Katherine J. Hare

Thymic epithelial cells provide Wnt signals to developing thymocytes

Interactions with thymic stromal cells are known to be critical for the development of Tu2004cells from progenitors entering the thymus, yet the molecular mechanisms of stromal cell function remain poorly understood. Accumulating evidence has highlighted the importance of β‐catenin‐mediated activation of Tu2004cell factor (TCF)/lymphoid enhancer factor (LEF) transcription during thymocyte development. As regulation of this signaling pathway is controlled by binding of soluble Wnt proteins to cell surface Frizzled (Fz) receptors, we studied components of Wnt/Fz‐mediated signaling in thecontext of stromal cell regulation of thymocyte development. We show that mRNA for a variety of Wnt family members, notably Wnt‐4, Wnt‐7a and 7b, and Wnt‐10a and 10b, are expressed by thymic epithelium rather then by thymocytes, while thymocytes demonstrate a developmentally regulated pattern of Fz receptor expression. Collectively these findings suggest (1) a functional role for Wnt‐producing thymic epithelium in determining TCF/LEF‐mediated transcriptional regulation in Fz‐bearing thymocytes, and (2) a role for defined Wnt‐Fz interactions at successive stages of thymocyte maturation. In support of this we show that separation of thymocytes from Wnt‐producing epithelial cells and the thymic microenvironment, triggers β‐catenin phosphorylation and degradation in thymocytes. Thus, sustained exposure to Wnt in the context of an intact stromal microenvironment is necessary for stabilization of β‐catenin‐mediated signaling in thymocytes.

An Essential Role for the IL-7 Receptor During Intrathymic Expansion of the Positively Selected Neonatal T Cell Repertoire

Intrathymic T cell development is a multistage process involving discrete phases of proliferation as well as differentiation. From studies on IL-7 or IL-7Rα-deficient mice, it is clear that the IL-7 receptor (IL-7R) plays a critical role during the initial stages of intrathymic CD4−8− precursor development. In contrast, the role of IL-7R in later stages of thymocyte development are unclear. Here, we have used various approaches to investigate directly the role of the IL-7R in thymocyte positive selection and the recently described phase of postselection proliferation. First, we show that positive selection involves selective up-regulation of IL-7Rα- and IL-7Rγ-chains, with the majority of CD4+ and CD8+ cells being IL-7R+. Second, MHC class II+ thymic epithelium—which drives postselection proliferation—expresses IL-7 mRNA. Finally, analysis of positive selection and postselection proliferation in thymocytes from IL-7Rα−/− neonates shows that positive selection occurs normally, whereas postselection expansion is drastically reduced. Thus, our data provide the first evidence that, as well as playing a role during early phases of thymic development, IL-7R mediates intrathymic expansion of positively selected thymocytes, which may aid in establishment of the neonatal peripheral T cell pool.

Positive selection of thymocytes: the long and winding road

Positive selection is a crucial stage in T-cell development because it is here that CD4+CD8+ cells bearing T-cell receptors that interact with self-major histocompatibility complex molecules are rescued from cell death, resulting in the generation of mature T cells. Here, Graham Anderson and colleagues review recent studies indicating that positive selection is a multistage process involving interactions with thymic epithelium.

Modeling TCR Signaling Complex Formation in Positive Selection

T cell receptor signaling in the thymus can result in positive selection, and hence progressive maturation to the CD4+8− or CD4−8+ stage, or induction of apoptosis by negative selection. Although it is poorly understood how TCR ligation at the CD4+8+ stage can lead to such different cell fates, it is thought that the strength of signal may play a role in determining the outcome of TCR signaling. In this study, we have characterized the formation of an active signaling complex in thymocytes undergoing positive selection as a result of interaction with thymic epithelial cells. Although this signaling complex involves redistribution of cell surface and intracellular molecules, reminiscent of that observed in T cell activation, accumulation of GM1-containing lipid rafts was not observed. However, enforced expression of the costimulatory molecule CD80 on thymic epithelium induced GM1 polarization in thymocytes, and was accompanied by reduced positive selection and increased apoptosis. We suggest that the presence or absence of CD80 costimulation influences the outcome of TCR signaling in CD4+8+ thymocytes through differential lipid raft recruitment, thus determining overall signal strength and influencing developmental cell fate.

Cutting Edge: A Chemical Genetic System for the Analysis of Kinases Regulating T Cell Development

To understand the regulatory activities of kinases in vivo requires their study across a biologically relevant window of activity. To this end, ATP analog-sensitive kinase alleles (ASKAs) specifically sensitive to a competitive inhibitor have been developed. This article tests whether ASKA technology can be applied to complex immunological systems, such as lymphoid development. The results show that when applied to reaggregate thymic organ culture, novel p56Lck ASKAs readily expose a dose-dependent correlation of thymocyte development with a range of p56Lck activity. By regulating kinase activity, rather than amounts of RNA or protein, ASKA technology offers a general means for assessing the quantitative contributions to immunology of numerous kinases emerging from genomics analyses. It can obviate the generation of multiple lines of mice expressing different levels of kinase transgenes and should permit specific biological effects to be associated with defined biochemical activities.

Induction of thymocyte positive selection does not convey immediate resistance to negative selection

The acquisition of functional competence represents a critical phase during intrathymic development of T cells. Thymocytes reaching this stage represent cells which have been positively selected on the basis of major histocompatibility complex reactivity, but which have also been purged of potentially autoreactive T‐cell receptor specificities by negative selection. While the developmental window in which thymocytes are subjected to positive selection is now well defined, the precise developmental timing of negative selection, in relation to positive selection events, is less clear. Moreover, the underlying mechanism allowing single‐positive thymocytes to respond to T‐cell receptor ligation by activation rather than death, remains controversial. Here we have analysed the developmental timing of negative selection in relation to positive selection, using measurement of thymocyte susceptibility to dendritic cell presentation of the superantigen staphylococcal enterotoxin B (SEB). We show that thymocytes which have received initial positive selection signals, namely CD4+u2003CD8+u2003CD69+ thymocytes, like their CD4+u2003CD8+u2003CD69− precursors, are susceptible to negative selection, indicating that induction of positive selection does not convey immediate resistance to negative selection. In contrast, newly generated CD4+u2003CD8−u2003CD69+ cells are not only resistant to deletion by SEB, but respond to SEB‐mediated T‐cell receptor‐ligation by activation, indicating that the acquisition of functional competence occurs at the newly generated CD4+u2003CD8−u2003CD69+ stage. Finally, by using direct retroviral infection of primary CD4+u2003CD8+ thymocytes, we also show that Notch‐1 activation in CD4+u2003CD8+ thymocytes does not correlate with, nor convey resistance to superantigen‐mediated negative selection. Thus, our data suggest that although Notch‐1 has been implicated in resistance to thymocyte apoptosis, the acquisition of resistance to negative selection occurs independently of Notch‐1 signalling.