TCRζ-driven pre-signaling organization of Lck in Rab11⁺ endosomes shapes TCR activation

T cell activation relies on the precise spatiotemporal regulation of T cell receptor (TCR) signaling at the immunological synapse, where vesicular trafficking coordinates the delivery of key signaling molecules. Endosomal pools of lymphocyte-specific protein tyrosine kinase (Lck) and its immediate substrate, the TCRζ chain, have been linked to TCR signaling competence, yet the mechanisms that regulate and coordinate their trafficking routes remain unresolved.

We simultaneously tracked the endosomal localization dynamics of endogenous Lck, ζ chain, and the TCR in unperturbed cells and under conditions that preserve the endosomal network integrity. Genetic modifications, confocal microscopy, quantitative phospho-flow analysis, and biochemical assays were used to map trafficking behaviors and signaling outputs. Datasets were analyzed using unpaired Student's t-tests.

We identified a previously unrecognized plasma membrane-resident pool of ζ that exists independently of the TCR complex yet remains competent for phosphorylation and ZAP-70 recruitment. This "standalone" ζ population orchestrates the mobilization of Lck and CD45 into Rab11-positive compartments, establishing pre-assembled, signaling-primed platforms prior to synapse formation. These structures can accommodate constitutively phosphorylated ζ and ζ-chain-associated protein kinase 70 (ZAP-70), which depend on the presence of endosomal Lck.

This work uncovers a mechanistic link between vesicular ζ and Lck pools and establishes their coordinated trafficking as a key determinant of TCR activation. Our findings indicate that vesicular preprimed assemblies can provide a reservoir of signaling mediators, serving to sustain TCR signaling without a requirement for continuous receptor engagement, thereby determining the threshold, sensitivity, and persistence of T cell responses. By revealing a TCR-uncoupled ζ population that seeds endosomal signaling platforms, our study advances current models of T cell activation and highlights an additional layer of regulation in proximal signaling events. These insights also have translational implications, particularly within the context of improving the efficiency, performance, and safety of T cell immunotherapies.