The Kinase Occupancy of T Cell Coreceptors Reconsidered

The sensitivity of the αβ T cell receptor (TCR) is enhanced by the coreceptors CD4 and CD8αβ, which are expressed primarily by cells of the helper and cytotoxic T cell lineages, respectively. The coreceptors bind to major histocompatibility complex (MHC) molecules and associate intracellularly with the Src-family kinase Lck, which catalyzes TCR phosphorylation during receptor triggering. Although coreceptor/kinase occupancy was initially believed to be high, a recent study suggested that most coreceptors exist in an Lck-free state, and that this low occupancy helps to effect TCR antigen discrimination. Here, using the same method, we found instead that the CD4/Lck interaction was stoichiometric (~100%) and that the CD8αβ/Lck interaction was substantial (~60%). We confirmed our findings in live cells using fluorescence cross-correlation spectroscopy (FCCS) to measure coreceptor/Lck codiffusion in situ. After introducing structurally guided mutations into the intracellular domain of CD4, we used FCCS to also show that stoichiometric coupling to Lck required an amphipathic α-helix present in CD4 but not CD8α. In double-positive cells expressing equal numbers of both coreceptors, but limiting amounts of kinase, CD4 outcompeted CD8αβ for Lck. In T cells, TCR signaling induced CD4/Lck oligomerization but did not affect the high levels of CD4/Lck occupancy. These findings help settle the question of kinase occupancy and suggest that the binding advantages that CD4 has over CD8 could be important when Lck levels are limiting.