The data presented in this study are a first view of synapses as they occur in naive cells in vivo. The fundamental findings are threefold. First, prolonged TCR clustering or cSMAC formation for naive T cells interacting with DCs was infrequent in vivo as well as in vitro. Second, TCR internalization can occur rapidly in the absence of stable cSMAC formation. Finally, T cells are able to actively aggregate TCRs, flux calcium, and internalize TCR clusters, even in a motile synapse.
These data create the need to reconsider the dichotomy between T cell migration versus TCR clustering and signaling as clearly they are not exclusive. In our opinion, there is a spectrum of behaviors associated with signaling that does not require the stability of a synapse. These behaviors include calcium influx and formation and internalization of TCR clusters. This is somewhat at odds with older studies that suggested that calcium signaling and TCR occupancy necessarily led to arrest (Negulescu et al., 1996
; Dustin et al., 1997
) but likely could be reconciled if one considers that some of those studies may have used conditions of stimulation or cell lines that were more sensitive to motility arrest and that the speed of migration does decrease, even in cells that maintain motility. Another factor that may play into differences is that our analysis of TCR dynamics in naive T cells was limited to CD8 T cells expressing a high affinity TCR. As a result, it is difficult to assess the universality of our results for other TCRs or for CD4 T cells versus CD8 T cells. However, the fact that we saw very similar results over a 2-log range of antigen doses and after in vivo immunization suggests that this effect is not limited to superphysiological antigen levels or conditions uniquely present in vitro.
The idea that synapses can be motile fits well with a body of emerging data suggesting that cells can signal while moving. The first evidence for this was observations of T cell activation by motile interactions with DCs in collagen matrices (Gunzer et al., 2000
). Subsequently, a series of in vivo observations in LNs provided evidence of early signaling resulting in CD69 up-regulation, T cell proliferation, and cytokine production after short-lived encounters (Mempel et al., 2004
; Miller et al., 2004
; Celli et al., 2005
). Recently, Dustin (2007)
has suggested the term kinapse to differentiate motile synapses from the stable cSMAC/peripheral SMAC–containing immunological synapse (Monks et al., 1998
; Grakoui et al., 1999
). Based on our data, it is notable that the appearance of signaling clusters, calcium influx, and synapse formation (tight membrane apposition) are similar under motile and nonmotile conditions and likely represent a continuum of signaling modes rather than a strict dichotomy. A likely reason for very highly stabilized membrane–membrane junctions leading to symmetrical cSMAC structures could be that in homogenous systems such as lipid bilayers or B cell APCs, T cells receive symmetrically equivalent signals from all borders of the synapse, so actomyosin-based motility is balanced inward toward the center of the cSMAC. In that regard, PKC-Θ–deficient T cells appear to lack this balance and are highly motile, even on bilayer substrates (Sims et al., 2007
). Bilayers and some APCs may create an abnormally large tight apposition between the T cell and APC, whereas T cell–DC junctions are more typically multifocal (for review see Doh and Krummel, 2010
). Such tight apposition might disfavor continued migration and allow assembly of a single large synapse structure that restricts remodeling and allows formation of highly localized internalization domains.
The brief nature of TCR clustering that we observed in naive cells in multiple settings is likely related to rapid TCR internalization. Because TCR internalization and downmodulation is a direct result of TCR signaling (Valitutti et al., 1997
; Liu et al., 2000
), the presence of TCR clustering and internalization provides perhaps the first direct evidence for TCR signaling by naive cells in the early and transient interactions after antigen recognition in vivo. It should be noted that we were unable to distinguish between surface clustering and internalized TCR just below the membrane because of the resolution of the imaging techniques used. As a result, there is a possibility that we overestimated the frequency and stability of surface cSMACs and underestimated the frequency and speed of internalization.
Based on several in vitro studies, it has been inferred that the cSMAC is likely to be the site of TCR internalization (Lee et al., 2003
; Varma et al., 2006
; Vardhana et al., 2010
); however, the spatiotemporal dynamics of these events have not previously been examined. Although in some cases we did observe TCR internalization occurring from the site of stable TCR surface clustering, in others, TCRs were internalized in the absence of detectable, stable, and prolonged surface clustering. In these cases, internalization likely occurred from sites of TCR microclusters that were below our limit of detection or after rapid aggregation of these microclusters at speeds faster than our rate of acquisition (2 min/frame). Based on these data demonstrating variability of the site of internalization, it will be interesting to see whether the mechanisms of TCR downmodulation and signal termination observed in the cSMACs of activated cells stimulated by passive antigen-presenting surfaces are also used by naive T cells stimulated by DCs. It is likely that in these settings, recruitment of LBPA would be more multifocal with heterogeneity of signal termination among microclusters in the same cell.
The overall motility seen during TCR signaling was slow, as would be expected during a motile interaction in which a T cell crawls along the surface of a DC or a network of DCs. In fact, TCR signaling inactivates the contractile machinery necessary for rapid motility by phosphorylating myosin IIA (Jacobelli et al., 2004
). Upon myosin IIA inhibition or depletion, T cells maintain an active lamellipodium and slide along in a slow, actin protrusion–dependent mode of motility (Jacobelli et al., 2009
). This mode of slow motility during TCR signaling might serve to ensure that the T cell can detect and integrate as many antigen signals as possible by probing as large of an antigen-presenting surface as possible.
Our in vivo imaging of TCR dynamics in the absence of cognate antigen showed transient clustering of the TCR without discernable T cell arrest or rounded morphology. We observed these clustering events at low frequencies, and they were much more transient than reports of antigen-independent receptor clustering observed in vitro (Revy et al., 2001
), likely caused by the three-dimensional environment in the LN. Our imaging was performed in regions of the T cell zone containing an extensive DC network, providing frequent access for antigen-independent interactions. Clustering events such as these may be stochastic, representing a contact with specialized APCs, or might be a random membrane dynamic that is independent of TCR engagement. An intriguing possibility is that in vivo, these result from interactions with eTACs (extra-thymic Aire-expressing cells) presenting self-antigens (Gardner et al., 2008
). Notably, eTACs are clustered in the interfollicular regions of the T zone in the LN, where we imaged the TCR.
The image acquisition and processing strategies we developed in this study have allowed us to analyze surface and intracellular TCR dynamics in naive cells in the LN for the first time by enhancing detection and discrimination of in vivo fluorescent signals. It is clear that the use of fluorescent probes that read out more than cellular positioning are the next hurdle to gain further insight from real-time analyses of cells in situ. The method we have described is significant in that it will allow for the in vivo analysis of the dynamics of other signaling molecules using fluorescent reporters, even when expressed at physiologically appropriate levels. This, in turn, will allow increased understanding of receptor and signaling dynamics within single cells in real time and in their native environments.
The development of real-time molecular imaging in situ has provided us with a view of TCR dynamics that represent the complexities of physiological interactions, including observations of motile signaling modes that might allow for optimal scanning of the T cell’s environment. Continued evolution of these imaging and analysis methods will help integrate signaling dynamics into the biology of complex systems.