T-cell development in human thymoma.

Human thymoma is derived from thymic epithelial cells and often associated with a large number of cortical thymocytes. Since thymic epithelial cells play key roles in T-cell development in the normal thymus, we hypothesized that the neoplastic epithelial cells of thymoma may support T-cell differentiation. We attempted to reconstitute the T-cell development in vitro by using neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on a proportion of CD4-CD8- cells in thymoma. These CD34+CD4-CD8- cells also expressed both IL-7R alpha-chain and common gamma-chain. Purified CD4-CD8- cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4+CD8+ cells via CD4 single positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. The CD34+CD4-CD8- cells purified from a normal thymus also differentiated to CD4+CD8+ cells in an allogeneic co-culture with the neoplastic epithelial cells of thymoma. In addition, a pleural dissemination from thymoma contained a large amount of cortical thymocytes. These results suggest that the neoplastic epithelial cells retain the function of thymic epithelium and can support T-cell development in thymomas.     

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.     

Human thymic epithelium and T cell development: current issues and future directions

The human thymus develops early in fetal gestation with morphologic maturity reached by the beginning of the second trimester. TE3+ cortical thymic epithelium is most likely derived from endodermal third pharyngeal pouch, while A2B5/TE4+ medullary and subcapsular cortical thymic epithelium is likely derived from third pharyngeal cleft ectoderm. Fetal liver and yolk sac CD7+, CD4-, CD8-, surface(s) CD3- T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression of other T lineage surface molecules on developing thymocytes. CD7+, CD4-, CD8-, sCD3- thymocytes give rise to T cells of both the TCR alpha beta and TCR gamma delta lineages. Human thymic epithelial cells produce numerous cytokines including IL1, IL6, TGF alpha, leukemia inhibitory factor (LIF), M-CSF, G-CSF and GM-CSF- molecules that likely play important roles in multiple stages of thymocyte selection, activation and differentiation. Important areas for future research on human thymic epithelium include study of lymphoid and non-lineage differentiation potentials of CD7+, CD4-, CD8-, sCD3- T cell precursors in response to TE-cell produced cytokines, study of the triggering signals of cytokine release within the thymic microenvironment, and study of TCR-MHC mediated TE-thymocyte interactions.     

Developmentally regulated interactions of human thymocytes with different laminin isoforms

The gene family of heterotrimeric laminin molecules consists of at least 15 naturally occurring isoforms which are formed by five different alpha, three beta and three gamma subunits. The expression pattern of the individual laminin chains in the human thymus was comprehensively analysed in the present study. Whereas laminin isoforms containing the laminin alpha1 chain (e.g. LN-1) were not present in the human thymus, laminin isoforms containing the alpha2 chain (LN-2/4) or the alpha5 chain (LN-10/11) were expressed in the subcapsular epithelium and in thymic blood vessels. Expression of the laminin alpha4 chain seemed to be restricted to endothelial cells of the thymus, whereas the LN-5 isoform containing the alpha3 chain could be detected on medullary thymic epithelial cells and weakly in the subcapsular epithelium. As revealed by cell attachment assays, early CD4- CD8- thymocytes which are localized in the thymus beneath the subcapsular epithelium adhered strongly to LN-10/11, but not to LN-1, LN-2/4 or LN-5. Adhesion of these thymocytes to LN-10/11 was mediated by the integrin alpha6beta1. During further development, the cortically localized CD4+ CD8+ thymocytes have lost the capacity to adhere to laminin-10/11. Neither do these cells adhere to any other laminin isoform tested. However, the more differentiated single positive CD8+ thymocytes which were mainly found in the medulla were able to bind to LN-5 which is expressed by medullary epithelial cells. Interactions of CD8+ thymocytes with LN-5 were integrin alpha6beta4-dependent. These results show that interactions of developing human thymocytes with different laminin isoforms are spatially and developmentally regulated.     

Cyclosporin A and anti-Ia antibody cause a maturation defect of CD4+8- cells in organ-cultured fetal thymus

The effects of anti-Ia antibodies and cyclosporin A (CsA) on phenotypic differentiation of murine thymocytes were assessed in organ-cultured fetal thymuses. Both agents specifically abrogated the generation of CD4+8- thymocytes. Immunohistochemical studies revealed that Ia antigen on the organ-cultured thymic epithelial cells did not disappear with the addition of the agents, although anti-Ia antibody was proved to bind to Ia antigen during the culture. On the other hand, CsA neither changed the expression of Ia nor bound to it. As CsA is known to block the signalling cascade initiated by perturbation of T-cell receptor (TcR), it is suggested that both the Ia expression in the thymus and the signalling via receptors on thymocytes, the signals presumably generated by TcR binding to class II MHC molecules, might be necessary for phenotypic differentiation of class II MHC-restricted T cells (CD4+8- cells), but not for class I MHC-restricted T cells (CD4-8+).     

Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) provide co-stimulation in positive selection along with survival of selected thymocytes

T-cell differentiation in the thymus depends on positive selection of CD4+CD8+ double positive (DP) thymocytes by thymic major histocompatibility complex (MHC) molecules. Positive selection allows maturation of only those thymocytes that are capable of self-peptide-MHC recognition. Thymocytes that fail to bind self-peptide-MHC die by apoptosis. An important question in thymocyte differentiation is whether co-stimulation is required for positive selection and on which cells co-stimulatory molecules may be expressed in the thymus. The vascular cell adhesion molecule (VCAM-1) and the intercellular cell adhesion molecule (ICAM-1) are known to be potent co-stimulatory molecules in activation of peripheral T-cells by interacting with the integrins VLA-4 and LFA-1, respectively. We were prompted to investigate whether VCAM-1 and ICAM-1 may also act as co-stimulators during selection of thymocytes. By using recombinant proteins of murine VCAM-1 and ICAM-1 fused to the Fc region of human IgG1 (rVCAM-1, rICAM-1) we examined the capacity of VCAM-1 and ICAM-1 to act as co-stimulatory molecules in positive selection in vitro. Triggering the CD3/TCR complex together with co-stimulation applied by rVCAM-1 or rICAM-1 induced the generation of CD4+ single positive (SP) thymocytes from CD4+CD8+ DP thymocytes whereas either signal alone did not result in generation of CD4+ SP thymocytes. VCAM-1 and ICAM-1 act therefore as co-stimulatory molecules in thymocyte positive selection in vitro. The generation of CD4+ SP cells is accompanied by cell survival both when it was co-stimulated with rVCAM-1 and with rICAM-1. Importantly we show here that VCAM-1 expression in the murine thymus is restricted to cortical F4/80 positive hematopoietic antigen presenting cells (hAPC) present exclusively in the cortex whereas expression of ICAM-1 has been reported on the epithelium both in cortex and medulla. This suggests that not only the cortical epithelium may use the co-stimulatory molecule ICAM-1 to mediate positive selection, but also cortical hAPCs may contribute to positive selection of thymocytes by using the co-stimulator VCAM-1.     

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 of early activation antigen (CD69) during human thymic development.

The novel early activation antigen, EA1, has been shown to be induced by mitogens, antigens and the tumour promoter, phorbol myristate acetate (PMA), on human lymphocytes. This antigen has been designated to be CD69. EA1 has also been shown to be expressed on thymocytes without exogenous activation stimuli. In order to characterize further the expression of EA1 on thymocytes, the ontogeny of its expression was studied. EA1 appeared between 7 and 9.5 weeks of gestation, after colonization of the thymic rudiment with CD7+ T cell precursors, but before the onset of compartmentalization of the thymus into cortical and medullary zones. After cortico-medullary differentiation, the majority of medullary thymocytes expressed EA1 while only a fraction of the cortical thymocytes expressed this antigen. In the fetal and post-natal cortex, EA1 expression appeared to cluster in the subcapsular cortex. EA1+ cells were also scattered throughout the inner cortex. By two-colour fluorocytometric analysis of post-natal thymocytes, it was shown that EA1 was expressed on 30 to 65% of thymocytes. EA1 was expressed on CD4+ CD8+ as well as on the more immature CD4- CD8- thymocytes. In contrast to circulating T cells, thymocytes were much less responsive to PMA stimulation for the expression of EA1. Molecular characterization showed that EA1 on thymocytes had the same structure as that of activated peripheral T cells. In addition, thymic EA1 was constitutively phosphorylated. Thus, EA1 expression is acquired early during thymic development after colonization of the thymic rudiment by CD7+ T cell precursors. However, the specific role that EA1 may play in the activation and function of developing thymocytes remains to be determined.     

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. ROS-associated 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.     

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.     

IL-7 is a growth and maintenance factor for mature and immature thymocyte subsets

The specific signals inducing the growth and maturation of thymocytes remain undefined. We show here that recombinant IL-7 induces growth of fetal and adult mouse CD4-8- thymocytes. IL-7 also induces a lower but significant response in CD4+8- and CD4-8+ thymocytes. Day 14 fetal thymic lobes cultured in IL-7 for 6 days show a 2-fold increase in cell number when compared to control cultures. The thymocyte subsets that proliferate in response to IL-7 can be maintained in culture for extended periods of time. CD4-8- thymocytes maintained in IL-7 did not change their phenotype with respect to CD4 and CD8 expression. In addition, we show that the combination of IL-7 plus IL-6 provides a potent growth stimulus for CD4+8- and CD4-8+ thymocytes. A cloned thymic epithelial cell, that can be induced to express MHC class II molecules, transcribes both IL-7 and IL-6 mRNA. A cloned thymic macrophage cell line produces IL-6 but no detectable IL-7 mRNA. The pattern of biological activities present in the supernatants of these cell lines is also presented. These observations suggest that the thymic epithelial and macrophage cell types may be an in vivo source of signals which mediate thymocyte development.     

A Novel Adhesion Molecule in the Murine Thymic Microenvironment: Functional and Biochemical Analysis

The rat monoclonal antibody (mAb) 4F1, raised against mouse thymic stromal cells, recognizes cortical epithelium in tissue sections of mouse thymus; however, in flow cytometry, activated leucocytes (T cells, B cells, and macrophages) and transformed thymocytes are also positive for the 4F1-antigen (4F1-Ag). Western blotting, under both reducing and nonreducing conditions, demonstrates that the molecule to which 4F1 binds is expressed in four forms, 29, 32, 40, and 43 kD, all of which carry N-linked carbohydrate; and that the structure is identical on epithelium and lymphocytes. The 4F1-Ag on cortical epithelium is partially sensitive to PI-PLC treatment, whereas on transformed epithelial and lymphoid cell lines, it was resistant to this enzyme. The molecule, therefore, may exist in both transmembrane and phosphoinositol-linked forms. In functional blocking experiments, mAb 4F1 gave inhibition of both T-cell proliferation in MLR and of cytotoxic T-cell killing of alloantigenic targets; it also blocked adhesion of transformed thymocytes to thymic epithelial cells in vitro. These molecular and functional characteristics suggest that the 4F1-Ag is a novel adhesion molecule that may be involved both in intrathymic T lymphocyte differentiation and in peripheral T-cell function.     

Novel origin of lpr and gld cells and possible implications in autoimmunity

The lpr and gld mutations are prime examples of single-gene defects associated with expansion of a unique double-negative (CD4-8-), T-cell receptor alpha:beta + cell population and heightened polyclonal and autoimmune responses. The exact origin of these autoimmunity-inducing/enhancing T cells remains controversial. Here, we review the characteristics of the lpr and gld mutations, and speculate on the possible relationship of these cells to normal thymic differentiation pathways. We argue that mounting evidence now supports the existence of a CD4/CD8-loss pathway of late thymic differentiation, responsible for the origin of both normal and lpr/gld double-negative alpha:beta + cells. We further speculate that downregulation of CD4 and CD8 accessory molecules on thymocytes with moderately autoreactive T-cell receptors is involved in selecting cells, including lpr/gld precursors for this pathway. Escape of a large number of such autoreactive cells from thymic elimination might be an important contributory factor to the pathogenesis of autoimmunity.     

Promiscuous gene expression in the thymus: the root of central tolerance

The thymus is a complex organ with an epithelium formed by two main cell types, the cortical thymic epithelial (cTECs) and medullary thymic epithelial cells (mTECs), referred to as stroma. Immature thymocytes arising from the bone marrow, macrophages and dendritic cells also populate the thymus. Thymocytes evolve to mature T cells featuring cell differentiation antigens (CDs), which characterize the phenotypically distinct stages, defined as double-negative (DN), double positive (DP) and single positive (SP), based on expression of the coreceptors CD4 and CD8. The thymus is therefore implicated in T cell differentiation and during development into T cells thymocytes are in close association with the stroma. Recent evidence showed that mTECs express a diverse set of genes coding for parenchymal organ specific proteins. This phenomenon has been termed promiscuous gene expression (PGE) and has led to the reconsideration of the role of the thymus in central T cell tolerance to self-antigens, which prevents autoimmunity. The evidence of PGE is causing a reanalysis in the scope of central tolerance understanding. We summarize the evidence of PGE in the thymus, focusing particularly the use of cDNA microarray technology for the broad characterization of gene expression and demarcation of PGE emergence during thymus ontogeny.     

The role of adenosine receptor engagement in murine fetal thymocyte development.

The role of adenosine receptor engagement in murine T-cell development was evaluated by culturing day 15-16 fetal thymic lobes in the presence of various concentrations of the adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine (NECA) or the adenosine receptor antagonist 8-phenyl-theophylline (8-PT) using the fetal thymic organ culture (FTOC) system. Before and 8 days after culture, thymocytes were isolated, counted, and analyzed for the expression of CD4 and CD8 T-cell differentiation molecules. Analysis of fresh thymocytes prior to culture showed that the majority of cells were of the CD4 single-positive or CD4+ CD8+ immature phenotype. Eight days after culture with media alone, 44% of cells were CD4+ and 23% were CD8+, and the number of viable thymocytes had increased from 1.7 x 10(5) to 2.2 x 10(5) cells per thymic lobe. A dose-dependent inhibition of maturation was observed in cultures with 8-PT, with greater than 85% inhibition at 50 microM. The double-positive thymocyte subset was most severely depleted. The number of cells obtained from cultures with NECA was also reduced, with about 65% inhibition at 50 microM, especially the CD8+ subset that was most severely affected. These results suggest that adenosine receptor engagement is required for normal T-cell differentiation and that adenosine receptor agonists and antagonists have distinct effects on thymocyte differentiation. An understanding of the cell-type-specific and developmental expression of adenosine receptors will help elucidate the mechanisms by which adenosine receptor engagement influences T-cell development.     

T cell receptor targeting to thymic cortical epithelial cells in vivo induces survival,activation and differentiation of immature thymocytes

We report that targeting of T cell receptors (TcR) to non-major histocompatibility complex (MHC) molecules on thymic cortical epithelial cells by hybrid antibodies in vivo and in fetal thymic organ cultures results in phenotypic and functional differentiation of thymocytes. A single pulse with hybrid antibodies rescues immature, CD4/8 double-positive thymocytes from their programmed death in vivo, induces expression of the early activation antigen CD69 followed by TcR up-regulation, concomitant down-regulation of CD8 or CD4 and their conversion to functional mature T cells by day 3. This temporal sequence of maturation only affects small thymocytes without co-induction of blastogenesis. TcR targeting to MHC class II-positive epithelial cells predominantly induces CD4-positive T cells. This generation of CD4 single-positive T cells occurs also in MHC class II-deficient mice and thus is independent of CD4-MHC class II interactions. Moreover, in the presence of a specific deleting antigen (Mls 1^a),TcR targeting results in transient activation of immature thymocytes, however, not in subsequent TcR (Vβ6) up-regulation and development of single-positive T cells. Our findings imply that TcR cross-linking to cortical epithelial cells is sufficient to confer a differentiation signal to immature thymocytes. Futhermore, this approach distinguishes two independent TcR-mediated intrathymic events: activation and subsequent deletion of the same thymocyte subset.     

Thymic Medulla Epithelial Cells Acquire Specific Markers by Post-Mitotic Maturation

The development of thymocyte subsets and of the thymic epithelium in SCID and RAG-2^-/– mice was monitored after normal bone-marrow-cell transfer. The kinetics of thymic reconstitution and their relationships with cell proliferation were investigated by using bromodeoxyuridine to detect DNA-synthesizing cells among lymphoid cells by 3-color flow cytometry, and in epithelial compartments by staining frozen sections. Thymocytes started to express CD8 and CD4 10 days after transfer, simultaneously with extensive proliferation. The first mature CD4⁺ single-positive cells were generated, from resting CD4⁺CD8⁺ cells after day 15. During this day 10–15 period, many epithelial cells positive for cortexspecific or panepithelial markers were labeled with BrdUrd after pulse-injection. Organized medullary epithelium also developed after day,15, that is, synchronously with the appearance of mature thymocytes, but medullary cells were never found BrdUrd⁺. These results suggest that, in these models, the reconstitution of the thymic epithelial network proceeds through expansion of preexisting cortical or undifferentiated cells and by later maturation (acquisition of specific markers) of medullary cells. This last process is dependent of the presence of mature thymocytes.     

Developmental changes predispose the fetal thymus to positive selection of CD4+CD8- T cells.

Selection of a competent T-cell repertoire is dependent on complex interactions between immature thymocytes and components of the thymic stroma. These events may be preserved in vitro by excising developing thymus rudiments and maintaining them under carefully controlled conditions in fetal thymus organ cultures (FTOC). Using this approach, we have shown that the ability of C57B1/6 thymi to sustain positive selection of mature CD4+CD8- cells is profoundly influenced by the day of gestation on which they are excised: while thymocytes from day 14 rudiments fail to progress beyond the CD4+CD8+ stage of the developmental pathway, day 15 and day 16 thymi support the differentiation of CD4+CD8- thymocytes. Importantly, day 16 thymocytes transferred to day 14 deoxyguanosine-treated rudiments are likewise arrested at the CD4+CD8+ stage, suggesting that the thymic microenvironment of day 14 rudiments, rather than the state of differentiation of the thymocytes they contain, is responsible for the block in positive selection. Our studies of the stromal elements of day 14 rudiments have, however, revealed no obvious deficiencies in the cell types represented, or their expression of class II major histocompatibility complex (MHC) determinants. Furthermore, we have been unable to circumvent the blockage in positive selection by the addition of certain cytokines expressed late during gestation. These results suggest that subtle changes occurring at day 15 of ontogeny render the thymic microenvironment capable of positive selection.