The immunoproteasome and thymoproteasome: functions, evolution and human disease.

The basic principle of adaptive immunity is to strictly discriminate between self and non-self, and a central challenge to overcome is the enormous variety of pathogens that might be encountered. In cell-mediated immunity, immunological discernment takes place at a molecular or cellular level. Central to both mechanisms of discernment is the generation of antigenic peptides associated with MHC class I molecules, which is achieved by a proteolytic complex called the proteasome. To adequately accomplish the discrimination between self and non-self that is essential for adaptive immunity and self-tolerance, two proteasome subtypes have evolved via gene duplication: the immunoproteasome and the thymoproteasome. In this Review, we describe various aspects of these immunity-dedicated proteasomes, from their discovery to recent findings.

TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8(+) T cells.

In the thymus, low-affinity T cell antigen receptor (TCR) engagement facilitates positive selection of a useful T cell repertoire. Here we report that TCR responsiveness of mature CD8(+) T cells is fine tuned by their affinity for positively selecting peptides in the thymus and that optimal TCR responsiveness requires positive selection on major histocompatibility complex class I-associated peptides produced by the thymoproteasome, which is specifically expressed in the thymic cortical epithelium. Thymoproteasome-independent positive selection of monoclonal CD8(+) T cells results in aberrant TCR responsiveness, homeostatic maintenance and immune responses to infection. These results demonstrate a novel aspect of positive selection, in which TCR affinity for positively selecting peptides produced by thymic epithelium determines the subsequent antigen responsiveness of mature CD8(+) T cells in the periphery.

A novel method to identify Post-Aire stages of medullary thymic epithelial cell differentiation.

Autoimmune regulator+ (Aire) medullary thymic epithelial cells (mTECs) play a critical role in tolerance induction. Several studies demonstrated that Aire+ mTECs differentiate further into Post-Aire cells. Yet, the identification of terminal stages of mTEC maturation depends on unique fate-mapping mouse models. Herein, we resolve this limitation by segmenting the mTEChi (MHCIIhi CD80hi ) compartment into mTECA/hi (CD24- Sca1- ), mTECB/hi (CD24+ Sca1- ), and mTECC/hi (CD24+ Sca1+ ). While mTECA/hi included mostly Aire-expressing cells, mTECB/hi contained Aire+ and Aire- cells and mTECC/hi were mainly composed of cells lacking Aire. The differential expression pattern of Aire led us to investigate the precursor-product relationship between these subsets. Strikingly, transcriptomic analysis of mTECA/hi , mTECB/hi , and mTECC/hi sequentially mirrored the specific genetic program of Early-, Late- and Post-Aire mTECs. Corroborating their Post-Aire nature, mTECC/hi downregulated the expression of tissue-restricted antigens, acquired traits of differentiated keratinocytes, and were absent in Aire-deficient mice. Collectively, our findings reveal a new and simple blueprint to survey late stages of mTEC differentiation.

PITHD1 is a proteasome-interacting protein essential for male fertilization.

The proteasome is a protein-degrading molecular complex that is necessary for protein homeostasis and various biological functions, including cell cycle regulation, signal transduction, and immune response. Proteasome activity is finely regulated by a variety of proteasome-interacting molecules. PITHD1 is a recently described molecule that has a domain putatively capable of interacting with the proteasome. However, it is unknown whether PITHD1 can actually bind to proteasomes and what it does in vivo Here we report that PITHD1 is detected specifically in the spermatids in the testis and the cortical thymic epithelium in the thymus. Interestingly, PITHD1 associates with immunoproteasomes in the testis, but not with thymoproteasomes in the thymus. Mice deficient in PITHD1 exhibit severe male infertility accompanied with morphological abnormalities and impaired motility of spermatozoa. Furthermore, PITHD1 deficiency reduces proteasome activity in the testis and alters the amount of proteins that are important for fertilization capability by the sperm. However, the PITHD1-deficient mice demonstrate no detectable defects in the thymus, including T cell development. Collectively, our results identify PITHD1 as a proteasome-interacting protein that plays a nonredundant role in the male reproductive system.

Rank signaling links the development of invariant γδ T cell progenitors and Aire(+) medullary epithelium.

The thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance. Here we showed that emergence of the first cohorts of Aire(+) mTECs at this key developmental stage, prior to αß T cell repertoire selection, was jointly directed by Rankl(+) lymphoid tissue inducer cells and invariant Vγ5(+) dendritic epidermal T cell (DETC) progenitors that are the first thymocytes to express the products of gene rearrangement. In turn, generation of Aire(+) mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire. Hence, our data attributed a functional importance to the temporal development of Vγ5(+) γδ T cells during thymus medulla formation for αß T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire(+) mTEC maturation.

TCR Affinity for In Vivo Peptide-Induced Thymic Positive Selection Fine-Tunes TCR Responsiveness of Peripheral CD8+ T Cells.

The affinity for TCR interactions with self-peptide/MHC complexes (pMHC) in the thymus critically affects immature thymocytes that newly express TCRs. Previous fetal thymus organ culture experiments have indicated that difference in the affinity for thymic TCR/pMHC interactions not only determines thymocyte fate between positive and negative selection, but also affects Ag responsiveness of positively selected thymocytes. In the current study, we examined whether TCR/pMHC affinity during positive selection in the thymus would further affect Ag responsiveness of mature T cells in the periphery. To do so, OVA peptide variants were in vivo administered to TAP1-deficient OT-I/TCR-transgenic mice in which T cell development was otherwise arrested at CD4+CD8+ thymocytes because of the lack of self-pMHC presentation in thymic APCs. We found that a group of peptide variants induced the transient generation of OT-I CD8+ T cells in the thymus and the periphery. We also noticed that the affinity threshold for positive and negative selection detected in adult mice in vivo was higher than that measured in fetal thymus organ culture experiments in vitro. Interestingly, we further found that the affinity for positively selecting peptides proportionally affected TCR responsiveness of peripheral naive CD8+ T cells. These results indicate that in vivo administration of a peptide can promote T cell selection in the thymus and the affinity for TCR/pMHC interaction during positive selection fine-tunes Ag responsiveness of peripheral T cells.

Identification of an Intronic Regulatory Element Necessary for Tissue-Specific Expression of Foxn1 in Thymic Epithelial Cells.

The thymus is critical for the establishment of the adaptive immune system and the development of a diverse T cell repertoire. T cell development depends upon cell-cell interactions with epithelial cells in the thymus. The thymus is composed of two different types of epithelial cells: cortical and medullary epithelial cells. Both of these express and critically depend on the transcription factor Foxn1 Foxn1 is also expressed in the hair follicle, and disruption of Foxn1 function in mice results in severe thymic developmental defects and the hairless (nude) phenotype. Despite its importance, little is known about the direct regulation of Foxn1 expression. In this study, we identify a cis-regulatory element (RE) critical for expression of Foxn1 in mouse thymic epithelial cells but dispensable for expression in hair follicles. Analysis of chromatin accessibility, histone modifications, and sequence conservation identified regions within the first intron of Foxn1 that possessed the characteristics of REs. Systematic knockout of candidate regions lead us to identify a 1.6 kb region that, when deleted, results in a near total disruption of thymus development. Interestingly, Foxn1 expression and function in the hair follicle were unaffected. RNA fluorescent in situ hybridization showed a near complete loss of Foxn1 mRNA expression in the embryonic thymic bud. Our studies have identified a genomic RE with thymic-specific control of Foxn1 gene expression.

Thymus machinery for T-cell selection.

An immunocompetent and self-tolerant pool of naive T cells is formed in the thymus through the process of repertoire selection. T cells that are potentially capable of responding to foreign antigens are positively selected in the thymic cortex and are further selected in the thymic medulla to help prevent self-reactivity. The affinity between T-cell antigen receptors expressed by newly generated T cells and self-peptide-major histocompatibility complexes displayed in the thymic microenvironments plays a key role in determining the fate of developing T cells during thymic selection. Recent advances in our knowledge of the biology of thymic epithelial cells have revealed unique machinery that contributes to positive and negative selection in the thymus. In this article, we summarize recent findings on thymic T-cell selection, focusing on the machinery unique to thymic epithelial cells.

Cellularity of Thymic Epithelial Cells in the Postnatal Mouse.

The molecular and cellular biology of thymic epithelial cells (TECs) often relies on the analysis of TECs isolated in enzymatically digested single-cell suspensions derived from mouse thymus. Many independent studies have reported that the estimated cellularity of total TECs isolated from one adult mouse is on the order of up to 105 However, these numbers appear extremely small given that the cellularity of total thymocytes exceeds 108 and that TECs play multiple roles in thymocyte development and repertoire formation. In the present study, we aimed to measure the numbers of ß5t-expressing cortical TECs and Aire-expressing medullary TECs in postnatal mouse thymus in situ without enzymatic digestion. The numbers of these TECs were manually counted in individual thymic sections and were three-dimensionally summed throughout the entire thymic lobes. The results show that the cellularity of total TECs in one 5-wk-old female mouse exceeds 106, containing ∼9 × 105 ß5t+ cortical TECs and ∼1.1 × 106 Aire+ medullary TECs. These results suggest that the use of conventional enzymatic digestion methods for the isolation of TECs may have resulted in the underestimation of the cellularity, and possibly the biology, of TECs.

Formation of the Intrathymic Dendritic Cell Pool Requires CCL21-Mediated Recruitment of CCR7+ Progenitors to the Thymus.

During αß T cell development in the thymus, migration of newly selected CD4+ and CD8+ thymocytes into medullary areas enables tolerance mechanisms to purge the newly selected αß TCR repertoire of autoreactive specificities. Thymic dendritic cells (DC) play key roles in this process and consist of three distinct subsets that differ in their developmental origins. Thus, plasmacytoid DC and Sirpα+ conventional DC type 2 are extrathymically derived and enter into the thymus via their respective expression of the chemokine receptors CCR9 and CCR2. In contrast, although Sirpα- conventional DC type 1 (cDC1) are known to arise intrathymically from immature progenitors, the precise nature of such thymus-colonizing progenitors and the mechanisms controlling their thymus entry are unclear. In this article, we report a selective reduction in thymic cDC1 in mice lacking the chemokine receptor CCR7. In addition, we show that the thymus contains a CD11c+MHC class II-Sirpα-Flt3+ cDC progenitor population that expresses CCR7, and that migration of these cells to the thymus is impaired in Ccr7-/- mice. Moreover, thymic cDC1 defects in Ccr7-/- mice are mirrored in plt/plt mice, with further analysis of mice individually lacking the CCR7 ligands CCL21Ser (Ccl21a-/- ) or CCL19 (Ccl19-/-) demonstrating an essential role for CCR7-CCL21Ser during intrathymic cDC1 development. Collectively, our data support a mechanism in which CCR7-CCL21Ser interactions guide the migration of cDC progenitors to the thymus for correct formation of the intrathymic cDC1 pool.

Development and developmental potential of cortical thymic epithelial cells.

The thymic cortex provides a microenvironment for the development and positive selection of immature T cells. Cortical thymic epithelial cells (cTECs), which structurally and functionally support the thymic cortical microenvironment, originate from endodermal epithelial progenitors that arise in the third pharyngeal pouch. Recent studies have revealed that thymic epithelial progenitors pass through a stage where the cells express cTEC-associated molecules prior to lineage separation into cTECs and medullary TECs (mTECs). Here, we review the molecular signatures of cTECs and highlight the development and developmental potential of cTECs.

Thymoproteasome and peptidic self.

Positive selection of T cells in the thymus is induced by low-affinity TCR recognition of self-peptide-MHC complexes expressed by cortical thymic epithelial cells (cTECs). cTECs express a specialized type of proteasomes, the thymoproteasome, which generates a unique spectrum of MHC class I-associated peptides and plays a critical role in thymic positive selection of CD8+ T cells. However, it remains unclear how the thymoproteasome contributes to the thymic positive selection. More than 30 years ago, the "peptidic self" hypothesis proposed that TCRs recognize MHC-presented peptides only, without interacting with MHC molecules, which turned out to be incorrect. Interestingly, however, by implying that a set of MHC-associated peptides forms immunological self, this hypothesis also predicted that positive selection in the thymus is the primary immune response to "foreign epitope" peptides during T cell development. The thymoproteasome-dependent unique self-peptides may create those foreign epitope peptides displayed in the thymus for positive selection of T cells.

Thymoproteasome shapes immunocompetent repertoire of CD8+ T cells.

How self-peptides displayed in the thymus contribute to the development of immunocompetent and self-protective T cells is largely unknown. In contrast, the role of thymic self-peptides in eliminating self-reactive T cells and thereby preventing autoimmunity is well established. A type of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (cTECs) and is required for the generation of optimal cellularity of CD8+ T cells. Here, we show that cTECs displayed thymoproteasome-specific peptide-MHC class I complexes essential for the positive selection of major and diverse repertoire of MHC class I-restricted T cells. CD8+ T cells generated in the absence of thymoproteasomes displayed a markedly altered T cell receptor repertoire that was defective in both allogeneic and antiviral responses. These results demonstrate that thymoproteasome-dependent self-peptide production is required for the development of an immunocompetent repertoire of CD8+ T cells.

Generation of Peptides That Promote Positive Selection in the Thymus.

To establish an immunocompetent TCR repertoire that is useful yet harmless to the body, a de novo thymocyte repertoire generated through the rearrangement of genes that encode TCR is shaped in the thymus through positive and negative selection. The affinity between TCRs and self-peptides associated with MHC molecules determines the fate of developing thymocytes. Low-affinity TCR engagement with self-peptide-MHC complexes mediates positive selection, a process that primarily occurs in the thymic cortex. Massive efforts exerted by many laboratories have led to the characterization of peptides that can induce positive selection. Moreover, it is now evident that protein degradation machineries unique to cortical thymic epithelial cells play a crucial role in the production of MHC-associated self-peptides for inducing positive selection. This review summarizes current knowledge on positive selection-inducing self-peptides and Ag processing machineries in cortical thymic epithelial cells. Recent studies on the role of positive selection in the functional tuning of T cells are also discussed.

Dynamic spatio-temporal contribution of single ß5t+ cortical epithelial precursors to the thymus medulla.

Intrathymic T-cell development is critically dependent on cortical and medullary thymic epithelial cells (TECs). Both epithelial subsets originate during early thymus organogenesis from progenitor cells that express the thymoproteasome subunit ß5t, a typical feature of cortical TECs. Using in vivo lineage fate mapping, we demonstrate in mice that ß5t(+) TEC progenitors give rise to the medullary TEC compartment early in life but significantly limit their contribution once the medulla has completely formed. Lineage-tracing studies at single cell resolution demonstrate for young mice that the postnatal medulla is expanded from individual ß5t(+) cortical progenitors located at the cortico-medullary junction. These results therefore not only define a developmental window during which the expansion of medulla is efficiently enabled by progenitors resident in the thymic cortex, but also reveal the spatio-temporal dynamics that control the growth of the thymic medulla.

Autoantigen-specific interactions with CD4+ thymocytes control mature medullary thymic epithelial cell cellularity.

Medullary thymic epithelial cells (mTECs) are specialized for inducing central immunological tolerance to self-antigens. To accomplish this, mTECs must adopt a mature phenotype characterized by expression of the autoimmune regulator Aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. The mechanisms that control mature Aire(+) mTEC development in the postnatal thymus remain poorly understood. We demonstrate here that, although either CD4(+) or CD8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mTEC population requires autoantigen-specific interactions between positively selected CD4(+) thymocytes bearing autoreactive T cell receptor (TCR) and mTECs displaying cognate self-peptide-MHC class II complexes. These interactions also involve the engagement of CD40 on mTECs by CD40L induced on the positively selected CD4(+) thymocytes. This antigen-specific TCR-MHC class II-mediated crosstalk between CD4(+) thymocytes and mTECs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mTEC population competent for ensuring central T cell tolerance.

The tumor necrosis factor family receptors RANK and CD40 cooperatively establish the thymic medullary microenvironment and self-tolerance.

Medullary thymic epithelial cells (mTECs) establish T cell self-tolerance through the expression of autoimmune regulator (Aire) and peripheral tissue-specific self-antigens. However, signals underlying mTEC development remain largely unclear. Here, we demonstrate crucial regulation of mTEC development by receptor activator of NF-kappaB (RANK) and CD40 signals. Whereas only RANK signaling was essential for mTEC development during embryogenesis, in postnatal mice, cooperation between CD40 and RANK signals was required for mTEC development to successfully establish the medullary microenvironment. Ligation of RANK or CD40 on fetal thymic stroma in vitro induced mTEC development in a tumor necrosis factor-associated factor 6 (TRAF6)-, NF-kappaB inducing kinase (NIK)-, and IkappaB kinase beta (IKKbeta)-dependent manner. These results show that developmental-stage-dependent cooperation between RANK and CD40 promotes mTEC development, thereby establishing self-tolerance.

The cytokine RANKL produced by positively selected thymocytes fosters medullary thymic epithelial cells that express autoimmune regulator.

The thymic medulla provides a microenvironment where medullary thymic epithelial cells (mTECs) express autoimmune regulator and diverse tissue-restricted genes, contributing to launching self-tolerance. Positive selection is essential for thymic medulla formation via a previously unknown mechanism. Here we show that the cytokine RANK ligand (RANKL) was produced by positively selected thymocytes and regulated the cellularity of mTEC by interacting with RANK and osteoprotegerin. Forced expression of RANKL restored thymic medulla in mice lacking positive selection, whereas RANKL perturbation impaired medulla formation. These results indicate that RANKL produced by positively selected thymocytes is responsible for fostering thymic medulla formation, thereby establishing central tolerance.

Journey through the thymus: stromal guides for T-cell development and selection.

Lympho-stromal interactions in multiple microenvironments within the thymus have a crucial role in the regulation of T-cell development and selection. Recent studies have implicated that chemokines that are produced by thymic stromal cells have a pivotal role in positioning developing T cells within the thymus. In this Review, I discuss the importance of stroma-derived chemokines in guiding the traffic of developing thymocytes, with an emphasis on the processes of cortex-to-medulla migration and T-cell-repertoire selection, including central tolerance.