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E J Jenkinson

Publications and source records attributed to E J Jenkinson.

At least 55 records · Page 3Linked to original sources
PubMed1994

Fetal thymic organ cultures.

Thymic organ cultures are currently the only system capable of supporting a full programme of T-cell development in vitro. Unmanipulated thymus lobes are useful for studying some aspects of T-cell development but are limited for studies on interactions between thymocytes and stromal cells by their cellular heterogeneity. However, techniques have now been developed for the association of defined stromal and lymphoid populations in organ culture where optimal conditions for the interaction are maintained. This approach is now being applied to study the role of individual stromal cell types in T-cell development and selection of the T cell receptor repertoire.

Animals↗
PubMed1994

Positive selection by purified MHC class II+ thymic epithelial cells in vitro: costimulatory signals mediated by B7 are not involved.

We have investigated the possibility that the costimulatory signals required for activation of mature T cells also play a role in providing differentiation signals for positive selection during T-cell development. We show that purified MHC Class II+ thymic epithelial cells are able to support positive selection in vitro but lack both the functional capacity to deliver costimulatory signals and expression of the costimulatory ligand B7. Our results suggest that the additional signals provided by costimulatory ligands are not required for TCR-mediated positive selection, although other ancillary signals provided by thymic epithelial cells may be involved.

Animals↗
PubMed1994

The role of macrophages and bone marrow-derived dendritic cells in the rejection of fetal thymus allografts.

Deoxyguanosine (dGuo)-treated fetal thymus lobes are capable of prolonged survival in histoincompatible recipients despite their expression of both class I and class II major histocompatibility complex (MHC) antigens. Although dGuo treatment has been directly shown to eliminate lymphocytes from the lobes its effect upon other marrow-derived passenger cells such as macrophages and dendritic cells is less well defined. Here we show that dGuo-treated CBA(H-2k) fetal thymus lobes allowed to develop under the renal capsule of immunoincompetent BALB/c (H-2d) mice for 3 weeks are depleted of donor-type dendritic cells in contrast to grafts of untreated lobes where donor-derived dendritic cells are still detectable at this time. Moreover, dGuo-treated thymus lobes underwent prompt allo-rejection if recolonized with donor-type dendritic cells prior to transplantation into immunocompetent recipients. Together with our observation that macrophages (or their precursors) survive dGuo treatment, these results suggest that the reduced immunogenicity of fetal thymus grafts seen following dGuo treatment is related to dendritic cell, rather than macrophage depletion.

Animals↗
PubMed1994

Developmental regulation of bcl-2 expression in the thymus.

An important factor in shaping the T-cell receptor (TcR) repertoire during thymocyte development is the susceptibility of double-positive (CD4+ CD8+) thymocytes to induction of apoptosis (negative selection) when the TcR is engaged by 'self'-antigens. Recent evidence has suggested that this susceptibility to apoptosis may be influenced by the expression of bcl-2, a proto-oncogene known to increase the resistance to apoptosis in various cell systems. Using a semi-quantitative polymerase chain reaction (PCR) technique in conjunction with staged embryonic material and purified thymocyte subpopulations we have investigated patterns of bcl-2 expression during normal T-cell development. Our results show that while bcl-2 alpha gene expression is readily detectable in immature CD3-CD4-CD8- thymocytes and in mature single-positive TcRhi cells, it is drastically reduced in TcR negative double-positive (CD3- CD4+ CD8+) cortical thymocytes of intermediate maturity. Careful mapping of bcl-2 alpha re-expression in relation to the onset of TcR expression within the population of embryonic thymocytes indicates that bcl-2 alpha is up-regulated as soon as TcR molecules are expressed on the surface of CD4+ CD8+ thymocytes. Therefore, thymocytes susceptible to apoptosis on TcR ligation express bcl-2 alpha mRNA suggesting that changing levels of bcl-2 expression are unlikely to be the only determinant regulating susceptibility to apoptosis in the thymus. The possible implications of these changes in bcl-2 expression regarding other facets of thymocyte development will be discussed.

Animals↗
PubMed1993

MHC class II-positive epithelium and mesenchyme cells are both required for T-cell development in the thymus.

T lymphocytes are produced in the thymus from precursors originating in the haemopoietic tissues. On entering the thymus, they undergo a programme of proliferation, T-cell receptor (TCR) gene rearrangement, differentiation and repertoire selection. Although the thymus provides a unique environment for these events, the role of the thymic stroma in regulating specific developmental stages is not well understood. We therefore devised an in vitro system to study the role of individual thymic stromal components in T-cell development. We report here that the development of TCR-CD4-CD8-T-cell precursors into TCR+ cells expressing CD4 and/or CD8 requires the presence of both major histocompatibility complex class II+ epithelial cells and fetal mesenchyme. The requirement for mesenchymal support can be mapped to the initial stages of intrathymic development because the later stages of maturation, from double-positive CD4+CD8+ thymocytes into single-positive CD4+ or CD8+ cells, can be supported by epithelial cells alone. We also show that the requirement for mesenchymal cells can be met by cells of the fibroblast line 3T3 (but not by supernatants from these cells). To our knowledge, these findings provide the first direct evidence that mesenchymal as well as epithelial cells are involved in T-cell development, and suggest that their involvement is stage-specific and likely to be dependent on short-range or contact-mediated interactions.

3T3 Cells↗
PubMed1993

Analysis of cytokine gene expression in subpopulations of freshly isolated thymocytes and thymic stromal cells using semiquantitative polymerase chain reaction.

Using a semi-quantitative polymerase chain reaction (PCR) technique we have examined the expression of a panel of cytokines during thymus development, localizing the expression to individual components of the thymic stroma and thymocytes at different maturational stages. The expression of interleukin (IL)-7, stem cell factor (SCF), IL-1 alpha and granulocyte-monocyte-colony-stimulating factor (GM-CSF) mRNA was mapped to individual stromal cell types, while the expression of IL-1 alpha and GM-CSF, along with interferon (IFN)-gamma and IL-4 was detected in the lymphoid compartment of fetal day (Fd) 14 thymus. The expression of lymphoid-specific cytokines genes was selectively down-regulated in thymocytes undergoing maturation. CD3-/lo4+8+ cells, representing an intermediate stage of thymocyte maturation, were devoid of cytokine gene expression. Their CD3+ progeny, on the other hand, expressed IFN-gamma mRNA, supporting the notion that positive selection of cells for further maturation induces the reexpression of some cytokine genes. The cytokine profiles of the various stromal components differed. Purified major histocompatibility complex class II+ cortical epithelial cells strongly expressed IL-7 and SCF, but only limited expression of IL-1 alpha and GM-CSF could be detected. Fetal mesenchyme, on the other hand, expressed SCF, IL-1 alpha and GM-CSF but not IL-7. The importance of these cytokine profiles in relation to T cell development is discussed.

Animals↗
PubMed1992

Apoptosis and T-cell repertoire selection in the thymus.

Thymic tolerance depends on induction of apoptosis (programmed cell death) in immature thymocytes by antigen/MHC complexes on dendritic cells (and possibly other bone marrow-derived APCs). Interactions with antigen/MHC complexes on thymic epithelial cells promote maturation of double-positive thymocytes to single-positive cells. However, the nature of the antigen/MHC complexes on thymic epithelial cells is unknown, and if the stromal cell interaction model as just outlined is correct, then presumably these complexes must be different from those presented on dendritic cells, otherwise all cells signaled for positive selection would be subject to negative selection by similar complexes on APCs. The nature of the signals provided by thymic epithelial cells versus dendritic cells is unknown and may provide a key to understanding the processes of positive and negative selection within the thymus. Clearly the intense selection of the T-cell repertoire within the thymus explains the high level of cell death observed within the immature thymocyte compartment. Such intensive selection shapes the T-cell repertoire in a way that provides an explanation for the genetic basis of immune responsiveness and for the susceptibility of certain individuals to autoimmunity.

Animals↗
PubMed1992

Studies on T cell maturation on defined thymic stromal cell populations in vitro.

We describe an in vitro system in which positive selection of developing T cells takes place on defined stromal cell preparations, which include major histocompatibility complex class II+ epithelial cells but exclude cells of bone marrow origin. In this system, maturation of double-positive T cell receptor negative (TCR-), CD4+8+ thymocytes into single-positive TCR+, CD4+ and CD8+ cells takes place together with the development of functional competence. As in vivo, this maturation is associated with the upregulation of TCR levels as cells progress from double-positive to single-positive status. We also show that class II+ epithelial cells in these cultures are less efficient than dendritic cells in mediating the deletion (negative selection) of V beta 8+ cells by the superantigen staphylococcal enterotoxin B. For the first time, this approach provides a model in which the cellular interactions involved in both positive and negative selection can be studied under controlled in vitro conditions.

Animals↗
PubMed1992

Inositol lipids and phosphates in the proliferation and differentiation of lymphocytes and myeloid cells.

It is established that receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate is an essential signalling reaction in the responses of many haemopoietic cells to stimuli: examples include platelet activation, antigen-driven initiation of cell proliferation in mature B and T lymphocytes and histamine release by mast cells, and chemotaxis and oxygen radical generation by neutrophils. However, the roles of inositol lipids and phosphates in the development of haemopoietic and immune cells are less well understood. This paper discusses three such situations: the sequential employment of phosphatidylinositol 4,5-bisphosphate hydrolysis and cyclic AMP accumulation as two signals essential to the action of the B lymphocyte-stimulatory cytokine interleukin 4; the involvement of antigen receptor-triggered inositol lipid hydrolysis in apoptotic elimination of immature anti-self T lymphocytes in the fetal mouse thymus; and the possible role of changes in the levels of abundant inositol polyphosphates in the differentiation of HL-60 promyelocytic cells and of normal human myeloid blast cells.

Bone Marrow Cells↗
PubMed1992

Effects of the thymic microenvironment on the response of thymocytes to stimulation.

We show that, in vitro, the response of thymocytes to certain stimuli, and their survival largely depend on the nature of the culture environment, i.e. whether thymocytes are stimulated within intact thymus lobes or in cell suspension. Exposure of isolated thymocytes to 12-O-tetra-decanoylphorbol 13-acetate (TPA)+ionomycin rapidly abolishes the expression of recombination-activating gene-1 (RAG-1) mRNA (3 h), down-regulates CD4 surface antigen expression (3 h), and enhances apoptosis (24 h). On the other hand, when thymocytes are cultured in intact lobes, TPA plus ionomycin down-regulate rather than abolish RAG-1 mRNA expression (3 h), have little effect on CD4 expression even following 24-h exposure, and only marginally induce apoptosis (24 h). Differences between the culture systems are less pronounced in response to anti-CD3 antibodies. Therefore, it appears that removing thymocytes from their thymic microenvironment makes the cells more susceptible to certain stimuli, possibly by altering their physiological status. In addition, it has been suggested that termination of RAG-1 expression can be linked to thymocyte selection processes. We found that the down-regulation of RAG-1 expression was not dependent on the induction of apoptosis, supporting a proposed link with positive selection.

Animals↗
PubMed1990

Alkaline phosphatase-fast red, a new fluorescent label. Application in double labelling for cell cycle analysis.

We have observed that the red reaction product of alkaline phosphatase immuno-conjugates and certain substrate preparations produces a brilliant red fluorescence that is visible by fluorescence microscopy using both fluorescein and rhodamine filter combinations. This provides a level of sensitivity greater than that obtained with other commonly used red fluorochromes or by inspection of the reaction product under bright field illumination. Of particular value, the reaction product is unaffected by the denaturing conditions required for the detection of incorporated nuclear BrdU with FITC conjugated anti-BrdU antibody and provides a simple and robust method for the simultaneous detection of cell proliferation and cell surface markers.

Alkaline Phosphatase↗
PubMed1990

Cell growth and gene rearrangement signals during the development of T lymphocytes within the thymus.

The thymus provides signals that control the proliferation and differentiation of T lymphocytes and select the repertoire of T-cell specificities. Antibodies to CD3 molecules inhibit full rearrangement of T-cell receptor beta chain genes in organ cultures of early embryo mouse thymus. Whether this effect is mediated through gamma delta CD3 expressing cells, which are present in small numbers at this stage, or through low amounts of CD3 on alpha beta precursor cells is unclear. A requirement for special gene rearrangement signals within the thymus is supported also by the observations that growth factors such as IL-2 and IL-4, although stimulating proliferation of precursor cells removed from the thymus, do not induce full T-cell receptor gene rearrangements. Recent studies show that newly formed thymic lymphocytes expressing alpha beta CD3 receptors are targets for negative selection (deletion) as a means of removing autoreactive cells. Signalling to immature thymocytes via the alpha beta CD3 complex induces the activation of endogenous endonucleases that cleave DNA into oligonucleosomal fragments. We suggest that the activation of this mechanism is the means by which autoreactive cells are removed.

Animals↗
PubMed1990

Newly generated thymocytes are not refractory to deletion when the alpha/beta component of the T cell receptor is engaged by the superantigen staphylococcal enterotoxin B.

It has been reported that, following the initial expression of the T cell receptor (TcR) alpha/beta, newly generated thymocytes pass through a developmental window characterized by ineffective coupling between the alpha/beta and CD3 components resulting in resistance to deletion (negative selection). However, we now provide evidence that the TcR alpha/beta on developing thymocytes is capable of delivering deletional signals in response to the superantigen staphylococcal enterotoxin B (SEB) as soon as the receptor is expressed. We also show that if TcR+ thymocytes are allowed to mature in organ cultures of embryonic thymus before SEB is added, they respond by proliferation giving rise to blast cells of CD4-CD8-, CD4+CD8- or CD4-CD8+ phenotypes.

Animals↗
PubMed1990

Developmentally regulated fetal thymic and extrathymic T-cell receptor gamma delta gene expression.

The gamma delta T-cell receptor (TCR) is the first TCR to be expressed in ontogeny in all vertebrates in which it has been examined thoroughly. Murine gamma delta cell-surface protein is detected by the fourteenth day of gestation. In this work, the activation of gamma delta RNA has been studied. Data indicate that the first TCR protein to appear in the thymus is encoded by gamma genes that are activated after cells colonize the thymus. However, the sequential appearance of different gamma delta TCR proteins during thymic ontogeny cannot be readily explained by differential temporal activation of V gamma genes in the thymus. There are distinct patterns of gamma and delta gene expression during fetal liver development and in the fetal gut (or tissue associated with it). Cells apparent in the liver of mice at birth express gamma delta cell-surface protein, but they disappear from the liver very soon afterward. One V gamma gene is rearranged and expressed prethymically. In addition, gamma gene expression is detectable in the livers of newborn athymic mice. Together, these observations indicate a thymic-independent pathway of activation of TCR genes.

Animals↗
PubMed1990

T-cell differentiation in thymus organ cultures.

Fetal thymus organ cultures support a full range of T-cell precursor differentiation in vitro, including TCR gene rearrangement and expression. This provides an accessible model system in which the intra-thymic regulation of T-cell development can be investigated. Thymus organ cultures can be manipulated by adding antibodies to block different cell surface components on stromal or lymphoid elements. In addition, it is possible to deplete thymus lobes of their lymphoid cells and recolonize them with T-cell precursors of a different MHC haplotype to produce chimeric lobes. Recolonization can also be achieved with defined numbers or types of precursor cells, including a single micro-manipulated cell. These approaches have been used to obtain information on the signals regulating intra-thymic proliferation, T-cell lineage relationships, antigen receptor diversification within the thymus and the cellular interactions and intracellular mechanisms regulating selection of the antigen receptor repertoire.

Fetus↗
PubMed1989

Apoptosis.

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Animals↗
PubMed1989

Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures.

The receptors found on most T lymphocytes bind to antigen presented on major histocompatibility complex proteins and consist of dimers of alpha- and beta-polypeptides associated with the invariant CD3 complex. A fully competent immune system requires a diverse array of T-cell antigen receptors (TCRs) with different specificities. This diversity is generated by rearrangement of TCR alpha- and beta-chain gene segments within the thymus where the receptors are first expressed. Any cells carrying self-reactive receptors must be eliminated, suppressed or inactivated so that destructive autoimmunity is avoided. Recently, compelling evidence has shown that one process involved in producing such self-tolerance is clonal deletion of autoreactive cells within the thymus by an as-yet-undefined mechanism. Here we show that engaging the CD3/TCR complex of immature mouse thymocytes with anti-CD3 antibodies produces DNA degradation and cell death through the endogenous pathway of apoptosis. Activation of this process in immature T cells by the binding of the TCR to self-antigens may therefore be the mechanism which produces clonal deletion and consequently self-tolerance.

Animals↗