Cortical Thymic Epithelial Cells Research Articles

Restricted Expression of the Thymoproteasome Is Required for Thymic Selection and Peripheral Homeostasis of CD8+ T Cells

The thymoproteasome subunit β5t is specifically expressed in cortical thymic epithelial cells (TECs) and generates unique peptides to support positive selection. In this study, using a mouse model ubiquitously expressing β5t, we showed that aberrant expression of self-peptides generated by β5t affects CD8+ Tcell homeostasis, including thymic selection and maintenance of the peripheral naive pool of CD8+ Tcells. In mice in which β5t was expressed both in cortical and medullary TECs, the abundance of CD8+ lineage thymocytes was reduced, and extra-thymic expression of β5t caused accumulation of CD8+ Tcells with the memory or exhausted phenotype and induced autoreactive Tcell responses. We found that thymoproteasomes are essential for positive selection but that the subsequent change in peptide repertoire in the medulla is also crucial for thymic selection and that β5t-derived peptide must be confined to the thymus to avoid autoimmunity in peripheral tissues.

Thymic Epithelial Cell Support of Thymopoiesis Does Not Require Klotho.

Age-related thymic involution is characterized by a decrease in thymic epithelial cell (TEC) number and function parallel to a disruption in their spatial organization, resulting in defective thymocyte development and proliferation as well as peripheral T cell dysfunction. Deficiency of Klotho, an antiaging gene and modifier of fibroblast growth factor signaling, causes premature aging. To investigate the role of Klotho in accelerated age-dependent thymic involution, we conducted a comprehensive analysis of thymopoiesis and peripheral T cell homeostasis using Klotho-deficient (Kl/Kl) mice. At 8 wk of age, Kl/Kl mice displayed a severe reduction in the number of thymocytes (10-100-fold reduction), especially CD4 and CD8 double-positive cells, and a reduction of both cortical and medullary TECs. To address a cell-autonomous role for Klotho in TEC biology, we implanted neonatal thymi from Klotho-deficient and -sufficient mice into athymic hosts. Kl/Kl thymus grafts supported thymopoiesis equivalently to Klotho-sufficient thymus transplants, indicating that Klotho is not intrinsically essential for TEC support of thymopoiesis. Moreover, lethally irradiated hosts given Kl/Kl or wild-type bone marrow had normal thymocyte development and comparably reconstituted T cells, indicating that Klotho is not inherently essential for peripheral T cell reconstitution. Because Kl/Kl mice have higher levels of serum phosphorus, calcium, and vitamin D, we evaluated thymus function in Kl/Kl mice fed with a vitamin D-deprived diet. We observed that a vitamin D-deprived diet abrogated thymic involution and T cell lymphopenia in 8-wk-old Kl/Kl mice. Taken together, our data suggest that Klotho deficiency causes thymic involution via systemic effects that include high active vitamin D levels.

CD31 Expression Defines Heterogeneity of Donor Plasmacytoid Dendritic Cell Populations That Home to the Thymus: Implications for Control of GvHD

Introduction: Plasmacytoid dendritic cells (pDC) are known to possess tolerogenic properties in allogeneic bone marrow transplantation (allo-BMT), but the relationship between pDC lineage and their modulation of graft-versus-host disease (GvHD) has not been defined. Recently, we have shown that murine treatment with the pleiotropic cytokine FMS-like Tyrosine Kinase 3 Ligand (Flt3L) increases pDC content in the marrow, and that allo-BMT of Flt3L-treated marrow (FBM) grafts leads to higher overall survival and reduced GvHD in lethally irradiated hosts. Furthermore, FBM pDC have increased potency in preventing GvHD on a cell-by-cell basis (Hassan, EBMT, 2018). Interestingly, FBM pDC expressed CD11b, a myeloid-specific marker that is not expressed on currently defined pDC. Lineage ontogeny of pDC can be tracked based upon exclusive expression of CD31 and Ly6C on myeloid-progenitor derived pDC, while pDC from lymphoid progenitors express RAG1 and Siglec H. We hypothesized that treatment of murine bone marrow donors with Flt3L, which enhances their GvHD-reducing activity, modifies the distribution of lineage-specific precursors of pDC in bone marrow, and that lineage-associated pDC will have distinct biological activity in allo-BMT.Methods and Results: To assess the effect of Flt3L on BM pDC, we treated C57BL/6 mice with Flt3L (CDX-301, 300 ug/kg) on days -1 and -4 relative to bone marrow harvest. Using flow cytometric analysis, we show that FBM pDC over-express myeloid-specific markers CD31, Ly6C and CD11b, but down-regulate the lymphoid-specific marker Siglec H (Figure 1). Moreover, we observed similar phenotypic trends for myeloid-specific markers in pDC from the marrow of RAG1KO mice, confirming the existence of a unique myeloid-derived population of pDC.To determine the effect of pDC linage on their gene expression, we performed Illumina RNA Sequencing on human-derived pDC from Flt3L-mobilized peripheral blood and from human bone marrow. Interestingly, we found that Flt3L-treated pDC overexpress PRSS16, which is known to be exclusively expressed on cortical thymic epithelial cells. Furthermore, using GFP-transgenic mice as a source of donor pDC in B6 -> B10.BR allo-BMT, we show via confocal microscopy that donor pDC selectively home to the recipient thymus (Figure 2). Moreover, flow cytometric analysis revealed that homing of FBM pDC to the thymus is not impaired, in spite of decreased CCR9 expression on FBM pDC in comparison to BM pDC. Because it is currently believed that CCR9 expression on pDC is necessary for their homing to the thymus, these observations denote that FBM pDC use a CCR9-independent homing strategy.Since CD31 expression is upregulated in FBM pDC with enhanced immune-regulatory activity in allo-BMT models, these data suggest that local expression of CD31 by donor pDC in the thymic microenvironment may be relevant to their ability to favorably regulate thymopoiesis and limit the development of alloreactive T cells.Conclusions: Our data poses a link between pDC lineage and control of post-transplant GvHD, possibly via regulation of positive and negative selection of donor-derived T cells in the thymus. Specifically, we identify a CD31+ myeloid-specific population of pDC that emerges in the marrow upon treatment with Flt3L and exhibits a genetic profile that resembles thymic epithelial cells as a candidate for adoptive cell therapy to prevent GvHD. [Display omitted] DisclosuresWaller:Pharmacyclics: Other: Travel Expenses, EHA, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kalytera: Consultancy; Celldex: Research Funding; Cambium Medical Technologies: Consultancy, Equity Ownership.

LAMTOR2 Is Dispensable for T Cell Receptor-Mediated Signaling of Thymocytes but Controls Medullary Thymic Epithelial Cells

Human LAMTOR2 deficiency is characterized by severe congenital neutropenia, growth failure, partial albinism, as well as B and T cell deficiencies (Bohn et al., Nat Med 2007). To determine the role of the endosomal adaptor LAMTOR2 in T cell development and homeostasis we used conditional knockout mouse models.Mx1-Cre-driven knockout of Lamtor2 resulted in reduction of thymus weight and total thymocyte numbers. Immunophenotyping revealed an impaired T cell development characterized by a partial block at the double negative CD4-CD8- T cell precursor stage after 7 and 21 days of poly I:C injection that induced deletion of the Lamtor2 gene. Since Mx1-Cre-driven knockout does not allow a discrimination between T cell intrinsic and extrinsic effects, we next generated pre-TCRα-iCre conditional knockout mice. In contrast to Mx1-Cre-Lamtor2fl/fl mice, mice with T cell-specific knockout of Lamtor2 showed normal frequencies of total thymocytes and T cell progenitor subsets. Furthermore, LAMTOR2-deficient thymocytes exhibited normal TCR signaling (p-ERK, p-LAT, p-LCK, p-PLCγ, Nur77) and internalization of TCRβ upon stimulation with anti-CD3ε +/- anti-CD28, indicating that LAMTOR2 in T cells is dispensable for thymocyte development.To assess whether T cell developmental defects in Mx1-Cre-Lamtor2fl/fl mice are caused by a dysfunctional thymic epithelium, we analyzed thymic epithelial cells (TECs) after 4 days of poly I:C injection by flow cytometry and detected a reduced ratio of CD45-EpCAM+UEA-1+Ly51- medullary TECs (mTECs) to CD45-EpCAM+UEA-1-Ly51+ cortical TECs in LAMTOR2-deficient mice. Further studies are underway to determine the role of LAMTOR2 in mTECs.Taken together, our findings show that LAMTOR2 is not required for TCR-mediated signaling but plays a critical role in controlling mTEC homeostasis. DisclosuresNo relevant conflicts of interest to declare.

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 30years 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.

FoxN1-dependent thymic epithelial cells promote T-cell leukemia development.

T-cell acute lymphoblastic leukemia (T-ALL) and T-lymphoblastic lymphomas (T-LBL) are aggressive malignancies of thymocytes. The role of thymic microenvironmental cells and stromal factors in thymocyte malignant transformation and T-ALL development remains little explored. Here, using the TEL-JAK2 transgenic (TJ2-Tg) mouse model of T-ALL/LBL, which is driven by constitutive JAK/STAT signaling and characterized by the acquisition of Notch1 mutations, we sought to identify stromal cell alterations associated with thymic leukemogenesis. Immunofluorescence analyses showed that thymic lymphomas presented epithelial areas characterized by keratin (Krt) 5 and Krt8 expression, adjacently to epithelial-free areas negative for Krt expression. Both areas contained abundant laminin (extracellular matrix) and ER-TR7+ (fibroblasts) CD31+ (endothelial) and CD11c+ (dendritic) cells. Besides Krt5, Krt-positive areas harbored medullary thymic epithelial cells (TECs) labeled by Ulex europaeus agglutinin-1. By performing flow cytometry and RNA sequencing analyses of thymic lymphomas, we observed an enrichment in medullary TEC markers in detriment of cortical TEC markers. To assess whether TECs are important for T-ALL/LBL development, we generated TJ2-Tg mice heterozygous for the FoxN1 transcription factor nude null mutation (Foxn1+/nu). Strikingly, in TJ2-Tg;Foxn1+/nu compound mice, both emergence of malignant cells in preleukemic thymi and overt T-ALL onset were significantly delayed. Moreover, in transplantation assays, leukemic cell expansion within the thymus of recipient Foxn1+/nu mice was reduced as compared with control littermates. Since thymopoesis is largely normal in Foxn1+/nu mice, these results indicate that FoxN1 haploinsufficiency in TECs has a more profound impact in thymic leukemogenesis.

Thymus-specific serine protease, a protease that shapes the CD4 T cell repertoire.

The lifespan of T cells is determined by continuous interactions of their T cell receptors (TCR) with self-peptide-MHC (self-pMHC) complexes presented by different subsets of antigen-presenting cells (APC). In the thymus, developing thymocytes are positively selected through recognition of self-pMHC presented by cortical thymic epithelial cells (cTEC). They are subsequently negatively selected by medullary thymic epithelial cells (mTEC) or thymic dendritic cells (DC) presenting self-pMHC complexes. In the periphery, the homeostasis of mature T cells is likewise controlled by the interaction of their TCR with self-pMHC complexes presented by lymph node stromal cells while they may be tolerized by DC presenting tissue-derived self-antigens. To perform these tasks, the different subsets of APC are equipped with distinct combination of antigen processing enzymes and consequently present specific repertoire of self-peptides. Here, we discuss one such antigen processing enzyme, the thymus-specific serine protease (TSSP), which is predominantly expressed by thymic stromal cells. In thymic DC and TEC, TSSP edits the repertoire of peptide presented by class II molecules and thus shapes the CD4 T cell repertoire.

Foxa1 and Foxa2 in thymic epithelial cells (TEC) regulate medullary TEC and regulatory T-cell maturation

The Foxa1 and Foxa2 transcription factors are essential for mouse development. Here we show that they are expressed in thymic epithelial cells (TEC) where they regulate TEC development and function, with important consequences for T-cell development. TEC are essential for T-cell differentiation, lineage decisions and repertoire selection. Conditional deletion of Foxa1 and Foxa2 from murine TEC led to a smaller thymus with a greater proportion of TEC and a greater ratio of medullary to cortical TEC. Cell-surface MHCI expression was increased on cortical TEC in the conditional Foxa1Foxa2 knockout thymus, and MHCII expression was reduced on both cortical and medullary TEC populations. These changes in TEC differentiation and MHC expression led to a significant reduction in thymocyte numbers, reduced positive selection of CD4+CD8+ cells to the CD4 lineage, and increased CD8 cell differentiation. Conditional deletion of Foxa1 and Foxa2 from TEC also caused an increase in the medullary TEC population, and increased expression of Aire, but lower cell surface MHCII expression on Aire-expressing mTEC, and increased production of regulatory T-cells. Thus, Foxa1 and Foxa2 in TEC promote positive selection of CD4SP T-cells and modulate regulatory T-cell production and activity, of importance to autoimmunity.

Fundamental parameters of the developing thymic epithelium in the mouse

The numbers of thymic epithelial cells (TECs) and thymocytes steadily increase during embryogenesis. To examine this dynamic, we generated several TEC-specific transgenic mouse lines, which express fluorescent proteins in the nucleus, the cytosol and in the membranes under the control of the Foxn1 promoter. These tools enabled us to determine TEC numbers in tissue sections by confocal fluorescent microscopy, and in the intact organ by light-sheet microscopy. Compared to histological procedures, flow cytometric analysis of thymic cellularity is shown to underestimate the numbers of TECs by one order of magnitude; using enzymatic digestion of thymic tissue, the loss of cortical TECs (cTECs) is several fold greater than that of medullary TECs (mTECs), although different cTEC subsets appear to be still present in the final preparation. Novel reporter lines driven by Psmb11 and Prss16 promoters revealed the trajectory of differentiation of cTEC-like cells, and, owing to the additional facility of conditional cell ablation, allowed us to follow the recovery of such cells after their depletion during embryogenesis. Multiparametric histological analyses indicate that the new transgenic reporter lines not only reveal the unique morphologies of different TEC subsets, but are also conducive to the analysis of the complex cellular interactions in the thymus.

Retinoic Acid Signaling in Thymic Epithelial Cells Regulates Thymopoiesis.

Despite the essential role of thymic epithelial cells (TEC) in T cell development, the signals regulating TEC differentiation and homeostasis remain incompletely understood. In this study, we show a key in vivo role for the vitamin A metabolite, retinoic acid (RA), in TEC homeostasis. In the absence of RA signaling in TEC, cortical TEC (cTEC) and CD80loMHC class IIlo medullary TEC displayed subset-specific alterations in gene expression, which in cTEC included genes involved in epithelial proliferation, development, and differentiation. Mice whose TEC were unable to respond to RA showed increased cTEC proliferation, an accumulation of stem cell Ag-1hi cTEC, and, in early life, a decrease in medullary TEC numbers. These alterations resulted in reduced thymic cellularity in early life, a reduction in CD4 single-positive and CD8 single-positive numbers in both young and adult mice, and enhanced peripheral CD8+ T cell survival upon TCR stimulation. Collectively, our results identify RA as a regulator of TEC homeostasis that is essential for TEC function and normal thymopoiesis.

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 β5t-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 β5t+ 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.

Dynamic regulation of NOTCH1 activation and Notch ligand expression in human thymus development.

T-cell development is a complex dynamic process that relies on ordered stromal signals delivered to thymus-seeding progenitors that migrate throughout different thymus microenvironments (TMEs). Particularly, Notch signaling provided by thymic epithelial cells (TECs) is crucial for T-cell fate specification and generation of mature T cells. Four canonical Notch ligands (Dll1, Dll4, Jag1 and Jag2) are expressed in the thymus, but their spatial distribution in functional TMEs is largely unknown, especially in humans, and their impact on Notch1 activation during T-lymphopoiesis remains undefined. Based on immunohistochemistry and quantitative confocal microscopy of fetal, postnatal and adult human and mouse thymus samples, we show that spatial regulation of Notch ligand expression defines discrete Notch signaling niches and dynamic species-specific TMEs. We further show that Notch ligand expression, particularly DLL4, is tightly regulated in cortical TECs during human thymus ontogeny and involution. Also, we provide the first evidence that NOTCH1 activation is induced in vivo in CD34+ progenitors and developing thymocytes at particular cortical niches of the human fetal and postnatal thymus. Collectively, our results show that human thymopoiesis involves complex spatiotemporal regulation of Notch ligand expression, which ensures the coordinated delivery of niche-specific NOTCH1 signals required for dynamic T-cell development.

A critical epithelial survival axis regulated by MCL-1 maintains thymic function in mice

AbstractT-cell differentiation is governed by interactions with thymic epithelial cells (TECs) and defects in this process undermine immune function and tolerance. To uncover new strategies to restore thymic function and adaptive immunity in immunodeficiency, we sought to determine the molecular mechanisms that control life and death decisions in TECs. Guided by gene expression profiling, we created mouse models that specifically deleted prosurvival genes in TECs. We found that although BCL-2 and BCL-XL were dispensable for TEC homeostasis, MCL-1 deficiency impacted on TECs as early as embryonic day 15.5, resulting in early thymic atrophy and T-cell lymphopenia, with near complete loss of thymic tissue by 2 months of age. MCL-1 was not necessary for TEC differentiation but was continually required for the survival of mature cortical and medullary TECs and the maintenance of thymic architecture. A screen of TEC trophic factors in organ cultures showed that epidermal growth factor upregulated MCL-1 via MAPK/ERK kinase activity, providing a molecular mechanism for the support of TEC survival. This signaling axis governing TEC survival and thymic function represents a new target for strategies for thymic protection and regeneration.

Reduced Numbers of Mature Medullary Thymic Epithelial Cells in SKG Mice.

Attenuated T cell receptor (TCR) signalling contributes to the susceptibility for autoimmunity as shown via mutants of PTPN22 and Zap70 genes. We here set out to investigate the effect of an attenuated TCR signal on the composition of the thymic epithelial cell (TEC) compartment. To that extent, we combined flow cytometry and histology and compared the TEC subpopulations of Zap70 wild type with SKG mutant mice. We found an increased cortical TEC compartment in SKG thymi at the expense of reduced numbers of mature medullary TECs and a 4.8-fold reduced medulla area. We also found reduced proportions of CD69+ -activated thymocytes among double-negative, double-positive and CD4- CD8+ single-positive stages, reduced absolute numbers of single-positive thymocytes, diminished expression of Lta and Ltb by CD4- CD8+ single-positive thymocytes and a diminished expression of Ccl19, a target gene of the lymphotoxin-b-receptor. While the reduced thymocyte numbers together with the attenuated TCR signal explain the diminished expression of lymphotoxins, the latter is required for an AIRE-independent expression of tissue-restricted antigens as well as attracting positively selected thymocytes to the medulla. Our results describe altered TEC compartments in SKG mice that are likely to support the development of autoimmunity.

The immunoproteasome subunit LMP7 is required in the murine thymus for filling up a hole in the Tcell repertoire.

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible variant of the proteasome which has been implicated in regulating inflammatory responses and antigen presentation. In the thymus, medullary thymic epithelial cells (mTECs) and cortical thymic epithelial cells (cTECs) do express different proteasome subunits exerting chymotrypsin-like activities suggesting distinct functions in thymic Tcell selection. Employing the lymphocytic choriomeningitis virus (LCMV) infection model, we could show that the immunoproteasome subunit LMP7 was absolutely required for the generation of LCMV GP118-125 -specific Tcells although the class I mediated presentation of GP118-125 was not dependent on LMP7. Using bone marrow chimeras and adoptive transfer of LMP7-deficient CD8+ Tcells into RAG1-deficient mice we show that LMP7-deficient mice lacked GP118-125 -specific Tcell precursors and that LMP7 was required in radioresistant cells - most likely thymic epithelial cells - to enable their selection. Since LMP7 is strongly expressed in negatively selecting mTECs but barely in positively selecting cTECs our data suggest that LMP7 was required to avoid excessive negative selection of GP118-125 -specific Tcell precursors. Taken together, this study demonstrates that the immunoproteasome is a crucial factor for filling up holes within the cytotoxic Tcell repertoire.

EphB receptors, mainly EphB3, contribute to the proper development of cortical thymic epithelial cells

ABSTRACTEphB and their ligands ephrin-B are an important family of protein tyrosine kinase receptors involved in thymocyte-thymic epithelial cell interactions known to be key for the maturation of both thymic cell components. In the present study, we have analyzed the maturation of cortical thymic epithelium in EphB-deficient thymuses evaluating the relative relevance of EphB2 and EphB3 in the process. Results support a relationship between the epithelial hypocellularity of mutant thymuses and altered development of thymocytes, lower proportions of cycling thymic epithelial cells and increased epithelial cell apoptosis. Together, these factors induce delayed development of mutant cortical TECs, defined by the expression of different cell markers, i.e. Ly51, CD205, MHCII, CD40 and β5t. Furthermore, although both EphB2 and EphB3 are necessary for cortical thymic epithelial maturation, the relevance of EphB3 is greater since EphB3−/− thymic cortex exhibits a more severe phenotype than that of EphB2-deficient thymuses.

Approach for analysis of human thymic epithelial cells

Background: The thymus is implicated as an organ that contributes to autoimmunity in myasthenia gravis (MG). The thymic parenchyma consists of the cortex and medulla, which are constructed by cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells (mTECs).

Immunoproteasome-selective and non-selective inhibitors: A promising approach for the treatment of multiple myeloma.

The ubiquitin-proteasome system (UPS) is the major non-lysosomal proteolytic system for the degradation of abnormal or damaged proteins no longer required. The proteasome is involved in degradation of numerous proteins which regulate the cell cycle, indicating a role in controlling cell proliferation and maintaining cell survival. Defects in the UPS can lead to anarchic cell proliferation and to tumor development. For these reasons UPS inhibition has become a significant new strategy for drug development in cancer treatment. In addition to the constitutive proteasome, which is expressed in all cells and tissues, higher organisms such as vertebrates possess two immune-type proteasomes, the thymoproteasome and the immunoproteasome. The thymoproteasome is specifically expressed by thymic cortical epithelial cells and has a role in positive selection of CD8+ T cells, whereas the immunoproteasome is predominantly expressed in monocytes and lymphocytes and is responsible for the generation of antigenic peptides for cell-mediated immunity. Recent studies demonstrated that the immunoproteasome has a preservative role during oxidative stress and is up-regulated in a number of pathological disorders including cancer, inflammatory and autoimmune diseases. As a consequence, immunoproteasome-selective inhibitors are currently the focus of anticancer drug design. At present, the commercially available proteasome inhibitors bortezomib and carfilzomib which have been validated in multiple myeloma and other model systems, appear to target both the constitutive and immunoproteasomes, indiscriminately. This lack of specificity may, in part, explain some of the side effects of these agents, such as peripheral neuropathy and gastrointestinal effects, which may be due to targeting of the constitutive proteasome in these tissues. In contrast, by selectively inhibiting the immunoproteasome, it may be possible to maintain the antimyeloma and antilymphoma efficacy while reducing these toxicities, thereby increasing the therapeutic index. This review article will be focused on the discussion of the most promising immunoproteasome specific inhibitors which have been developed in recent years. Particular attention will be devoted to the description of their mechanism of action, their structure-activity relationship, and their potential application in therapy.

In vivo lineage tracing reveals Axin2-expressing, long-lived cortical thymic epithelial progenitors in the postnatal thymus.

In the thymus, cortical and medullary thymic epithelial cells (TECs) are instrumental for generating a repertoire of functional T cells. Hence, there has been much interest in the ontogeny of TECs. While medullary TEC (mTEC) and bipotent progenitors have been identified, the existence of a cortical TEC (cTEC) progenitor remains ambiguous. In this study, we used lineage tracing based on a target gene of the Wnt pathway, Axin2. We found that Axin2 initially labels cells in both the cortical and medullary compartments. Using Axin2-CreERT2 mice to track the fate of labelled cells, we identified long-lived cortical TEC progenitors that give rise to expanding clones and contribute to homeostasis in postnatal thymus. In contrast, no clonal expansion was found in the medullary or in the K5K8-double positive compartments. The identification of cTEC progenitors and their regulation by Wnt signaling have important implications for our understanding of thymus physiology during homeostasis and TEC-related disorders.

Progressive Changes in CXCR4 Expression That Define Thymocyte Positive Selection Are Dispensable For Both Innate and Conventional \u03b1\u03b2T-cell Development

The ordered migration of immature thymocytes through thymic microenvironments generates both adaptive MHC restricted αβT-cells and innate CD1d-restricted iNKT-cells. While several chemokine receptors and ligands control multiple stages of this process, their involvement during early thymocyte development often precludes direct analysis of potential roles during later developmental stages. For example, because of early lethality of CXCR4−/− mice, and stage-specific requirements for CXCR4 in thymus colonisation and pre-TCR mediated selection, its role in thymic positive selection is unclear. Here we have examined CXCR4-CXCL12 interactions during the maturation of CD4+CD8+ thymocytes, including downstream stages of iNKT and αβT-cell development. We show CXCL12 expression is a common feature of cortical thymic epithelial cells, indicating widespread availability throughout the cortex. Moreover, CXCR4 expression by CD4+CD8+ pre-selection thymocytes is progressively downregulated following both MHC and CD1d-restricted thymic selection events. However, using CD4Cre-mediated deletion to bypass its involvement in CD4−CD8− thymocyte development, we show CXCR4 is dispensable for the maintenance and intrathymic positioning of CD4+CD8+ thymocytes, and their ability to generate mature αβT-cells and CD1d-restricted iNKT-cells. Collectively, our data define dynamic changes in CXCR4 expression as a marker for intrathymic selection events, and show its role in T-cell development is restricted to pre-CD4+CD8+ stages.