T cell receptor-mediated signaling events in CD4⁺CD8⁺ thymocytes undergoing thymic selection: Requirement of calcineurin activation for thymic positive selection but not negative selection

The goal of this study was to identify the differences of intracellular signals between the processes of thymic positive and negative selection. The activation of calcineurin, a calcium⁻ and calmodulindependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4⁺ CD8⁺ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FKS06 treatment blocked the generation of mature TCRhishCD4+CDS⁻ and TCRhishCD4-CD8⁺ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (R.AG-1) transcription and the dowuregnlation of CD4 and CD8 expression were inhibited by FKS06 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation did not appear to be required for negative selection based on the observations that negative selection of TCRαβ T cells in the H-2 b male thymus (a negative selecting environment) was not inhibited by in vivo treatment with FKS06 and that there was no rescue of the endogenous superantigen-mediated clonal deletion of VB6 and V/511 thymocytes in FK506-treated CBA/J mice. DNA fragmentation induced by TCR activation of DP thymocytes in vitro was not affected by FKS06. In addition, different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes.