The observations described above were made at relatively late time-points (more than 10 min after conjugate formation). Experiments with conjugates fixed at various time-points and studies utilizing time-lapse microscopy have demonstrated that IS formation is a dynamic process involving a number of intermediate structures. Early images of CD4+ T cells forming ISs with supported planar bilayers demonstrated that the majority of TCR–pMHC interactions exist at the periphery of the interface at early time-points, but consolidate in the cSMAC within 5 min, while the opposite pattern occurs for LFA-1–ICAM-1.6 The large phosphatase CD45 is present in the centre of the contact zone at early time-points,13 but is cleared from the cSMAC by 7 min post-conjugation.13,14 However, experiments using supported planar bilayers as APCs demonstrated that under some experimental conditions CD45 does accumulate at the cSMAC in addition to the strong peripheral accumulation.15,16 The spatiotemporal location of CD45 relative to the TCR during IS formation is of interest because, while dephosphorylation of the Src-family kinase Lck is critical for the initiation of a signal through the TCR, CD45 is also capable of dephosphorylating activated components of the proximal TCR signalling complex.17 The conflicting data obtained for CD45 localization in experiments using different types of APC (planar bilayers and B lymphoma cells in this case) demonstrate the important effect that experimental parameters can have on results and conclusions, and the hazard in generalizing results derived from one experimental system. The influence of the type of APC on IS structure is discussed in detail below.
In an experimental tour de force, Wülfing et al. used live cell imaging to examine the spatiotemporal patterns of 30 green fluorescent protein (GFP)-conjugated signalling sensors and found that the TCR and a group of proximal signalling molecules, including zeta-chain associated protein kinase of 70 kDa (ZAP-70), linker for activation of T cells (LAT), phospholipase C-γ and PKC-θ, are rapidly recruited to the cSMAC,18 in agreement with previous results.7,13 This large accumulation of TCR-proximal signalling molecules in the cSMAC could be interpreted as evidence that IS formation is required for the initiation of signalling; however, this is not the case because phosphorylated signalling proteins, including Lck and ZAP-70, are found at the T cell–APC interface prior to the formation of mature, bull’s-eye ISs.13,19 In fact, the majority of phosphorylated signalling molecules are found in the periphery of the T cell–APC contact.19 This result was explained by the discovery that TCR microclusters, containing 40–150 TCR molecules, form immediately upon contact with planar bilayers containing pMHC and ICAM-1.20 The microclusters exclude CD45 and contain activated signalling molecules, including pLck, pZAP-70 and pLAT, as well as CD28 and PKC-θ,16,20–22 and are reminiscent of the small signalling clusters seen when Jurkat cells are introduced to anti-TCR coated coverslips.23 These microclusters move centripetally in an F-actin and myosin motor-dependent fashion, resulting in the formation of a TCR-rich cSMAC.16,20,24,25 However, as the TCR microclusters move towards the centre of the interface, they become dissociated from the TCR-proximal signalling molecules, as well as CD28 and PKC-θ.20,22 New microclusters are continuously generated in the periphery and move centripetally, even after the formation of a mature, bull’s-eye IS,20,21 while the presence of a marker for multivesicular bodies and the endosomal sorting complex required for transport I ubiquitin-recognition complex at the cSMAC indicates that this is a site of active TCR down-modulation.16,26 Thus, IS formation is best viewed as a two-stage process. Stage I occurs immediately upon T cell–APC conjugation and involves the formation and coalescence of TCR microclusters, resulting in a large-scale, actin-dependent rearrangement of receptors, downstream signalling molecules and adhesion molecules into SMACs. Stage II is characterized by relative stability in the macro-structure of the IS and the centripetal movement of newly generated microclusters from the periphery to the cSMAC where signalling is extinguished and the TCR is down-modulated.