TCR triggering occurs on interstitially motile T cells undergoing constant morphological remodeling and crawling at speeds of approximately 10 µm/min (Miller et al. 2002
). In vitro, the actinomyosin cytoskeleton reacts to local integrin levels to allow T cells to adopt one of two crawling modes (Jacobelli et al. 2009
). In the faster “walking” mode, T cells create relatively small contacts to grab surfaces and bound from one contact to the next (A). In a mesenchymal mode, T cells place large portions of their membrane into contact with the surfaces they are surveying. These large, contiguous contacts translate as the T cell moves forward, giving rise to a “moving walkway” of T-cell membrane that enables extensive scanning of the underlying surface. Although these modes are only presumed to occur in vivo, recent evidence indicates that T cells employ a “millipede” type motility to move along the blood vessel endothelium that is similar to the walking mode seen in vitro (Shulman et al. 2009
). The need for the fast mode may be most pronounced when navigating between loose adherence zones on follicular reticular cell fibers in the lymph nodes, which T cells appear to use to help guide their circuit from afferent to efferent vessels (Bajenoff et al. 2008
). Furthermore, transitions between modes are hinted at by the variety of cell shapes and speeds observed within the T-cell zone (Miller et al. 2002
). The actin cytoskeletal mechanics of these motility modes are an essential parameter for TCR triggering, because TCR-pMHC ligation logically requires tight juxtaposition between domains on the T-cell surface that bear TCRs and membrane regions on the APC that display agonist pMHC complexes. By establishing the rate of T-cell scanning across APC membranes and the extent of the T cell-APC contact area, the actin cytoskeleton dictates the time window in which a TCR will bind a specific pMHC complex.
Figure 1. The actin meshwork of the motile T cell establishes a mechanical regime of TCR triggering. (A) T cells (TC) display various modes of motility depending on the adhesive properties of their microenvironment. To achieve high velocities, T cells create series (more ...)
In addition to establishing the transience of membrane-membrane interactions, the cytoskeleton also facilitates T cells’ exceptional responsiveness to TCR engagement. From the host perspective, it is undesirable for T cells to fail to activate following encounters with antigens, no matter how limiting the dose. TCRs, therefore, must be very sensitive—and there is no doubt that they are sensitive: CD8-positive T cells are potentially capable of responding to a single agonist peptide (Sykulev et al. 1996
), whereas 1–10 pMHCs can activate CD4 T cells thanks to their coreceptor (Irvine et al. 2002
). At a naive glance, the ideal mechanism to achieve such sensitivity might seem obvious: A receptor should combine a very fast association rate with a very slow dissociation rate for agonist ligands. Studying the measured binding and unbinding kinetics of some model TCRs quickly shows that not to be the case for TCRs (Davis et al. 2003
). It appears that T cells instead take advantage of rapid unbinding of pMHCs to sequentially trigger multiple TCRs, (Valitutti et al. 1995
; Lanzavecchia et al. 1999
). In effect, the successive ligation of TCRs serves as a gain, turning each pMHC into many activated receptors. The development of mathematical models has helped to explain how T cells have adapted to TCR biochemical properties to achieve both fidelity and sensitivity (McKeithan 1995
; Coombs et al. 2002
; Wedagedera and Burroughs 2006
But what sort of biomechanical processes can we imagine that would facilitate this mechanism of triggering TCRs? Conformational change models of TCR triggering fell into disfavor because of an apparent lack of structural data to support large scale structural alterations following pMHC binding (Ding et al. 1999
; Willcox et al. 1999
; Degano et al. 2000
). However, evidence for rearrangements between members of the TCR complex or within various TCR subunits has been described (Krogsgaard et al. 2003
; Kim et al. 2009
). These studies provide clues to how information about the TCR binding partner is transmitted across the T-cell membrane, but don’t immediately address how the TCR translates this into a signaling output. Under the kinetic-segregation model, T cells use the intercellular spacing to exclude inhibitory molecules from the domain of triggered TCRs (van der Merwe et al. 2000
). By creating zones of close contact in which TCRs can bind to pMHCs, integral membrane phosphatases with large ectodomains that antagonize TCR phosphorylation can be excluded. Specificity is achieved because relatively weak interactions, such as between TCRs and self-peptide MHCs, dissociate more rapidly, and the phosphorylated TCRs diffuse out of the close contact zone. This allows inhibitory phosphatase activities to deactivate the released TCRs. In contrast, relatively stable TCR-pMHC interactions would tether the TCR in the zone of tight contact longer, providing a window to assemble an activated, phosphatase-resistant signalosome. Although lipid rafts might explain how TCRs and other prosignaling factors remain tethered within the area of close contact, it is not clear how the weak adhesive strength from a limited number of pMHC-TCR interactions could maintain a closely spaced junction between the two cellular membranes at the size scale required for the kinetic-segregation model (Burroughs et al. 2006
). To this end, mechanical pressure exerted by the actin cytoskeleton of the leading edge may be instrumental in pressing the apposed cell membranes together, squeezing proteins with larger ectodomains away from the tightest contact (B). Consistent with this idea, increasing the length of the pMHC extracellular domain impacted TCR segregation from CD45 and antagonized receptor triggering (Choudhuri et al. 2005
). Kinetic-segregation might partially explain why integrin-mediated adhesion enhances TCR signaling even on bilayer systems (Grakoui et al. 1999
; Porter et al. 2002
; Suzuki et al. 2007
), as the longer LFA1-ICAM pairs would establish zones of looser contact to accommodate bulky phosphatases.
It is important to note that one of the phosphatases associated with Lck inhibition and TCR deactivation, CD45, also appears to positively regulate Lck by dephosphorylating an inhibitory phosphotyrosine of Lck (Biffen et al. 1994
; Stone et al. 1997
). As a result, it would appear that CD45 must have access to TCR signalosomes near the time of triggering; otherwise, phosphorylation of the Lck inhibitory site could inactivate Lck and cause a triggering failure. Balancing the activating and inhibitory potential of CD45, then, may come down to timing access and exclusion through actin-mediated force generation. Following dephosphorylation of the inhibitory site of Lck, CD45 might be rapidly driven from the zone of close contact as integrin-mediated actin polymerization presses the cell membranes together, preventing it from dephosphorylating the active site phosphotyrosine of Lck (Thomas and Brown 1999
). Spatiotemporal analysis of CD45 at T-APC interfaces indicated that CD45 was colocalized with Lck in young cSMACs and segregated from Lck at more mature interfaces (Freiberg et al. 2002
). So far, though, only limited evidence for the sort of microscale exclusion of CD45 envisioned by the kinetic-segregation model has been presented (Choudhuri et al. 2005
; Choudhuri et al. 2009
). Furthermore, the importance of the extracellular domain to signaling is not obvious, and, in fact, smaller extracellular domains of CD45 appear to positively correlate with coreceptor association and antigen receptor signaling (Leitenberg et al. 1996
; Shenoi et al. 1999
; Trowbridge and Thomas 2003
). Therefore, a variation on this apposition-induced triggering mechanism could be that the force pressing the membranes together induces the bending of TCR complexes in TCR-pMHC pairs, revealing sites on CD3 for phosphorylation. Weaker, nonagonist pMHC-TCR interactions would release before phosphorylation sites expose, allowing the TCR to relax into the untriggered, resting conformation.
Ma and others recently proposed a very intriguing modified conformational change model (Ma et al. 2008
) hypothesizing that actin cytoskeleton generated shear force parallel to the synapse, rather than a force perpendicular to TCRs, facilitates receptor triggering. In this receptor deformation model, they argue that the stress on TCR-pMHC pairs induced by spreading over an APC essentially analyzes TCR-pMHCs interactions: Strong agonists provide a sufficiently high resistance to bond rupture to induce a deformation of the TCR upon actin cytoskeletal induced stress. That deformation is interpreted as an authentic antigen encounter only if the deformation is large enough or sufficiently persistent to lead to signalosome assembly on a deformed TCR (C). This hypothesis is particularly interesting when viewed in the context of a mixture of integrin and TCR signaling. As the T cells crawl over an APC, LFA-1 stimulation will be temporally coincident with initial TCR-pMHC ligation. The background of integrin signaling can facilitate dynamic actin cytoskeletal polymerization that will generate the shear force needed to test recently bound TCRs. As a result, this model lends itself to a very elegant mechanical explanation of why integrin cosignaling is required to sustain TCR signaling—beyond the adhesion strength supplied to hold the T-APC couple together, it drives the shear induced bond rupture testing with actin remodeling (C). This fits nicely with observations from Takashi Saito’s group. They found that LFA1 signaling induced an actin cloud at the center of contact sites between T cells and APCs in the absence of antigen (Suzuki et al. 2007
). The formation of this cloud lowered the threshold for T-cell activation after subsequent antigen exposure.
It is not clear if the sheer-induced TCR-pMHC bond testing would require the assembly of a nascent signalosome with signaling factors providing specific binding sites for actin filaments. The cytoskeleton could exert force on a TCR without a specific molecular coupling linking receptors to filaments through general steric interactions. By first assembling a nascent actin-linked signalosome around the receptor, though, the cytoskeleton could apply force to the TCR-pMHC bond no matter what direction the filament moved relative to the TCR. If the receptor deformation testing mechanism is engaged after the assembly of an initial signalosome, a secondary proof-reading mechanism is needed to explain how the signalosomes of TCRs bound to weak agonists, which fail the sheer test, can be reverted. SHP-1 phosphatase regulates a negative feedback loop that deactivates TCR signaling initiated by weak agonists, whereas ERK can stabilize Lck modification and stabilize signalosomes (Stefanova et al. 2003
). Combining this observation with the receptor deformation model, ERK may be recruited specifically to those receptors that have undergone deformation, exposing a binding site for ERK or an ERK-binding intermediate.
This model can also explain why soluble multivalent, but not monomeric, pMHCs can stimulate T cells (Boniface et al. 1998
; Cochran et al. 2000
), which is sometimes taken as evidence for a multimerization model of TCR triggering. For single TCRs ligated to soluble pMHCs, the added mass of the bound pMHC is unlikely to distort the TCR conformation under a pulling force applied by actin. When two pMHCs are bound to nearby TCRs, though, small deviations in the directions or magnitudes of the forces applied to the TCRs could induce conformation changes in one or both receptors. This model might even be able to explain the “pseudodimer” mechanism (Krogsgaard et al. 2005
), if the added stability of the low-affinity member of the pseudodimer can increase the rupture strength of the high-affinity counterpart just enough to induce a conformational change.
An intriguing recent observation indicates that the interaction of untriggered TCRs with the actin cytoskeleton increases in response to calcium release (Dushek et al. 2008
). In T cells stimulated to release calcium with ionomycin, rather than antigenic stimulation of TCRs, F-actin increased and TCR mobility decreased. Although the decrease in TCR mobility could be described by diffusion trapping, the authors argue that a model combining diffusion trapping with receptor-actin binding better explained the observed changes in TCR mobility. Even if we dismiss the actin binding component, diffusion trapping alone of untriggered TCRs has significant implications for the biology TCRs. Migrating T cells are most sensitive to TCR triggering at the leading edge (Negulescu et al. 1996
), site of the filamentous actin-rich lamellipod and lamella (Pollard and Borisy 2003
). Trapping TCRs might prepare them for signalosome assembly by placing them in proximity to a number of effectors of TCR signaling, or facilitate formation of the cytoskeletal linkages that will test pMHC interactions through the receptor deformation mechanism. It would appear, then, that TCR triggering mechanisms are adapted to achieve maximum sensitivity within the micro-environmental context of the actin cytoskeleton of motile T cells. This suggests an additional benefit for scanning T cells to continuously slide over APC surfaces—it maintains the dynamic actin meshwork that facilitates TCR triggering. Following triggering, it would be expected that TCR signalosome and microcluster life cycles are likewise coordinated with the morphological rearrangements of continuous motility.