Discrimination between maintenance- and differentiation-inducing signals during initial and intermediate stages of positive selection

As well as signaling through the alpha beta T cell receptor complex, positive selection of immature CD4(+)8(+) thymocytes involves additional ill-defined accessory interactions provided by thymic epithelial cells. Here, we have isolated CD4(+)8(+) thymocytes at a pre-positive selection stage of development (TCR- CD69(-) 4(+)8(+) cells), or after initiation of positive selection (CD69(+) 4(+)8(+) cells), from mice where the normal lifespan of thymocytes is extended by the presence of a bcl-2 transgene, to allow us to discriminate between requirements for maintenance and differentiation signals during positive selection. We find that MHC class II+ thymic epithelial cells drive positive selection of TCR- CD69(-) 4(+)8(+) bcl-2 tg thymocytes to the CD4(+) and CD8(+) stage, while no such mature subsets are observed when thymocytes are cultured alone or with major histocompatibility complex (MHC) class II+ salivary epithelial cells. However, CD4(+)8(+) cells remain in such cultures in considerable numbers, and retain the potential for positive selection if re-cultured with thymic epithelium, suggesting that thymic epithelial cells provide specific differentiation-inducing signals for positive selection. In contrast, intermediate CD69(+) 4(+)8(+) thymocytes show some capacity for phenotypic conversion In the absence of thymic stromal cells although strikingly the single-positive CD4(+) and CD8(+) cells generated are not functionally competent. Finally, we show that prior culture of thymic epithelial cells under monolayer conditions abrogates their ability to support the initiation of positive selection suggesting that the epithelial cell molecules necessary for the provision of differentiation signals during positive selection are down-regulated under such conditions.