Thymic shared antigen-2: a novel cell surface marker associated with T cell differentiation and activation

Thymic shared Ag-2 (TSA-2) is a 28-kDa, glycophosphatidylinitosol-linked cell surface molecule expressed on various T cell and thymic stromal cell subsets. It is expressed on most CD3-CD4-CD8-, CD4+CD8+, and CD3highCD4-CD8+ thymocytes but is down-regulated on approximately 40% of CD3highCD4+CD8- thymocytes. Expression on peripheral TCR-alphabeta+ T cells is similar to that of CD3+ thymocytes, although a transient down-regulation occurs with cell activation. Consistent with the recent hypothesis that emigration from the thymus is an active process, recent thymic emigrants are primarily TSA-2-/low. TSA-2 expression reveals heterogeneity among subpopulations of CD3highCD4+CD8- thymocytes and TCR-gamma delta+ T cell previously regarded as homogenous. The functional importance of TSA-2 was illustrated by the severe block in T cell differentiation caused by adding purified anti-TSA-2 mAb to reconstituted fetal thymic organ culture. While each CD25/CD44-defined triple-negative subset was present, differentiation beyond the TN stage was essentially absent, and cell numbers of all subsets were significantly below those of control cultures. Cross-linking TSA-2 on thymocytes caused a significant Ca2+ influx but no increase in apoptosis, unless anti-TSA-2 was used in conjunction with suboptimal anti-CD3 mAb. Similar treatment of mature TSA-2+ T cells had no effect on cell survival or proliferation. This study reveals TSA-2 to be a functionally important molecule in T cell development and a novel indicator of heterogeneity among a variety of developing and mature T cell populations.     

T cell development in PU.1-deficient mice.

These studies address the role of PU.1 in T cell development through the analysis of PU.1-/- mice. We show that the majority of PU.1-/- thymocytes are blocked in differentiation prior to T cell commitment, and contain a population of thymocyte progenitors with the cell surface phenotype of CD44+, HSAbright, c-kitint, Thy-1-, CD25-, Sca-1-, CD4-, and CD8-. These cells correspond in both number and cell surface phenotype with uncommitted thymocyte progenitors found in wild-type fetal thymus. RT-PCR analysis demonstrated that PU.1 is normally expressed in this early progenitor population, but is down-regulated during T cell commitment. Rare PU.1-/- thymi, however, contained small numbers of thymocytes expressing markers of T cell commitment. Furthermore, almost 40% of PU.1-/- thymi placed in fetal thymic organ culture are capable of T cell development. Mature PU. 1-/- thymocytes generated during organ culture proliferated and produced IL-2 in response to stimulation through the TCR. These data demonstrate that PU.1 is not absolutely required for T cell development, but does play a role in efficient commitment and/or early differentiation of most T progenitors.     

Expression and function of the UM4D4 antigen in human thymus

UM4D4 is a newly identified T cell surface molecule, distinct from the Ag receptor and CD2, which is expressed on 25% of peripheral blood T cells, resting or activated. Monoclonal anti-UM4D4 is mitogenic for T cells and T cell clones. Since alternative activation pathways independent of Ag/MHC recognition may be important in thymic differentiation, the expression and function of UM4D4 was examined in human thymus. UM4D4 was found on the surface of 6% of thymocytes. All thymocyte subsets contained UM4D4+ cells but expression was greatest on thymocytes that were CD1- (12%), CD3+ (11%) and especially CD4-CD8- (18%). CD3+CD4- CD8- cells, most of which bear the gamma delta-receptor, were greater than or equal to 50% + for UM4D4. Moreover, anti-UM4D4 was comitogenic for thymocytes together with PMA or IL-2. Anti-UM4D4 also reacted strongly with a subset of thymic epithelial cells in both cortex and medulla. Dual color fluorescence microscopy, with anti-UM4D4 and antibodies to other thymic epithelial Ag, showed UM4D4 expression on neuroendocrine thymic epithelium but not on thymic fibrous stroma. Thus, UM4D4 is expressed on, and represents an activation pathway for, a subset of thymic T cells. In addition, this determinant, initially identified as a novel T cell activating molecule, is broadly expressed by neuroendocrine thymic epithelium. Although the function of UM4D4 on the thymic epithelial cells is not yet clear, it is possible that UM4D4 represents a pathway for the functional activation of a subset of the thymic epithelium as well as a subset of thymocytes, thus playing a dual role in T cell differentiation.     

Human thymic epithelial cells inhibit IL-15- and IL-2-driven differentiation of NK cells from the early human thymic progenitors

T/NK progenitors are present in the thymus; however, the thymus predominantly promotes T cell development. In this study, we demonstrated that human thymic epithelial cells (TEC) inhibit NK cell development. Most ex vivo human thymocytes express CD1a, indicating that thymic progenitors are predominantly committed to the T cell lineage. In contrast, the CD1a(-)CD3(-)CD56(+) NK population comprises only 0.2% (n = 7) of thymocytes. However, we observed increases in the percentage (20- to 25-fold) and absolute number (13- to 71-fold) of NK cells when thymocytes were cultured with mixtures of either IL-2, IL-7, and stem cell factor or IL-15, IL-7, and stem cell factor. TEC, when present in the cultures, inhibited the increases in the percentage (3- to 10-fold) and absolute number (3- to 25-fold) of NK cells. Furthermore, we show that TEC-derived soluble factors inhibit generation of NK-CFU and inhibit IL15- or IL2-driven NK cell differentiation from thymic CD34(+) triple-negative thymocytes. The inhibitory activity was found to be associated with a 8,000- to 30,000 Da fraction. Thus, our data demonstrate that TEC inhibit NK cell development from T/NK CD34(+) triple negative progenitors via soluble factor(s), suggesting that the human thymic microenvironment not only actively promotes T cell maturation but also controls the development of non-T lineage cells such as the NK lineage.     

An immuno-electron-microscopic study of human thymic B cells

Thymic B cells are a constituent of normal human thymic medulla. They are supposed to play a role in T cell maturation. Thymic B cells have been characterized morphologically and immunohistochemically at the light-microscopic level. Their ultrastructural appearance in vivo has not been demonstrated. Six normal infantile thymi were immunolabelled with the pan-B cell marker CD20 using a pre-embedding technique and viewed at the electron-microscopic level. Cells expressing CD20 had long cytoplasmic processes. They were all ”asteroid” in shape and in close contact with thymocytes. Also, their long cytoplasmic processes intermingled with cytoplasmic processes of cells that were presumed to be interdigitating reticulum cells (IDC) based on morphological criteria. Thymic B cells may act in concert with IDC during T cell maturation. Received: 20 October 1995 / Accepted: 10 January 1996     

Induction of thymocyte proliferation by supernatants from a mouse thymic epithelial cell line

The thymic stroma plays a critical role in the generation of T lymphocytes by direct cell-to-cell contacts as well as by secreting growth factors or hormones. The thymic epithelial cells, responsible for thymic hormone secretion, include morphologically and antigenically distinct subpopulations that may exert different roles in thymocyte maturation. The recent development of thymic epithelial cell lines provided an interesting model for studying thymic epithelial influences on T cell differentiation. Treating mouse thymocytes by supernatants from one of TEC line (IT-76M1), we observed an induction of thymocyte proliferation and an increase in the percentages of CD4-/CD8- thymocytes. This proliferation was largely inhibited when thymocytes were incubated with IT-76M1 supernatants together with an anti-thymulin monoclonal antibody, but could be enhanced by pretreating growing epithelial cells by triiodothyronine. We suggest that among the target cells for thymulin within the thymus, some putative precursors of early phenotype might be included.     

Epithelial cell-specific laminin 5 is required for survival of early thymocytes

The gene LamC2 encoding the gamma2 chain of laminin 5, an epithelial cell-specific extracellular matrix protein, was identified in a PCR-based subtracted cDNA library from mouse thymic stromal cells. The mRNA existed in two alternative forms (5.1 and 2.4 kb). The full-length message was highly expressed in SCID thymus and in a nurse cell line, but not in other thymic epithelial cell lines, while the short form was more widely expressed. In situ hybridization and immunohistochemical staining revealed laminin 5 expression mostly in the subcapsular region of the adult thymus. Addition to fetal thymic organ cultures of a cell adhesion-blocking mAb to the alpha3 chain of laminin 5 interrupted T cell development. There was a 40% reduction in the total yield of thymocytes, and the most profound decrease (75-90%) was seen in the CD25+CD44+ and CD25+CD44-subsets of the CD4-CD8- double negative fraction. Most of the surviving double negative thymocytes expressed Sca-1, and there were significant increases in the number of cells with CD69 expression and in the fraction of annexin V-stained cells. None of these changes were observed with a nonblocking anti-laminin alpha3 chain mAb. These results suggest that the interaction between double negative thymoctyes and laminin 5 made by subcapsular epithelial cells is required for the survival and differentiation of mouse thymocytes.     

Thymic stromal cell clone with nursing activity supports the growth and differentiation of murine CD4+8+ thymocytes in vitro

Thymic stromal cell clone, TNC-R3.1 cell, was established from spontaneous AKR/J mouse thymoma. TNC-R3.1 cell, which has the similar properties to thymic nurse cells, formed a unique complex with normal thymocyte subpopulations. Flow cytometry analysis demonstrated that CD4+8+ and CD4-8- immature thymocytes preferentially interacted with TNC-R3.1 stromal cell clone. CD4+8+ thymocytes, which interacted with TNC-R3.1 stromal cell clone, contained a higher proportion of large size and cycling T cells than did noninteracting CD4+8+ thymocytes. As is generally accepted, CD4+8+ thymocytes did not respond to any stimulation such as IL-2, anti-CD3 mAb (2C11), or IL-2 plus 2C11. However, culture of isolated CD4+8+ thymocytes on TNC-R3.1 stromal cell monolayer in the presence of suboptimal dose of IL-2 induced a significant cell growth. Moreover, the addition of 2C11 and IL-2 into this coculture system resulted in a dramatic increase of the proliferative response of thymocytes. Flow cytometry analysis showed the proliferating cells on TNC-R3.1, which originated from CD4+8+ thymocytes, were mostly TCR-alpha beta+ CD3+CD4-8+ T cells. These results provide in vitro evidence that CD4+8+ thymocytes are at an intermediate stage of T cell maturation and TNC-R3.1 stromal cell clone induces the growth and differentiation of CD4+8+ thymocytes into CD4-8+ T cells.     

Altered thymocyte development resulting from expressing a deleting ligand on selecting thymic epithelium.

The maturation of CD4+8- and CD4-8+ thymocytes from CD4+8+ thymocytes is dependent on the mandatory interaction of their alpha beta TCR with selecting ligands expressed on thymic epithelial cells (TE). This is referred to as positive selection. The deletion of CD4+8+ thymocytes that express autospecific TCR (negative selection) is mediated primarily by bone marrow-derived cells. Previous studies have shown that TE is relatively ineffective in mediating the deletion of CD4+8- thymocytes expressing autospecific TCR but TE can render them anergic, i.e., nonresponsive, to the self Ag. The mechanism by which anergy is induced in these cells is unknown. In this study, we used thymocytes expressing a transgenic TCR specific for the male Ag presented by H-2Db class I MHC molecules to examine how expression of the deleting ligand by TE affects thymocyte development and phenotype. The development of female TCR-transgenic thymocytes was examined in irradiated male hosts or in female hosts that had received male fetal thymic epithelial implants. It was observed that the development of transgenic-TCR+ thymocytes was affected in mice with male TE. CD4+8+ thymocytes with reduced CD8 expression and markedly enhanced transgenic TCR expression accumulated in mice with male TE. Development of CD4-8+ thymocytes was also affected in these mice in that fewer were present and they expressed an intermediate CD8 coreceptor level. These CD4-8+ thymocytes expressed a high level of the transgenic TCR, retained the ability to respond to anti-TCR antibodies, but were nonresponsive to male APC. However, the maturation of CD4+8- thymocytes, which are also derived from CD4+8+ precursor cells, was relatively unaffected. In an in vitro assay for assessing negative selection, male TE failed to delete CD4+8+ thymocytes expressing the transgenic TCR under conditions where they were efficiently deleted by male dendritic cells. Collectively these results support the conclusion that male TE was inefficient in mediating deletion. Furthermore, expression of the deleting ligand on thymic epithelium interferes with the maturation of functional male-specific T cells and results in the accumulation of CD4+8+ and CD4-8+ thymocytes expressing a lower level of the CD8 coreceptor but a high level of the transgenic TCR.     

Peripheral T cell lymphopenia and concomitant enrichment in naturally arising regulatory T cells: the case of the pre-Talpha gene-deleted mouse

In pre-Talpha (pTalpha) gene-deleted mice, the positively selectable CD4+ CD8+ double-positive thymocyte pool is only 1% that in wild-type mice. Consequently, their peripheral T cell compartment is severely lymphopenic with a concomitant increase in proportion of CD25+ FoxP3+ regulatory T cells. Using mixed bone marrow chimeras, where thymic output was 1% normal, the pTalpha(-/-) peripheral T cell phenotype could be reproduced with normal cells. In the pTalpha(-/-) thymus and peripheral lymphoid organs, FoxP3+ CD4+ cells were enriched. Parabiosis experiments showed that many pTalpha(-/-) CD4+ single-positive thymocytes represented recirculating peripheral T cells. Therefore, the enrichment of FoxP3+ CD4+ single-positive thymocytes was not solely due to increased thymic production. Thus, the pTalpha(-/-) mouse serves as a model system with which to study the consequences of chronic decreased thymic T cell production on the physiology of the peripheral T cell compartment.     

Acquisition of non-MHC restricted cytotoxic function by IL 2 activated thymocytes with an "immature" antigenic phenotype

Culture of human thymocytes in interleukin 2 (IL 2) results in the generation of cytotoxic T lymphocytes (CTL) that kill tumor cell targets without major histocompatibility complex (MHC) restriction. Thymic non-MHC restricted CTL expressed Leu-19 antigen, but were generated from thymic precursor cells that lacked expression of Leu-19. In contrast, short term culture in Il 2 of peripheral blood lymphocytes depleted of Leu-19+ lymphocytes did not result in the generation of cytotoxic activity. IL 2 was necessary and sufficient for the generation of cytotoxic thymocytes and induction of Leu-19 antigen expression. Thymic non-MHC restricted CTL were generated from precursor cells expressing CD1, an antigen present on the majority of thymocytes. Furthermore, cytotoxic activity was detected in IL 2 cultured thymocyte populations with an "immature" antigenic phenotype, i.e. CD1+ and CD4+, CD8+. Upon subsequent culture, thymic non-MHC restricted CTL lost expression of CD1, and developed an antigenic phenotype similar to peripheral blood non-MHC-restricted CTL, suggesting that peripheral non-MHC-restricted CTL may originate from these thymic precursors.     

Early block in maturation is associated with thymic involution in mammary tumor-bearing mice

We previously reported that mice implanted with mammary tumors show a progressive thymic involution that parallels the growth of the tumor. The involution is associated with a severe depletion of CD4+8+ thymocytes. We have investigated three possible mechanisms leading to this thymic atrophy: 1) increased apoptosis, 2) decreased proliferation, and 3) disruption of normal thymic maturation. The levels of thymic apoptosis were determined by propidium iodide and annexin V staining. A statistically significant, but minor, increase in thymic apoptosis in tumor-bearing mice was detected with propidium iodide and annexin V staining. The levels of proliferation were assessed by in vivo labeling with 5'-bromo-2'-deoxyuridine (BrdU). The percentages of total thymocytes labeled 1 day following BrdU injection were similar in control and tumor-bearing mice. Moreover, the percentages of CD4-8- thymocytes that incorporated BrdU during a short term pulse (5 h) of BrdU were similar. Lastly, thymic maturation was evaluated by examining CD44 and CD25 expression among CD4-8- thymocytes. The percentage of CD44+ cells increased, while the percentage of CD25+ cells decreased among CD4-8- thymocytes from tumor-bearing vs control animals. Together, these findings suggest that the thymic hypocellularity seen in mammary tumor bearers is not due to a decreased level of proliferation, but, rather, to an arrest at an early stage of thymic differentiation along with a moderate increase in apoptosis.     

Development of CD4+ macrophages from intrathymic T cell progenitors is induced by thymic epithelial cells

It was recently demonstrated that there are CD4(+) macrophages, which exhibit strong phagocytic activity, in the thymus. They are suggested to play an important role for the elimination of apoptotic thymocytes. However, the origin and nature of CD4(+) macrophages in the thymus remain unexplored. In this study, we describe that the most immature intrathymic progenitors (CD25(-)/CD44(+)/FcR(+)) give rise to CD4(+) macrophages by oncostatin M-responsive thymic epithelial cells (ORTEC) in an IL-7-dependent manner. Neither conditioned medium of ORTEC nor a mixture of cytokines induced CD4(+) macrophages, and oncostatin M receptor was not expressed in thymocytes, suggesting that the development of CD4(+) macrophages from the immature thymocytes requires a direct interaction with ORTEC. These results collectively suggest that the development of CD4(+) macrophages from the intrathymic T cell progenitors is induced by thymic epithelial cells.     

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

EphB 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.     

The majority of CD4+8- thymocytes are functionally immature.

The thymus is the major site of T cell development and repertoire selection. During these processes, T cells segregate into two subsets that express either CD4 or CD8 accessory molecules, the phenotype of peripheral T cells. Analysis of CD4+8- thymocytes revealed that the majority of these cells express the heat-stable Ag (HSA) but not the nonclassical class I Ag, Qa-2. This HSA+, Qa-2- phenotype is similar to that of the less mature, CD4+8+ thymocytes. The remaining CD4+8- thymocytes possess the HSA-, Qa-2+ phenotype of peripheral T cells. To determine whether the Qa-2-, CD4+8- thymic subset is fully mature, we have analyzed the functional status of these CD4+8- subpopulations. The results indicate that only those thymocytes which express Qa-2 are fully responsive to anti-TCR stimulation in a manner analogous to peripheral T cells. The Qa-2- subset is nonresponsive to stimulation by anti-TCR antibodies that have been immobilized to plastic, even in the presence of lymphokines or syngeneic APC. This subset is, however, capable of proliferating to allogeneic cells or to anti-TCR on the surface of syngeneic APC, although not to the levels achieved by Qa-2+ thymocytes. Thus, the Qa-2- subset appears to require additional interactions which are not necessary for peripheral T cells or Qa-2+ thymocytes. Relevant to this issue, the Qa-2+ thymocyte subset does not appear until relatively late in development, and does not reach adult frequencies until several weeks after birth. These results would suggest that there is a progression from HSA+, Qa-2- to HSA-, Qa-2+ which parallels the maturation of functional responsiveness. These findings are important to understanding T cell selection since thymocytes with such a decreased responsiveness may have a differential capacity for tolerance induction. The results presented suggest that the bulk of CD4+8- thymocytes are not fully mature and that Qa-2 may serve as a marker for T cells with a more complete functional competence.     

CD83 expression influences CD4+ T cell development in the thymus

T lymphocyte selection and lineage commitment in the thymus requires multiple signals. Herein, CD4+ T cell generation required engagement of CD83, a surface molecule expressed by thymic epithelial and dendritic cells. CD83-deficient (CD83-/-) mice had a specific block in CD4+ single-positive thymocyte development without increased CD4+CD8+ double- or CD8+ single-positive thymocytes. This resulted in a selective 75%-90% reduction in peripheral CD4+ T cells, predominantly within the naive subset. Wild-type thymocytes and bone marrow stem cells failed to differentiate into mature CD4+ T cells when transferred into CD83-/- mice, while CD83-/- thymocytes and stem cells developed normally in wild-type mice. Thereby, CD83 expression represents an additional regulatory component for CD4+ T cell development in the thymus.     

The role of the Ets2 transcription factor in the proliferation,maturation, and survival of mouse thymocytes

In this study, we investigated the effects of Ets2 expression on the proliferation, maturation, and survival of thymocytes by establishing transgenic mice that specifically express Ets2 or a dominant negative form of Ets2, Deltaets2, in the thymus. We show that, in young animals, there are fewer T cells in Deltaets2 transgenic thymi and that the maturation of these T cells is affected at the CD4(-)CD8(-) double-negative to CD4(+)CD8(+) double-positive transition compared with wild-type littermate mice. Partial recovery in the number of thymocytes and full T cell maturation are restored with increasing age of Deltaets2 transgenic animals. However, thymocytes from adult Deltaets2 transgenic mice cultured ex vivo are more sensitive to cell death and to glucocorticoid-induced apoptosis than are T cells from control littermate mice. We also show that T cells from adult ets2 transgenic mice proliferate faster than their wild-type littermates. The proliferation and survival of these T cells are clearly affected upon apoptotic signals: glucocorticoid-induced apoptosis induces T cells from ets2 transgenic mice to continue to proliferate in vivo and to survive better ex vivo than T cells from control littermates. It has been shown that c-Myc expression is required for thymic proliferation and improves thymocyte survival of dexamethasone-treated animals. We show that the expression of c-Myc, an Ets2 target, is elevated in T cells freshly isolated from thymi of ets2 transgenic mice pretreated with dexamethasone. Together, these results show that Ets2 plays a role in the proliferation and survival of thymocytes, implicating a Myc-dependent pathway.     

Differential expression and regulation of cyclooxygenase isozymes in thymic stromal cells

Prostaglandins (PGs) are lipid-derived mediators of rapid and localized cellular responses. Given the role of PG in supporting thymic T cell development, we investigated the expression of the PG synthases, also known as cyclooxygenases (COX)-1 and -2, in the biosynthesis of PGs in thymic stromal cell lines. The predominant isozyme expressed in cortical thymic epithelial cells was COX-1, while COX-2 predominated in the medulla. IFN-gamma up-regulated expression and activity of COX-2 in medullary cells, in which COX-2 was expressed constitutively. In contrast, IFN-gamma down-regulated COX-1 activity, but not expression, in cortical cells. Stromal cells support T cell development in the thymus, although the mediators of this effect are unknown. Selective inhibition of COX-2, but not COX-1, blocked the adhesion of CD4+CD8+ and CD4+CD8- thymocytes to medullary cell lines. No effect of the inhibitors was observed on the interactions of thymocytes with cortical epithelial lines. These data further support the differential regulation of COX-1 and COX-2 expression and function in thymic stromal cells. PGs produced by COX-2 in the medullary thymic stroma may regulate the development of thymocytes by modulating their interaction with stromal cells.