Interactions between stromal cells and thymocytes play a crucial role in T cell development. The thymic stroma is complex and consists of epithelial cells derived from the pharyngeal region during development, together with macrophages and dendritic cells of bone marrow origin. In addition, fibroblasts and matrix molecules permeate the whole framework. It is now apparent that these individual stromal components play specialized roles at different stages of T cell differentiation. Thus, at the early CD4-8- stage of development, T cell precursors require fibroblast as well as epithelial cell interactions. Later, at the CD4+8+ stage, as well as providing low avidity TCR/MHC-peptide interactions, thymic epithelial cells have been shown to possess unique properties essential for positive selection. Dendritic cells, on the other hand, are probably efficient mediators of negative selection, but they may not be solely responsible for this activity. Alongside the functional roles of stromal cells, considerable progress is being made in unraveling the nature of the signaling pathways involved in T cell development. Identification of the pre-T cell receptor (pre-TCR) and associated signaling molecules marks an important advance in understanding the mechanisms that control gene rearrangement and allelic exclusion. In addition, a better understanding of the signaling pathways that lead to positive selection on the one hand and negative selection on the other is beginning to emerge. Many issues remain unresolved, and some are discussed in this review. What, for example, is the nature of the chemotactic factor(s) that attract stem cells to the thymus? What is the molecular basis of the essential interactions between early thymocytes and fibroblasts, and early thymocytes and epithelial cells? What is special about cortical epithelial cells in supporting positive selection? These and other issues are ripe for analysis and can now be approached using a combination of modern molecular and cellular techniques.

TTAGTGGCGGGATCTATC SS LVAGSI (VJ35.2/ Ji32. 7) Ct AGCAGC ATAGCTGGCGGT SS IAGG (Vi36/ Ji3 2.3) • CDR3 usage in human MBP 88-99 specific T-cell line 1. t CDR3 usage in rat spinal cord derived T-cell clones specific for MPB 85-99 (ref. 2). t Clone derived from a human tonsil cDNA library1 3. § Non-cytolytic mouse T-cell clone specific for influenza virus strain A/PR8/34 (ref. 15). II CDR3 usage in rat lymph node derived T-cell clone specific for MBP 85-99 (ref. 2). ~ G-+S substitution in Ji32.7.

SummaryUsing a novel system that supports positive selection in vitro, we have investigated the cellular requirements for this process by testing the ability of individual thymic and nonthymic stromal cell types to support the maturation of CD4 + CD8 + thymocytes into CD4 + or CD8 + T cells. We show that thymic cortical epithelial ceils are unique in their ability to mediate this maturation, and suggest that in addition to TCR ligation, these cells supply specific signals for positive selection. Moreover, by demonstrating positive sdection on ECDI (1-ethyl-3-[Ydimethyl-aminopropyl]-carbodiimide)-fixed epithelial ceils in this system, we provide direct evidence that the provision of these signals involves interactions with epithelial ceil surface molecules rather than the release of soluble factors. Signaling through the TCP, complex on immature CD4 +CD8 § thymocytes can lead to two distinct devdopmental fates: positive selection, which results in further maturation, or negative selection, which results in cell death by apoptosis (1-3). One explanation for this paradox is that additional signals regulate the outcome of TCP, ligation, and that for positive selection, these are provided by interactions with thymic epithdial ceils (4, 5). Recently, a specific requirement for interaction with thymic epithelial ceils has been questioned by studies that demonstrate positive selection on various cell types, including fibroblasts and hemopoietic cells, that are introduced into the thymus in vivo (6-9). However, whereas these studies indicate that positive selection does not require recognition of peptide-MHC complexes unique to thymic epithelial ceils, they do not exclude the possibility that endogenous epithelial ceils are providing additional differentiation signals essential for positive selection and consequent maturation.In this study, we have used a novel in vitro system for positive sdection, based on reaggregate organ cultures (10,11), to analyze the ability of individual stromal ceil types to support the maturation of CD4 +CD8 + thymocytes in the absence of any other thymic dements. We show that the ability to support the maturation of CD4 § CD8 + ot/3TCR 1~ thymocytes into CD4 + 8-and CD4-8 + cells bearing high levels of oe/~TCR is a property unique to thymic cortical epithelium, and suggest that these ceils provide specific differentiation signals for positive selection. Furthermore, we demonstrate that ECDI (1-ethyl-3-[Ydimethyl-aminopropyl]-carbodiimide-fixed, metabolically inert, thymic epithdial cells can support the maturation of CD4 + CD8 + ceils and thus provide direct evidence that positive selection by these cells involves interaction with surface molecules rather than the release of soluble factors. Materials and Methods

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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.