The structure of immunological synapses formed between murine naive T cells and mature dendritic cells has been subjected to a quantitative analysis. Immunofluorescence images of synapses formed in the absence of antigen show a diffuse synaptic accumulation of CD3 and LFA-1. In electron microscopy, these antigen-free synapses present a number of tight appositions (cleft size *15 nm), all along the synapse. These tight appositions cover a significantly larger surface fraction of antigen-dependent synapses. In immunofluorescence, antigen-dependent synapses show multiple patches of CD3 and LFA-1 with a variable overlap. A similar distribution is observed for PKCh and talin. A concentric organization characteristic of prototypical synapses is rarely observed, even when dendritic cells are paralyzed by cytoskeletal poisons. In T-DC synapses, the interaction surface is composed of several tens of submicronic contact spots, with no large-scale segregation of CD3 and LFA-1. As a comparison, in T-B synapses, a central cluster of CD3 is frequently observed by immunofluorescence, and electron microscopy reveals a central tight apposition. Our data show that it is inappropriate to consider the concentric structure as a "mature synapse" and multifocal structures as immature.

Physiologically, Ag detection by T cells occurs at the immunological synapse (IS) formed at the interface with an APC. CD5 is considered as an inhibitory molecule for Ag receptor-mediated signals in T cells. However, the influence of CD5 at the IS on synapse formation and functioning has not yet been reported. We demonstrate here that CD5 is recruited and tightly colocalized with CD3 in different human and murine IS. Following transfection in a CD5-negative T cell line of CD5 fused to the green fluorescent protein, we show that CD5 recruitment includes a fast Ag-independent and a slower Ag-dependent component. In video-imaging recordings of doubly transfected cells, the movements of CD3 and CD5 show similar kinetics, and the amount of CD3 recruited to the synapse is unaffected by CD5 expression. Moreover, APC-T cell adhesion is unchanged in CD5-expressing cells. Despite this, the extent of tyrosine phosphorylation at the synapse and the amplitude of calcium responses induced by Ag recognition are both decreased by CD5. These inhibitions increase with CD5 membrane levels. They also requires the pseudo-immunoreceptor tyrosine-based activation motif expressed in the cytoplasmic domain of the molecule. Thus, CD5 is rapidly recruited at the IS and lowers the T cell response elicited by Ag presentation by targeting downstream signaling events without affecting IS formation.