Biomedical subjects

E J Jenkinson

Publications and source records attributed to E J Jenkinson.

At least 19 recordsLinked to original sources

PubMed1999

Factors regulating stem cell recruitment to the fetal thymus.

Colonization of the thymic rudiment during development is initiated before vascularization so that hemopoietic precursors must leave the pharyngeal vessels and migrate through the perithymic mesenchyme to reach the thymus, suggesting that they may be responding to a gradient of chemoattractant factors. We report that diffusible chemoattractants are produced by MHC class II+ epithelial cells of the fetal thymus, and that the response of precursors to these factors is mediated via a G protein-coupled receptor, consistent with factors being members of the chemokine family. Indeed, a number of chemokine receptors are expressed by thymic precursors, and several chemokines are also expressed by thymic epithelial cells. However, these chemokines are also expressed in a tissue that is unable to attract precursors, although the thymus expressed chemokine, TECK, is expressed at higher levels in thymic epithelial cells and we show that it has chemotactic activity for isolated thymic precursors. Neutralizing Ab to TECK, however, did not prevent thymus recolonization by T cell precursors, suggesting that other novel chemokines might be involved in this process. In addition, we provide evidence for the involvement of matrix metalloproteinases in chemoattractant-mediated T cell precursor recruitment to the thymus during embryogenesis.

Animals↗

PubMed1999

CD69 expression discriminates MHC-dependent and -independent stages of thymocyte positive selection.

In the thymus, phenotypically and functionally mature single positive cells are generated from immature CD4+8+ precursors by a process known as positive selection. Although this event is known to involve alphabetaTCR ligation by peptide/MHC complexes expressed on thymic stromal cells, it is clear that positive selection is a multistage process involving transition through an intermediate CD4+8+69+ phase as well as subsequent postselection phases. By analyzing the development of preselection CD4+8+69- and intermediate CD4+8+69+ thymocytes in the presence of MHC class I-deficient, MHC class II-deficient, and MHC double-deficient thymic stromal cells, we investigated the role of MHC molecules at three distinct points during positive selection. Although the initiation of positive selection is critically dependent upon MHC interactions, we find the that later stages of maturation, involving the differentiation of CD4+8- and CD4-8+ cells from CD4+8+69+ thymocytes, occur in the absence of MHC molecules. Moreover, an analysis of the postselection proliferation of newly generated CD4+8- and CD4-8+ thymocytes shows that this also occurs independently of MHC molecules. Thus, our data provide direct evidence that, although positive selection is a multistage process initiated by TCR-MHC interactions, continuation of this process and subsequent postselection events are independent of ongoing engagement of the TCR.

Animals↗

PubMed1999

A novel method of cell separation based on dual parameter immunomagnetic cell selection.

We describe a novel method of cell purification involving two stage immunomagnetic selection which permits isolation of cells based on a second cell surface marker without the need for removal of beads used in the first selection step. This approach takes advantage of the size differences between commercially available immunomagnetic beads and/or particles and their differing properties in terms of attraction to magnetic fields of various strengths. The first stage of separation involves positive selection of cells using the Miltenyi MiniMacs system, utilising 50 nm MicroBeads and a MiniMacs magnet. Cells obtained from this procedure--still rosetted with 50 nm MicroBeads--can then be subjected to further positive or negative selection using either streptavidin M280 or anti-rat M450 Dynabeads, without the need for prior bead removal, since the strength of the magnetic field of the Dynal separator is sufficient to attract the larger Dynabeads but not the MicroBeads. Here, we show that this system can be used to isolate a number of cell types including very rare target cell populations such as haemopoietic stem cells, using two different surface markers without perturbing subsequent functional capacity.

Animals↗

PubMed1999

Studies on the phenotype of migrant thymic stem cells.

Stem cells first enter the thymus around the 11th to 12th days of gestation in BALB/c mouse embryos. The phenotype of these stem cells has been difficult to determine because their entry occurs when the thymic primordium is very small and involves too few stem cells to allow studies by flow cytometry. We have been able to microdissect the thymus from embryos during this stage and immunophenotype cells in sections using a sensitive tyramide amplification system. Our results show that migrant stem cells express CD45, c-kit, CD44, CD34 and alpha4 integrin, but other markers such as CD62L, CD25, Thy-1.2, CD3epsilon, alpha5 integrin and RAG-1 expression are detected only after stem cell entry. These results should help to improve the isolation and characterization of migrant thymic stem cells.

Animals↗

PubMed1999

In vitro models of T cell development.

A number of in vitro systems are currently being used to study both thymocyte development and thymic stromal cell function. However, the usefulness of dispersed culture systems is limited since they often involve disruption of interactions within the normal three-dimensional architecture of the thymus in vivo which are critical for normal development to proceed. In contrast, Fetal Thymus Organ Culture (FTOC) provides an experimental system where such interactions are maintained, thereby allowing in vitro access to key aspects of thymocyte development. More recently, Reaggregate Thymus Organ Cultures (RTOCs) have allowed detailed analysis of thymic stromal cell function, while retroviral transfection of thymocyte subsets under FTOC conditions provides a rapid means to investigate thymocyte development at the molecular level. Current use of the FTOC approach is summarised here, and where appropriate is compared to the use of dispersed culture systems.

Animals↗

PubMed1998

Differential effects of peptide diversity and stromal cell type in positive and negative selection in the thymus.

Thymocyte positive selection results in maturation to the single-positive stage, while negative selection results in death by apoptosis. Although kinetic analyses indicate only 3-5% of CD4+ 8+ cells reach the single-positive stage, the balance of positive and negative selection and the nature and quantity of cells mediating maximal negative selection are uncertain. Here, using a system where the number and type of stromal cells and thymocytes can be controlled, we investigated the maturation of CD4+ 8+ thymocytes in the presence or absence of thymic epithelium and dendritic cells (DC) from wild-type (wt) and H-2M(-/-) mice expressing different peptide arrays. We find that titration of wt DC into reaggregates of wt epithelium has a dramatic effect on the number of CD4+ cells generated, with 1% DC causing a maximal 80% reduction. Moreover, while addition of 1% wt DC into cultures of H-2M(-/-) epithelium causes a 90% reduction in CD4+ cells, no effect was observed when similar numbers of wt thymic epithelium were added. Collectively, these data provide the first accurate indication of the quantity and quality of stromal cells required for maximal negative selection in the thymus, demonstrate the importance of peptide diversity in T cell selection, and highlight a large degree of overlap between positive and negative selection events.

Animals↗

PubMed1998

Use of explant technology in the study of in vitro immune responses.

The establishment of in vitro culture systems provides an accessible means to study events within the immune system. In contrast to either dispersed suspension or two-dimensional monolayer culture, the explantation of tissue fragments under organ culture conditions is, to date, the only method which allows essential three-dimensional cellular interactions to be maintained under conditions which permit controlled experimental manipulation in vitro. Recent modifications of explant technology, particularly within the area of fetal thymic organ culture, now allow the controlled reassociation of defined cellular subsets and manipulation of gene expression, under conditions where the functioning of both lymphoid and stromal cell types closely resembles that in vivo.

Immunity↗

PubMed1998

Identification of a developmentally regulated phase of postselection expansion driven by thymic epithelium.

To investigate events following the initiation of positive selection, we have used reaggregate organ cultures to follow the maturation of purified CD4+8+69+ thymocytes; these thymocytes represent a subpopulation of thymocytes which have already received positive selection signals. Using a dilution analysis of an FITC-based membrane-binding dye, 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester, to allow a quantitative measure of proliferation, we show that while newly selected CD4+ and CD8+ cells are nondividing, both subsets subsequently undergo a wave of postpositive selection proliferation involving multiple cell divisions. Moreover, in the presence of fetal stromal cells, postselection expansion is more extensive in newborn thymocytes compared with adult thymocytes, suggesting that this phase of expansion is developmentally regulated. We also show that proliferation of CD4+ and CD8+ cells is seen in reaggregates of purified MHC class II+ thymic epithelial cells, while CD4+ and CD8+ cells generated from bcl-2 transgenic CD4+8+69+ thymocytes in the absence of stromal cell support survive but do not proliferate; this observation indicates that MHC class II+ thymic epithelial cells are both necessary and sufficient to mediate this wave of cell division. Finally, the maturation of CD4+8+69+ thymocytes and the subsequent proliferation of CD4+ and CD8+ cells occur in the presence of MHC-mismatched thymic stromal cells, suggesting that the later stages of positive selection and the associated postselection events do not depend on interactions with the same peptide/MHC complexes responsible for initiation.

Animals↗

PubMed1998

Cellular interactions in the thymus regulate the protein kinase C signaling pathway.

We provide evidence that thymocytes receive signals from the thymic microenvironment which regulate the protein kinase C (PKC) signaling pathway. Thus, phorbol 12-myristate 13-acetate (PMA) causes a PKC-dependent down-regulation of CD4 expression and induces apoptosis in isolated thymocytes but has little effect on thymocytes maintained within intact thymic lobes or in reaggregate lobes containing purified thymocytes with either thymic or non-thymic stromal cells. Moreover, compact pellets of thymocytes alone are protected from the effects of PMA. This protection is maintained when the compacted thymocytes are rigorously depleted of MHC class II-expressing cells. We conclude that signals arising from thymocyte-thymocyte contact control the utilization of the PKC cascade. These observations have implications for thymocyte signaling in general as well as for the interpretation of studies carried out on thymocyte suspensions.

Animals↗

PubMed1997

Fibroblast dependency during early thymocyte development maps to the CD25+ CD44+ stage and involves interactions with fibroblast matrix molecules.

We have investigated the role of specific components of the thymic stroma during development of CD4-8-T cell precursors by separating and reaggregating precursor subsets with individual or combinations of stromal cells. We show that while the development of CD25+ 44+ precursors is dependent upon a combination of major histocompatibility complex (MHC) class II+ thymic epithelial cells and fibroblasts, their direct descendants, CD25+ 44- precursors, develop to the CD4+ 8+ stage in the presence of MHC class II+ thymic epithelial cells alone. Thus, CD25+ 44+ precursors are the last developmental stage to be dependent upon fibroblast support. In addition, while metabolically inactive, 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide (ECDI)-treated fibroblasts retain the ability to promote T cell development, prior treatment with hyaluronidase abrogates this effect, suggesting that fibroblast-associated extracellular matrix components are the key elements involved. In support of this, we show that fibroblasts are located in cortical regions of the thymus where T cell precursors are known to reside, and that these fibroblasts are associated with an extensive extracellular matrix not found on thymic epithelial cells. Finally, antibodies to alpha 4 integrin and CD44 interfere with the efficiency with which CD4+ 8+ cells are generated from CD25+ 44+ precursors in reaggregate cultures and also reduce the binding of the latter to 3T3 fibroblasts, suggesting these molecules play a role in bringing T cell precursors into contact with fibroblast-associated extracellular matrix.

3T3 Cells↗

PubMed1997

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

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

Animals↗

PubMed1997

Anti-alpha 4 integrin antibody induces apoptosis in murine thymocytes and staphylococcal enterotoxin B-activated lymph node T cells.

We have shown that an antibody (9C10) to the alpha 4 integrin induces apoptosis in murine immature CD4+ CD8+ thymocytes and in activated (but not resting) mature lymph node T cells. In both cases, apoptosis is blocked by the highly selective protein kinase C (PKC) inhibitor Ro31.8425, suggesting that 9C10 induces signalling through the alpha 4 integrin resulting in PKC activation leading to apoptosis. Overall, our results indicate the potential role of the alpha 4 integrin-mediated interactions in apoptosis induction during T-cell development and following mature T-cell activation.

Animals↗

PubMed1996

Intracellular signaling pathways involved in the induction of apoptosis in immature thymic T lymphocytes.

We describe a novel technique for studying the signaling pathways that control thymocyte negative selection which maintains the essential interactions between thymocytes and thymic stromal cells. Bisected lobes from newborn mouse thymus are maintained in organ culture for up to 36 h, and the thymocytes analyzed by flow cytometry. Inclusion of [3H]inositol during culture allows measurements of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2) hydrolysis and inositol phosphate accumulation. Using this technique we have compared the thymocyte responses induced by anti-CD3, anti-Fas, Con A, and beta-adrenergic stimulation. We show that PtdIns(4,5)P2 hydrolysis precedes anti-CD3-induced thymocyte apoptosis, but not the apoptosis induced by anti-Fas. In contrast, Con A stimulates PtdIns(4,5)P2 hydrolysis, but does not induce thymocyte apoptosis. Anti-CD3, anti-Fas, and Con A all fail to change thymic cAMP levels, but beta-adrenergic stimulation causes a large increase in intracellular cAMP, and agents that elevate cAMP induce thymocyte apoptosis. Inhibition of protein synthesis (with cycloheximide or emetine) prevents the apoptosis induced by anti-CD3 and elevated cAMP, but not that induced by anti-Fas, whereas protease inhibition (with 3,4-dichloroisocoumarin or N(alpha)-tosyl-phenylalanine chloromethyl ketone) prevents the apoptosis caused by all of the effective stimuli. These results offer three important conclusions. First, activation of a variety of different signaling pathways can bring about thymocyte apoptosis. Second, ligation of the thymocyte TCR/CD3 complex provokes PtdIns(4,5)P2 hydrolysis, but signaling through this pathway alone does not necessarily lead to apoptosis. Third, by whichever signaling pathway the response is initiated, the activity of one or more protease enzymes appears to form an essential component in the final common pathway leading to apoptosis.

Adrenergic beta-Agonists↗

PubMed1996

Involvement of LFA-1/ICAM-2 adhesive interactions and PKC in activation-induced cell death following SEB rechallenge.

Ligation of T-cell receptor (TCR) causes mature T cells to proliferate or, on re-exposure to antigen, can cause them to die by activation-induced cell death (AICD). In proliferative responses, costimulatory and adhesive interactions are required and activation of protein kinase C (PKC) has been shown to be essential. Whether or not interactions involving costimulatory signals and PKC have a role in facilitating AICD remains unclear. Here we have examined the role of CD28/B7 and leucocyte function associated antigen-1 (LFA-1)/intracellular adhesion molecule (ICAM) mediated interactions in AICD triggered by staphylococcal enterotoxin B (SEB) in murine lymph node T cells. We show that, after a primary proliferative response to SEB, LFA-1/ICAM-2 adhesive interactions can play a part in AICD following SEB rechallenge, while B7 and ICAM-1 mediated interactions are not essential for this process. In addition, using a highly selective PKC inhibitor, Ro31.8425, we show that PKC activation is essential for the regulation of AICD by SEB rechallenge.

Animals↗

PubMed1996

Cellular interactions in thymocyte development.

Interactions between stromal cells and thymocytes play a crucial role in T cell development. The thymic stroma is complex and consists of epithelial cells derived from the pharyngeal region during development, together with macrophages and dendritic cells of bone marrow origin. In addition, fibroblasts and matrix molecules permeate the whole framework. It is now apparent that these individual stromal components play specialized roles at different stages of T cell differentiation. Thus, at the early CD4-8- stage of development, T cell precursors require fibroblast as well as epithelial cell interactions. Later, at the CD4+8+ stage, as well as providing low avidity TCR/MHC-peptide interactions, thymic epithelial cells have been shown to possess unique properties essential for positive selection. Dendritic cells, on the other hand, are probably efficient mediators of negative selection, but they may not be solely responsible for this activity. Alongside the functional roles of stromal cells, considerable progress is being made in unraveling the nature of the signaling pathways involved in T cell development. Identification of the pre-T cell receptor (pre-TCR) and associated signaling molecules marks an important advance in understanding the mechanisms that control gene rearrangement and allelic exclusion. In addition, a better understanding of the signaling pathways that lead to positive selection on the one hand and negative selection on the other is beginning to emerge. Many issues remain unresolved, and some are discussed in this review. What, for example, is the nature of the chemotactic factor(s) that attract stem cells to the thymus? What is the molecular basis of the essential interactions between early thymocytes and fibroblasts, and early thymocytes and epithelial cells? What is special about cortical epithelial cells in supporting positive selection? These and other issues are ripe for analysis and can now be approached using a combination of modern molecular and cellular techniques.

Animals↗

PubMed1995

Positive selection of thymocytes involves sustained interactions with the thymic microenvironment.

CD4+8+ cortical thymocytes are critically dependent upon interaction with the thymic epithelium to undergo positive selection and maturation into single-positive CD4+ or CD8+ cells. Here we investigate further the nature of this interaction and provide evidence that positive selection requires sustained, rather than "single hit," interaction with thymic stromal cells. We also show that calcineurin-mediated signaling in thymocytes is required for the initial stages of positive selection, but is not essential throughout the period of thymocyte dependence on stromal cell contact during positive selection. In addition, we show that double-positive thymocytes that have initiated positive selection (CD69+4+8+) and newly generated single-positive (CD69+4+) cells differ markedly in response to the same stimulus through the TCR. The former undergo deletion, whereas the latter proliferate, indicating that a critical change in response to TCR ligation occurs within the narrow developmental window between these two stages.

Animals↗

PubMed1995

Stimulation of thymocytes before and after positive selection results in the induction of different NF-kappa B/Rel protein complexes.

Positive selection triggers the differentiation of immature CD4+8+TCRlow thymocytes into TCRhigh single-positive CD4+ or CD8+ cells and is associated with major changes in gene expression. However, little is known about the DNA binding factors controlling these fundamental changes. Here we have examined NF-kappa B/Rel subunit expression and DNA-binding activity in developing thymocytes before and after the induction of positive selection. We show that positive selection is accompanied by the strong up-regulation of c-rel mRNA expression and the constitutive activation of p50/p65 and p50/c-Rel NF-kappa B/Rel complexes, confirming the activation-like status of cells undergoing positive selection. Moreover, CD69+ cells that have initiated positive selection (but not their preselection CD4+8+TCR- precursors) respond to stimulation by the preferential activation of c-Rel-containing DNA-binding complexes. Because the different NF-kappa B/Rel dimers have distinct transcriptional activities and binding site preferences, this preferential activation of c-Rel-containing DNA-binding complexes may well have implications for the changes in gene expression and functional response associated with positive selection.

Amino Acid Sequence↗

PubMed1995

Intracellular signaling events during positive and negative selection of CD4+CD8+ thymocytes in vitro.

We have used in vitro models of thymocyte positive and negative selection in conjunction with selective inhibitors of the TCR-mediated signaling cascade to investigate the intracellular signaling events that mediate these processes. We report that Ro 31.8425, a potent and selective inhibitor of protein kinase C, which blocks the activation of mature T cells in a dose-dependent fashion, has no effect on either positive or negative selection of CD4+8+ thymocytes. In contrast, cyclosporin A fails to prevent negative selection, but inhibits positive selection through a direct effect on developing thymocytes, rather than through the perturbation of stromal cell support. Thus, our data suggest that positive and negative selection may operate via distinct intracellular signaling pathways.

Animals↗