Nel C. Moore

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.

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.

Formation of nuclear anomalies in rat intestine by benzidine and its biliary metabolites

Administration of benzidine (BZ) by intraperitoneal injection (i.p.) to rats (0-100 mg/kg) produced, after 24 h, a dose-dependent formation of nuclear anomalies (micronuclei, pyknotic and karyorrhectic nuclei) in intestinal epithelial cells analysed both in isolated cell suspensions and in the intestinal crypts in tissue sections. When bile collected (0-4 h) from rats treated with BZ (150 mg/kg, i.p.) was infused into the duodenum of recipient rats, nuclear anomalies were observed in mucosal epithelial cells, after 24 h, with a similar distribution to that in rats given BZ by i.p. injection. The formation of nuclear anomalies in the intestine is in accord with the intestinal carcinogenic effect of BZ and is, at least partially, dependent on exposure of epithelial cells to biliary metabolites of BZ.

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.

THYMOCYTE-STROMAL-CELL INTERACTIONS AND T-CELL SELECTION

Several key advances have been made since 1992, notably in teh area of signalling in early T-cell development and in the regulation of T-cell selection. Future goals include a fuller understanding of the roles of stromal cells and matrix molecules in T-cell development and further elucidation of the diverse signalling pathways involved. The ultimate challenge will be to generate T cells from stem cells under conditions where all of the developmental cues that earmark thymopoiesis are defined.

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 higly selective PKC inhibitor, Ro31.8425, we show that PKC activation is essential for the regulation of AICD by SEB rechallenge.