DP thymocytes express substantially less TCR on their cell surface than do single-positive thymocytes and peripheral T cells. It has been suggested that this enables DP thymocytes to distinguish more sensitively between quantitative differences in signalling initiated by TCR interaction with different peptide–MHC complexes, something that is thought to be important in determining whether a DP thymocyte is positively selected, negatively selected or dies of neglect. Results of previous studies led Myers and colleagues to hypothesize that SLAP and CBL might function in the same molecular pathway to actively regulate TCR expression by DP thymocytes. So, they compared TCR and CD3 expression by DP thymocytes from mice lacking both SLAP and CBL, as well as from mice lacking either SLAP or CBL and from wild-type mice. Expression of TCRβ and CD3ε was substantially higher on the cell surface of Sla−/−, Cbl−/− and Sla−/−Cbl−/− DP thymocytes than on wild-type DP thymocytes, and the increase in TCRβ and CD3ε expression was similar for all three mutant cell types. Similarly, degradation of TCRζ was markedly impaired in Sla−/−, Cbl−/− and Sla−/−Cbl−/− DP thymocytes compared with wild-type DP thymocytes.
Consistent with this genetic evidence that placed SLAP and CBL in the same molecular pathway, the expression of cell-surface CD3ε and intracellular TCRζ was downregulated in Jurkat T cells that had been engineered to express both SLAP and CBL but not in cells engineered to express only SLAP or CBL alone. This downregulation of CD3ε and TCRζ expression in Jurkat T cells was abrogated if SLAP lacked its myristoylation site, its SRC homology 2 (SH2) domain or its carboxyl terminus, or if CBL lacked its RING (really interesting new gene)-finger domain (the domain of CBL that has E3 ubiquitin-ligase activity). Further analysis showed that expression of SLAP and CBL, but not SLAP or CBL alone, in Jurkat T cells induced ubiquitylation of TCRζ.
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