John J. T. Owen

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 selection1. Although the thymus provides a unique environment for these events, the role of the thymic stroma in regulating specific developmental stages is not well understood2. 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 CDS requires the presence of both major histocom-patibility 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 fib rob last 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.

Development of the Thymus Requires Signaling Through the Fibroblast Growth Factor Receptor R2-IIIb

Mice deficient for fibroblast growth factor (Fgf)R2-IIIb show a block in thymic growth after embryonic day 12.5, a stage that just precedes its detection in thymic epithelial cells. Fgf7 and Fgf10, the main ligands for FgfR2-IIIb, are expressed in the mesenchyme surrounding the thymic epithelial primordium, and Fgf10-deficient mice also exhibit impaired thymic growth. Hence, Fgf signaling is essential for thymic epithelial proliferation. In addition to the proliferative block, most thymic epithelial cells fail to progress from an immature cytokeratin 5-positive to a cytokeratin 5-negative phenotype. Nevertheless, sufficient epithelial cell differentiation occurs in the severely hypoplastic thymus to allow the development of CD4/CD8-double-positive thymocytes and a very small number of single-positive thymocytes expressing TCRs.

Alkaline phosphatase-fast red, a new fluorescent label: Application in double labelling for cell surface antigen and 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.

A single micromanipulated stem cell gives rise to multiple T-cell receptor gene rearrangements in the thymus in vitro

The extensive range of specificities of T-cell receptors is generated, as for immunoglobulins, by rearrangement of genetic information1. Much valuable information about rearrangement processes has been inferred by comparing DNA from (monoclonal) lymphoid lines with germ-line DNA2,3 and, for B cells, from rearrangements in some Abelson murine leukaemia virus-transformed cell lines4,5. However, because it is difficult to isolate and grow precursor populations, it has not proved possible to study rearrangements occurring in normal untransformed cells in vitro. Here we show that a single T-cell precursor colonizing an alymphoid thymus lobe in organ culture can generate multiple receptor β-chain gene rearrangements. These observations provide unequivocal evidence for the intra-thymic diversification of the T-cell repertoire. They also offer the possibility of investigating rearrangement and its control in the clonal progeny of a single normal T-cell precursor without the perturbations involved in the use of viral transformation or the production of T-cell hybridomas.

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 α4 integrin, but other markers such as CD62L, CD25, Thy‐1.2, CD3ϵ, α5 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.

Studies on thymic epithelial cells in vitro

Thymic epithelial cells are unique in their ability to support positive selection and are essential throughout thymocyte development. Here, we describe a technique for measuring the proliferation of thymic epithelial cells by flow cytometry using a combination of BrdU and pancytokeratin labelling, and we examine the effects of different in vitro culture strategies on thymic epithelial cell function. We find that at d15 gestation, 74% (+/- 0.4%) of thymic epithelial cells are in cycle, which declines to 63% (+/- 1.3%) by d16, and to 34% (+/- 1.9%) by d18. This decline in proliferation is also found in organ cultures and in cultures depleted of lymphoid cells by 2-dGuo, suggesting that the cell cycle status of thymic epithelial cells is independent of the lymphoid population. When cultured in vitro as 3-dimensional aggregates, purified MHC class II+ thymic epithelial cells retain the ability to support thymocyte maturation. In contrast, 2-dimensional monolayer culture abrogates the ability of these cells to support positive selection, causes a reduction in whn gene expression and reduces their ability to re-form coherent reaggregate structures. Intact lobes and 3-dimensional aggregates are therefore the best way of maintaining thymic epithelial cell function and gene expression in vitro.

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 α4 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 α4 integrin resulting in PKC activation leading to apoptosis. Overall, our results indicate the potential role of the α4 integrin‐mediated interactions in apoptosis induction during T‐cell development and following mature T‐cell activation.

Measurement of tumor immunity in vitro with 125 i-iododeoxyuridine- -labeled target cells.

SUMMARY Easily quantitated methods which employ 125IUdR to label target cells are presented for assaying tumor immunity in vitro. With these methods killing or inhibition of growth of tumor cells can be measured.

The Role of Mesenchyme in Thymus Development

We have reviewed the evidence that thymic mesenchymal cells and their progeny thymic fibroblasts play an important role in early T-cell development. Although it is possible that mesenchyme plays an inductive role in thymic epithelial morphogenesis, we have presented evidence to suggest that there is a direct effect of mesenchyme and fibroblasts on T-cell development. Moreover the association of these cell types with an ECM raises the possibility that the latter might be important in integrin and/or cytokine presentation especially during the CD4(-)8- phase of T-cell development.

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.