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.     

Function of CD44(Pgp-1) homing receptor in human T cell precursors

T cell precursors migrate from extrathymic hematopoietic tissues and differentiate after encountering the thymic microenvironment. We asked whether human T cell precursors express the CD44(Pgp-1/gp90HR) class of homing receptors that have been implicated in the traffic of hematopoietic cells, such as lymphocyte entry to peripheral lymphoid organs. Flow cytometry and immunoprecipitation studies demonstrate that CD7+34+, CD1-2-3-4-8-14-16-20- cells in bone marrow and thymus, which have been shown to exhibit features of T cell precursors, bear CD44. Immunohistological studies show that clusters of thymocytes in the subcapsular and the inner cortex and most medullary thymocytes are clearly CD44+, whereas the expression of CD44 is selectively downregulated in CD3- and CD3low functionally incompetent cortical thymocytes. The expression of CD44 is not restricted to T cell precursors but also occurs in thymic stroma, which bear a different molecular species of CD44. CD44-specific antibodies exert stimulatory effects on T cell precursors, a process that is dependent on stromal cells. We postulate that CD44 might be an adhesion molecule for precursor homing to thymus and that it participates in cell-to-cell interactions within the thymic environment.     

Demonstration of phenotypic abnormalities of thymic epithelium in thymoma including two cases with abundant Langerhans cells.

A panel of monoclonal antibodies that phenotypically define stages of normal human thymic epithelial (TE) cell maturation was used to compare thymic epithelium of nine thymomas with hyperplastic thymic epithelium in myasthenia gravis (MG) and thymic epithelium of normal thymuses. It has been shown previously that normal thymic epithelial cells express antigens of early TE cell maturation (A2B5, TE-4) throughout thymic ontogeny and acquire antigens 12/1-2, TE8, and TE-15 at 14 to 16 weeks of fetal gestation. Hyperplastic MG thymic epithelial cells expressed TE antigens in phenotypic patterns similar to that seen in normal postnatal thymus, ie, TE in subcapsular cortex and medulla was TE4+, A2B5+, and 12/1 - 2+ and Hassall's bodies were reactive with antibodies TE8 and TE15. In contrast, thymic epithelium in primary mediastinal thymomas was TE4+, A2B5+, TE8-, and greater than 75% of thymoma epithelium was 12/1 - 2-, a thymic epithelial phenotype similar to that seen on normal fetal thymic epithelium at 14 to 16 weeks fetal gestation. In one subject with a mature epithelial histologic pattern, thymoma epithelium was found to be strongly TE8+, a phenotype suggestive of a later stage of TE maturation. Lymphocytes in five of seven thymomas with immature thymic epithelial cells predominantly expressed immature thymocyte phenotype while two thymomas with immature epithelial phenotype showed a predominance of Langerhans cells and surrounding lymphocytes expressing a mature phenotype. Lymphocytes in the thymoma with differentiated epithelial cells expressed a mature thymocyte phenotype. Thus, in thymomas of varying histologic types, phenotypic abnormalities of thymic epithelium are present; these phenotypic abnormalities may reflect abnormal thymic epithelial maturation.     

Early deletion and late positive selection of T cells expressing a male-specific receptor in T-cell receptor transgenic mice

The ontogeny of T cells in T-cell receptor (TCR) transgenic mice, which express a transgenic alpha beta heterodimer, specific for the male (H-Y) antigen in association with H-2Db, was determined. The transgenic alpha chain was expressed on about 10% of the fetal thymocytes on day 14 of gestation. About 50% of day-15 fetal thymocytes expressed both alpha and beta transchains and virtually all fetal thymocytes expressed the transgenic alpha beta heterodimer by day 17. The early expression of the transgenic TCR on CD4-8- thymocytes prevented the development of gamma delta cells, and led to accelerated growth of thymocytes and an earlier expression of CD4 and CD8 molecules. Up to day 17, no significant differences in T-cell development could be detected between female and male thymuses. By day 18 of gestation, the male transgenic thymus contained more CD4-8- thymocytes than the female transgenic thymus. The preponderance of CD4-8- thymocytes in the male transgenic thymus increased until birth and was a consequence of the deletion of the CD4+8+ thymocytes and their CD4-8+ precursors. By the time of birth, the male transgenic thymus contained half the number of cells as the female transgenic thymus. The deletion of autospecific precursor cells in the male transgenic mouse began only at day 18 of gestation, despite the fact that the ligand could already be detected by day 16. The preferential accumulation of CD4-8+ T cells, which expressed a high density of the transgenic TCR, occurred only after birth and was obvious in 6-week-old female thymus. These data support the hypothesis that the positive selection of T cells expressing this transgenic heterodimer may involve two steps, i.e., the commitment of CD4+8+ thymocytes to the CD4-8+ lineage following the interaction of the transgenic TCR with restricting major histocompatibility molecules, followed by a slow conversion of CD4+8+ thymocytes into CD4-8+ T cells. In normal mice, the precursors of CD4+8+ and single positive thymocytes have the CD4-8- CD3-J11d+ (or M1/69+) phenotype. Because of the early expression of the transgenic alpha beta heterodimer, this population was not detected in adult transgenic mice. All CD4-8- M1/69+ cells expressed the transgenic receptor associated with CD3 and could be readily grown in media containing T-cell lectins and interleukin 2.     

Interleukin 7 preferentially supports the growth of gamma delta T cell receptor-bearing T cells from fetal thymocytes in vitro.

Murine fetal thymus cells were cultured with various interleukins (IL-1, 2, 3, 4, 5, 6, and 7) in the absence or presence of phorbol 12-myristate 13-acetate (PMA), and it was found that only IL-4 and IL-7 induced a prominent proliferative response in the presence of PMA. A large proportion of cells grown in the cultures of fetal thymus cells (days 15 and 17 of gestation) stimulated with PMA plus IL-4 or with PMA plus IL-2 remained CD4-CD8-. In marked contrast, nearly 70% of the cells generated in the cultures of the same fetal thymocytes stimulated with PMA plus IL-7 expressed CD8 on their surface. Approximately 30% of these cells expressed TCR gamma, delta, whereas TCR alpha beta+ cells were virtually undetectable. The cells grown in cultures stimulated with PMA plus IL-7 comprised three populations: CD4-Lyt-2-3-, CD4-Lyt-2 + Lyt-3- and CD4-Lyt-2 + Lyt-3+, and that TCR gamma delta+ T cells were found in all three populations. It was also found that the addition of IL-7 in the culture of adult CD4-CD8- thymocytes on the monolayer of a thymic stromal cell line, which selectively promotes the generation of alpha beta T cells, resulted in the generation of gamma delta T cells. These results strongly suggest that IL-7 plays an important role in the development of gamma delta T cells.     

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.     

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.     

Tetrachlorodibenzo-p-Dioxin (TCDD) Inhibits Differentiation and Increases Apoptotic Cell Death of Precursor T-Cells in the Fetal Mouse Thymus

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes thymic atrophy as well as alterations in thymocyte maturity in mice. Multiple mechanisms for thymic hypocellularity have been suggested, and include an increase in thymocyte apoptosis, a maturation arrest of thymocyte development, inhibited thymocyte proliferation, and a diminution of seeding of the thymus by the hematopoietic progenitors in the fetal liver or adult bone marrow. Fetal mice are highly sensitive to hypocellularity induction by TCDD when the chemical is administered during the window of thymic development, between days 10 and 18 of gestation. Treatment of pregnant C57Bl/6 mice in the present experiments with doses of 5 or 10 mu g/kg TCDD by oral gavage on gestation days 14 and 16 severely depressed day 18 thymic cellularity. Histopathologic evaluation of day 18 fetal thymi showed disruption of the normal organ architecture with loss of clear distinction between cortical and medullary regions after TCDD. A decrease in thymocyte density was noted in all regions, and was most dramatic in the cortical zones where pyknotic cells were increased by TCDD treatment. Using day 18 thymocyte suspensions and flow cytometry, the marker 7-AAD showed a decrease in viable thymocytes from TCDD-treated fetal mice, and a concomitant and dose-related increase of thymocytes in early apoptosis. Specifically, relative to control, thymocytes from the 5 and 10 mug/kg TCDD exposure groups displayed 1.9% and 5.3% respective increases in early apoptotic cells. When thymocytes were co-identified by CD4 and CD8 cell surface antigen expression, the enhanced apoptosis occurred in the CD4(+)CD8(+) phenotype with no significant apoptosis seen in the CD4(-)CD8(-), CD4(+)CD8(-), or CD4(-)CD8(+) thymocytes. Given the rapid clearance of apoptotic cells from the thymus, these histopathologic and cytometric data suggest increased thymocyte apoptosis contributes to fetal thymic atrophy after TCDD exposure.     

A bone marrow-derived stroma cell line, ST2, can support the differentiation of fetal thymocytes from the CD4−CD8−double negative to the CD4+CD8+ double positive differentiation stage in vitro

T-cell precursors differentiate into mature T cells predominantly in the thymus. However, it has also been reported that T-cell precursors mature in extrathymic organs such as the liver, bone marrow, or intestines. In order to investigate the nature of the extrathymic microenvironment that supports T-cell maturation, we examined the effect of a bone marrow-derived stroma cell line, ST2, on T-cell precursors by using a reaggregate thymic organ culture (RTOC) system. We found that ST2 cells supported the differentiation of fetal thymocytes at day 14.5 of gestation from a CD4- CD8- double negative (DN) to a CD4+ CD8+ double positive (DP) differentiation stage in a manner similar to that observed in thymus. Anti-interleukin-7 receptor (IL-7R) and anti-c-kit antibodies blocked the growth of thymocytes in RTOC with ST2 cells, but did not inhibit the generation of DP thymocytes. These data indicate that a bone marrow-derived stroma cell, ST2, which supports B-cell differentiation, is also able to support T-cell development and may constitute one of the microenvironmental components for extrathymic T-cell development.     

Gamma delta T cells expressing CD8 or CD4low appear early in murine foetal thymus development.

Three-colour flow cytometry was used to study the distribution of TCR gamma delta+ cells among CD4+CD8-, CD4-CD8+, CD4+CD8+, and CD4-CD8- cell populations during thymic development. Thymocytes were obtained either directly from embryos at different stages of gestation (ex vivo) or from organ cultures maintained in vitro. In both cases, TCR gamma delta+ cells were found predominantly among the double negative (CD4-CD8-) and CD8 single positive subsets. These cells were actively dividing as demonstrated by 7 amino actinomycin D (7AAD) labelling. A small population of TCR gamma delta+ cells expressing low levels of CD4 was identified early and transiently (days 15-18) during development, but this subset was rare in the adult thymus. In newborn mice, adult mice, and late during organ culture, TCR gamma delta+ cells were found mainly within the CD4-CD8- compartment of thymocytes, although a minor population of CD8+ cells (5-10%) bearing gamma delta receptor was routinely observed. In contrast, few gamma delta cells were contained among the CD4+CD8+ subset at any timepoint studied. These data highlight differences between the ontogeny of alpha beta and gamma delta cells in the thymus, and suggest that a CD4+CD8+ intermediate may not be a requisite for the intrathymic differentiation of murine gamma delta T cells.     

Delineation of chicken thymocytes by CD3-TCR complex, CD4 and CD8 antigen expression reveals phylogenically conserved and novel thymocyte subsets.

To further define the relationship between thymocyte subsets and their developmental sequence, multi-parameter flow cytometry was used to determine the distribution of the CD3-TCR complex and the accessory molecules CD4 and CD8 on chicken thymocytes. As in mammals, adult thymocytes could be subdivided into CD3-, CD3lo, and CD3hi staining populations. CD4 and CD8 distribution on such populations revealed the presence of CD3-CD4+CD8- and CD3-CD4-CD8+ thymocytes, putative precursors to CD4+CD8+ cells, detectable in the adult and at high frequency during ontogeny. Of particular interest was the existence of CD3lo expression on CD4+CD8- and CD4-CD8+, and in some instances, on CD4-CD8- thymocytes. Such phenotypes are not easily detectable in the mammalian thymus but were readily observed in both adult and embryonic chicken thymus from 16 days of embryogenesis. Further analysis of the TCR lineage of these CD3lo cells revealed that they were essentially all of the alpha beta TCR type. Mature CD3hi thymocytes were found within the CD4+CD8+ and CD4+CD8- and CD4-CD8+ subsets. Both alpha beta and gamma delta TCR lineage thymocytes were detected within all CD4- and CD8-defined subsets, thus identifying novel thymocyte subsets in the chicken thymus, namely alpha beta TCR+CD4-CD8- and gamma delta TCR+ CD4+CD8- cells. Hence, this analysis of chicken thymocytes, while confirming the phylogenically conserved nature of the thymus, has revealed novel T cell subsets, providing further insight into the complexity of mainstream thymocyte maturation pathways.     

Lineage relationships of the fetal thymocyte subset that expresses the β chain of the interleukin-2 receptor

The β chain (p75) of the interleukin-2 (IL-2) receptor (IL-2R) is expressed on up to 5-7% of fetal thymocytes on day 16 of gestation, declining thereafter to a minute proportion of less than 1% around birth, and of 1-2% of adult thymocytes. A significant part of fetal IL-2R β⁺ thymocytes are γδ cells. The precursor-progeny relationships of fetal DL-2Rβ⁺ thymocytes to the αδ T cell lineage have not been previously studied, nor has their position within the developmental sequence been determined. Here we show that IL-2Rβ is expressed on a subset of very immature cells, along with high amounts of Pgp1 and FCγRII/III, partially preceding the expression of intracellular CD3?. IL-2-Rβ disappears before expression of IL-2Rα. IL-2Rβ⁺ cells, purified by sorting on day 15 of gestation, efficiently reconstituted fetal thymic lobes depleted of lymphoid cells by treatment with desoxyguanosine. They developed into T cell receptor (TCR)αβ⁺, TCRγδ⁺, and CD4/CD8 double- and single-positive cells in similar proportions as did sorted IL-2Rα⁺ day 15 fetal thymocytes. These data suggest that IL-2Rβ expression marks a short period of very early thymocyte development, perhaps immediately after entry into the thymus.     

Expression of high molecular weight isoforms of CD45 by mouse thymic progenitor cells

We have studied the expression of isoforms of CD45 (leukocyte common antigen, LCA) among T cell precursors using the organ culture system of Jenkinson et al. (Eur. J. Immunol. 1982. 12: 583). These experiments show that cells capable of recolonizing alymphoid embryonic thymic lobes in vitro can be detected in the thymus of fetal and adult mice and are enriched when thymocytes are depleted of cells bearing CD4 or CD8. These data are consistent with results from in vivo experiments of Fowlkes et al. (J. Exp. Med. 1985. 162: 802) indicating that T cell precursors lie within the double-negative (CD4-CD8-) compartment. No precursors were detected among the reciprocal populations of cells bearing CD4 and/or CD8 (single and double positives). Double-negative cell fractions were then divided on the basis of reactivity with monoclonal antibodies RA3-2C2 and RA3-3A1. These antibodies recognize the high molecular weight species of the LCA or, more accurately, a product defined by exon A of the CD45 gene. Recolonizing cells were found predominantly in the CD45RA+ (RA3-2C2 and RA3-3A1 reactive) fraction of double-negative thymocytes; CD45RA- enriched populations had increased efficiency of recolonization and CD45RA- depleted populations had decreased ability to recolonize as compared with the whole CD4-CD8- fraction. To clarify whether progenitors enriched in the CD45RA+ fraction were capable of giving rise to mature CD4+, CD8+ and CD4+ CD8+ cells, we analyzed the progeny of lobes seeded with CD4-CD8-CD45RA+ fractions. After 7-9 days in organ culture the proportion of CD4+, CD8+ or CD4+ CD8+ cells had increased to 35.2%, 18.6% and 23.7%, respectively (mean of five experiments), indicating that progenitors among the CD45RA+ population were indeed multipotent. These results suggest that the majority of T stem cells in the thymus are among thymocytes that express the CD45RA molecule, an hypothesis supported by our finding that removal of CD45RA-expressing cells (using complement and antibody) eliminated recolonizing capacity of thymic cell fractions.     

Microenvironmental regulation of T cell development in the thymus

T cell development in the thymus occurs through a series of events beginning with thymic colonization by migrant precursors and ending with the emigration of functionally competent CD4+ and CD8+ T cells to the periphery. It is well accepted that signals through the pre-T cell receptor (pre-TCR) and alpha-beta TCR (alphabetaTCR) complex play pivotal roles in the maturation of CD4-8- and CD4+8+ thymocytes, respectively. It is clear that stromal cells constituting the thymic microenvironment provide non-TCR-mediated interactions which are essential for several developmental events. Examples of such will be discussed here in relation to early and late events in T cell development.     

Characterization of T cell precursor activity in the murine fetal thymus: evidence for an input of T cell precursors between days 12 and 14 of gestation

In the mouse, the number and the differentiation potential of thymic migrants remain controversial. A fetal thymic organ culture under limiting dilution conditions allowed us to show a 130-fold increase in the numbers of T cell precursors in the embryonic thymus between days 12 and 14 of gestation. A comparative analysis of the most immature thymocytes at these two stages revealed that: (1) CD44(+)CD25(-) (DN1) thymocytes at 14 days post coitum (dpc) efficiently differentiate into mature T cells both in vivo and in vitro; (2) 12dpc thymocytes exhibit a low frequency of T cell precursors and were unable to generate a detectable progeny after in vivo intrathymic transfer. A 48-h organ culture of 12dpc thymic lobes did neither correct the low frequency of T cell precursors nor the absence of expression of T cell-specific genes observed in 12dpc thymocytes. We thus concluded that a fraction of recent thymic immigrants contribute to the observed properties in DN1 14dpc thymocytes. We show that increasing numbers of T cell precursors migrate to the thymus from 11 to 14 dpc. We propose that the first thymic immigrants do not contribute significantly to T cell generation which depends on the subsequent colonization by cells with a high T cell precursor potential.     

Ontogeny of T-cell precursors: a model for the initial stages of human T-cell development

Although most investigators agree that the CD4- CD8- CD3- thymocyte subset represents the most immature intrathymic T cell capable of repopulating the thymus in vivo, little is known of the earliest stages of human T-cell development. Using mAbs to hematopoietic and T-cell lineage molecules in quantitative immunofluorescence studies, new insight has been gained regarding the phenotype of human T-cell precursors before and after colonization of human thymic rudiment. In this article, Barton Haynes and colleagues discuss the sequential expression of CD7, CD4, CD8, CD3, CD2, CD1, CD45, TCR gamma delta and TCR alpha beta, and propose a model defining the stages of T-cell precursors during fetal ontogeny.     

Effects of irradiation, glucocorticoid and FK506 on cell-surface antigen expression by rat thymocytes: a three-colour flow cytofluorometric analysis.

The expression of T-cell antigen receptor (TCR) alpha beta was investigated in rat CD4- CD8- thymocytes during thymic reconstitution after the exposure of animals to irradiation or glucocorticoid. The effect of the immunosuppressant FK506 on the expression of TCR alpha beta in rat CD4- CD8- thymocytes was also examined. The percentage of CD4- CD8- thymocytes constituted 2.6% of total thymocytes and that of CD4- CD8- TCR alpha beta high cells constituted 12.6% of CD4- CD8- thymocytes in normal adult Lewis rats. The percentage of CD4- CD8- TCR alpha beta high cells increased during thymic reconstitution after irradiation, and maximally constituted 28.6% of CD4- CD8- thymocytes on day 7. Similar results were obtained during thymic reconstitution after glucocorticoid treatment. In contrast, continuous treatment with FK506 for 7 days markedly decreased not only the percentages of CD4+ CD8- TCR alpha beta high and CD4- CD8+ TCR alpha beta high thymocytes, but also that of CD4- CD8- TCR alpha beta high thymocytes. These results indicate that rat CD4- CD8- thymocytes contain a subpopulation of mature (TCR alpha beta high) cells. The possible implications of the existence of this subpopulation with regard to thymocyte differentiation and maturation are discussed.     

In vivo dynamics of CD4-8- thymocytes. Proliferation, renewal and differentiation of different cell subsets studied by DNA biosynthetic labeling and surface antigen detection

The proliferative status of CD4-8- thymocyte subsets was determined by in vivo or in vitro labeling with bromodeoxyuridine (BrdUrd), a nonreutilized thymidine analogue detectable with a monoclonal antibody, simultaneously with relevant surface proteins. An actively cycling subset [J11d+, interleukin 2 receptor-positive (IL 2R+)] was defined, besides a relatively resting one (J11d-, Pgp1+, T cell receptor-positive). Continuous per os administration of BrdUrd showed that 85% only of CD4-8- thymocytes were labeled in 6 days confirming the existence of a relatively long-term resting subset. By contrast, CD4+8+ thymocytes were all labeled in 3-4 days. Observation of labeled CD4-8- cells after pulse labeling showed an immediate decrease of their absolute number per thymus, confirming their low autorenewal capacity. However, a small number of labeled cells which were hydroxyurea or colchicine resistant remained CD4-8- for several days and progressively acquired surface expression of IL 2R. IL 2R expression by cycling CD4-8- cells during thymus regeneration after antimitogenic drug treatment was rapid, but very transient. According to these results most CD4-8- thymocytes appear as largely engaged in a proliferation-dependent differentiative process, and do not behave as true stem cells. Consequently, this subset is principally renewed by thymic immigration of exogenously produced resting cells. However, a tenfold expansion of CD4-8- cells was found in the fetal and regenerating thymus, suggesting two proliferative phases during intrathymic CD4-8- cell maturation, the first one yielding to cell expansion and the second to cell differentiation. A tentative evaluation of daily cell immigration is proposed starting with the determination of the number of cells beginning DNA synthesis each day. A global model is finally discussed by confronting our kinetic results with the known reconstitution capacities of CD4-8- thymocyte subsets.     

Expression and function of CD2 during murine thymocyte ontogeny

CD2, originally recognized as the sheep erythrocyte receptor of human T cells, has been implicated in early T cell development in the thymus. In this report, we examined the expression and functional role of CD2 during murine thymocyte ontogeny by using monoclonal antibodies to murine CD2. Surface expression of CD2 was first detected in Thy-1+ fetal thymocytes at day 14 of gestation and it progressively increased during CD4-CD8- phenotype. Surface IL 2 receptor (CD25) expression was readily detected in surface CD2- cells at day 13 of gestation and the majority of CD2+ cells appeared to be generated from CD25+ cells thereafter. In adult CD4-CD8- thymocytes, the expression of CD2 and CD25 was mutually exclusive. These results indicate that surface CD2 expression is not a prerequisite for CD25 induction during murine thymocyte ontogeny. This was further confirmed by fetal thymus organ culture in which anti-murine CD2 mAb was included. The antibody treatment led to a suppressed CD2 expression on thymocytes; however, there was no effect on the appearance of CD25. Moreover, no influence on the development of mature CD3+ thymocytes was observed after fetal thymus organ culture in the presence of anti-murine CD2 mAb, and a substantial number of CD3+CD2- cells was demonstrated in fetal and adult CD4-CD8- thymocytes. These findings argue against the functional relevance of CD2 expression during early T cell development as proposed in humans.     

Notch ligands potentiate IL‐7‐driven proliferation and survival of human thymocyte precursors

Notch and IL‐7 are both well‐characterized factors involved in T‐cell development. In contrast to the mouse model, their precise requirements in the differentiation and/or proliferation of various stages of human thymic development have not been fully explored. Here, we demonstrate that IL‐7 alone is sufficient to induce the differentiation of ex vivo purified CD34⁺ triple negative (TN) surface (s) CD3^? CD4^?CD8^? (CD3^?CD4^?CD8^?), CD4 immature single positive (ISP) (sCD3^?CD4⁺CD8^?) and double positive (DP) (sCD3^?CD4⁺CD8⁺) human thymic precursors to mature DP expressing sCD3 (sCD3⁺CD4⁺CD8⁺). We show that activation of Notch signaling by its ligands Delta‐1 or Delta‐4 potentiates IL‐7‐driven proliferation and survival of CD34⁺ TN and to a lesser extent of CD4⁺ ISP precursors. This effect of Notch is related to a sustained induction of IL‐7 receptor α chain expression on thymocytes through a decreased methylation of its gene promoter. Thus, we show here that proliferation and differentiation of T‐cell precursors are differentially modulated by IL‐7 depending on the presence or absence of external signals. These results may have important implications for the clinical use of this cytokine as a strategy aimed at improving immune restoration.