Thymocytes and RelB-dependent medullary epithelial cells provide growth-promoting and organization signals,respectively, to thymic medullary stromal cells

The thymic medulla is composed of distinct epithelial cell subsets, defined in this report by the reactivity of two novel antibodies, 95 and 29, raised against mouse thymic epithelial cell lines. These antibodies were used to probe the development of medulla in wild-type or mutant thymuses. In CD3σ-deficient mice where thymocyte maturation is arrested at the CD4^? CD8^? stage, few scattered 95⁺ and 29⁺ epithelial cells are found. When few mature thymocytes develop as in CD3-ζ/η mice, expansion and organization of 95⁺ but not 29⁺ cells, becomes detectable. In RelB-deficient mice, T cell maturation proceeds normally but negative selection is inefficient due to the lack of thymic medulla and dendritic cells. Strikingly, 29⁺ epithelial cells are absent and 95⁺ medullary epithelial cells are scattered throughout the thymus, intermingling with CDR1⁺ cortical epithelium. In chimeric mice lacking only dendritic cells, the corticomedullary junction persists and both 95⁺ and 29⁺ epithelial cells are localized in the medulla. These results suggest that two types of signals are required for development of thymic medulla. A growth signal depends upon the presence of maturing thymocytes, but organization of the thymic medulla requires the presence of activated 29⁺ medullary epithelial cells.     

Discrimination between maintenance- and differentiation-inducing signals during initial and intermediate stages of positive selection

As well as signaling through the αβ T cell receptor complex, positive selection of immature CD4⁺ 8⁺ thymocytes involves additional ill-defined accessory interactions provided by thymic epithelial cells. Here, we have isolated CD4⁺ 8⁺ thymocytes at a pre-positive selection stage of development (TCR^? CD69^? 4⁺ 8⁺ cells), or after initiation of positive selection (CD69⁺ 4⁺ 8⁺ cells), from mice where the normal lifespan of thymocytes is extended by the presence of a bcl-2 transgene, to allow us to discriminate between requirements for maintenance and differentiation signals during positive selection. We find that MHC class II⁺ thymic epithelial cells drive positive selection of TCR^? CD69^? 4⁺ 8⁺ bcl-2 tg thymocytes to the CD4⁺ and CD8⁺ stage, while no such mature subsets are observed when thymocytes are cultured alone or with major histocompatibility complex (MHC) class II⁺ salivary epithelial cells. However, CD4⁺ 8⁺ cells remain in such cultures in considerable numbers, and retain the potential for positive selection if re-cultured with thymic epithelium, suggesting that thymic epithelial cells provide specific differentiation-inducing signals for positive selection. In contrast, intermediate CD69⁺ 4⁺ 8⁺ thymocytes show some capacity for phenotypic conversion in the absence of thymic stromal cells although strikingly the single-positive CD4⁺ and CD8⁺ cells generated are not functionally competent. Finally, we show that prior culture of thymic epithelial cells under monolayer conditions abrogates their ability to support the initiation of positive selection, suggesting that the epithelial cell molecules necessary for the provision of differentiation signals during positive selection are down-regulated under such conditions.     

Downregulated Expression of Ly-6-ThB on Developing T Cells Marks CD4+CD8+ Subset Undergoing Selection in the Thymus

Interaction of TCRs on CD4⁺CD8⁺ immature T cell with MHC-peptide complexes on stromal cells is required for positive and negative selection in the thymus. Identification and characterization of a subpopulation of CD4⁺CD8⁺ thymocytes undergoing selection in the thymus will aid in understanding the mechanisms underlying lineage commitment and thymic selection. Herein, we describe the expression of Ly-6 ThB on developing thymocytes. The majority of CD4⁺CD8⁺ thymocytes express Ly-6 ThB at high levels. Its expression is downregulated in a subset of CD4⁺CD8⁺ thymocytes as well as in mature CD4⁺CD8^- and CD4^-CD8⁺ T cells. More importantly, interaction of TCR/coreceptor with the self-MHC-peptide contributes to the downregulation of ThB expression on developing thymocytes. These findings indicate that downregulation of ThB on CD4⁺CD8⁺ thymocytes identifies a unique subset (CD4+CD8+ThB^neg–low) of thymocytes that has received the initial signals for thymic selection but have not yet downregulated the CD4 and CD8 cell surface expression. In addition, these results also indicate that a high frequency (Ÿ20–40%) of CD4⁺CD8⁺ immature thymocytes receive these initial signals during thymic selection.     

Different Subpopulations of Developing Thymocytes are Associated with Adherent (Macrophage) or Nonadherent (Dendritic) Thymic Rosettes

Thymic rosettes (ROS), structures consisting of thymic lymphoid cells attached to a central stromal cell, were isolated from mouse thymus by collagenase digestion and unit-gravity elutriation. The ROS were then separated into those where the stromal cells were either macrophage-like (M-ROS) or dendritic cell-like (D-ROS), on the basis of the differences in adherence properties or in the level of MAC-1 surface antigen. The ROS were then dissociated and the thymocyte content analyzed by immunofluorescent staining and flow cytometry. M-ROS and D-ROS differed in thymocyte composition, although the major component of both was the CD4⁺CD8⁺ cortical thymocyte. D-ROS were enriched in thymocytes expressing high levels of surface T-cell antigen receptor (TcR) and the associated CD3 complex, and these included both CD4⁺CD8⁺CD3⁺⁺ and CD4^-CD8⁺CD3⁺⁺ mature thymocytes. M-ROS were enriched in CD4^-CD8^- thymocytes and had a reduced content of thymocytes expressing high TcR-CD3 levels; they nevertheless contained some mature thymocytes, but only of the CD4⁺CD8^-CD3⁺⁺ category. Several lines of evidence indicated that the mature thymocytes in ROS were cells recently formed in the cortex, and were not from the medullary pool. ROSassociated mature thymocytes expressed lower levels of H-2K than free, mature thymocytes. The CD4⁺CD8⁺CD3⁺⁺ subpopulation, believed to be a developmental intermediate between cortical thymocytes and mature T cells, was present in both ROS populations. Further, late intermediates leading to both mature T-cell categories were evident in D-ROS, but only those leading to CD4⁺CD8^-CD3⁺⁺ T cells were evident in M-ROS. The results are compatible with a role for ROS in TcR-specificity selection and in the final maturation steps in the thymic cortex.     

Using thymus anatomy to dissect T cell repertoire selection

The thymic development of CD4⁺T cells incorporates the opposing processes of positive and negative selection to produce mature lymphocytes which respond to foreign peptides in the context of self-major histocompatibility complex class II molecules. Here, we present a model in which these events occur in two temporally and anatomically distinct steps. We propose that an initial positive selection step is mediated exclusively by thymic cortical epithelium. Sub- sequently, all those thymocytes which have been positively selected will interact with medullary epithelium and bone marrow-derived cells. Those thymocytes reacting with excess affinity or avidity to these antigen presenting cells will be negatively selected. Although acknowledging the importance of differential signalling to the developing thymocyte, we will emphasize the centrality of the phenotype of the tissues which comprise the thymus.     

The murine homolog of human Ep-CAM,a homotypic adhesion molecule,is expressed by thymocytes and thymic epithelial cells

In this report, we demonstrate that gp40, a molecule previously shown to be expressed by thymic epithelial cell lines in vitro and by thymic epithelial cells in vivo, is the murine homolog of human Ep-CAM, a calcium-independent homotypic adhesion molecule. gp40 is also expressed at low levels by thymocytes and peripheral T cells. In the adult thymus, gp40 expression was inversely related to the state of thymocyte maturation, with the highest levels associated with CD4^? CD8^? and CD4⁺CD8⁺ thymocyte populations. Ultrastructural immunohistochemistry revealed gp40 localization to areas of thymocyte/epithelial contact and demonstrated that gp40 is also expressed by thymic dendritic cells. During fetal development, thymocytes at days 14–16 of gestation expressed high levels of gp40. At later stages, the observed decline in the frequency of gp40⁺ cells and levels of expression correlated with the emergence of αβ⁺ thymocytes by day 18 of gestation. In short-term cultures, stimulation of unfractionated adult thymocytes with concanavalin A increased gp40 expression, particularly among CD3^hi and CD3^int thymocyte populations. This demonstration that Ep-CAM, initially considered to be expressed primarily by epithelial cells, is also expressed by thymocytes, T cells and antigen-presenting cells, raises the possibility that Ep-CAM may contribute to adhesive interactions between thymocytes and epithelial cells or dendritic cells, either in the context of thymocyte development or peripheral T cell trafficking and function.     

Human intrathymic T cell differentiation

The human thymus develops early on in fetal gestation with morphologic maturity reached by the beginning of the second trimester. Endodermal epithelial tissue from the third pharyngeal pouch gives rise to TE3+ cortical thymic epithelium while ectodermal epithelial tissue from the third pharyngeal cleft invaginates and splits during development to give rise to A2B5/TE4+ medullary and subcapsular cortical thymic epithelium. Fetal liver CD7+ T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression on thymocytes of other T lineage surface molecules. Human thymic epithelial cells grown in vitro bind to mature and immature thymocytes via CD2 and CD11a/CD18 (LFA-1) molecules on thymocytes and by CD58 (LFA-3) and CD54 (ICAM-1) molecules on thymic epithelial cells. Thymic epithelial cells produce numerous cytokines including IL1, IL6, G-CSF, M-CSF, and GM-CSF--molecules that likely are important in various stages of thymocyte activation and differentiation. Thymocytes can be activated via several cell surface molecules including CD2, CD3/TCR, and CD28 molecules. Finally, CD7+ CD4-CD8- CD3- thymocytes give rise to T cells of both the TCRab+ and TCR gd+ lineages.     

Absence of thymus crosstalk in the fetus does not preclude hematopoietic induction of a functional thymus in the adult

Cortical and medullary thymic epithelial cells provide essential signals for a normal programme of T‐cell development. Current models of thymus development suggest that thymocyte‐derived signals play an important role in establishing thymic microenvironments, a process termed thymus crosstalk. Studies on CD3εtg26 mice lacking intrathymic T‐cell progenitors provided evidence that normal development of the thymic cortex depends upon thymocyte‐derived signals. Importantly, the reported failure to effectively reconstitute adult CD3εtg26 mice raised the possibility that such crosstalk must occur within a developmental window, and that closure of this window during the postnatal period renders thymic epithelium refractory to crosstalk signals and unable to effectively impose T‐cell selection. We have re‐investigated the timing of provision of crosstalk in relation to development of functional thymic microenvironments. We show that transfer of either fetal precursors or adult T‐committed precursors into adult CD3εtg26 mice initiates key parameters of successful thymic reconstitution including thymocyte development and emigration, restoration of cortical and medullary epithelial architecture, and establishment of thymic tolerance mechanisms including maturation of Foxp3⁺ Treg and autoimmune regulator‐expressing medullary epithelium. Collectively, our data argue against a temporal window of thymocyte crosstalk, and instead demonstrates continued receptiveness of thymic epithelium for the formation of functionally competent thymic microenvironments.     

Thymic Stromal-Cell Abnormalities and Dysregulated T-Cell Development in IL-2-Deficient Mice

The role that interleukin-2 (IL-2) plays in T-cell development is not known. To address this issue, we have investigated the nature of the abnormal thymic development and autoimmune disorders that occurs in IL-2-deficient (IL-2^–/–) mice. After 4 to 5 weeks of birth, IL-2^–/– mice progressively develop a thymic disorder resulting in the disruption of thymocyte maturation. This disorder is characterized by a dramatic reduction in cellularity, the selective loss of immature CD4^-8^- (double negative; DN) and CD4⁺8⁺ (double positive; DP) thymocytes and defects in the thymic stromal-cell compartment. Immunohistochemical staining of sections of thymuses from specific pathogen-free and germ-free IL-2^–/– mice of various ages showed a progressive ,loss of cortical epithelial cells, MHC class II-expressing cells, monocytes, and macrophages. Reduced numbers of macrophages were apparent as early as week after birth. Since IL-2^–/– thymocyte progenitor populations could mature normally on transfer into a normal thymus, the thymic defect in IL-2^–/– mice appears to be due to abnormalities among thymic stromal cells. These results underscore the role of IL-2 in maintaining functional microenvironments that are necessary to support thymocyte growth, development, and selection.     

Expression of CTLA-4 (CD152) on human medullary CD4+ thymocytes

CTLA-4 (CD152) is a T cell surface receptor with sequence homology to the co-stimulatory molecule CD28. The molecule, which is essential for the inhibitory regulation of the immune response, becomes transiently expressed on mature T cells after stimulation in vitro. In situ, CTLA-4⁺ T cells are enriched in the light zones of the germinal centers in human peripheral lymphoid organs. In this study we have studied expression of CTLA-4 in human thymus in situ. CTLA-4 was expressed on about one third of CD4⁺/CD8^–/CD1^– medullary thymocytes. CTLA-4 was acquired by a subset of immature (CD1⁺) thymocytes and lost from the mature (CD1^–) subpopulation within 48 h of cell culture, suggesting that the expression on medullary thymocytes is transient. The demonstration of CTLA-4 on a substantial subpopulation of mature CD4⁺ thymocytes adds a new dimension to the understanding of this important molecule. When contemplating application of anti-CTLA-4 for therapy its potential influence on T cell maturation has to be taken into account. Received: 5 January 1998     

BST2/Tetherin is constitutively expressed on human thymocytes with the phenotype and function of Treg cells

In contrast to peripheral plasmacytoid DCs (pDCs), thymic pDCs constitutively express low levels of IFN‐α. This leads to induction of interferon secondary genes (ISGs) in medullary thymocytes, raising the question whether IFN‐α may play a role in T‐cell development. When characterizing further differences between peripheral and thymic pDCs, we found that thymic pDCs have a phenotype consistent with an “activated signature” including expression of TNF‐α and bone marrow stromal cell antigen 2 (BST2), but no expression of ILT7. Given that BST2 is induced by IFN‐α, and IFN‐α secretion is controlled by interaction between ILT7 and BST2, this regulatory pathway is apparently lost in thymic pDCs. Further, we also show that BST2 is constitutively expressed on a subset of medullary thymocytes at the mRNA and protein level reflecting a history of IFN‐α transduced signals. The majority of BST2⁺ thymocytes express CCR5 rendering them prevalent targets for R5‐tropic HIV infection. Moreover, BST2⁺ thymocytes express Foxp3 and CD25, consistent with the phenotype of natural Treg cells, and exert suppressive activity as they impair the proliferation of autologous CD3⁺ thymocytes. Collectively, our results suggest that low levels of IFN‐α secreted by thymic pDCs play an important role in the development of natural Treg cells.     

Thymic microenvironment reconstitution after postnatal human thymus transplantation

A functional thymus develops after cultured thymus tissue is transplanted into subjects with complete DiGeorge anomaly. To gain insight into how the process occurs, 7 post-transplantation thymus biopsy tissues were evaluated. In 5 of 7 biopsies, the thymus appeared to be predominantly cortex with thymocytes expressing cortical markers. Unexpectedly, the epithelium expressed both cortical [cortical dendritic reticulum antigen 2 (CDR2)] and medullary [cytokeratin (CK) 14] markers. Early medullary development was suggested by epithelial cell adhesion molecule (EpCAM) reactivity in small areas of biopsies. Two other biopsies had distinct mature cortex and medulla with normal restriction of CK14 to the medulla and subcapsular cortex, and of CDR2 to cortex. These data are consistent with a model in which thymic epithelium contains CK14+ "progenitor epithelial cells". After transplantation these cells proliferate as CK14+CDR2+ thymic epithelial cells that are associated with cortical thymocytes. Later these cells differentiate into distinct cortical and medullary epithelia.     

Thymic lymphoma cells as a model for complex-formation between thymocytes and thymic medullary epithelial cells

The formation of complexes between thymocytes and thymic stromal elements is known to be involved in T cell differentiation. We have previously described one type of lympho-stromal interaction involving CD4+ CD8+ thymocytes and a medullary epithelial cell line (E-5). In this study we report the potential for complex formation of two different thymic lymphoma cell lines (Ti-6 and RDM-4). Ti-6 cells were shown to adhere to the E-5 cells, while RDM-4 cells were totally incompetent. Phenotypic characterization of these cell lines suggests that immature thymocytes are not capable of forming complexes with medullary epithelium and that a certain level of differentiation is required to do so. Comparison of their phenotypes showed that the possibility of some classical T cell surface markers being the receptor for the E-5 ligand can be dismissed.     

The influence of FK-506 on the thymus: an immunophenotypic and structural analysis.

The influence of the powerful new immunosuppressant FK-506 on the thymus was investigated in Sprague-Dawley rats that were immunized with sheep erythrocytes and treated with FK-506 (1 mg/kg/day i.m.) for 7 days. Suppression of humoral immunity in drug-treated animals was accompanied by reductions in circulating lymphocytes bearing activation markers (interleukin-2 receptor beta-chain and OX40, activated CD4+ cells) and by striking thymic medullary atrophy. There were, however, no significant differences in thymic weights or in thymocyte numbers between experimental and control groups during the period of FK-506 administration. Reduction of the medullary compartment was visualized immunohistochemically, by decreases in major histocompatibility complex (MHC) class I- and MHC class II-positive cells and in CD37+ (mature medullary) thymocytes. Flow cytometric analysis of thymocytes showed that FK-506 induced increases in bright, Thy-1.1+ cells and in numbers of CD4+ and CD8+ thymocytes, whilst CD37+ cells were less numerous than in controls. Percentages of MHC class I- and MHC class II-positive cells varied little throughout the course of FK-506 administration. Evidence of selective damage to medullary epithelial cells, attributable to FK-506, was found at both the light and electron microscopic levels, whilst thymic macrophages in drug-treated rats displayed features of enhanced phagocytic activity, including ingestion of damaged epithelial cells. These FK-506-induced abnormalities were reversed within 14 days of drug withdrawal. These findings suggest that, like cyclosporin A, FK-506 reversibly disrupts the thymic microenvironment and may interfere with the function/maturation of T lymphocytes.     

Long-term thymic reconstitution by peripheral CD4 and CD8 single-positive lymphocytes

Significant immigration of peripheral T cells into SCID thymus was observed following reconstitution with normal Peyer's patch, mesenteric lymph node or peripheral lymph node cells. Immunohistologic and flow cytometric analyses reveal that T cells from these tissues are found in the thymus for as long as 177 days and can account for up to 67% of intrathymic cells. The returning cells express the CD3/Tcell receptor α/β complex, indicative of mature cells, and are equally divided among helper (CD4⁺ CD8 ) and cytotoxic (CD4^?/CD8⁺) phenotypes. The immigration of peripheral T cells is not accompanied by the appearance of immature, double-positive (CD4⁺CD8⁺) thymocytes as seen in similar reconstitutions using bone marrow. Taken together, these results suggest that peripheral T cells from a variety of lymphoid organs may regularly re-enter the thymus and, thus, possibly play a role in normal thymic development.     

Developmentally regulated expression of P-glycoprotein (multidrug resistance) activity in mouse thymocytes

P-glycoprotein (P-gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells. The activity of P-gly in mature peripheral lymphocytes is lineage specific, with CD8⁺ T cells and natural killer (NK) cells expressing high levels as compared to CD4⁺ T cells and B cells. We have now investigated P-gly activity in immature and mature subsets of mouse thymocytes. Our data indicate that P-gly activity is undetectable in immature CD4^?8^? and CD4⁺8⁺ thymocyte subsets. Among mature thymocytes, P-gly activity is absent in the CD4⁺ subset but present in the more mature (HSA^low) fraction of CD8⁺ cells. Furthermore, while thymic CD4^?8^? T cell receptor (TCR) γδ cells have little P-gly activity, a minor subset of CD4^?8^? or CD4⁺ TCR αβ⁺ thymocytes bearing the NK1.1 surface marker expresses high levels of P-gly activity. Collectively, our results indicate that P-gly activity arises late during thymus development and is expressed in a lineage-specific fashion.     

Linomide Enhances Apoptosis in CD4+CD8+ Thymocytes

Linomide, a quinoline-3-carboxamide, has a pleiotropic immune modulating capacity and inhibits development as well as progression of disease in animal models of autoimmunity. Linomide treatment of mice resulted in a dramatic, dose-dependent decrease of the thymic cell number shortly after the start of administration. Flow cytometric analysis revealed that the major thymocyte subset, the early immature type CD4⁺CD8⁺ thymocytes, were reduced in number by 75%, mature CD4⁺CD8^? or CD4^?CD8⁺ thymocytes were less sensitive to treatment. The polyclonal T cell activator Con A (Concanavalin A) was used together with IL-2 to evaluate the potential proliferative responsiveness of ex vivo thymocytes. Thymocytes from mice treated with Linomide exhibited a more vigorous proliferation than control cultures. An effect shown to not only be due to the enrichment of mature thymocytes in the cultures from Linomide treated animals, but also when purified, mature thymocytes (CD4⁺CD8^? and CD4^?CD8⁺) were cultured with Con A and IL-2, these cells responded with a significantly enhanced proliferation. In vivo Linomide treatment did not result in increased plasma concentrations of corticosterone and treatment of adrenalectomized mice resulted in a reduction of thymocytes which was comparable to the effect in intact mice, indicating that glucocorticoids (GC) are not major mediators of Linomide-induced thymocyte deletion. In addition to this, and supporting a glucocorticoid independent mode of action, Linomide treatment of thymocytes in vitro resulted in a significant increase in the number of apoptotic cells, specifically in the CD4⁺CD8⁺ subset, implicating apopotosis as one component in the course of thymocyte reduction. In addition to this, in vivo treatment with Linomide resulted in an identical pattern to that seen in vitro in that there was significantly increased apoptosis only in the CD4⁺CD8⁺. These data indicate that Linomide modifies thymocyte development using a glucocorticoid independent pathway and results in the increased apoptosis of the CD4⁺CD8⁺ subset.     

Bone morphogenetic protein-2/4 signalling pathway components are expressed in the human thymus and inhibit early T-cell development

T-cell differentiation is driven by a complex network of signals mainly derived from the thymic epithelium. In this study we demonstrate in the human thymus that cortical epithelial cells produce bone morphogenetic protein 2 (BMP2) and BMP4 and that both thymocytes and thymic epithelium express all the molecular machinery required for a response to these proteins. BMP receptors, BMPRIA and BMPRII, are mainly expressed by cortical thymocytes while BMPRIB is expressed in the majority of the human thymocytes. Some thymic epithelial cells from cortical and medullary areas express BMP receptors, being also cell targets for in vivo BMP2/4 signalling. The treatment with BMP4 of chimeric human-mouse fetal thymic organ cultures seeded with CD34+ human thymic progenitors results in reduced cell recovery and inhibition of the differentiation of human thymocytes from CD4- CD8- to CD4+ CD8+ cell stages. These results support a role for BMP2/4 signalling in human T-cell differentiation.     

Expression of the HPV16E7 oncoprotein by thymic epithelium is accompanied by disrupted T cell maturation and a failure of the thymus to involute with age

Transgenic mice expressing the E7 protein of HPV16 from the keratin 14 promoter demonstrate increasing thymic hypertrophy with age. This hypertrophy is associated with increased absolute numbers of all thymocyte types, and with increased cortical and medullary cellularity. In the thymic medulla, increased compartmentalization of the major thymic stromal cell types and expansion of thymic epithelial cell population is observed. Neither an increased rate of immature thymocyte division nor a decreased rate of immature thymocyte death was able to account for the observed hypertrophy. Thymocytes with reduced levels of expression of CD4 and/or CD8 were more abundant in transgenic (tg) mice and became increasingly more so with age. These thymic SP and DP populations with reduced levels of CD4 and/or CD8 markers had a lower rate of apoptosis in the tg than in the non-tg mice. The rate of export of mature thymocytes to peripheral lymphoid organs was less in tg animals relative to the pool of available mature cells, particularly for the increasingly abundant CD4lo population. We therefore suggest that mature thymocytes that would normally die in the thymus gradually accumulated in E7 transgenic animals, perhaps as a consequence of exposure to a hypertrophied E7-expressing thymic epithelium or to factors secreted by this expanded thymic stromal cell population. The K14E7 transgenic mouse thus provides a unique model to study effects of the thymic epithelial cell compartment on thymus development and involution.     

Recent thymic emigrants are distinct from most medullary thymocytes

In the mouse thymus, newly formed single positive (SP) cells spend an average of 14 days in the thymic medulla. During this time, phenotypic and functional maturation occurs with down-regulation of CD69 and heat stable antigen (HSA), and up-regulation of Qa-2. Very little is known about the final steps that allow or direct these T cells to emigrate and join the recirculating peripheral T cell pool. Currently available data suggest that not all recent thymic emigrants (RTE) complete this maturational sequence in the medulla and that emigration may occur at any time during the medullary maturation stage. In this study, we have compared adhesion and activation marker expression on SP thymocytes, RTE and peripheral T cells to determine more precisely which SP medullary thymocytes are exported. Although RTE were heterogeneous for HSA and Qa-2 expression, they were quite uniform with regard to the expression of other molecules. In contrast to medullary SP thymocytes, most RTE were L-selectin^high and CD69^?. In addition, CD4⁺ CD8^? and CD4^? CD8⁺ RTE were phenotypically distinct from each other in that the former were β₇ integrin^?/low, CD45RB^intermediate and CD45RC^?, while the latter were β₇ integrin^high, CD45RB^high and CD45RC^low. These phenotypes were comparable to only a minor (as little as 6 %) subpopulation of medullary SP thymocytes. Overall, the data indicate that export of cells from the medullary pool of SP thymocytes is not random, but that a series of maturational events within the SP stage are necessary before export can occur.