Regulation of RAG‐1 and CD69 expression in the thymus during positive and negative selection

Successful interaction of the T cell receptor (TCR) 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 α‐chain gene rearrangements (Borgulya, P., Kishi, H., Uematsu, Y. and von Boehmer, H., Cell. 1992. 69: 529‐‐537; Brändle, D., Müller, C., Rülicke, 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 TCR‐MHC interactions of thymocytes with thymic stromal cells. Firstly, transgenic (Tg) mice expressing a virus‐specific MHC class I (H‐2Db)‐restricted TCR were injected with anti‐Db 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 24 h 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 (H‐2d) down‐regulated RAG‐1 expression after antigen‐induced TCR 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.