Functionally diverse thymic medullary epithelial cells interplay to direct central tolerance.

Medullary thymic epithelial cells (mTECs) are essential for the establishment of self-tolerance in T cells. Promiscuous gene expression by a subpopulation of mTECs regulated by the nuclear protein Aire contributes to the display of self-genomic products to newly generated T cells. Recent reports have highlighted additional self-antigen-displaying mTEC subpopulations, namely Fezf2-expressing mTECs and a mosaic of self-mimetic mTECs including thymic tuft cells. In addition, a functionally different subset of mTECs produces chemokine CCL21, which attracts developing thymocytes to the medullary region. Here, we report that CCL21+ mTECs and Aire+ mTECs non-redundantly cooperate to direct self-tolerance to prevent autoimmune pathology by optimizing the deletion of self-reactive T cells and the generation of regulatory T cells. We also detect cooperation for self-tolerance between Aire and Fezf2, the latter of which unexpectedly regulates thymic tuft cells. Our results indicate an indispensable interplay among functionally diverse mTECs for the establishment of central self-tolerance.

Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium.

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here, we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny in mice. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.

Developmental conversion of thymocyte-attracting cells into self-antigen-displaying cells in embryonic thymus medulla epithelium.

Thymus medulla epithelium establishes immune self-tolerance and comprises diverse cellular subsets. Functionally relevant medullary thymic epithelial cells (mTECs) include a self-antigen-displaying subset that exhibits genome-wide promiscuous gene expression promoted by the nuclear protein Aire and that resembles a mosaic of extrathymic cells including mucosal tuft cells. An additional mTEC subset produces the chemokine CCL21, thereby attracting positively selected thymocytes from the cortex to the medulla. Both self-antigen-displaying and thymocyte-attracting mTEC subsets are essential for self-tolerance. Here we identify a developmental pathway by which mTECs gain their diversity in functionally distinct subsets. We show that CCL21-expressing mTECs arise early during thymus ontogeny. Fate-mapping analysis reveals that self-antigen-displaying mTECs, including Aire-expressing mTECs and thymic tuft cells, are derived from CCL21-expressing cells. The differentiation capability of CCL21-expressing embryonic mTECs is verified in reaggregate thymus experiments. These results indicate that CCL21-expressing embryonic mTECs carry a developmental potential to give rise to self-antigen-displaying mTECs, revealing that the sequential conversion of thymocyte-attracting subset into self-antigen-displaying subset serves to assemble functional diversity in the thymus medulla epithelium.

Endogenous CCL21-Ser deficiency reduces B16-F10 melanoma growth by enhanced antitumor immunity.

The chemokine CCL21 regulates immune and cancer cell migration through its receptor CCR7. The Ccl21a gene encodes the isoform CCL21-Ser, predominantly expressed in the thymic medulla and the secondary lymphoid tissues. This study examined the roles of CCL21-Ser in the antitumor immune response in Ccl21a-knockout (KO) mice. The Ccl21a-KO mice showed significantly decreased growth of B16-F10 and YUMM1.7 melanomas and increased growth of MC38 colon cancer, despite no significant difference in LLC lung cancer and EO771 breast cancer. The B16-F10 tumor in Ccl21a-KO mice showed melanoma-specific activated CD8+ T cell and NK cell infiltration and higher Treg counts than wild-type mice. B16-F10 tumors in Ccl21a-KO mice showed a reduction in the positive correlation between the ratio of regulatory T cells (Tregs) to activated CD8+ T cells and tumor weight. In Ccl21a-KO tumor, the intratumoral Tregs showed lower co-inhibitory receptors TIM-3 and TIGIT. Taken together, these results suggest that endogenous CCL21-Ser supports melanoma growth in vivo by maintaining Treg function and suppressing antitumor immunity by CD8+ T cells.

The thymoproteasome in shaping the CD8+ T-cell repertoire.

The thymoproteasome is a type of proteasome expressed specifically in thymic cortical epithelial cells. Thymoproteasome affects antigen processing of major histocompatibility complex (MHC)-I-associated peptides and optimizes positive selection of CD8+ T cells. However, it remains unanswered whether and how thymoproteasome-dependent MHC-I-associated self-peptides contribute to positive selection of cortical thymocytes. This short piece discusses the potential mechanisms of thymoproteasome contribution to positive selection of MHC-I-restricted CD8+ T cells.

Embryonic keratin19+ progenitors generate multiple functionally distinct progeny to maintain epithelial diversity in the adult thymus medulla.

The thymus medulla is a key site for immunoregulation and tolerance, and its functional specialisation is achieved through the complexity of medullary thymic epithelial cells (mTEC). While the importance of the medulla for thymus function is clear, the production and maintenance of mTEC diversity remains poorly understood. Here, using ontogenetic and inducible fate-mapping approaches, we identify mTEC-restricted progenitors as a cytokeratin19+ (K19+) TEC subset that emerges in the embryonic thymus. Importantly, labelling of a single cohort of K19+ TEC during embryogenesis sustains the production of multiple mTEC subsets into adulthood, including CCL21+ mTEClo, Aire+ mTEChi and thymic tuft cells. We show K19+ progenitors arise prior to the acquisition of multiple mTEC-defining features including RANK and CCL21 and are generated independently of the key mTEC regulator, Relb. In conclusion, we identify and define a multipotent mTEC progenitor that emerges during embryogenesis to support mTEC diversity into adult life.

Large-Scale Isolation of Mouse Thymic Epithelial Cells.

The thymus is compartmentalized into the cortex and the medulla. Cortical and medullary thymic epithelial cells (TECs) characterize T cell-producing and T cell-selecting functions of cortical and medullary microenvironments in the thymus. Enzymatic digestion of the thymus and flow cytometric isolation of TECs and their subpopulations are useful for molecular and cellular characterization of TECs. However, the cellularity of cTECs and mTECs isolated from mouse thymus is limited. In this chapter, we describe the method for isolation of a large number of TECs using enlarged mouse thymus, which enables biochemical and proteomic analysis of TEC subpopulations.

Tissue-specific proteasomes in generation of MHC class I peptides and CD8+ T cells.

Thymoproteasomes and immunoproteasomes are two types of tissue-specific proteasomes, which contribute to the production of major histocompatibility complex (MHC) class I (MHC-I)-associated peptides that are important for the development and function of CD8+ cytotoxic T cells. Thymoproteasomes are specifically expressed by cortical thymic epithelial cells and are important for MHC-I-dependent positive selection of developing thymocytes, whereas immunoproteasomes are abundant in many other cells, including hematopoietic cells and medullary thymic epithelial cells. Here we summarize the role of these two tissue-specific proteasomes, focusing on their functions in the development of CD8+ T cells in the thymus.

Recycling of memory B cells between germinal center and lymph node subcapsular sinus supports affinity maturation to antigenic drift.

Infection or vaccination leads to the development of germinal centers (GC) where B cells evolve high affinity antigen receptors, eventually producing antibody-forming plasma cells or memory B cells. Here we follow the migratory pathways of B cells emerging from germinal centers (BEM) and find that many BEM cells migrate into the lymph node subcapsular sinus (SCS) guided by sphingosine-1-phosphate (S1P). From the SCS, BEM cells may exit the lymph node to enter distant tissues, while some BEM cells interact with and take up antigen from SCS macrophages, followed by CCL21-guided return towards the GC. Disruption of local CCL21 gradients inhibits the recycling of BEM cells and results in less efficient adaption to antigenic variation. Our findings thus suggest that the recycling of antigen variant-specific BEM cells and transport of antigen back to GC may support affinity maturation to antigenic drift.

Fine-tuning of ß-catenin in mouse thymic epithelial cells is required for postnatal T-cell development.

In the thymus, the thymic epithelium provides a microenvironment essential for the development of functionally competent and self-tolerant T cells. Previous findings showed that modulation of Wnt/ß-catenin signaling in mouse thymic epithelial cells (TECs) disrupts embryonic thymus organogenesis. However, the role of ß-catenin in TECs for postnatal T-cell development remains to be elucidated. Here, we analyzed gain-of-function (GOF) and loss-of-function (LOF) of ß-catenin highly specific in mouse TECs. We found that GOF of ß-catenin in TECs results in severe thymic dysplasia and T-cell deficiency beginning from the embryonic period. By contrast, LOF of ß-catenin in TECs reduces the number of cortical TECs and thymocytes modestly and only postnatally. These results indicate that fine-tuning of ß-catenin expression within a permissive range is required for TECs to generate an optimal microenvironment to support postnatal T-cell development.

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.

Peptides for T cell selection in the thymus.

Major histocompatibility complex (MHC)-associated peptides generated and displayed by antigen-presenting cells in the thymus are essential for the generation of functional and self-tolerant T cells that protect our body from various pathogens. The peptides displayed by cortical thymic epithelial cells (cTECs) are generated by unique enzymatic machineries including the thymoproteasomes, and are involved in the positive selection of self-protective T cells. On the other hand, the peptides displayed by medullary thymic epithelial cells (mTECs) and thymic dendritic cells (DCs) are involved in further selection to establish self-tolerance in T cells. Although the biochemical nature of the peptide repertoire displayed in the thymus remains unclear, many studies have suggested a thymus-specific mechanism for the generation of MHC-associated peptides in the thymus. In this review, we summarize basic knowledge and recent advances in MHC-associated thymic peptides, focusing on the generation and function of thymoproteasome-dependent peptides specifically displayed by cTECs.

Specific impact of ß5t on proteasome subunit composition in cortical thymic epithelial cells.

ß5t is a cortical thymic epithelial cell (cTEC)-specific component of the thymoproteasome, which is essential for the optimal production of functionally competent CD8+ T cells. Our recent analysis showed a specific impact of ß5t on proteasome subunit composition in cTECs, supporting the possibility that the thymoproteasome optimizes CD8+ T cell development through the production of MHC-I-associated unique self-peptides in cTECs. However, a recent article reports that ß5t regulates the expression of hundreds of cTEC genes and affects both CD4+ and CD8+ thymocytes by causing oxidative stress in thymocytes. The authors further analyze our published data and describe that they confirm their conclusions. Here, we examine the issues that they raise and conclude that, rather than regulating hundreds of genes in cTECs, ß5t has a highly specific impact in cTECs on proteasome subunit composition. This Matters Arising Response article addresses the Apavaloaei et al. (2021) Matters Arising paper, published concurrently in Cell Reports.

Thymoproteasome optimizes positive selection of CD8+ T cells without contribution of negative selection.

Functionally competent and self-tolerant T cell repertoire is shaped through positive and negative selection in the cortical and medullary microenvironments of the thymus. The thymoproteasome specifically expressed in the cortical thymic epithelium is essential for the optimal generation of CD8+ T cells. Although how the thymoproteasome governs the generation of CD8+ T cells is not fully understood, accumulating evidence suggests that the thymoproteasome optimizes CD8+ T cell production through the processing of self-peptides associated with MHC class I molecules expressed by cortical thymic epithelial cells. In this review, we describe recent advances in the mechanism of thymoproteasome-dependent generation of CD8+ T cells, focusing on the process of cortical positive selection independent of apoptosis-mediated negative selection.

Radiation inducible MafB gene is required for thymic regeneration.

The thymus facilitates mature T cell production by providing a suitable stromal microenvironment. This microenvironment is impaired by radiation and aging which lead to immune system disturbances known as thymic involution. Young adult thymus shows thymic recovery after such involution. Although various genes have been reported for thymocytes and thymic epithelial cells in such processes, the roles of stromal transcription factors in these remain incompletely understood. MafB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is a transcription factor expressed in thymic stroma and its expression was induced a day after radiation exposure. Hence, the roles of mesenchymal MafB in the process of thymic regeneration offers an intriguing research topic also for radiation biology. The current study investigated whether MafB plays roles in the adult thymus. MafB/green fluorescent protein knock-in mutant (MafB+/GFP) mice showed impaired thymic regeneration after the sublethal irradiation, judged by reduced thymus size, total thymocyte number and medullary complexity. Furthermore, IL4 was induced after irradiation and such induction was reduced in mutant mice. The mutants also displayed signs of accelerated age-related thymic involution. Altogether, these results suggest possible functions of MafB in the processes of thymic recovery after irradiation, and maintenance during aging.

The Role of Proteasomes in the Thymus.

The thymus provides a microenvironment that supports the generation and selection of T cells. Cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs) are essential components of the thymic microenvironment and present MHC-associated self-antigens to developing thymocytes for the generation of immunocompetent and self-tolerant T cells. Proteasomes are multicomponent protease complexes that degrade ubiquitinated proteins and produce peptides that are destined to be associated with MHC class I molecules. cTECs specifically express thymoproteasomes that are essential for optimal positive selection of CD8+ T cells, whereas mTECs, which contribute to the establishment of self-tolerance in T cells, express immunoproteasomes. Immunoproteasomes are also detectable in dendritic cells and developing thymocytes, additionally contributing to T cell development in the thymus. In this review, we summarize the functions of proteasomes expressed in the thymus, focusing on recent findings pertaining to the functions of the thymoproteasomes and the immunoproteasomes.

Medullary stromal cells synergize their production and capture of CCL21 for T-cell emigration from neonatal mouse thymus.

The release of newly selected αßT cells from the thymus is key in establishing a functional adaptive immune system. Emigration of the first cohorts of αßT cells produced during the neonatal period is of particular importance, because it initiates formation of the peripheral αßT-cell pool and provides immune protection early in life. Despite this, the cellular and molecular mechanisms of thymus emigration are poorly understood. We examined the involvement of diverse stromal subsets and individual chemokine ligands in this process. First, we demonstrated functional dichotomy in the requirement for CCR7 ligands and identified CCL21, but not CCL19, as an important regulator of neonatal thymus emigration. To explain this ligand-specific requirement, we examined sites of CCL21 production and action and found Ccl21 gene expression and CCL21 protein distribution occurred within anatomically distinct thymic areas. Although Ccl21 transcription was limited to subsets of medullary epithelium, CCL21 protein was captured by mesenchymal stroma consisting of integrin α7+ pericytes and CD34+ adventitial cells at sites of thymic exit. This chemokine compartmentalization involved the heparan sulfate-dependent presentation of CCL21 via its C-terminal extension, explaining the absence of a requirement for CCL19, which lacks this domain and failed to be captured by thymic stroma. Collectively, we identified an important role for CCL21 in neonatal thymus emigration, revealing the importance of this chemokine in initial formation of the peripheral immune system. Moreover, we identified an intrathymic mechanism involving cell-specific production and presentation of CCL21, which demonstrated a functional synergy between thymic epithelial and mesenchymal cells for αßT-cell emigration.

The thymoproteasome hardwires the TCR repertoire of CD8+ T cells in the cortex independent of negative selection.

The thymoproteasome expressed specifically in thymic cortical epithelium optimizes the generation of CD8+ T cells; however, how the thymoproteasome contributes to CD8+ T cell development is unclear. Here, we show that the thymoproteasome shapes the TCR repertoire directly in cortical thymocytes before migration to the thymic medulla. We further show that the thymoproteasome optimizes CD8+ T cell production independent of the thymic medulla; independent of additional antigen-presenting cells, including medullary thymic epithelial cells and dendritic cells; and independent of apoptosis-mediated negative selection. These results indicate that the thymoproteasome hardwires the TCR repertoire of CD8+ T cells with cortical positive selection independent of negative selection in the thymus.

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.

Epigenetic Protection of Vertebrate Lymphoid Progenitor Cells by Dnmt1.

DNA methylation is a universal epigenetic mechanism involved in regulation of gene expression and genome stability. The DNA maintenance methylase DNMT1 ensures that DNA methylation patterns are faithfully transmitted to daughter cells during cell division. Because loss of DNMT1 is lethal, a pan-organismic analysis of DNMT1 function is lacking. We identified new recessive dnmt1 alleles in medaka and zebrafish and, guided by the structures of mutant proteins, generated a recessive variant of mouse Dnmt1. Each of the three missense mutations studied here distorts the catalytic pocket and reduces enzymatic activity. Because all three DNMT1 mutant animals are viable, it was possible to examine their phenotypes throughout life. The consequences of genome-wide hypomethylation of DNA of somatic tissues in the Dnmt1 mutants are surprisingly mild but consistently affect the development of the lymphoid lineage. Our findings indicate that developing lymphocytes in vertebrates are sensitive to perturbations of DNA maintenance methylation.