Generation of T-cell function in organ culture of foetal mouse thymus. II. Mixed lymphocyte culture reactivity.

The generation of MLC reactive cells in organ cultures of embryonic mouse thymus is described. At least 4 days in culture are required before MLC reactive thymocytes are detectable in this system, which is in marked contrast to the short incubation period required for the expression of Thy-1 on T-cell precursors in other systems. Our data demonstrates the generation of T-cell subsets independent of influences from peripheral lymphoid organs, and in the absence of B cells or their products. One very important application of this technique is in the investigation of the mechanism of T-cell tolerance to self.     

Differentation of foetal mouse thymus. Ultrastructure of organ cultures and of subcapsular grafts

Thymuses removed from 12–13-day-old foetal (C57×CBA)F₁ mice, were grown in organ culture for up to 25 days. Initially the thymuses consisted predominantly of undifferentiated epithelial cells and some large lymphoblasts. The epithelial cells differentiated rapidly but epithelial cells in mitosis were seen throughout the entire culture period. The lymphoblasts, however, showed a restricted period of proliferation between the 2nd and 10th days of culture which resulted in the production of large numbers of typical small lymphocytes. Many of these cells died in situ but even after 25 days many apparently viable small lymphocytes were present.

Some 14-day cultures were implanted beneath the kidney capsule of young neonatally thymectomized or sham thymectomized syngeneic mice. These grafts lost their complement of small lymphocytes within the first 24 hours but their epithelial cells rapidly started to proliferate and the grafts became infiltrated with large lymphoblasts. After 1 week a small thymus was reformed and after 6–10 weeks up to 50 mg of thymic tissue could be obtained from each graft. Electron microscopy showed that the cortex of the graft was indistinguishable from a normal thymus but the medulla was small and contained few characteristic medullary epithelial cells.

     

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.     

Effects of cyclosporin A on T-cell development in organ-cultured foetal thymus.

The effects of cyclosporin A (CsA) on T-cell development were assessed in an organ culture of murine foetal thymus. Applying three-colour flow cytometric analysis, we showed that the agent inhibits the development of mature CD3/T-cell receptor alpha beta (TcR alpha beta)+ cells both in CD4+8- and CD4-8+ populations. CD4-8- cells appeared to be accumulated by CsA. We examined the heterogeneity of CD4-8- cells generated in the organ culture, and defined five subpopulations by the expression of the cell-surface molecules CD3/TcR, J11d and CD25. It has been demonstrated that only the CD3/TcR alpha beta+ J11d- CD25- subpopulation is susceptible to the suppressive effects of CsA among CD4-8- cells, whereas all the other four subpopulations, including CD3/TcR gamma delta+ cells, are resistant. Thus, all of the TcR alpha beta-bearing cells, including CD4-8- cells but none of the TcR alpha beta- cells, are CsA sensitive. Because it is known that CsA inhibits the TcR-mediated signalling events in mature T cells and that signallings mediated via the interaction of TcR with major histocompatibility complex (MHC) molecules on thymic stroma cells are crucial for thymic selection of T cells, these results indicate that TcR alpha beta-bearing CD4-8- cells but not TcR gamma delta-bearing CD4-8- cells undergo thymic positive selection.     

The effects of interleukin 2 on early and late thymocyte differentiation in foetal thymus organ culture

The effects of interleukin 2 (IL-2) on in vitro T cell development in organ cultures of day 14 foetal mouse thymus were investigated. Over a 12 day culture period IL-2 was found to inhibit the development of each of the major thymocyte subpopulations, defined by CD4 and CD8 expression. Since each subpopulation was reduced in number, a common precursor to these subsets was thought to be inhibited by IL-2. Indeed, subsequent analyses of subsets of CD4- CD8- cells, characterized by expression of HSA, Pgp-1, and IL-2R, demonstrated that CD4- CD8- cells expressing IL-2R and their postulated progeny were reduced in IL-2-treated cultures. The number of cells reputed to be the immediate precursor of CD4- CD8- IL-2R+ cells, i.e. CD4- CD8- Pgp-1+IL-3R- cells, was not change by treatment with IL-2. Interestingly, however, numbers of the most immature subset of CD4- CD8- cells, identified as having the HSA1oPgp-1+IL-2R- and CD3- phenotype, increased in IL-2-treated cultures and was the only population of cells to do so. We also document the failure to detect any LAK cell activity against syngeneic thymocytes in IL-2-treated cultures and therefore hypothesize that the inhibition of T cell differentiation in these cultures is due to an arrest in differentiation of CD4- CD8- cells bearing the IL-2R.     

Tolerance of self induced in thymus organ culture.

F liver protein occurs in serum at low concentration, and therefore induces tolerance of self only in T cells. T cells which mature in cultured thymus lobes in the absence of this protein become reactive towards it but can be prevented from doing so by exposure to the protein while in culture. The threshold of tolerance induction for this soluble antigen is estimated in this way at approximately 1 microgram/ml, which is slightly less than the threshold of response of primed T cells in a proliferation assay. Freshly isolated thymocytes do not display reactivity to self-F protein, indicating that T cells normally become tolerant while still within the thymus.     

The effects of plasma from patients with Graves' disease on foetal mouse hearts in organ culture.

Plasma, obtained during plasma exchange therapy, from 3 euthyroid patients with Graves'' disease and severe progressive exophthalmos induced an increase in heart rate and then early death when applied to foetal mouse hearts maintained in isolated organ culture. All plasma samples which induced an increase in foetal heart rate had high titres of thyroid stimulating immunoglobulins. Plasma samples obtained after exchange had a much diminished effect. These studies may indicate a previously unrecognized non-thyroidal action of the abnormal immunoglobulins associated with Graves'' disease and suggest that chronic thyroid heart disease may be due, at least in part, to the effect of these immunoglobulins especially when not associated with elevated thyroid hormones concentrations.     

Coxsackievirus B4 infection of murine foetal thymus organ cultures

The infection of foetal thymus with coxsackievirus B4 (CV-B4) E2 has been studied ex vivo by using CD-1 mice on foetal day 14, as a ready source of organs for experimentation to investigate the hypothesis of the role of thymic viral infections in the pathogenesis of type 1 diabetes. The replication of CV-B4 E2 in murine foetal thymus organ cultures has been demonstrated by evaluating the levels of positive- and negative-stranded viral RNA in cells by using a real-time quantitative RT-PCR method and by determining titres of infectious viral particles in culture supernatants for 7 days post-infection (p.i.). Staining of tissue sections with an anti-cytokeratin antibody and haematoxylin-eosin showed that CV-B4 infection had no visible effect on cell survival and organ integrity. Cell counts in mock- and virus-infected foetal thymus organ cultures increased from day 1 through day 7, and live cell numbers were comparable in both conditions as shown by Trypan blue exclusion test and 7-amino-actinomycin D staining of thymocytes. Compared with controls on day 7 p.i., cytofluorometric analyses on cells from CV-B4 E2-infected foetal thymus organ cultures displayed a marked increase in the percentage of the most immature CD3(-)CD4(-)CD8(-) thymocytes, and a decrease in the percentage of immature CD3(-)CD4(+)CD8(+) cells, together with an increase in the percentage of mature CD3(+)CD4(+) and CD3(+)CD8(+) cells. These data show that CV-B4 E2 disturbs T-cell maturation and differentiation processes in infected murine foetal thymus organ cultures and provide evidence of a suitable system to investigate the effect of viruses in T-cell differentiation.     

Human foetal kidney explant in serum-free organ culture

Summary The purpose of the work was to develop an in vitro model for the study of human kidney development. Human metanephric explants from foetuses 10–18 weeks of gestational age were cultured in serum-free Leibovitz L-15 medium without hormones. Under the current minimal conditions for growth, the system permitted to maintain the renal tissues in culture for periods up to 9 days, although no evident sign of morphological differentiation was observed. However, during the studied period the overall architecture of the explants was preserved as well as the ultrastructural features of cytoplasmic organelles. The incorporation of ³H-thymidine and ³H-leucine indicated that DNA and protein synthesis was maintained or increased. Glycoprotein synthesis evaluated by ³H-glucosamine incorporation and radioautography continued in mesangium as well as in glomerular and tubular basement membranes. Alkaline phosphatase, -glutamyltranspeptidase (brush border) and catalase (peroxisomes) activities remained histochemically active. The proposed organ culture system appears as a reliable and promising model that will provide basic data on the morphology and functional characteristics of the developing kidney. Since it is achieved in a completely controlled environment, it will permit to study the role of growth factors and hormones in proliferation and differentiation of the cell populations during development of the human foetal kidney.     

Organ culture studies of nude mouse thymus.

There is evidence that peripheral lymphoid organs of nude mice, born from homozygous matings, contain a small proportion of theta-positive lymphocytes indicating that nude mice may not be totally devoid of T cell function. It has been suggested that such lymphocytes may develop within the dysplastic nude thymus itself. While this suggestion receives no support from morphological studies, it has been claimed that on explanation to organ culture the developing nude thymus becomes lymphoid. In this present study we confirm the presence of theta-positive lymphocytes in peripheral lymphoid tissues of homozygous nude mice born of nude parents. However, when we have organ-cultured nude thymus, explained from homozygous nude embryos at days 13, 14, 16 and 18 of gestation, we have found no histological sign of lymphopoiesis nor have we detected any theta-positive cells in such cultured material. On the contrary, the nude thymus in vitro develops into the polycystic structure characteristic of the adult nude thymus. We conclude that the small number of theta-positive cells present in the periphery result from extrathymic differentiation.     

Modulation of T-cell differentiation in murine fetal thymus organ cultures

The effects of IL-1, IL-2, and a panel of monoclonal antibodies to thymic stroma and/or thymocytes, on T-cell differentiation in murine fetal thymus organ cultures were followed. Day-14 fetal thymic lobes were cultured for up to 12 days in the presence of IL-1 and/or IL-2 at concentrations of 100 U/ml. Development of all the major subpopulations defined by CD4 and CD8 expression was inhibited by IL-2, however the degree of inhibition was greatest for CD4+CD8- and CD4+CD8+ subsets. IL-1 alone caused only minor shifts in the subpopulations, but when added together with IL-2 the inhibitory effects of IL-2 were markedly enhanced. Analyses of subsets of CD4-CD8- cells demonstrated that the inhibition was most dramatic at the IL-2R positive and subsequent stages of CD4-CD8- differentiation. Interestingly, the putative precursors of IL-2R+CD4-CD8- increased in the presence of IL-2. In preliminary studies the organ culture system was used to examine the effects of a panel of antibodies to thymocytes and/or thymic stromal cells. Out of 14 antibodies tested, MTS 35 and MTS 37 have caused relative increases in the CD8 and CD4 single-positives, respectively. Both antibodies also induced increases in the percentages of CD4-CD8- cells and decreases in the percentages of CD4+CD8+ cells.     

The effects of plasma from patients with Graves' disease on foetal mouse hearts in organ culture

Plasma, obtained during plasma exchange therapy, from 3 euthyroid patients with Graves' disease and severe progressive exophthalmos induced an increase in heart rate and then early death when applied to foetal mouse hearts maintained in isolated organ culture. All plasma samples which induced an increase in foetal heart rate had high titres of thyroid stimulating immunoglobulins. Plasma samples obtained after exchange had a much diminished effect. These studies may indicate a previously unrecognized non-thyroidal action of the abnormal immunoglobulins associated with Graves' disease and suggest that chronic thyroid heart disease may be due, at least in part, to the effect of these immunoglobulins especially when not associated with elevated thyroid hormones concentrations.     

Generation of cells expressing cytoplasmic and/or surface T-cell receptor beta chains during the development of mouse fetal thymus.

We have used a double immunofluorescence technique to identify newly formed cells expressing cytoplasmic and/or surface T-cell receptor beta chains in mouse embryo thymus. In addition, we have localized these cells to the developing thymic cortex by immunoperoxidase labelling of frozen sections. The first positive cells appear in small numbers in the thymus at Day 15 of gestation and express cytoplasmic but not surface beta chains (C beta+). At Day 16 of gestation, cells appear that express small capped areas of surface beta chains as well as cytoplasmic beta chains (C beta+S beta+ cells). Just prior to birth, a cohort of cells appears that express surface beta chains but no cytoplasmic beta chains detectable by the methods employed (S beta+ cells). These results and vincristine mitosis-blocking studies suggest that a proliferating population of cortical thymocytes accumulate cytoplasmic beta chains before giving rise to T cells that express surface beta chains (presumably in association with alpha chains). When a monoclonal antibody to beta chains is present in organ cultures of embryonic thymus, C beta+ thymocytes continue to be generated but there is an absence of S beta+ cells. The latter appear after the cultures are transferred to fresh medium not containing the antibody.     

Onset of promiscuous gene expression in murine fetal thymus organ culture

T-cell differentiation and induction of tolerance to self-antigens occurs mainly in the thymus. Thymic stromal cells, specifically medullary thymic epithelial cells, express a diverse set of genes encoding parenchymal organ-specific proteins. This phenomenon has been termed promiscuous gene expression (PGE) and has been implicated in preventing organ-specific autoimmunity by inducing T-cell tolerance to self antigens. Early thymopoiesis and the critical factors involved in T-cell differentiation can be reproduced in vitro by murine fetal thymus organ culture (FTOC), which mimics the natural thymic microenvironment. To evaluate the occurrence of PGE in FTOC, gene expression profiling during in vitro thymic development in BALB/c mice was performed using a set of nylon cDNA microarrays containing 9216 sequences. The statistical analysis of the microarray data (sam program) revealed the temporal repression and induction of 57 parenchymal and seven lymphoid organ-specific genes. Most of the genes analysed are repressed during early thymic development (15-17 days post-coitum). The expression of the autoimmune regulator (AIRE) gene at 16 days post-coitum marks the onset of PGE. This precedes the induction of parenchymal organ genes during the late developmental phase at 20 days post-coitum. The mechanism of T-cell tolerance induction begins during fetal development and continues into adulthood. Our findings are significant because they show a fine demarcation of PGE onset, which plays a central role in induction of T-cell tolerance.     

Analysis of the cytotoxic function of the thymocytes generated in a 14-day old mouse fetal thymus organ culture with high dose of IL-2

The target cell specificity of LAK cells generated by addition of rIL-2 to thymic rudiments of 14 day old BALB/C embryo's was compared to the lytic potential of adult BALB/C splenic LAK cells. We used four tumor cell lines as targets, one NK-susceptible (YAC-1) and three NK-resistant tumors (C1300, P815 and EL4), which were all killed by splenic LAK cells. It is shown that fetal thymic LAK cells display a lower lytic activity, while EL4 tumor cells are not killed at all. By elimination of the CD8 positive cells from both the adult splenic and the fetal thymic LAK cells, we demonstrate that the fetal thymic LAK activity is mainly exerted by CD4-CD8- cells, whereas part of the cytotoxicity of splenic LAK cells is due to CD8+ cells.