Biomedical subjects

S M Denning

Publications and source records attributed to S M Denning.

29 records · Page 2Linked to original sources

PubMed1989

Immature human thymocytes can be driven to differentiate into nonlymphoid lineages by cytokines from thymic epithelial cells.

The signals and cellular interactions required for hematopoietic stem-cell commitment to the T lineage are unknown, yet are central to understanding the early stages of normal T-cell development. To study the differentiative capacity of T-cell precursors, we isolated CD4-, CD8-, surface(s) CD3- thymocytes from postnatal human thymuses and determined their capacity to differentiate into lymphoid and nonlymphoid lineages in vitro. We found that CD4-, CD8-, sCD3- thymocytes, which differentiated in the presence of T-cell conditioned medium plus interleukin 2 into T cells expressing the gamma delta receptor for antigen, were capable of differentiating into myeloid or erythroid lineages in the presence of either 5637 bladder carcinoma cell line conditioned medium plus recombinant human erythropoietin or human thymic epithelial cell conditioned medium. Thymic epithelial cell conditioned medium was as effective as 5637 supernatant plus erythropoietin in inducing myeloerythroid differentiation in the CD4-, CD8-, sCD3- thymocytes. Sixty-eight +/- 14% of CD4-, CD8-, sCD3- thymocytes underwent nonlymphoid differentiation within 4 days in culture with 5637 supernatant plus erythropoietin. Twenty-six +/- 4% of freshly isolated CD4-, CD8-, sCD3- cells were CD34+, and clonal granulocyte/macrophage, granulocyte/erythrocyte/monocyte/megakaryocyte, and T-cell progenitors were found in both CD34+ and CD34- subsets of CD4-, CD8-, sCD3- thymocytes. Thus, cells within the human CD4-, CD8-, sCD3- thymocyte subset can give rise to gamma delta+ T cells as well as to cells of myeloerythroid lineages. Moreover, CD34+, CD4-, CD8-, sCD3- cells can give rise to clonal T-cell progenitors as well as to clonal myeloid progenitors.

Antibodies, Monoclonal↗

PubMed1989

The gene SCL is expressed during early hematopoiesis and encodes a differentiation-related DNA-binding motif.

We have identified the human gene, SCL. We discovered this gene because of its involvement in a chromosomal translocation associated with the occurrence of a stem cell leukemia manifesting myeloid and lymphoid differentiation capabilities. Here we report the sequence of a cDNA for the normal SCL transcript, as well as for an aberrant fusion transcript produced in the leukemic cells. Although different at their 3' untranslated regions, both cDNAs predict a protein with primary amino acid sequence homology to the previously described amphipathic helix-loop-helix DNA binding and dimerization motif of the Ly1-1, myc, MyoD, immunoglobulin enhancer binding, daughterless, and achaete-scute families of genes. For these cDNAs, at least two different 5' ends are predicted, both of which retain this putative DNA binding domain and predict proteins in the range of 20-30 kDa. SCL mRNA is observed in "early" hematopoietic tissues. Taken together, these studies lead to the speculation that SCL plays a role in differentiation and/or commitment events during hematopoiesis.

Amino Acid Sequence↗

PubMed1989

Removal of fibroblasts from human epithelial cell cultures with use of a complement fixing monoclonal antibody reactive with human fibroblasts and monocytes/macrophages.

A complement fixing IgM monoclonal antibody (1B10) that reacts with surface membrane molecules of human fibroblasts, tissue macrophages, and peripheral monocytes was produced. In Western blot analysis of detergent extracts of cultured human foreskin fibroblasts, antibody 1B10 detected protein bands of Mr 43,000 and 72-80,000. We used the 1B10 antibody with complement to eliminate most 1B10 positive nonepithelial cells from thymic epithelial (TE) cell cultures, thereby allowing us to grow highly enriched populations of human TE cells.

Antibodies, Monoclonal↗

PubMed1988

Analysis of expression of CD2, CD3, and T cell antigen receptor molecules during early human fetal thymic development.

To define early stages of T cell maturation during human fetal thymic development, we have used mAb reactive with CD2, CD3, and TCR molecules in indirect immunofluorescence assays on a series of early human fetal thymic specimens. Using a technique of quantitating the relative proportions of fluorescent-positive cells present in tissue sections, we found at 8.5 wk of gestational age after arrival of CD7+ T cell precursors into the thymic rudiment, 60% of thymic CD7+ cells were CD2+, 4% were CD3+ and none was TCR-delta+ or TCR beta+. Moreover, cells reactive with anti-CD2 antibodies against T11(2) and T11(3) epitopes of CD2 as well as thymic stromal cells expressing the CD2 ligand, lymphocyte function associated Ag-3, were also present at 8.5 wk. From 9.5 wk to birth TCR beta+ cells increased to include greater than 90% of all CD7+ cells while TCR-delta+ cells fell from a peak of 11% of CD7+ cells at 9.5 wk to 1% of CD7+ cells at birth. These data suggest that epitopes of CD2 molecules are expressed early on during fetal thymic development. Moreover, these data suggest that CD7+, CD2+, cytoplasmic CD3+ T cell precursors in man give rise to both TCR-delta+ T cells as well as to T cells expressing TCR-alpha beta.

Antibodies, Monoclonal↗

PubMed1988

Purified lymphocyte function-associated antigen-3 (LFA-3) activates human thymocytes via the CD2 pathway.

Defining the cellular and molecular mechanisms of interaction of developing thymocytes with nonlymphoid cells of the thymic microenvironment is critical for understanding normal thymus function. We have previously shown that the CD2/LFA-3 adhesion pathway is important in the interaction of thymocytes with a variety of LFA-3+ nonlymphoid thymic microenvironment cell types. Moreover, T cell activation via the CD2 (alternative, Ag independent) pathway is considered an important mechanism for intrathymic T cell proliferation. To study the relevance of CD2/LFA-3 interactions to human thymocyte activation, we have used purified LFA-3 Ag in several in vitro assays of thymocyte proliferation. Whereas LFA-3 Ag alone did not induce thymocyte proliferation, LFA-3 Ag in combination with the anti-CD2 antibody, CD2.1, and rIL-2 induced marked thymocyte proliferation. Additionally, the anti-CD28 antibody, Kolt2, could substitute for rIL-2, resulting in thymocyte activation induced by LFA-3 Ag in combination with antibodies CD2.1 and Kolt2. In both triggering systems, LFA-3 induced thymocyte activation was dependent upon the concentration of LFA-3 Ag. LFA-3 Ag-dependent thymocyte activation was directed primarily toward CD1-, mature thymocytes. Finally, intact SRBC that express the sheep homolog of LFA-3, T11TS, in combination with antibody CD2.1 and rIL-2 could also induce thymocyte activation. These data suggest that interaction of LFA-3 molecules with thymocyte CD2 molecules may provide a component of the stimulus for normal intrathymic thymocyte activation leading to thymocyte proliferation.

Animals↗

PubMed1988

A novel activation pathway for mature thymocytes. Costimulation of CD2 (T,p50) and CD28 (T,p44) induces autocrine interleukin 2/interleukin 2 receptor-mediated cell proliferation.

Prior studies have shown that thymocytes, unlike peripheral T cells, do not proliferate in response to mitogenic combinations of anti-CD2 mAbs. The present study demonstrated that stimulation by a mitogenic anti-CD2 combination (9-1 plus 9.6) with anti-CD28 induced vigorous thymocyte proliferation in the absence of exogenous IL-2. This thymocyte proliferation was IL-2 dependent as shown by the complete inhibition using anti-IL-2-R mAbs. Induction of IL-2-R transcripts was detected in thymocytes stimulated by the anti-CD2 antibody combination alone or the anti-CD2 combination plus anti-CD28 antibody. However, induction of IL-2 transcripts was observed only in thymocytes triggered jointly by the anti-CD2 combination plus anti-CD28 antibodies. The double-negative (CD4-8-) or CD1+ thymocytes isolated by sorting or by panning were unresponsive to CD2/CD28 triggering. The same mitogenic signal could induce vigorous proliferation of thymocytes with a mature phenotype, i.e., CD3+CD4+ or CD3+CD8+ thymocytes. Immunofluorescence studies demonstrated that the majority of CD3+ thymocytes were CD28+, and most of the CD28+ cells were located in the medullary compartment of thymus. These results indicated that the T cell lineage surface molecules CD28 and CD2 are involved in the regulation of expansion and further differentiation of mature thymocytes.

Antibodies, Monoclonal↗

PubMed1988

Human thymic epithelial cells directly induce activation of autologous immature thymocytes.

To study the role that epithelial cells of the thymic microenvironment play in promoting activation of immature CD7+, CD2+, CD4-, CD8- (double-negative) human thymocytes, we have isolated thymocyte subsets from normal postnatal thymus and have cocultured autologous double-negative thymocytes with pure populations of thymic epithelial (TE) cells. We report that TE cells directly activate double-negative thymocytes to proliferate and that TE cells enhance the ability of double-negative thymocytes to proliferate in response to stimulation with exogenous interleukin 2. Activated double-negative thymocytes that proliferated in vitro in the presence of TE cells and interleukin 2 remained double-negative after 23 days in culture. Moreover, TE-cell culture supernatants in the absence of intact TE cells contain interleukin 1, interleukin 3, and granulocyte/macrophage-colony-stimulating factor activity for human bone marrow cells and can activate double-negative thymocytes to proliferate. Antibodies against interleukin 1 and against granulocyte/macrophage-colony-stimulating factor inhibited TE-cell-induced thymocyte activation. These data indicate that one role of TE cells in vivo may be to activate double-negative thymocytes to proliferate.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1988

Differentiation of human T cells.

This article discusses the ontogeny of human T cells along with the relationship of normal T-cell maturation to the development of various malignant T-cell syndromes. The impact of monoclonal antibody technology, the discovery of the T-cell receptor for antigen, and the discovery of mechanisms of thymocyte-thymic microenvironment interactions on the analysis of human T-cell development are emphasized.

Antibodies, Monoclonal↗

PubMed1987

Monoclonal antibodies to CD2 and lymphocyte function-associated antigen 3 inhibit human thymic epithelial cell-dependent mature thymocyte activation.

Recent study of human thymocyte-thymic epithelial (TE) cell interactions has demonstrated that thymocytes bind to TE cells, and a consequence of this binding is the provision of accessory cell signals by TE cells for phytohemagglutinin (PHA)-induced mature thymocyte activation. In this paper we report on studies of the molecules involved in TE cell-dependent mature thymocyte activation. TE-thymocyte interactions necessary for PHA-induced thymocyte activation were inhibited by monoclonal antibodies against the cluster of differentiation (CD)2 antigen on thymocytes and lymphocyte function-associated (LFA)-3 antigen on TE cells. Inhibition of TE accessory cell signals by antibodies against CD2 (alpha CD2) and LFA-3 (alpha LFA-3) antigens occurred early on during thymocyte activation and prevented thymocyte interleukin 2 receptor expression. Further, alpha CD2 and alpha LFA-3 inhibited PHA-induced thymocyte activation in whole thymic explant cultures suggesting a significant role of the CD2 and LFA-3 antigens in thymocyte activation when accessory cell signals for PHA-induced thymocyte triggering were delivered by cells within an intact thymic microenvironment.

Antibodies, Monoclonal↗

PubMed1987

Human thymic epithelial cells function as accessory cells for autologous mature thymocyte activation.

Using human thymocytes and autologous thymic epithelial (TE) cells grown in vitro in long-term culture, we have found TE cells can function as accessory cells for mitogen-induced mature thymocyte activation. Tritiated thymidine incorporation, blast formation, and protein synthesis were all induced in accessory cell-depleted thymocytes by autologous TE cells in the presence of suboptimal concentrations of PHA. After 3 days of mitogen stimulation of thymocyte-TE cell cocultures in vitro, thymocyte blasts bound to TE cells and 77 +/- 4% (mean +/- SEM) of TE cells acquired expression of major histocompatibility complex (MHC) class II (DR) antigen. TE accessory cell function for thymocyte activation was dependent on the number of TE cells added to thymocyte cultures, was not dependent on TE cell division, but did require TE cell protein synthesis. In thymocyte separation experiments, the predominant cell type responding to PHA in the presence of TE cells was T6- mature (stage III) thymocytes. Thus, human TE cells are capable of providing signals that lead to mature thymocyte activation.

Antigen-Presenting Cells↗

PubMed1986

Human thymocytes bind to autologous and allogeneic thymic epithelial cells in vitro.

The thymus plays a critical role in the generation of immunocompetent T lymphocytes. In the thymus, lymphocytes are in close contact with epithelial cells, and this contact is necessary for T-cell maturation. Using cultured human thymic epithelial (TE) cells, we have found that human thymocytes bind to human TE cells in vitro. Thymocytes bound to both allogeneic and autologous TE cells and to the epidermoid carcinoma cell line A431 but did not bind to epidermal keratinocytes or to thymic fibroblasts. Thymocyte binding to TE cells was trypsin- and cytochalasin B-sensitive. Indirect immunofluorescence assays showed that both mature (T6-, T3+) and immature (T6+, T3-) thymocytes bound TE cells. In our system, TE-thymocyte binding was not inhibited by antibodies to class I or class II major histocompatibility antigens. In vitro binding of thymocytes to TE cells may represent a correlate of in vivo TE-thymocyte interactions and provides a model system for the study of human intrathymic T-lymphocyte maturation and activation.

Antigens, Differentiation, T-Lymphocyte↗