A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry

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Induction of apoptosis in fibroblasts by c-myc protein

Fas antigen is a cell-surface protein that mediates apoptosis. It is expressed in various tissues including the thymus and has structural homology with a number of cell-surface receptors, including tumour necrosis factor receptor and nerve growth factor receptor. Mice carrying the lymphoproliferation (lpr) mutation have defects in the Fas antigen gene. The lpr mice develop lymphadenopathy and suffer from a systemic lupus erythematosus-like autoimmune disease, indicating an important role for Fas antigen in the negative selection of autoreactive T cells in the thymus.

Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis

The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis.

The p53 tumour suppressor gene is the most widely mutated gene in human tumorigenesis. p53 encodes a transcriptional activator whose targets may include genes that regulate genomic stability, the cellular response to DNA damage, and cell-cycle progression. Introduction of wild-type p53 into cell lines that have lost endogenous p53 function can cause growth arrest or induce a process of cell death known as apoptosis. During normal development, self-reactive thymocytes undergo negative selection by apoptosis, which can also be induced in immature thymocytes by other stimuli, including exposure to glucocorticoids and ionizing radiation. Although normal negative selection involves signalling through the T-cell receptor, the induction of apoptosis by other stimuli is poorly understood. We have investigated the requirement for p53 during apoptosis in mouse thymocytes. We report here that immature thymocytes lacking p53 die normally when exposed to compounds that may mimic T-cell receptor engagement and to glucocorticoids but are resistant to the lethal effects of ionizing radiation. These results demonstrate that p53 is required for radiation-induced cell death in the thymus but is not necessary for all forms of apoptosis.

p53 is required for radiation-induced apoptosis in mouse thymocytes

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.

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

On the basis of reports that deoxyguanosine is selectively toxic for adult T lymphocytes, the usefulness of this compound in the production of lymphocyte-depleted embryonic thymus rudiments in an in vitro organ culture system was investigated. The results showed that a period of exposure to deoxyguanosine causes depletion of the lymphoid cells while the stromal elements continue to survive, with many of the cells showing an epithelial morphology and expression of I region products of the major histocompatibility complex (MHC). When associated with either fetal liver or another thymus fragment as a source of T cell precursors in transfilter experiments, these "empty" thymuses become recolonized, enabling the production of chimeric thymus with stromal and lymphoid cells of different haplotypes. In combination with functional assays, this system offers an entirely in vitro approach to questions relating to the repertoire potential of T cell precursors from different sources and the role of the thymus in tolerance and MHC restriction.

Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: application in the production of chimeric thymus rudiments.

There is much interest in early T-cell development, particularly in relation to the diversification of the T-cell receptor repertoire1,2 and the elucidation of the lineage relationships between T-cell populations in the thymus and peripheral lymphoid organs (reviewed in refs 3, 4). However, the requirements for the growth of the earliest thymic T-cell precursor in 13–14-day mouse embryo thymus in isolation from the thymic environment are unknown. Proliferation and maturation of such cells are not sustained either in the presence of monolayers of thymic stromal cells (refs. 5, 6 and see below) or by the addition of interleukin-2 (IL-2), despite the expression of receptors for this growth factor on a proportion of thymocytes displaying the immature Thy 1+ Lyt-2−L3T4− phenotype in the embryonic thymus7,8. In contrast, when maintained within the intact thymic environment in organ cultures9,10, 13–14-day thymic stem cells do show a pattern of surface marker and functional development similar to that seen in vivo, suggesting that short-range growth signals, perhaps necessitating direct contact with organized epithelial cells, are required. We have shown, by exploiting the selective toxicity of deoxyguanosine (dGuo) for early T cells, that this organ culture system can be manipulated to produce alymphoid lobes that can be recolonized from a source of precursors in a transfilter system11. We now show that recolonization of alymphoid lobes can also be achieved by association with T-cell precursors in hanging drops, allowing recolonization by exposure to defined numbers of precursors, including a single micromanipulated stem cell. Analysis of T-cell marker expression in these cultures shows that a single thymic stem cell can produce progeny of distinct phenotypes, suggesting that these marker-defined populations are not derived from separate pre-thymic precursors, but arise within the thymus.

A single stem cell can recolonize an embryonic thymus, producing phenotypically distinct T-cell populations

Herein we have investigated the ability of antigen to induce thymocyte death by apoptosis on the basis that this may be the mechanism for the deletion of autoreactive cells during T cell development. We show that the ability of the bacterial "superantigen" staphylococcal enterotoxin B to cause specific depletion of V beta 8+ cells when added to thymus organ cultures is accompanied by DNA degradation into oligonucleosomal fragments, indicating that depletion involves apoptosis. Our results provide the first direct evidence that antigen-induced apoptosis can be triggered in developing T cells.

Antigen-induced apoptosis in developing T cells: a mechanism for negative selection of the T cell receptor repertoire

Foreign tissues grafted into healthy recipients are usually rejected by the hosts' immune system largely by means of major histocompatibility complex (MHC) products expressed on donor cells1. During ontogeny, developing T lymphocytes acquire tolerance to self-MHC antigens and the thymus has been considered as the most likely site for the abrogation of self-reactive clones2. We demonstrate here that embryonic thymus lobes, when organ cultured in the presence of deoxyguanosine, which is toxic to proliferating embryonic thymic lymphocytes but does not affect the epithelial framework, when transplanted to the kidney capsule of normal healthy histoincompatible mice, are not rejected despite their continued expression of both class I and class II donor MHC products3 but do not induce tolerance. This suggests that immunogenicity is not solely a function of MHC antigen expression but is also influenced by the type of cell upon which the antigens are expressed and, if the thymus is involved in the induction of tolerance to self-MHC products, this is a function of a component other than the epithelium, perhaps thymic dendritic cells.

Rosetta Kingston

Papers

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

Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: application in the production of chimeric thymus rudiments.

A single stem cell can recolonize an embryonic thymus, producing phenotypically distinct T-cell populations

Antigen-induced apoptosis in developing T cells: a mechanism for negative selection of the T cell receptor repertoire

Successful transplantation across major histocompatibility barrier of deoxyguanosine-treated embryonic thymus expressing class II antigens