Expression of the p56 lck Y505F Mutation in CD45-Deficient Mice Rescues Thymocyte Development

CD45 is a transmembrane protein tyrosine phosphatase uniquely expressed by cells of the immune system (38). For T cells, CD45 is required for T-cell antigen receptor (TCR) signal transduction (17, 31). CD45-deficient T cells are impaired in their ability to initiate signaling in response to either antigen or TCR cross-linking. The inefficient TCR-mediated signaling in CD45-deficient cells correlates with an eight- to ninefold increase in tyrosine phosphorylation at the inhibitory site in the Src family member p56lck (Lck) (10, 34). These results suggest that CD45 regulates Lck by dephosphorylating the inhibitory site. Since it is thought that Lck is the primary kinase responsible for initiating signals through the antigen receptor, the inability to activate Lck in CD45-deficient cells appears to account for the inefficient signaling through the TCR. However, while all CD45-deficient T-cell lines exhibit increased tyrosine phosphorylation of Lck at the inhibitory site, there is discordance with regard to kinase activity; some cell lines have increased kinase activity (8, 12, 13), while others have decreased kinase activity (10, 14, 26, 27, 33, 34). The basis for this difference is unknown. Furthermore, the paradox of inefficient signaling through the TCR despite enhanced Lck kinase activity is unexplained. These contradictory findings raise the possibility that the block in TCR signaling in CD45-deficient cells is not due to the failure to dephosphorylate the inhibitory site in Lck but is attributable to a different mechanism. In principle, expression of Lck Y505F in CD45-deficient cells should rescue TCR signaling. However, expression of Lck Y505F in CD45-deficient cell lines results in rapid internalization of the TCR, thereby preventing an assessment of the ability to rescue signaling (13). CD45-deficient mice exhibit a block in thymocyte development at the stage at which cells express both CD4 and CD8 coreceptor proteins. This is the stage in thymocyte development at which newly rearranged TCRs are expressed and signaling thresholds are determined to eliminate cells that are autoreactive or fail to generate sufficient signals. Cells that signal appropriately develop further to express either CD4 or CD8 and are released to the periphery, where they recognize antigen in the context of major histocompatibility complex class II or class I molecules, respectively. As such there are subsequently few mature T cells in the periphery of CD45-deficient mice (9, 15). As predicted by studies of cell lines, the small number of T cells that accumulate in the periphery fail to efficiently signal through the TCR. The phenotype of CD45-deficient mice is consistent with the idea that CD45 is required for TCR signaling. However, whether this is due to the regulation of Lck by CD45 has not been determined. The importance of Lck in thymocyte development and TCR function has been demonstrated by the development of transgenic and gene-ablated mice. Mutation of the inhibitory tyrosine phosphorylation site to phenylalanine (Y505F) results in a constitutively active kinase (4, 5). Expression of the Lck Y505F mutant as a transgene driven by the lck proximal promoter results in a block in thymocyte development due to premature signaling and a subsequent failure to rearrange the TCR β-chain (1, 2, 18). Developing thymocytes do not progress past the CD4+ CD8+ double-positive stage and do not up-regulate expression of the CD3 component of the TCR. However, development can be rescued by the simultaneous transgenic expression of a TCR β-chain which, obviously, has already been rearranged (7). Further, expression of the Lck Y505F transgene overcomes the block in thymocyte development observed in Rag-deficient mice and permits progression from CD4− CD8− double-negative to CD4+ CD8+ double-positive cells (6). The generation of Lck-deficient mice has demonstrated that Lck expression is critical for signal transduction through the pre-TCR that is required during the development of CD4+ CD8+ double-positive thymocytes from their CD4− CD8− double-negative precursors (7, 24, 30). These experiments have emphasized that Lck plays a role in the TCR signaling that is required for thymocyte development. To examine whether the regulation by CD45 of the inhibitory tyrosine phosphorylation site in Lck is critical to thymocyte development, we generated CD45-deficient mice that express Lck Y505F under the control of the lck proximal promoter (1). To overcome the block in thymocyte development caused by Lck Y505F, the DO11.10 TCR was also expressed (25). Expression of Lck Y505F in the presence of the DO11.10 TCR relieves the requirement for CD45 in thymocyte development. Our results indicate that the inhibitory site in Lck is a critical substrate of CD45 in vivo. Dephosphorylation of Lck Y505 is necessary for the signaling through the TCR that is required for thymocyte development and response to antigen.