REGULATION OF RAG-1 AND CD69 EXPRESSION IN THE THYMUS DURING POSITIVE AND NEGATIVE SELECTION

Successful interaction of the T cell receptor (TPR) with major histocompatibility complex (MHC) molecules during thymic selection down-regulates the expression of the recombination activating genes (RAG)-1 and -2 in cortical thymocytes and thereby prevents further endogenous TCR alpha-chain gene rearrangements (Borgulya, P., Kishi, H., Uematsu,Y. and von Boehmer, H., Cell. 1992. 69: 529-537; Brandle, D., Muller, C., Rulicke,T., Hengartner, H. and Pircher, H., Proc. Natl. Acad. Sci. USA 1992. 89: 9529-9533). To address the question whether down-regulation of RAG-1 activity represents an irreversible process we have blocked TPR-MHC interactions of thymocytes with thymic stromal cells. Firstly, transgenic (Tg) mice expressing a virus-specific MHC class I (H-2D(b))-restricted TCR were injected with anti-D-b or anti-CD8 monoclonal antibodies and RAG-1 expression was examined by in situ hybridization on thymus sections. The results show that cortical thymocytes up-regulated RAG-1 expression within 24h after antibody administration. Secondly, immature thymocytes from TCR Tg mice were released from the thymic microenvironment and cultured in vitro for 14 h in single-cell suspension. The amount of RAG-1 mRNA was increased sixfold in cultured cells when compared to freshly isolated thymocytes. In addition, we show that immature thymocytes from TCR transgenic mice bearing non-selective MHC molecules (11-2(d)) down-regulated RAG-1 expression after antigen-induced TPR engagement. Cytofluorometric analysis further revealed that surface expression of CD69 on immature thymocytes inversely correlated with RAG-1 expression during positive and negative selection processes.