Loss-of-function mutations of Dynamin 2 promote T-ALL by enhancing IL-7 signalling.

Mutations in the DYNAMIN2 (DNM2) gene are frequently detected in human acute T-cell lymphoblastic leukemia (T-ALL), although the mechanisms linking these mutations to disease pathogenesis remain unknown. Using an ENU-based forward genetic screen for mice with erythroid phenotypes, we identified a heterozygous mouse line carrying a mutation in the GTPase domain of Dnm2 (Dnm2V265G) that induced a microcytic anemia. In vitro assays using the V265G mutant demonstrated loss of GTPase activity and impaired endocytosis that was comparable to other DNM2 mutants identified in human T-ALL. To determine the effects of DNM2 mutations in T-ALL, we bred the Dnm2V265G mice with the Lmo2 transgenic mouse model of T-ALL. Heterozygous Dnm2 mutants lacking the Lmo2 transgene displayed normal T-cell development, and did not develop T-ALL. In contrast, compound heterozygotes displayed an accelerated onset of T-ALL compared with mice carrying the Lmo2 oncogene alone. The leukemias from these mice exhibited a more immature immunophenotype and an expansion in leukemic stem cell numbers. Mechanistically, the Dnm2 mutation impaired clathrin-mediated endocytosis of the interleukin (IL)-7 receptor resulting in increased receptor density on the surface of leukemic stem cells. These findings suggest that DNM2 mutations cooperate with T-cell oncogenes by enhancing IL-7 signalling.

The Notch ligand DLL4 specifically marks human hematoendothelial progenitors and regulates their hematopoietic fate.

Notch signaling is essential for definitive hematopoiesis, but its role in human embryonic hematopoiesis is largely unknown. We show that in hESCs the expression of the Notch ligand DLL4 is induced during hematopoietic differentiation. We found that DLL4 is only expressed in a sub-population of bipotent hematoendothelial progenitors (HEPs) and segregates their hematopoietic versus endothelial potential. We demonstrate at the clonal level and through transcriptome analyses that DLL4(high) HEPs are enriched in endothelial potential, whereas DLL4(low/-) HEPs are committed to the hematopoietic lineage, albeit both populations still contain bipotent cells. Moreover, DLL4 stimulation enhances hematopoietic differentiation of HEPs and increases the amount of clonogenic hematopoietic progenitors. Confocal microscopy analysis of whole differentiating embryoid bodies revealed that DLL4(high) HEPs are located close to DLL4(low/-) HEPs, and at the base of clusters of CD45+ cells, resembling intra-aortic hematopoietic clusters found in mouse embryos. We propose a model for human embryonic hematopoiesis in which DLL4(low/-) cells within hemogenic endothelium receive Notch-activating signals from DLL4(high) cells, resulting in an endothelial-to-hematopoietic transition and their differentiation into CD45+ hematopoietic cells.

The differentiation stage of p53-Rb-deficient bone marrow mesenchymal stem cells imposes the phenotype of in vivo sarcoma development.

Increasing evidence suggests that mesenchymal stem/stromal cells (MSCs) carrying specific mutations are at the origin of some sarcomas. We have reported that the deficiency of p53 alone or in combination with Rb (Rb(-/-) p53(-/-)) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53(-/-) and Rb(-/-)p53(-/-) MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53(-/-) and Rb(-/-)p53(-/-) ASCs. In addition, gene expression profiling revealed transcriptome similarities between p53- or Rb-p53-deficient BM-MSCs/ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem to determine the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate toward the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors, which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development.

Rapid and sensitive method for determining free amino acids in honey by gas chromatography with flame ionization or mass spectrometric detection.

This paper describes a rapid, sensitive and specific method for determination of free amino acids in honey involving a new reaction of derivatization and gas chromatography (GC) with flame ionization (FID) and mass spectrometric (MS) detection. The method allows the determination of 22 free amino acids in honey samples in a short time: 8 and 5 min for GC-FID and GC-MS, respectively. Quantitation was performed using Norvaline as internal standard, with detection limits ranging between 0.112 and 1.795 mg/L by GC-FID and between 0.001 and 0.291 mg/L by GC-MS in the selected-ion monitoring mode. The method was validated and applied to a set of 74 honey samples belonging to four different botanical origins: eucaliptus, rosemary, orange and heather. The statistical treatment of data shows a correct classification of different origins over 90%.

Some hints concerning the shape of T-cell receptor structures.

Several models are proposed for T-cell antigen receptor (TCR) assembly and structure. However, there is little experimental data favouring directly either one or the other(s). The minimal complex appears to be composed of a TCRalphabeta/CD3deltaepsilon,gammaepsilon/zeta2 structure but at the cell membrane, multimers of this minimal structure may be formed. Quantitative cytofluometry has suggested three CD3epsilon chains for two TCRbeta (or TCRdelta) chains/complex. Such data should be repeated with monoclonal antibodies (MoAb) against extracellular (EC) parts of CD3delta or CD3gamma chains. In the present review, we have compared the TCR/CD3 assembly of pre-TCR, TCRgammadelta and TCRalphabeta containing complexes, and analysed the reactivity of antibodies (Abs) against the EC part of CD3delta chains. Our data suggest an alternative assembly pathway and structure of TCR/CD3 complexes.

Differential developmental regulation and functional effects on pre-TCR surface expression of human pTalpha(a) and pTalpha(b) spliced isoforms.

Functional rearrangement at the TCRbeta locus leads to surface expression on developing pre-T cells of a pre-TCR complex composed of the TCRbeta-chain paired with the invariant pre-TCRalpha (pTalpha) chain and associated with CD3 components. Pre-TCR signaling triggers the expansion and further differentiation of pre-T cells into TCRalphabeta mature T cells, a process known as beta selection. Besides the conventional pTalpha transcript (termed pTalpha(a)), a second, alternative spliced, isoform of the pTalpha gene (pTalpha(b)) has been described, whose developmental relevance remains unknown. In this study, phenotypic, biochemical, and functional evidence is provided that only pTalpha(a) is capable of inducing surface expression of a CD3-associated pre-TCR complex, which seems spontaneously recruited into lipid rafts, while pTalpha(b) pairs with and retains TCRbeta intracellularly. In addition, by using real-time quantitative RT-PCR approaches, we show that expression of pTalpha(a) and pTalpha(b) mRNA spliced products is differentially regulated along human intrathymic development, so that pTalpha(b) transcriptional onset is developmentally delayed, but beta selection results in simultaneous shutdown of both isoforms, with a relative increase of pTalpha(b) transcripts in beta-selected vs nonselected pre-T cells in vivo. Relative increase of pTalpha(b) is also shown to occur upon pre-T cell activation in vitro. Taken together, our data illustrate that transcriptional regulation of pTalpha limits developmental expression of human pre-TCR to intrathymic stages surrounding beta selection, and are compatible with a role for pTalpha(b) in forming an intracellular TCRbeta-pTalpha(b) complex that may be responsible for limiting surface expression of a pTalpha(a)-containing pre-TCR and/or may be competent to signal from a subcellular compartment.

An endoplasmic reticulum retention function for the cytoplasmic tail of the human pre-T cell receptor (TCR) alpha chain: potential role in the regulation of cell surface pre-TCR expression levels.

The pre-T cell receptor (TCR), which consists of a TCR-beta chain paired with pre-TCR-alpha (pTalpha) and associated with CD3/zeta components, is a critical regulator of T cell development. For unknown reasons, extremely low pre-TCR levels reach the plasma membrane of pre-T cells. By transfecting chimeric TCR-alpha-pTalpha proteins into pre-T and mature T cell lines, we show here that the low surface expression of the human pre-TCR is pTalpha chain dependent. Particularly, the cytoplasmic domain of pTalpha is sufficient to reduce surface expression of a conventional TCR-alpha/beta to pre-TCR expression levels. Such reduced expression cannot be attributed to qualitative differences in the biochemical composition of the CD3/zeta modules associated with pre-TCR and TCR surface complexes. Rather, evidence is provided that the pTalpha cytoplasmic tail also causes a reduced surface expression of individual membrane molecules such as CD25 and CD4, which are shown to be retained in the endoplasmic reticulum (ER). Native pTalpha is also observed to be predominantly ER localized. Finally, sequential truncations along the pTalpha cytoplasmic domain revealed that removal of the COOH-terminal 48 residues is sufficient to release a CD4-pTalpha chimera from ER retention, and to restore native CD4 surface expression levels. As such a truncation in pTalpha also correlates with enhanced pre-TCR expression, the observed pTalpha ER retention function may contribute to the regulation of surface pre-TCR expression on pre-T cells.

Beta-selection is associated with the onset of CD8beta chain expression on CD4(+)CD8alphaalpha(+) pre-T cells during human intrathymic development.

T-cell precursors that undergo productive rearrangements at the T-cell receptor (TCR) beta locus are selected for proliferation and further maturation, before TCRalpha expression, by signaling through a pre-TCR composed of the TCRbeta chain paired with a pre-TCRalpha (pTalpha) chain. Such a critical developmental checkpoint, known as beta-selection, results in progression from CD4(-) CD8(-) double negative (DN) to CD4(+) CD8(+) double positive (DP) TCRalphabeta(-) thymocytes. In contrast to mice, progression to the DP compartment occurs in humans via a CD4(+) CD8(-) intermediate stage. Here we show that the CD4(+) CD8(-) to CD4(+) CD8(+) transition involves the sequential acquisition of the alpha and beta chains of CD8 at distinct maturation stages. Our results indicate that CD8alpha, but not CD8beta, is expressed in vivo in a minor subset of DP TCRalphabeta(-) thymocytes, referred to as CD4(+) CD8alphaalpha(+) pre-T cells, mostly composed of resting cells lacking cytoplasmic TCRbeta chain (TCRbeta(ic)). In contrast, expression of CD8alphabeta heterodimers was selectively found on DP TCRalphabeta(-) thymocytes that express TCRbeta(ic) and are enriched for cycling cells. Interestingly, CD4(+) CD8alphaalpha(+) pre-T cells are shown to be functional intermediates between CD4(+) CD8(-) TCRbeta(ic)(-) and CD4(+) CD8alphabeta(+) TCRbeta(ic)(+) thymocytes. More importantly, evidence is provided that onset of CD8beta and TCRbeta(ic) expression are coincident developmental events associated with acquisition of CD3 and pTalpha chain on the cell surface. Therefore, we propose that the CD4(+) CD8alphaalpha(+) to CD4(+) CD8alphabeta(+) transition marks the key control point of pre-TCR-mediated beta-selection in human T-cell development.

Conformational and biochemical differences in the TCR.CD3 complex of CD8(+) versus CD4(+) mature lymphocytes revealed in the absence of CD3gamma.

Mature CD4(+) and CD8(+) T lymphocytes are believed to build and express essentially identical surface alphabeta T-cell receptor-CD3 (TCR.CD3) complexes. However, TCR.CD3 expression has been shown to be more impaired in CD8(+) cells than in CD4(+) cells when CD3gamma is absent in humans or mice. We have addressed this paradox by performing a detailed phenotypical and biochemical analysis of the TCR.CD3 complex in human CD3gamma-deficient CD8(+) and CD4(+) T cells. The results indicated that the membrane TCR.CD3 complex of CD8(+) T lymphocytes was conformationally different from that of CD4(+) lymphocytes in the absence of CD3gamma. In addition, CD8(+), but not CD4(+), CD3gamma-deficient T lymphocytes were shown to contain abnormally glycosylated TCRbeta proteins, together with a smaller, abnormal TCR chain (probably incompletely processed TCRalpha). These results suggest the existence of hitherto unrecognized biochemical differences between mature CD4(+) and CD8(+) T lymphocytes in the intracellular control of alphabetaTCR. CD3 assembly, maturation, or transport that are revealed when CD3gamma is absent. Such lineage-specific differences may be important in receptor-coreceptor interactions during antigen recognition.

Measles virus infection induces terminal differentiation of human thymic epithelial cells.

Measles virus infection induces a profound immunosuppression that may lead to serious secondary infections and mortality. In this report, we show that the human cortical thymic epithelial cell line is highly susceptible to measles virus infection in vitro, resulting in infectious viral particle production and syncytium formation. Measles virus inhibits thymic epithelial cell growth and induces an arrest in the G0/G1 phases of the cell cycle. Moreover, we show that measles virus induces a progressive thymic epithelial cell differentiation process: attached measles virus-infected epithelial cells correspond to an intermediate state of differentiation while floating cells, recovered from cell culture supernatants, are fully differentiated. Measles virus-induced thymic epithelial cell differentiation is characterized by morphological and phenotypic changes. Measles virus-infected attached cells present fusiform and stellate shapes followed by a loss of cell-cell contacts and a shift from low- to high-molecular-weight keratin expression. Measles virus infection induces thymic epithelial cell apoptosis in terminally differentiated cells, revealed by the condensation and degradation of DNA in measles virus-infected floating thymic epithelial cells. Because thymic epithelial cells are required for the generation of immunocompetent T lymphocytes, our results suggest that measles virus-induced terminal differentiation of thymic epithelial cells may contribute to immunosuppression, particularly in children, in whom the thymic microenvironment is of critical importance for the development and maturation of a functional immune system.

Identification of a late stage of small noncycling pTalpha- pre-T cells as immediate precursors of T cell receptor alpha/beta+ thymocytes.

During thymocyte development, progression from T cell receptor (TCR)beta to TCRalpha rearrangement is mediated by a CD3-associated pre-TCR composed of the TCRbeta chain paired with pre-TCRalpha (pTalpha). A major issue is how surface expression of the pre-TCR is regulated during normal thymocyte development to control transition through this checkpoint. Here, we show that developmental expression of pTalpha is time- and stage-specific, and is confined in vivo to a limited subset of large cycling human pre-T cells that coexpress low density CD3. This restricted expression pattern allowed the identification of a novel subset of small CD3(-) thymocytes lacking surface pTalpha, but expressing cytoplasmic TCRbeta, that represent late noncycling pre-T cells in which recombination activating gene reexpression and downregulation of T early alpha transcription are coincident events associated with cell cycle arrest, and immediately preceding TCRalpha gene expression. Importantly, thymocytes at this late pre-T cell stage are shown to be functional intermediates between large pTalpha+ pre-T cells and TCRalpha/beta+ thymocytes. The results support a developmental model in which pre-TCR-expressing pre-T cells are brought into cycle, rapidly downregulate surface pre-TCR, and finally become small resting pre-T cells, before the onset of TCRalpha gene expression.

Enhanced green fluorescent protein as an efficient reporter gene for retroviral transduction of human multipotent lymphoid precursors.

Owing to its autofluorescence properties, green fluorescent protein (GFP) has aroused increasing interest as a marker system for many research applications. In this study we investigated the suitability of the "enhanced" GFP (EGFP), a mutant version of GFP optimized for flow cytometry and microscopy detection, as a reporter gene for retroviral transduction protocols. EGFP was shown to display a bright and stably maintained emission pattern in transfected GP+envAm12 packaging cells. Stable fluorescent emission was observed as well after transduction in NIH 3T3 fibroblasts and in the human Jurkat T cell line, in which EGFP was shown to confer no deleterious effect or growth disadvantage on the expressing cells. Moreover, EGFP expression could be detected after short-term retroviral exposure, thus allowing a rapid and quantitative retroviral titering assay, alternative to the standard colony-formation procedure. Most importantly, we showed the feasibility of EGFP as a marker gene in retroviral-mediated transduction of primary lymphoid precursors. In particular, transduction of CD34+CD1- human thymocytes by short-term cocultivation yielded up to 30% of EGFP-expressing cells, while maintaining CD34 expression levels. Finally, when cultured under multicytokine-supported conditions, such transduced intrathymic progenitors were shown to efficiently generate lymphoid-related dendritic cells, which displayed a distinct EGFP expression. Therefore, because of its rapid and easy detectability and its nontoxic characteristics, EGFP proves itself to be a valuable reporter gene by allowing the transduction of multipotential progenitors and by being compatible with the developmental programs of lymphoid lineage generation.

Identification of a common developmental pathway for thymic natural killer cells and dendritic cells.

Current data support the notion that the thymus is seeded by a yet uncommitted progenitor cell able to generate T cells, B cells, natural killer (NK) cells, and dendritic cells (DCs). We assess in this report the developmental relationship of DCs and NK cells derived from a small subset of CD34(+) human postnatal thymocytes that, like the earliest precursors in the fetal thymus, display low CD33 surface expression. Culture of these isolated CD34(+) CD33(lo) thymic progenitors with a mixture of cytokines, including interleukin-7 (IL-7), IL-1alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, results in predominant generation of DCs. However, the addition of IL-2 to the cytokine mixture leads to the simultaneous development of DCs and NK cells. Both developmental pathways progress through a transient population of CD34(+)CD44(bright) CD5(lo/-)CD33(+) large-sized cells, distinct from small-sized T-lineage precursors, that contain bipotential NK/DC progenitors. These data provide evidence of linked pathways of NK cell and DC development from intrathymic precursors and suggest that NK cells and DCs branch off the T lineage through a common intermediate progenitor.

CD10 is expressed on human thymic epithelial cell lines and modulates thymopentin-induced cell proliferation.

Thymic hormones such as thymopoietin (TP) have been shown to regulate thymocyte differentiation and lymphocyte activation. However, it is not known whether thymopoietin affects thymic epithelial cell (TEC) functions. In this study we have examined the effect of a five amino acid active peptide (TP5), corresponding to amino acids 32-36 of TP, on the proliferation of nontransformed clones of human TEC. Our results indicate that TP5 induced reinitiation of DNA synthesis and potentiated fetal calf serum (FCS)-induced cell growth in postnatal and fetal-derived human TEC. We also found that TEC lines express high levels of endopeptidase 24.11, a cell-surface metallopeptidase also known as the CD10 antigen. We show that TP5 is cleaved by CD10 at the surface of TEC lines, indicating that this endopeptidase may regulate TP5-induced TEC proliferation. Phosphoramidon, a specific endopeptidase 24.11 inhibitor, consistently acts in synergy with TP5 to enhance FCS-induced TEC growth. Hence, we conclude that 1) TP5 alone or in combination with FCS supports the growth of TEC lines, and 2) TEC lines express high levels of CD10, which regulates TP5-induced TEC proliferation by acting as a thymic peptide degrading enzyme.

Herpesvirus saimiri immortalization of alpha beta and gamma delta human T-lineage cells derived from CD34+ intrathymic precursors in vitro.

Herpesvirus saimiri (HVS), an agent that can infect many human cell types, has been shown to immortalize selectively TCR alpha beta + CD3+ T lymphocytes. Human T cell precursors defined as CD34+CD3-CD4-CD8- were isolated from thymic samples and exposed to HVS in the presence of either IL-2 or IL-7. Cultures lacking the virus were non-viable by day 15. Test cultures, in contrast, showed a sustained proliferative activity lasting > 5 months, allowing the phenotypical and molecular analysis of the cellular progeny. In the presence of IL-7, TCR alpha beta + cells with three different phenotypes (mainly CD4+CD8-, but also CD4+CD8+ and CD4-CD8+) were immortalized, whereas no TCR gamma delta + cells were recovered. Kinetic studies showed that the expansion of immortalized TCR alpha beta + cells was preceded by a gradual loss of CD34+ cells followed by a transient accumulation of two distinct cell subsets: first CD1+CD4+CD3- cells and then CD4+CD8+ thymocytes. This resembles early phenotypic changes occurring during normal intrathymic T cell development. In the presence of IL-2, in contrast, only TCR gamma delta + cells were immortalized (mainly CD4-CD8+, but also CD4-CD8-). The results show that HVS can be used to read the CD3+ cellular outcome of T cell differentiation assays, including gamma delta + CD4-CD8+, gamma delta + CD4-CD8-, alpha beta + CD4+CD8-, alpha beta + CD4-CD8+ and alpha beta + CD4+CD8+ T cells. A clear role for different cytokines (IL-2 for gamma delta + cells, IL-7 for alpha beta + cells) in early T cell commitment was also apparent.

Expression of functionally active alpha 4 beta 1 integrin by thymic epithelial cells.

We have investigated the expression and function of the VLA-4 heterodimer alpha 4 beta 1, a member of the beta 1 integrin subfamily, on human thymic epithelial cells (TEC) derived from cortical epithelium. The expression of the alpha 4 integrin chain was studied in four different cloned TEC lines derived from either fetal or post-natal human thymus by both flow cytometry and immunoprecipitation techniques with anti-alpha 4 MoAbs. All different cell lines assayed expressed significant levels of alpha 4, as revealed by their reactivity with MoAbs specific for distinct alpha 4 epitopes. The alpha 4 subunit expressed by TEC was associated to beta 1 but not to beta 1 chain, and displayed the characteristic 80/ 70 kD pattern of proteolytic cleavage. The VLA-4 integrin in these cells was constitutively active in terms of adhesiveness to both fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In addition, this heterodimer localized to punctate regions of the cell in the area of contact with the substratum, named point contacts assessed by staining with the anti-beta 1 activation epitope 15/7 MoAb. According to the cortical origin of the TEC lines expressing VLA-4, human thymus sections stained with different anti-alpha 4 antibodies revealed the presence of cortical, and in smaller numbers medullary epithelial cells bearing alpha 4 integrin. The expression of alpha 4 in the thymus was also found in both adult and fetal rats, in which epithelial cells were also specifically stained. Altogether, our data show that VLA-4 is an additional component of the integrin repertoire of TEC, and suggest that it could have an important role in thymus epithelial cell-thymocyte interactions.

Regulation of pre-T cell receptor (pT alpha-TCR beta) gene expression during human thymic development.

In murine T cell development, early thymocytes that productively rearrange the T cell receptor (TCR) beta locus are selected to continue maturation, before TCR alpha expression, by means of a pre-TCR alpha- (pT alpha-) TCR beta heterodimer (pre-TCR). The aim of this study was to identify equivalent stages in human thymocyte development. We show here that variable-diversity-joining region TCR beta rearrangement and the expression of full-length TCR beta transcripts have been initiated in some immature thymocytes at the TCR alpha/beta- CD4+CD8- stage, and become common in a downstream subset of TCR alpha/beta- CD4+CD8+ thymocytes that is highly enriched in large cycling cells. TCR beta chain expression was hardly detected in TCR alpha/beta- CD4+CD8- thymocytes, whereas cytoplasmic TCR beta chain was found in virtually all TCR alpha/beta- CD4+CD8+ blasts. In addition, a TCR beta complex distinct from the mature TCR alpha/beta heterodimer was immunoprecipitated only from the latter subset. cDNA derived from TCR alpha/beta- CD4+CD8+ blasts allowed us to identify and clone the gene encoding the human pT alpha chain, and to examine its expression at different stages of thymocyte development. Our results show that high pT alpha transcription occurs only in CD4+CD8- and CD4+CD8+ TCR alpha/beta- thymocytes, whereas it is weaker in earlier and later stages of development. Based on these results, we propose that the transition from TCR alpha/beta- CD4+CD8- to TCR alpha/beta- CD4+CD8+ thymocytes represents a critical developmental stage at which the successful expression of TCR beta promotes the clonal expansion and further maturation of human thymocytes, independent of TCR alpha.

The development of T and non-T cell lineages from CD34+ human thymic precursors can be traced by the differential expression of CD44.

In addition to T-lineage cells, a small proportion of hematopoietic non-T cells are present in the human postnatal thymus. However, the origin of this minor non-T cell thymic compartment is presently unknown. In this study we have analyzed the developmental potential of the earliest human intrathymic precursors, characterized as CD34+ cells expressing intermediate levels of CD44. We show that these CD34+CD44int thymocytes cultured with interleukin 7 were able to develop simultaneously into both T- and non-T (monocytes and dendritic cells) -lineage cells. Both developmental pathways progress through a CD1+CD4+ intermediate stage, currently believed to be the immediate precursor of double positive thymocytes. However, separate progenitors for either T or non-T cells could be characterized within CD1+CD4+ thymocytes by their opposite expression of CD44. Downregulated levels of CD44 identified CD1+CD4+ T-lineage precursors, whereas CD44 upregulation occurred on CD1+CD4+ intermediates that later differentiated into non-T cells. Therefore, commitment of human early intrathymic precursors to either T or non-T cell lineages can be traced by the differential expression of the CD44 receptor.

Establishment and characterization of cloned human thymic epithelial cell lines. Analysis of adhesion molecule expression and cytokine production.

The thymic stromal microenvironment is required for the generation of immunocompetent T lymphocytes. However, the different thymic stromal cell types have not been fully characterized and their roles regarding T-cell development are not completely understood. To address the phenotypic characteristics of the epithelial component of the human thymic microenvironment as well as its functional involvement in T-cell development, we have established cloned thymic epithelial cell (TEC) lines from fetal and postnatal human thymuses by an explant technique, repeated subculture, and limiting dilution cloning. These cloned TEC lines were shown to be derived from cortical epithelium and to express a number of cell-surface molecules including CD40, major histocompatibility complex (MHC) HLA-ABC and HLA-DR antigens, homing-associated cell-adhesion molecule (H-CAM), intercellular adhesion molecule-1 (ICAM-1), leukocyte function-associated antigen 3 (LFA-3), and beta 1 subfamily integrins. Finally, both postnatal and fetal TEC clones were shown to produce interleukin-1 alpha (IL-1 alpha), IL-6, and IL-7. These well-defined cloned TEC lines may provide useful tools for the study of TEC biology and for the understanding of the precise role played by TEC in human T-cell development.