Thymic Cortical Epithelium Research Articles

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.

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.

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.

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.

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.

The thymic cortical epithelium determines the TCR repertoire of IL-17-producing γδT cells.

The thymus provides a specialized microenvironment in which distinct subsets of thymic epithelial cells (TECs) support T-cell development. Here, we describe the significance of cortical TECs (cTECs) in T-cell development, using a newly established mouse model of cTEC deficiency. The deficiency of mature cTECs caused a massive loss of thymic cellularity and impaired the development of αβT cells and invariant natural killer T cells. Unexpectedly, the differentiation of certain γδT-cell subpopulations-interleukin-17-producing Vγ4 and Vγ6 cells-was strongly dysregulated, resulting in the perturbation of γδT-mediated inflammatory responses in peripheral tissues. These findings show that cTECs contribute to the shaping of the TCR repertoire, not only of "conventional" αβT cells but also of inflammatory "innate" γδT cells.

Retraction. The requirements for natural Th17 cell development are distinct from those of conventional Th17 cells.

CD4+ T helper 17 (Th17) cells play a critical role in the adaptive immune response against extracellular pathogens. Most studies to date have focused on understanding the differentiation of Th17 cells from naive CD4+ T cells in peripheral effector sites. However, Th17 cells are present in the thymus. In this study, we demonstrate that a population of Th17 cells, natural Th17 cells (nTh17 cells), which acquire effector function during development in the thymus before peripheral antigen exposure, shows preferential usage of T cell receptor Vβ3. nTh17 cells are dependent on major histocompatibility complex (MHC) class II for thymic selection, yet unlike conventional CD4+ T cells, MHC class II expression on thymic cortical epithelium is not sufficient for their development, rather expression on medullary epithelium is necessary. Differential signaling requirements for IL-17 priming further distinguish nTh17 from conventional Th17 cells. Collectively, our findings define a Th17 population, poised to rapidly produce cytokines, that is developmentally distinct from conventional Th17 cells and that potentially functions at the interface of innate and adaptive immunity.

A T Cell Receptor Signaling Mutant Reveals Two Distinct Populations of IL-17 Producing CD4+ T Cells

Abstract 186Interleukin-17 (IL-17) producing CD4+ T cells (Th17 cells) are essential for immune responses in mucosal and epithelial sites which are the first line of host defense. Th17 cells play a critical role in the pathogenesis of many inflammatory and autoimmune diseases, and the role of IL-17 and Th17 cells in cancer has recently become the focus of extensive investigation. Most studies to date have focused on elucidating the cell extrinsic requirements for differentiation of Th17 cells from naïve CD4+ T cells in peripheral effector sites. Here we report an unconventional population of Th17 cells, “natural Th17 cells” (nTh17), that acquire effector function during development in the thymus, thereby distinguishing them from conventional Th17 cells which require antigen encounter and differentiation in the periphery. We show that these nTh17 cells are present and indeed develop in the thymus using fetal thymic organ culture. nTh17 cells express surface markers consistent with an innate and/or activated phenotype and their development is dependent on selection by MHC class II in the thymus. Yet unlike conventional CD4+ T cells, MHC class II expression on thymic cortical epithelium is not sufficient for their development, rather expression on medullary epithelium is necessary. In addition, T cell receptor (TCR) repertoire analysis of nTh17 cells revealed unique characteristics in TCR gene usage compared to conventional Th17 cells. A mouse model with a mutation in the TCR signaling protein SLP76 (SLP76 Y145F mice) further highlights the difference between the two distinct Th17 populations. SLP76 Y145F mice have increased numbers of nTh17 cells in the thymus compared to wild-type mice. However, peripheral naïve CD4+ T cells from these mice showed severely defective IL-17 production when cultured in vitro under conditions promoting Th17 cell differentiation. This defect was reflected in vivo as CD4+ T cells in the small intestinal lamina propria of SLP76 Y145F mice fail to produce IL-17. Using mixed radiation bone marrow chimeras, we found that the aberrant Th17 phenotype in the thymus and periphery of SLP76 Y145F mice is cell-intrinsic. Finally, adoptive transfer of purified nTh17 cells into RAG-deficient host mice revealed preferential homing of nTh17 cells to thymus and lung compared to other comparison/competitive populations that were co-transferred. Taken together, our data suggest a distinct population of Th17 cells that have characteristics of innate lymphocytes that function at the interface between innate and adaptive immunity. Understanding the biology of nTh17 cells will provide insight into the recently identified Th17 cells in human thymi and umbilical cord blood. Disclosures:No relevant conflicts of interest to declare.

The requirements for natural Th17 cell development are distinct from those of conventional Th17 cells

CD4(+) T helper 17 (Th17) cells play a critical role in the adaptive immune response against extracellular pathogens. Most studies to date have focused on understanding the differentiation of Th17 cells from naive CD4(+) T cells in peripheral effector sites. However, Th17 cells are present in the thymus. In this study, we demonstrate that a population of Th17 cells, natural Th17 cells (nTh17 cells), which acquire effector function during development in the thymus before peripheral antigen exposure, shows preferential usage of T cell receptor Vβ3. nTh17 cells are dependent on major histocompatibility complex (MHC) class II for thymic selection, yet unlike conventional CD4(+) T cells, MHC class II expression on thymic cortical epithelium is not sufficient for their development, rather expression on medullary epithelium is necessary. Differential signaling requirements for IL-17 priming further distinguish nTh17 from conventional Th17 cells. Collectively, our findings define a Th17 population, poised to rapidly produce cytokines, that is developmentally distinct from conventional Th17 cells and that potentially functions at the interface of innate and adaptive immunity.

Alterations of the Medullary Epithelial Compartment in the Aire-Deficient Thymus: Implications for Programs of Thymic Epithelial Differentiation

A widely held model of thymic epithelial differentiation is based on patterns of keratin expression, where a K8(+)K5(+) progenitor gives rise to K8(+)K5/K14(-) cortical thymic epithelium (CTEC), and medullary thymic epithelium (MTEC) are K8(-)K5(+)K14(+). The thymic phenotype of p63-deficient mice indicates that p63 is an important regulator of proximal stages of thymic epithelial differentiation. In this study, we have examined several features of the thymic medullary compartment in wild-type and Aire-deficient thymi in an effort to integrate the proapoptotic activity of Aire with these different perspectives of TE differentiation. Patterns of keratin and p63 expression by MTEC described here are difficult to reconcile with postmitotic MTEC that express a K8(-)K14(+) phenotype and suggest that the patterns of p63 and keratin expression reflecting differentiation programs of other epithelial tissues provide a useful framework for revising models of TE differentiation. Alterations of the Aire(-/-) MTEC compartment included reduced expression of p63, increased frequency of MTEC expressing truncated Aire protein, and shifts in the pattern of keratin expression and epithelial morphology. These data suggest a scenario where cellular targets of Aire-mediated apoptosis are postmitotic MTEC that have not yet completed their terminal differentiation program. According to this view, the minor population of globular K8(+)K14(-/low) MTEC observed in the Aire(+/+) thymus and significantly expanded in the Aire(-/-) thymic medulla represent end-stage, terminally differentiated MTEC. These Aire-dependent alterations of the MTEC compartment suggest that the activity of Aire is not neutral with respect to the program of MTEC differentiation.

Clonal analysis reveals a common progenitor for thymic cortical and medullary epithelium

The thymus provides an essential environment for the development of T cells from haemopoietic progenitors. This environment is separated into cortical and medullary regions, each containing functionally distinct epithelial populations that are important at successive stages of T-cell development and selection. However, the developmental origin and lineage relationships between cortical and medullary epithelial cell types remain controversial. Here we describe a clonal assay to investigate the developmental potential of single, individually selected, thymic epithelial progenitors (marked with enhanced yellow fluorescent protein) developing within the normal architecture of the thymus. Using this approach, we show that cortical and medullary epithelial cells share a common origin in bipotent precursors, providing definitive evidence that they have a single rather than dual germ layer origin during embryogenesis. Our findings resolve a long-standing issue in thymus development, and are important in relation to the development of cell-based strategies for thymus disorders and the possibility of restoring function of the atrophied adult thymus.

Thymic cortical epithelium induces self tolerance

Because of its role in positive selection, the ability of cortical epithelium to induce tolerance is controversial. On the one hand, experiments with transplanted thymuses showed that the recipients were functionally tolerant of all the antigens expressed by the cells of those thymuses, including cells of the cortical epithelium. On the other, the keratin 14 (K14) transgenic mouse strain, which expresses MHC class II on cortical epithelium under the control of the K14 promoter, does not seem to be tolerant of the transgenic MHC molecule. Here we tested whether the lack of tolerance in the K14 mouse might be more apparent than real. We found that K14 mice are indeed completely tolerant of K14 cortical thymic epithelium, whereas they remain reactive to tissues that express the same MHC allele under normal genetic control. These results establish the ability of cortical epithelium to induce central tolerance, and impinge on several of the models concerning positive selection of newly developing T cells.

Developing a new paradigm for thymus organogenesis.

The mature thymic epithelium is complex, with two major compartments — the cortex and the medulla — each containing several functionally distinct epithelial-cell types. There is considerable debate as to the embryonic origins of these different thymic epithelial-cell subpopulations. The textbook view is a dual origin, with cortical thymic epithelium arising from the ectoderm and medullary thymic epithelium originating in the endoderm. However, the literature has been divided on this issue since it was first considered. In this review, we discuss recent embryological, functional, genetic and molecular data that collectively support a new model of thymus organogenesis and patterning.

Expression of soma-restricted proteolipid/DM20 proteins in lymphoid cells

A new family of the myelin proteolipid protein (PLP/DM20) gene products, srPLP/DM20, has been identified recently in thymus and brain. In the central nervous system, srPLP/DM20 products are not localized in the myelin membrane, unlike their classic PLP/DM20 counterparts. In the immune system, the classic PLP/DM20 products appear to be expressed predominantly in thymic cortical epithelium. In this study, we examined the cellular expression of sr-PLP/DM20 proteolipids in lymphoid tissues and cells by immunohistochemistry, FACS analysis and RT-PCR. We found that in contrast to the classic PLP/DM20 products, sr-proteins are mainly expressed in developing thymocytes in thymus and in T- and B-lymphocytes in spleen. These results are of importance in our further understanding, not only the different role of these new PLP gene products in central and peripheral tolerance, but also the function of such products in lymphocyte biology.

Selective absence of CD8+ TCRalpha beta+ intestinal epithelial cells in transgenic mice expressing beta2-microglobulin-associated ligands exclusively on thymic cortical epithelium.

Whereas interactions between the TCRalpha beta and self MHC:peptide complexes are clearly required for positive selection of mature CD4(+) and CD8(+) T cells during intrathymic development, the role of self or foreign ligands in maintaining the peripheral T cell repertoire is still controversial. In this report we have utilized keratin 14-beta2-microglobulin (K14-beta2m)-transgenic mice expressing beta2m-associated ligands exclusively on thymic cortical epithelial cells to address the possible influence of TCR:ligand interactions in peripheral CD8(+) T cell homeostasis. Our data indicate that CD8(+) T cells in peripheral lymphoid tissues are present in normal numbers in the absence of self MHC class I:peptide ligands. Surprisingly, however, steady state homeostasis of CD8(+) T cells in the intestinal epithelium is severely affected by the absence of beta2m-associated ligands. Indeed TCRalpha beta(+) IEL subsets expressing CD8alpha beta or CD8alpha alpha are both dramatically reduced in K14-beta2m mice, suggesting that the development, survival or expansion of CD8(+) IEL depends upon interaction of the TCR with MHC class I:peptide or other beta2m-associated ligands elsewhere than on thymic cortical epithelium. Collectively, our data reveal an unexpected difference in the regulation of CD8(+) T cell homeostasis by beta2m-associated ligands in the intestine as compared to peripheral lymphoid organs.

Antigen presentation by keratinocytes directs autoimmune skin disease.

The antigen-presenting cells that initiate and maintain MHC class II-associated organ-specific autoimmune diseases are poorly defined. We now describe a new T cell antigen receptor (TCR) transgenic (Tg) model of inflammatory skin disease in which keratinocytes activate and are the primary target of autoreactive CD4(+) T cells. We previously generated keratin 14 (K14)-A(beta)b mice expressing MHC class II only on thymic cortical epithelium. CD4(+) T cells from K14-A(beta)b mice fail to undergo negative selection and thus have significant autoreactivity. The TCR genes from an autoreactive K14-A(beta)b CD4 hybridoma were cloned to produce a TCR Tg mouse, 2-2-3. 2-2-3 TCR Tg cells are negatively selected in WT C57BL6 mice but not in 2-2-3K14-A(beta)b mice. Interestingly, a significant number of mice that express both the K14-A(beta)b transgene and the autoreactive 2-2-3 TCR spontaneously develop inflammatory skin disease with mononuclear infiltrates, induction of MHC class II expression on keratinocytes, and T helper 1 cytokines. Disease can be induced by skin inflammation but not solely by activation of T cells. Thus, cutaneous immunopathology can be directed through antigen presentation by tissue-resident keratinocytes to autoreactive TCR Tg CD4(+) cells.

In vitro evidence for participation of DEC-205 expressed by thymic cortical epithelial cells in clearance of apoptotic thymocytes

Binding of apoptotic cells was compared after incubation of thymocytes with two clones of murine thymic stromal cells to which CD4(+)/CD8(+) thymocytes attach. With the BA/10, but not the BA/2, clone, thymocytes with apoptotic morphology were bound irreversibly. These tightly bound thymocytes were further identified as apoptotic in terms of active caspase-3 and DNA fragmentation assayed in situ. FACS analysis indicated that the apoptotic thymocytes are at an early double-positive stage and results with mice mutant for the Fas gene showed that the Fas-Fas ligand system is not involved. Comparison of BA/10 and BA/2 cells showed that the former, but not the latter, can be induced to express CDR-1 antigen which is characteristic of cortical epithelial thymic stroma and constitutively express DEC-205, a surface protein common to cortical thymic epithelium and dendritic cells. Antibody NLDC-145 that is specific for the DEC-205 protein strongly reduced the number of stromal cells with bound apoptotic thymocytes. Preincubation of thymocytes in dexamethasone dramatically increased the number of bound apoptotic cells, indicating that the thymic cortical epithelial cells can participate in clearance of apoptotic thymocytes through involvement of DEC-205.

Expression, Genomic Structure and Mapping of the Thymus Specific Protease Prss16: A Candidate Gene for Insulin Dependent Diabetes Mellitus Susceptibility

PRSS16 is a serine protease specifically expressed by epithelial cells in the thymic cortex. The human gene is encoded on 6p21.3-p22 where recent linkage analysis has identified an association with insulin dependent diabetes mellitus (IDDM) susceptibility independent of HLA-DR3. To further investigate its potential role in autoimmunity, we characterized the mouse orthologue, Prss16. The genomic structure of Prss16 shows conservation with the human gene in size, number of exons and chromosomal location. Mapping of Prss16 places it on mouse chromosome 13 centromeric of thesatin locus. This region is comparable to the PRSS16 region on human chromosome 6 and has also been linked to quantitative trait locus for IDDM in the nonobese diabetic mouse. Similar to the human gene, Prss16 expression is highly specific in the mouse with expression limited to the cortical thymic epithelium. Notably, embryonic expression coincides with population of the thymic anlage with T-cell precursors and initiation of T-cell development. We also show that NOD and New Zealand Black mice, which have a disrupted thymic architecture and autoimmune phenotype, have lower levels of Prss16 expression compared to C57BL/6 mice. These findings support the role of Prss16 in T-cell development and susceptibility to autoimmunity in the mouse.

Altered T cell development in human thymoma is related to impairment of MHC class II transactivator expression induced by interferon-gamma (IFN-γ)

Thymoma is known to contain CD4+CD8+ T cells, indicating that neoplastic epithelial cells of thymoma have a function as thymic cortical epithelium. However, it has been shown that there is an impairment of CD4+ T cell development in thymoma and that IFN-gamma-induced HLA-DR expression on cultured thymic epithelial cells (TEC) derived from thymoma is decreased when compared with the normal thymus. MHC class II transactivator (CIITA) is known to play a critical role in IFN-gamma-induced MHC II expression. In this study, we attempted to elucidate whether CIITA is responsible for the impaired up-regulation of MHC II molecules in response to IFN-gamma in thymoma TEC. A quantitative reverse transriptase-polymerase chain reaction examination revealed that the induced level of CIITA was significantly lower in thymoma TEC than in normal TEC. The induced levels of invariant chain (Ii) and HLA-DR in thymoma TEC were correlated with CIITA expression. The proportion of CD3+ cells in the CD4+CD8- subset in thymoma was also correlated with CIITA expression. A gel mobility shift assay however, revealed translocation of STAT1 to the nucleus in thymoma as well as normal TEC. Intercellular adhesion molecule-1 was up-regulated in the thymoma TEC to a level similar to normal TEC in response to IFN-gamma. These results indicate that impaired up-regulation of HLA-DR in response to IFN-gamma results from insufficient induction of CIITA, but not from the signal from IFN-gamma receptor to the nucleus. The abnormal regulation of HLA-DR expression caused by impaired induction of CIITA may affect CD4+ T cell development in thymoma.

Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4(+)25(+) immunoregulatory T cells.

CD4(+)25(+) T cells are a unique population of immunoregulatory T cells which are critical for the prevention of autoimmunity. To address the thymic selection of these cells we have used two models of attenuated thymic deletion. In K14-A(beta)(b) mice, major histocompatibility complex (MHC) class II I-A(b) expression is limited to thymic cortical epithelium and deletion by hematopoietic antigen-presenting cells does not occur. In H2-DMalpha-deficient mice, MHC class II molecules contain a limited array of self-peptides resulting in inefficient clonal deletion. We find that CD4(+)25(+) T cells are present in the thymus and periphery of K14-A(beta)(b) and H2-DMalpha-deficient mice and, like their wild-type counterparts, suppress the proliferation of cocultured CD4(+)25(-) effector T cells. In contrast, CD4(+)25(+) T cells from MHC class II-deficient mice do not suppress responder CD4(+) T cells in vitro or in vivo. Thus, development of regulatory CD4(+)25(+) T cells is dependent on MHC class II-positive thymic cortical epithelium. Furthermore, analysis of the specificities of CD4(+)25(+) T cells in K14-A(beta)(b) and H2-DMalpha-deficient mice suggests that a subset of CD4(+)25(+) T cells is subject to negative selection on hematopoietic antigen-presenting cells.