Cross-linking of T-cell receptors on double-positive thymocytes induces a cytokine-mediated stromal activation process linked to cell death

To investigate molecular events associated with the intrathymic process of negative selection, we established an in vivo system using an anti-CD3 epsilon monoclonal antibody to induce synchronous apoptosis in the thymus of AND T-cell receptor (TCR) transgenic RAG-2(-/-) mice in a non-selecting haplotype, This model eliminates endogenous negative selection as well as gene activation in the mature thymocyte compartment, offering an ideal source of tester (anti-CD3 epsilon-treated) and driver (untreated) thymus RNA for representational difference analysis (RDA), Fourteen mRNA sequences that are up-regulated in the thymuses of such mice 2-6 h after anti-CD3 epsilon treatment were identified, Surprisingly, the majority of these transcripts were derived from stromal cells rather than the TCR-crosslinked CD4(+)CD8(+)TCR(low) thymocytes including the macrophage products IL-1, the chemokine Mig and the transcription factor LRG-21. IFN-gamma secretion from the CD4(+)CD8(+)TCR(low) thymocytes regulates macrophage Mig production, Three other cytokines (IL-4, GM-CSF and TNF-alpha), known to activate a variety of stromal cells, are also induced in the same thymocyte population undergoing apoptosis, Expression of a TNF-alpha-inducible gene, B94, in stromal cells after TCR ligation further supports the notion of cross-talk between thymocytes and stroma, Thus, TCR-triggered immature thymocytes elaborate cytokines which may regulate the delivery of further signals from stromal cells required for apoptosis.