T cell activation relies on the T cell receptor (TCR), a multiprotein complex located at the plasma membrane that recognizes peptides presented by major histocompatibility complex molecules. This complex associates the clonotypic αβ heterodimers with the CD3ε, γ, and δ chains, as well as with a ζ-chain dimer commonly referred to as CD3ζ. The latter, encoded by the CD247 gene, carries six of the ten immunoreceptor tyrosine-based activation motifs (ITAMs) of the entire complex and plays a decisive role in signal amplification when TCR stimulation remains suboptimal. TCR endocytosis has long been regarded as a mechanism for terminating signaling. This work challenges that interpretation by showing that, following internalization, the receptor continues to signal from a specialized endosomal compartment that is essential to lymphocyte functions.
Mechanistically, the authors establish that TCR engagement triggers internalization of the TCR–CD3ζ complex through a clathrin-dependent pathway, while maintaining CD3ζ-borne signaling. This internalization occurs in endosomal vesicles characterized by the presence of the insulin-regulated aminopeptidase (IRAP) and the SNARE protein Syntaxin 6. To characterize this compartment and the intracellular localization of the ζ chain, the study combined several imaging approaches in human Jurkat T cells and in primary mouse T cells: confocal microscopy, total internal reflection fluorescence (TIRF) microscopy to quantify microaggregates at the membrane, fluorescence lifetime imaging microscopy (FLIM), and proximity ligation assays designed to reveal protein interactions.
Destabilization of this compartment by IRAP deletion produces a counterintuitive effect: membrane expression of the TCR–CD3ζ complex increases, even as overall CD3ζ-borne signaling is compromised. The integrity of this compartment further appears indispensable for T cell activation and survival following suboptimal TCR stimulation. To assess the functional significance of this mechanism, the team used mice carrying a T cell–restricted IRAP deletion, proliferation assays on transgenic OT1 lymphocytes stimulated with peptides of varying affinities, and immunization with an adeno-associated viral vector expressing ovalbumin. These mice proved unable to mount effective polyclonal antitumor responses.
Taken together, these data reveal a previously unsuspected role for IRAP-dependent endosomal TCR signaling in T cell activation, and call for reconsidering receptor endocytosis not as a mere termination of the signal, but as a step that fully contributes to the lymphocyte response.