The CD4 and CD8 co-receptors play important functions in both thymocyte development as well as in cellular functions such as lymphokine production, cell-mediated cytotoxicity and proliferation. We, as well as others, have observed that the level of Lck kinase activity induced upon CD4 or CD8 co-receptor crosslinking does not correlate with the ability of the co-receptor to enhance IL-2 production. These data question the importance of the Lck kinase activity associated with CD4 and CD8. We have studied the role of Lck kinase activity associated with the CD8 co- receptor in thymocyte development. Mice with a """"""""knock-out"""""""" of the CD8beta chain have >80% fewer single positive (SP) CD8 thymocytes and peripheral CD8 T cells. When we studied the Lck kinase activity induced upon CD9 crosslinking in these mice we observed that their Lck activity was markedly reduced. In fact, the level of CD8 associated Lck kinase activity was proportional to the percent of CD8 SP thymocytes that developed. Thus it appears that the CD8beta chain is required for Lck kinase activity and this kinase activity is required for efficient differentiation from double positive (DP) to SP CD8 thymocytes. To test the role of the Lck kinase activity associated with CD8 we have generated a transgene with the cDNA encoding the extracellular and transmembrane domains of murine CD8alpha ligated to Lck (CD8alpha-Lck). This chimera was inserted into the CD2 minigene and it has been expressed in sv129 mice and the following specific aims are proposed. Mice expressing this transgene will be bred to CD8beta-/- mice to determine whether expression of the transgene will reconstitute SP CD8 development. If differentiation is reconstituted then we will express a transgene with a kinase dead Lck, CD8alpha-Lck(R273), to determine whether the kinase activity is critical. If the CD8alpha-Lck transgene fails to reconstitute beta-/- mice then they will be bred to CD8alpha-/- mice, which have endogenous CD8beta to determine if the transgene can reconstitute development in the presence of the beta chain. If CD8alpha- Lck reconstitutes SP CD8 development then we will analyze the biochemical and cellular functions of the cells expressing this transgene. Similar experiments are planned to determine if a CD4-lck transgene expressed in CD4-/- mice can reconstitute SP CD4 thymocyte differentiation. Because of the limited number of cells available to perform biochemical studies from mice expressing the transgene we will express CD8alpha-Lck in a T cell hybridoma and identify tyrosine phosphorylated substrates in this model system. This approach should allow more targeted biochemical experiments using thymocytes from transgenic mice.
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