diseases that clinically manifest with antibody deficiency, infection susceptibility, and autoimmunity. Heterozygous mutations in the gene encoding the tumor necrosis factor receptor superfamily member TACI are associated with CVID and autoimmune manifestations, whereas two mutated alleles prevent autoimmunity. To assess how the number of TACI mutations affects B cell activation and tolerance checkpoints, we analyzed healthy individuals and CVID patients carrying one or two TACI mutations. We found that TACI interacts with the cleaved, mature forms of TLR7 and TLR9 and plays an important role during B cell activation and the central removal of autoreactive B cells in healthy donors and CVID patients. However, only subjects with a single TACI mutation displayed a breached immune tolerance and secreted antinuclear antibodies (ANAs). These antibodies were associated with the presence of circulating B cell lymphoma 6–expressing T follicular helper (Tfh) cells, likely stimulating autoreactive B cells. Thus, TACI mutations may favor CVID by altering B cell activation with coincident impairment of central B cell tolerance, whereas residual B cell responsiveness in patients with one, but not two, TACI mutations enables autoimmune complications.     

A comparative study on roles of natural killer T cells in two diet-induced non-alcoholic steatohepatitis-related fibrosis in mice

BackgroundImmune responses are important in the progression of non-alcoholic fatty liver disease (NAFLD). Natural killer T (NKT) cells are main components of the innate immune system that modulate immunity. However, the role of NKT cells in NAFLD remains controversial.ObjectiveWe aimed to investigate the role of NKT cells in non-alcoholic steatohepatitis (NASH)-related fibrosis in fast food diet (FFD)- and methionine choline-deficient (MCD) diet-induced mouse models.MethodsHepatic NKT cells were analysed in wild-type (WT) and CD1d-/- mice fed FFD or MCD diets. Hepatic pathology, cytokine profiles and liver fibrosis were evaluated. Furthermore, the effect of chronic administration of α-galactosylceramide (α-GalCer) on liver fibrosis was investigated in both FFD- and MCD-treated mice.ResultsFFD induced a significant depletion of hepatic NKT cells, thus leading to mild to moderate NASH and early-stage fibrosis, while mice fed MCD diets developed severe liver inflammation and progressive fibrosis without a significant change in hepatic NKT cell abundance. FFD induced a similar liver fibrogenic response in CD1d-/- and WT mice, while MCD induced a higher hepatic mRNA expression of Col1α1 and TIMP1 as well as relative fibrosis density in CD1d-/- mice than WT mice (31.8 vs. 16.3, p = .039; 40.0 vs. 22.6, p = .019; 2.24 vs. 1.59, p = .036). Chronic administration of α-GalCer induced a higher hepatic mRNA expression of TIMP1 in MCD-treated mice than controls (36.7 vs. 14.9, p = .005).ConclusionNKT cells have protective roles in NAFLD as the disease progresses. During diet-induced steatosis, mild to moderate NASH and the early stage of fibrosis, hepatic NKT cells are relatively depleted, leading to a proinflammatory status. In severe NASH and the advanced stage of liver fibrosis, NKT cells play a role in inhibiting the NASH-related fibrogenic response. Chronic administration of α-GalCer induces NKT cell anergy and tolerance, which may play a role in promoting the liver fibrogenic response.     

Expression of the retinoblastoma protein RbAp48 in exocrine glands leads to Sj?gren's syndrome–like autoimmune exocrinopathy

Although several autoimmune diseases are known to develop in postmenopausal women, the mechanisms by which estrogen deficiency influences autoimmunity remain unclear. Recently, we found that retinoblastoma-associated protein 48 (RbAp48) induces tissue-specific apoptosis in the exocrine glands depending on the level of estrogen deficiency. In this study, we report that transgenic (Tg) expression of RbAp48 resulted in the development of autoimmune exocrinopathy resembling Sjögren''s syndrome. CD4⁺ T cell–mediated autoimmune lesions were aggravated with age, in association with autoantibody productions. Surprisingly, we obtained evidence that salivary and lacrimal epithelial cells can produce interferon-γ (IFN-γ) in addition to interleukin-18, which activates IFN regulatory factor-1 and class II transactivator. Indeed, autoimmune lesions in Rag2^−/− mice were induced by the adoptive transfer of lymph node T cells from RbAp48-Tg mice. These results indicate a novel immunocompetent role of epithelial cells that can produce IFN-γ, resulting in loss of local tolerance before developing gender-based autoimmunity.Autoimmune disease is controlled by environments that include gene variants or various cytokines (1, 2). It can increase susceptibility to autoimmunity by affecting the overall reactivity and quality of the cells of the immune system. There is an autoimmune disease specific for certain organs in the body, involving a response to an antigen expressed only in those organs. Antigen/organ specificity is affected by antigen presentation and recognition, antigen expression, and the state and response of the target organs (3, 4), which are maintained by a local immune system termed here “local tolerance.”Many mechanisms protect tissues from autoimmune damage. These include relative isolation from the immune system and inhibition of the function of invading lymphocytes. For example, the eye has barriers to T cell infiltration and produces immunosuppressive cytokines, such as TGF-β (5). Constitutive expression of Fas ligand within the privileged site might also prevent immune-mediated damage by eliminating Fas-expressing T cells (6). Although they have yet to be well demonstrated in spontaneous animal models or human disease, genetic effects at the level of tissue protection are therefore to be expected. Autoimmune organ damage can be mediated by CD4⁺ T cells, which play a crucial role in the development of autoimmunity (7–9). MHC class II alleles are probably involved in autoimmune disease because different alleles have different abilities to present peptides from target cells to autoreactive CD4⁺ T cells (10, 11). Certain class II alleles might predispose to autoimmunity by increasing positive selection or decreasing negative selection of autoreactive T cells in the thymus. They might also act by inhibiting selection in the thymus of the regulatory CD4⁺ T cells that are thought to prevent autoantigen-specific responses. Evidence for the local tolerance hypothesis is provided by the observation that autoimmune diseases are often tissue specific and sometimes involve antibodies against a restricted set of antigens, thereby prompting us to accept this most simple explanation for the initiation of autoimmunity. The loss of local tolerance is considered to result from the combined effect of different environmental factors. MHC class II genes are constitutively expressed only on hematopoietic cells involved in antigen presentation (dendritic cells, macrophages, B cells, and cortical thymic epithelial cells), but can be aberrantly induced by inflammatory stimuli on many other cell types (such as endothelial cells, hepatocytes, β cells of the pancreas, and thyrocytes) (12, 13). Although it has been implicated in allograft rejection (14), and subsequently in autoimmunity, it is still unknown whether to initiate autoimmunity class II molecules have to be expressed on professional APCs within secondary lymphoid organs or on nonhematopoietic cells of the target organ itself.It has been suggested that estrogenic action is responsible for the strong female preponderance of many autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjögren''s syndrome (SS) (15, 16). Recent evidence suggests that apoptosis plays a key role in the physiology and pathogenesis of various autoimmune diseases, including SS (17–21). We have demonstrated that estrogenic action influences target epithelial cells through Fas-mediated apoptosis in a murine model for SS (21). Recently, we found that tissue-specific apoptosis in the exocrine glands spontaneously occurring in estrogen-deficient mice may contribute to the development of autoimmune exocrinopathy (22). Searching for the role of estrogen deficiency in the development of autoimmunity, we have recently identified retinoblastoma-associated protein 48 (RbAp48) gene specific for estrogen deficiency–dependent apoptosis in the exocrine glands, and transgenic expression of RbAp48 gene induced tissue-specific apoptosis in the exocrine glands (23). In this transgenic mouse model, we propose a possible clear and defined ab initio relationship between aberrant exposure of MHC class II molecules on IFN-γ–producing epithelial cells and disease development (i.e., autoimmune exocrinopathy).     

CD4 T Cell Tolerance to Human C-reactive Protein, an Inducible Serum Protein, Is Mediated by Medullary Thymic Epithelium

Inducible serum proteins whose concentrations oscillate between nontolerogenic and tolerogenic levels pose a particular challenge to the maintenance of self-tolerance. Temporal restrictions of intrathymic antigen supply should prevent continuous central tolerization of T cells, in analogy to the spatial limitation imposed by tissue-restricted antigen expression. Major acute-phase proteins such as human C-reactive protein (hCRP) are typical examples for such inducible self-antigens. The circulating concentration of hCRP, which is secreted by hepatocytes, is induced up to 1,000-fold during an acute-phase reaction. We have analyzed tolerance to hCRP expressed in transgenic mice under its autologous regulatory regions. Physiological regulation of basal levels (<10⁻⁹ M) and inducibility (>500-fold) are preserved in female transgenics, whereas male transgenics constitutively display induced levels. Surprisingly, crossing of hCRP transgenic mice to two lines of T cell receptor transgenic mice (specific for either a dominant or a subdominant epitope) showed that tolerance is mediated by intrathymic deletion of immature thymocytes, irrespective of widely differing serum levels. In the absence of induction, hCRP expressed by thymic medullary epithelial cells rather than liver-derived hCRP is necessary and sufficient to induce tolerance. Importantly, medullary epithelial cells also express two homologous mouse acute-phase proteins. These results support a physiological role of “ectopic” thymic expression in tolerance induction to acute-phase proteins and possibly other inducible self-antigens and have implications for delineating the relative contributions of central versus peripheral tolerance.     

Conditional inactivation of Fbxw7 impairs cell-cycle exit during T cell differentiation and results in lymphomatogenesis

Cell proliferation is strictly controlled during differentiation. In T cell development, the cell cycle is normally arrested at the CD4⁺CD8⁺ stage, but the mechanism underlying such differentiation-specific exit from the cell cycle has been unclear. Fbxw7 (also known as Fbw7, Sel-10, hCdc4, or hAgo), an F-box protein subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle, such as c-Myc, c-Jun, cyclin E, and Notch. FBXW7 is often mutated in a subset of human cancers. We have now achieved conditional inactivation of Fbxw7 in the T cell lineage of mice and found that the cell cycle is not arrested at the CD4⁺CD8⁺ stage in the homozygous mutant animals. The mutant mice manifested thymic hyperplasia as a result of c-Myc accumulation and eventually developed thymic lymphoma. In contrast, mature T cells of the mutant mice failed to proliferate in response to mitogenic stimulation and underwent apoptosis in association with accumulation of c-Myc and p53. These latter abnormalities were corrected by deletion of p53. Our results suggest that Fbxw7 regulates the cell cycle in a differentiation-dependent manner, with its loss resulting in c-Myc accumulation that leads to hyperproliferation in immature T cells but to p53-dependent cell-cycle arrest and apoptosis in mature T cells.