To the editor:

We read with interest the article by Huppa et al.1 that shows prolonged and sustained interaction between T cells and antigen-presenting cells (APCs) is required for full T cell activation. As part of this study, the authors examined the spatial relationship between the T cell receptor (TCR)-CD3 complex and early signaling markers at the immunological synapse. TCR trafficking was monitored with immunofluorescence microscopic analysis of cyan fluorescent protein (CFP)-tagged CD3ζ (CD3ζ-CFP), whereas TCR-derived signals (PI3K activity) were monitored by a yellow fluorescent protein (YFP)-tagged pleckstrin homology (PH) domain of the serine-threonine kinase Akt (PH(AKT)-YFP). The authors showed rapid colocalization of CD3ζ-CFP and PH(AKT)-YFP at the T cell–APC interface after B cell contact. Although PH(AKT)-YFP subsequently remained at the interface, CD3ζ-CFP was internalized into a central cluster of vesicles. In supplemental studies, the authors assessed TCR internalization by flow cytometric analyses of surface TCRβ. These data showed rapid internalization of most TCRs after activation. The authors therefore concluded that removal of activated TCRs from the T cell–APC interface had no great effect on synapse-associated PI3K activity, but these data do not justify this conclusion.

The TCR is a multichain, heteromeric structure composed of a variable antigen-binding (αβ) domain and noncovalently associated invariant signal-transducing complexes, the CD3 (γ, δ and ε) and ζ chains2. Although loss of CD3ζ chain from the T cell–APC interface is evident in the studies by Huppa et al., there are no data presented demonstrating a similar loss of TCRs from the immune synapse. This is important, given previous work showing surface CD3ζ can partition to intracellular compartments independently of other TCR chains3,4,5. Furthermore, sustained exposure of T cells to antigen in vivo can induce the specific loss of CD3ζ due to enhanced lysosomal degradation6. It is possible, therefore, that the dual-color, three-dimensional video microscopy of sustained antigenic stimulation in the paper by Huppa and colleagues has in fact caught the CD3ζ chain in the act of dissociating from the rest of the TCR.

Although we do not dispute the central conclusion of this paper, which documents very well the dynamics of synapse-associated CD3ζ-CFP after T cell–APC conjugate formation, the current data provide no insights into the dynamics of synapse-associated TCRs. This distinction, far from being trivial, will influence future analyses of molecular interactions among T cell and APC signaling proteins that partition to the immune synapse.