How exactly force is applied to the TCR is an important question. As already mentioned, the TCR can interact both directly and indirectly with the actin cytoskeleton. The direct association of CD3ζ is mediated by two stretches of basic amino acids, and mutation of these residues results in decreased synapse formation and T cell activation (116). Interestingly, these same amino acid stretches also mediate binding of the CD3ζ to the negatively charged inner leaflet of the plasma membrane, limit the phosphorylation of ITAMs (141, 142), and are required for synaptic recruitment of CD3 (143). The dual role of the basic stretch suggests a possible competition of binding for the basic residues in the CD3ζ cytoplasmic domain, with binding to the inner leaflet acting as a negative regulator for activation, and binding to the actin cytoskeleton acting as a positive regulator. It also raises the possibility that following ligand binding, force exerted by the actin cytoskeleton on CD3ζ may physically disrupt the association of CD3 chains with the plasma membrane, helping to expose the ITAMs for subsequent phosphorylation (**Figure 3D**, model 2). Similar binding of the CD3ϵ chain to the plasma membrane also restricts phosphorylation of ITAMs within the ϵ chain by Lck (144). Though no direct CD3ϵ/F-actin interaction has been discovered, it is known that CD3ϵ can bind directly to Nck following TCR engagement prior to detectable phosphorylation of ITAMs, and the Nck-binding site is exposed following TCR engagement, and independently of TCR signal initiation (131, 145). This interaction is critical for the initiation of TCR triggering at very early steps, since mutating the residues involved in Nck binding or blocking the interaction with cell permeant peptides results in greatly diminished phosphorylation of CD3ζ, CD3ϵ, and Zap70, reduced recruitment of CD3 to the synapse, and inhibition of proliferation and effector function (146, 147). Nck is linked to actin polymerization through recruitment and binding of N-WASp and WASp (148, 149). Since an N-WASpmediated linkage between actin and p130 Cas has been proposed to cause the force-dependent activation of p130 Cas (150), it is likely that the connection of CD3ϵ to the F-actin network through Nck can transduce a similar force. Therefore, CD3ϵ ITAM phosphorylation could be regulated by actin-generated force in a way similar to the one proposed for CD3ζ.

An alternate model involving normal force stems from recent work by Liu et al. demonstrating that TCR/pMHC interactions show catch-bond behavior (111). Since many of the theoretical mechanisms for catch-bond formation require an accompanying conformational change (151), this study provides strong circumstantial evidence for the existence of an as-yet undefined conformational change at the site of TCR/pMHC interaction. In the study by Liu et al., a tensile normal force was applied by retraction of an extracellular probe bearing pMHC, though it has been theorized that a similar normal force can be generated internally through the action of the F-actin network (118). In this case, the F-actin flow at the IS would pull on the TCR, inducing a conformational change in the ectodomain that would strengthen the interaction with bound pMHC complexes (**Figure 3D**, model 3). Even if this conformational change does not initiate signaling, it could enhance the probability of TCR triggering, as in the kinetic proofreading model.

# Regulation of Integrin Function by Cytoskeletal Forces

In T cell/APC contacts, integrins are primarily responsible for the adhesive interactions that maintain cell–cell contact (152, 153). Each integrin consists of an α and a β subunit, paired as shown in **Figure 4A**. In T cells, integrins are required for firm adhesion to endothelium during diapedesis and for formation of stable T cell/APC interactions, resulting in T cell activation or effector function. As such, integrins must function in a variety of extracellular environments, even under the extraordinary strain placed on the integrin–ligand bonds by the shear flow in blood vessels. Furthermore, integrin activation must be tightly regulated to prevent improper lymphocyte function. In general, integrins are regulated at two distinct levels: affinity (the strength of interaction between each individual integrin molecule and its ligand) and valency (integrin density at the cell–cell interface). Both valency and affinity contribute to adhesion (154). Therefore, the overall strength of interaction, or avidity, is a product of valency, affinity, and relative contact area (155). In T cells, intracellular signals emanating from chemokine receptors or the TCR have been shown to increase the activation state of integrins on the cell surface. This "inside out" signaling can result in either changes in valency or affinity, and a large body of work has accumulated defining the relevant biochemical pathways (155–157). Recently, new data have emerged demonstrating the regulation of integrin activation through applied forces (158–160). In the following section, the signaling pathways governing integrin activation at the IS will be covered, with a particular focus on the role of cytoskeletal forces in initiating and sustaining changes in integrin valency and affinity.

The αLβ2 (CD11a/CD18) integrin LFA-1 is expressed exclusively in leukocytes and is essential for T-cell trafficking and IS formation. In resting T cells, LFA-1 is maintained in an inactive, bent conformation with very low ligand-binding capacity (**Figure 4D**). Signaling through TCR and CD28 results in the activation of the small GTPase Rap1 downstream of PLCγ, PKCθ, and CrkL (161–164) (**Figure 2**). Following Rap1 activation, talin is recruited to the IS through the action of RIAM, which links talin to

### Comrie and Burkhardt Cytoskeletal Control of Immunoreceptor Triggering

### Figure 4 | Continued

(A) Known α and β integrin chains and pairings. Integrin pairs expressed in leukocytes are depicted in red. (B) Domain structure of the integrin LFA-1. The α chain consists of an intracellular tail (cyan), a transmembrane domain (orange), two calf and one thigh domains (gray), and a β-propeller domain (purple) with an inserted ligand-binding I domain (pink). The β chain consists of an intracellular tail (cyan), a transmembrane region (orange), a β tail domain (brown), four EGF repeats (yellow), a hybrid domain (blue) with an inserted βI domain (green), and a PSI domain (red). (C) Retrograde actin flow drives LFA-1 into the IS from the cell periphery. This increases local concentrations of LFA-1, thereby increasing the valency of the interaction and strengthening cell–cell adhesion. (D) LFA-1 can exist in roughly three conformations: a bent, low affinity conformation; an extended intermediate affinity conformation; and an extended conformation, where the hybrid domain on the β chain is swung outward, allowing for downward movement of the α7 helix in the βI domain. This downward movement allows the βI domain to bind an internal ligand in the αI domain, causing downward movement of the αI domain α7 helix and opening of the ligand-binding site. These changes generate a high affinity, extendedopen conformation. Maintenance of this conformation at the IS is dependent on ongoing actin flow, presumably because connection of the β chain intracellular domain to the dynamic F-actin network is enough to drive swing-out of the hybrid domain. The resulting force-dependent increase in affinity would promote and augment changes induced by ligand binding. (E) In addition to regulating LFA-1 affinity for ligand, applied force can also strengthen the connection of LFA-1 to the underlying actin cytoskeleton. Talin, a key protein that links integrins to the actin network, can stretch upon the application of force. This stretching reveals up to 11 cryptic vinculin-binding sites. Vinculin, itself an actin-binding protein, then binds to the exposed sites and reinforces linkage to the F-actin network.

the membrane targeting CAAX domain of activated Rap1 (165). Recruitment of talin to the IS is required for LFA-1 affinity and valency modulation as well as conjugate formation (166). Binding of the talin head domain to the cytoplasmic domain of the integrin β chain causes alterations to the β transmembrane domain, thereby relieving interactions between the α and β chains that maintain the bent conformation. This process allows unfolding of LFA-1 and the adoption of an intermediate conformation with 10-fold increased affinity for ligand over the bent conformation (167–175). This "switch-blade" like unfolding occurs in the presence of activating antibodies or ligand-mimetic peptides and exposes epitopes that report on integrin activation (176, 177). Importantly, overexpression of the talin head domain is enough to result in extension of the majority of LFA-1 molecules on the cell surface, but it does not fully rescue cell adhesion, suggesting that the actin-binding domain of talin is essential for full LFA-1-mediated cell adhesion (166, 178). In addition to talin, other proteins, including members of the kindlin family of adaptors, are known to bind to the cytoplasmic domains of integrin β chains and link them to the cytoskeleton, further emphasizing that cytoskeletal linkage is essential for proper integrin function (160, 179).

The role of the cytoskeleton in mediating changes in LFA-1 valency is not straightforward. Early studies proposed a mechanism whereby cytoskeletal restraints limited the mobility of LFA-1 in resting cells, thus preventing clustering. Upon activation, cytoskeletal restraints were released, allowing the free diffusion and coalescence of LFA-1, thus increasing valency (180–182). In this model, the increased association of high affinity LFA-1 with the cytoskeleton limits the ability to support firm adhesion (183). In support of this idea, low dose Cytochalasin D increases LFA-1 mobility and clustering and increases cell adhesion to ICAM-1-coated surfaces. These changes do not induce, and function independently of, changes to integrin affinity and conformational change (154, 184). Importantly, later studies demonstrated that the integrin clustering mediated by actin depolymerization only occurs in the presence of ligand, and suggested a trapping mechanism aided by the increased diffusivity of LFA-1 in the absence of the cytoskeleton (184). This would indicate that LFA-1 interaction with the cytoskeleton limits valency and is in contradiction with the finding that talin, the main link between LFA-1 and the cytoskeleton, is required for LFA-1 synaptic accumulation (166). Further complicating the picture is the observation that transport of microclusters containing LFA-1 and ICAM-1 at the IS is dependent on an intact actomyosin network (4, 185). One of the key confounding factors in this literature is that the studies that identified the actin cytoskeleton as a negative regulator of LFA-1 valency were not carried out within the context of an IS. In an IS setting, actin retrograde flow can actively draw LFA-1 into the IS, increasing the valency of LFA-1/ICAM-1 interactions (**Figure 4C**).

In addition to the regulation of valency, integrin avidity can be regulated at the level of affinity. Changes in integrin affinity are generally associated with conformational changes (158) (**Figure 4D**). As previously mentioned, "inside out" regulation of integrin extension mediates the transition from the low affinity to the high affinity conformation. Conformational change from the intermediate to the high affinity state results in a further 100-fold increase in affinity for ligand and has been proposed to be mediated by forces generated by the T cell actin cytoskeleton (159, 175). Structural changes associated with integrin activation have been characterized using activating mutations and antibodies (176, 186–188). Typically, integrin activation and ligand binding are associated with a lateral swing-out of the hybrid domain and downward movement of the α7 helix in the βI domain. This induces the high affinity conformation of the βI domain and has been shown to occur through a series of conformational intermediates (189). In αI domain-containing integrins such as LFA-1, the activated βI domain binds an invariant glutamate residue in the C-linker region between the αI- and β-propeller region. This results in downward movement of the αI domain α7 helix and adoption of the extended open, high affinity, αI domain. Importantly, antibodies that stabilize the extended and extendedopen conformations greatly increase LFA-1's affinity for ligand, resulting in a near 1000-fold dynamic affinity range from the bent to the extended-open conformations (175, 190). Furthermore, shortening of the C-linker region to mimic the downward motion exerted by the βI domain results in constitutively active LFA-1 (187). Steered molecular dynamic simulations have demonstrated that conversion between different integrin conformations can occur through the application of physical forces. Pulling on the headpiece or on bound ligand can overcome interactions between the hybrid and β-tail domains that help maintain the bent conformation, resulting in integrin extension. Importantly, forces applied to the headpiece were not sufficient in these simulations to induce the opening of the headpiece or separation of the integrin legs (191). Interestingly, similar simulations have shown that a tensile force applied parallel to the membrane on the β cytoplasmic tail can be propagated along the β chain, resulting in hybrid domain swing-out and affinity modulation (188). Since talin binds to the integrin β cytoplasmic domain, any force applied on LFA-1 through talin's linkage to the retrograde actin flow at the IS would result in a similar tensile force, and should mediate integrin affinity maturation (**Figure 4E**). Intriguingly, it is known that high affinity LFA-1 is more tightly bound to the actin cytoskeleton than intermediate or low affinity LFA-1, supporting the idea that linkage to the underlying cytoskeleton is involved in conformational regulation (192). Our recent work has demonstrated that, indeed, the force provided by retrograde actin flow is critical for maintaining LFA-1 in the high affinity conformation, ligand binding, and clustering of LFA-1 at the IS (193). Thus, connection of LFA-1 to the dynamic actin network provides the force required to initiate integrin recruitment to and clustering within the IS, thereby increasing valency, and also provides the force to induce conformational change to the high affinity state (**Figure 4**, green arrows).

Consistent with the prediction that force can enhance LFA-1 affinity, integrins engage in catch-bond interactions (194–196). As with other adhesion molecules, such as selectins, integrin bond lifetime increases as tensile normal force is applied, until a threshold known as critical force is reached, where bonds are rapidly ruptured (151, 197). Importantly, blocking binding of the αI internal ligand by the open βI domain inhibits catch-bond behavior, suggesting that conformational change initiated by hybrid domain swing-out is required to initiate catch-bond interactions. Furthermore, it has been shown that α5β1 and LFA-1 bond lifetimes are increased following a short, transient, period of high force application. For LFA-1/ICAM-1 interactions, loading and then unloading of applied force stabilizes the integrin/ligand bond, increasing the average bond lifetime from 1.5 to over 35 seconds (198).

So far, we have discussed the mechanosenstive aspects of LFA-1 regulation with a focus on integrin–ligand interactions. Importantly, the connection between the T cell and the APC mediated by the integrin–ligand bond is only as strong as the weakest link in the pathway (199). Whereas catch-bond interactions between integrins and their ligands exhibit increased affinity with the application of force, the links that connect integrins to the cytoskeleton are thought to behave as more conventional slip bonds, where force decreases bond lifetime. Nonetheless, the talin-mediated linkage of LFA-1 to the actin cytoskeleton is regulated by the application of force. Once talin binds to the integrin β tail through its head domain and to F-actin through its rod domain, actin–myosin-mediated force pulls on talin. This causes talin to unfold like an uncoiling spring, thereby exposing up to 11 cryptic vinculin-binding sites (200, 201) (**Figure 4E**). Binding of vinculin to the talin rod domain then allows vinculin to bind to F-actin and enforces the integrin linkage to the actin cytoskeleton (160, 201–204). Although this process is reversible, such that loss of force leads to diminished vinculin binding, vinculin binding can stabilize the unfolded conformation of talin (205). In T cells, vinculin is recruited to the IS and is required for talin recruitment and conjugate formation, suggesting that the destabilized talin– F-actin bond is not enough to maintain LFA-1 activation (206). Thus, talin–vinculin binding represents another force-dependent step in the pathway leading to integrin activation, and another mechanism through which cell adhesion can be enhanced by F-actin flow (**Figure 4E**).

Integrin-mediated outside-in signaling has been mentioned earlier as a driver of multiple pathways of T cell activation. Importantly, the conformational changes that mediate LFA-1 affinity maturation are also required for outside-in signal initiation. Blocking LFA-1 affinity maturation leads to decreased IL-2 production and T cell proliferation (207). Likewise, inhibition of the separation of the transmembrane domains through addition of an inter-subunit disulfide bond results in the loss of outside-in induced stress fiber formation and cell spreading in CHO cells (208). Conversely, affinity modulation through the addition of conformational change-inducing antibodies results in the same pattern of outside-in tyrosine phosphorylation as actual ligand binding. This suggests that integrin conformational changes are necessary and sufficient to induce outside-in signaling. Therefore, forces on the integrin–ligand bond that induce and stabilize integrin conformational change are likely to also be required to initiate and sustain outside-in signal transduction.

Given the accumulating evidence that physical force exerted by the actin cytoskeleton drives conformational changes that mediate LFA-1 activation and stabilize this active conformation, we must re-evaluate our understanding of TCR-mediated integrin activation. Under this new paradigm, forces generated by the retrograde flow of the T cell actin cytoskeleton act as a key component of inside-out signaling and are a critical allosteric regulator of integrin activation at the IS.

## Regulation of CD28 Signaling by the F-Actin Network

As with the TCR and LFA-1, there is strong evidence that F-actin contributes to costimulatory signaling at the IS. Here, we will focus on CD28, although signaling through CD2 and other costimulatory molecules also involves the actin cytoskeleton (52–55, 209–211). Microclusters of CD28 form concomitantly with TCR microclusters and then segregate into their own domain outside of the cSMAC, but the role of F-actin in the formation and centralization of these microclusters is unknown. Somewhat surprisingly, CD28 microclusters only require the presence of ligand to form and centralize and will do so even in the absence of the CD28 cytoplasmic tail, though differences in the rates were not addressed (212). This suggests that CD28 microcluster formation is primarily the result of kinetic segregation. If so, F-actin dynamics could contribute to this process, as diagrammed in **Figure 5**. Regardless of the effects of F-actin on CD28 microclusters, several studies indicate that propagation of signals downstream of CD28 is dependent on F-actin dynamics. First, the F-actin-uncapping protein Rltpr, which interacts with CD28, is absolutely required for CD28 signaling. Rltpr-deficient mice mimic the phenotype of CD28 knockout mice, and Rlptr is required for the CD28-mediated focusing of PKCθ and Carma 1 within the central region of the IS (49). Second, Tan et al. showed in thymocytes that activation of Src kinases by acute inhibition of Csk recapitulates many early signaling events in the TCR signaling pathway, but does not allow elevation of intracellular Ca2+ or ERK phosphorylation (45). Intriguingly, this blockade in

scaffolding protein and an essential CD28 effector that can undergo force-dependent conformational change. Force causes the extension of the second rod domain of filamin A, eliminating binding sites that exist in the relaxed protein, and exposing a different set of binding sites that only exist in the extended protein. Thus, under tension, FLNa can release low force-binding partners and recruit new proteins to the CD28 signaling complex.

signaling could be overcome by perturbing the actin cytoskeleton or by stimulating CD28-mediated F-actin rearrangement. These data support a model in which cortical actin forms a functional barrier between active PLC-γ1 and its substrate, and engagement of CD28 remodels actin architecture to allow signaling to proceed. Finally, it has been shown that costimulatory signaling by CD28 can induce greater force on stimulatory surfaces than TCR triggering alone (presumably through regulation of the F-actin network). This additional force is not applied through CD28 itself. Instead, force is applied through the TCR, at least in the absence of integrin engagement (138). Perhaps CD28 costimulation can lead to greater forces on other mechanosensitive receptors, including integrins. This idea is consistent with the finding that CD80 and CD86 on DCs increase the strength of cell–cell interactions with a responding T cell (213).

CD28 interacts with the F-actin cross-linking protein filamin A (FLNa) through the PxxPP motif in the CD28 cytoplasmic tail and domains 10–12 of FLNa. FLNa is recruited to the IS in a CD28-dependent manner following TCR stimulation, and CD28 interaction with FLNa is required for T cell costimulation (214). FLNa is a large, rod-like protein that is composed of an N-terminal actin-binding domain and 24 Ig-like domains. Ig-like domains 1–15 are referred to as Rod 1, while domains 16–23 make up Rod 2, with the 24th domain allowing for homodimerization. In addition to binding actin, FLNa is a prolific scaffolding protein with over 90 known binding partners, including intracellular signaling molecules, receptors, ion channels, transcription factors, and adhesion proteins (215, 216). Critically, many of these interactions can be regulated through the application of either external or internally generated force. Cryptic binding sites in the compact Rod 2 domain are exposed, and binding sites in the normal state are abolished following the application of force. This could occur through stretching of the Rod 2 domain under conditions where the F-actin network is under stress (216–218). This is particularly relevant at the IS, where the robust F-actin flow is likely to apply considerable stress to the network and may represent an important force-dependent aspect of CD28-mediated costimulation (**Figure 5**). Indeed, the mechanical regulation of FLNabinding partners sets up several potential signaling mechanisms. Molecules that are recruited under force could allow for localized signal activation and signal amplification. Conversely, molecules that are released following force application could act as soluble signaling factors, exerting their function on areas distant from the IS. FLNa has been shown to be important for PKCθ recruitment to, and NF-κB-inducing kinase (NIK) activation at, the IS. Interestingly, while NIK is constitutively associated with FLNa, interaction with PKCθ requires CD3/CD28 signaling. Thus, it will be interesting to see if FLNa-mediated CD28 recruitment of PKCθ is force dependent.

# Force-Based Activation of Other Mechanosensitive Molecules at the IS

The preceding sections address force-induced activation of surface expressed receptors and their ligands, but F-actin-generated forces affect cytoplasmic molecules as well. One prime example is CasL, a lymphocyte-specific member of the Crk-associated substrate (Cas) family of proteins. Members of the Cas family contain a highly conserved Src kinase substrate domain with multiple phosphorylatable YxxP motifs. For the non-hematopoietic isoform p130Cas, it has been shown that these motifs are exposed by mechanical stretching of the protein (219, 220). Stretching of p130Cas and exposure/phosphorylation of the Crk-binding site is dependent on integrin binding to an immobilized substrate. In T cells, phosphorylation of CasL allows binding to Crk and the associated GEF C3G, leading to the activation of the small GTPase Rap1. Since Rap1 activation induces the recruitment of talin and the affinity maturation of multiple integrins, it seems likely that CasL functions in a positive feedback loop linking mechanical forces on engaged integrins to additional integrin activation.

Two independent studies have demonstrated that myosin contractility is required for maximal CasL phosphorylation, though in both studies there was significant CasL phosphorylation left at the IS following myosin inhibition (221, 222). Interestingly, phosphorylation of Cas within the substrate domain is largely independent of myosin contractility, but completely dependent on F-actin polymerization (150). It is therefore likely that even in the absence of myosin contractility, continued polymerizationdriven F-actin flow at the IS provides sufficient force to drive stretch-dependent Cas/CasL activation.

# Cytoskeletal Forces May Create Signaling Rich and Poor Zones at the IS

Different mechanosensitive receptors can apply varying amounts of force on their ligands (223), and the maximum amount of force that can be applied is only as strong as the weakest link in the complex that links ligand to receptor and receptor to the actin network (199). Because of this, the force that allows for the greatest signaling for each receptor could be very different depending on the strength of these varying molecular interactions. In terms of the IS, TCR–pMHC catch-bonds can withstand a maximum force of roughly 10 pN. Unfolding of domain 20 in the CD28-binding protein FLNa occurs with the application of roughly 15 pN (224), while the integrin–ligand bonds are capable of withstanding maximum force of 30 pN (111, 194). Even the linkage of vinculin to the talin rod domains has a maximum force (~25 pN) that can be applied before unbinding occurs, since the talin helices become unstable (225). Since we have shown that the F-actin network slows as it moves toward the center of the IS, peak force is likely to decrease concomitantly [this assumes that force is directly proportional to the rate of F-actin flow, though reality may be more complex (199)]. If so, then signaling from force-resistant molecules would be initiated and sustained in regions of high and moderate F-actin dynamics (i.e., dSMAC and pSMAC regions), while molecular interactions with low force resistance would only occur in areas of moderate F-actin dynamics (i.e., the pSMAC region). This could set up intrinsic areas of maximal signaling for each receptor as microclusters form and traverse the IS. Additionally, since F-actin dynamics are poor or non-existent in the cSMAC, and the cSMAC represents an area of low force generation during the stable phase of IS formation (138), this region should not support force-dependent signaling. In keeping with this model, TCR microclusters retain their phosphorylation in the pSMAC, but become poorly phosphorylated in the cSMAC (77). Thus, the distribution of F-actin-generated forces at the IS may serve both to initiate signaling and to limit ongoing signaling by sweeping microclusters into areas of poor F-actin dynamics. In keeping with this, limiting the centralization of microclusters and maintaining them in the peripheral F-actinrich compartments of the IS enhance microcluster lifetime and signaling (77). Additionally, for molecules that engage in catchbond molecular interactions, the bond strength and half-life may also be regulated across the IS radius, with stronger interactions occurring in areas of higher F-actin-generated force, as long as that force is not above the rupture force. To really understand how changing actin rates and actin-generated forces affect T cell signaling differentially across the IS, careful measurements of traction forces on different molecules across the IS radius will be required.

# THE CONTRIBUTION OF THE DENDRITIC CELL CYTOSKELETON TO T CELL PRIMING

Although a great deal is known about the mechanisms through which T cell signaling affects actin dynamics on the T cell side of the IS, and *vice versa*, much less is known about the APC side of the synapse. The APC has long been assumed to be a passive partner in IS organization, and until recently, little attention has been paid to the possible contribution of the APC cytoskeleton. However, recent evidence indicates that at least for DCs, the F-actin network plays a critical role in regulating IS-associated signaling events leading to T cell activation.

# DCs Form Barriers to Lateral Diffusion and Control Synaptic Patterns

One area where the DC cytoskeleton is likely to play an active role is in regulating IS formation and structure (226). T cells responding to B cells or stimulatory supported lipid bilayers form the classical mature synapse with a characteristic annular pSMAC and cSMAC pattern (227, 228). This shows that in the absence of barriers to ligand mobility, TCR and LFA-1 microclusters will be driven toward the IS center in a T cell autonomous fashion. The DC/T cell IS lacks this annular pattern and is instead characterized by multiple patches of protein segregation. Even at late time points, there is no central accumulation of CD3 (229). Therefore, it is highly likely that the DC forms barriers to the free diffusion of T cell ligands that are either cytoskeletal or topological in nature. Importantly, these two possibilities are not mutually exclusive and the lateral mobility of some proteins could be regulated through linkage to the DC actin cytoskeleton, while others could be restricted by topological barriers. In either case, the DC actin cytoskeleton would play a crucial role in defining and maintaining these diffusive barriers. The existence of barriers at the DC/T cell IS has important implications for the mechanical regulation of signaling. As detailed below, we found that the T cell actin cytoskeleton activates mechanosensitive molecules, such as integrins, by applying force to the receptor–ligand bond, while barriers to diffusion provided by the DC cytoskeleton provide a retentive counter force on the ligand, thereby increasing tension at the molecular level. Through a similar mechanism, regulation of ligand mobility could prevent microcluster centralization and deactivation, thereby enhancing T cell activation. Just as upregulation of T cell ligands enhances T cell priming by mature DCs, control of molecular mobility would serve as a second mechanism through which DCs could modulate their T cell stimulatory capacity.

# The DC Cytoskeleton Plays a Critical Role in T Cell Priming

The DC F-actin network has been observed to polarize toward a cognate T cell in an MHC-dependent manner (230), and treatment of DCs with actin depolymerizing agents impairs the DCs ability to prime T cell responses (231). Polarization of the DC actin network is mediated by Rho-family GTPases and is required for proper conjugate formation, IL-2 production, and T cell proliferation (232–234). Similarly, the ARP2/3 complex activator WASp promotes the maintenance of T cell–DC interactions; WASp-deficient DCs exhibit fewer and shorter-lived contacts with cognate T cells, and a diminished ability to prime T cell proliferation (235, 236). Although it is clear that the F-actin network on the DC side of the synapse plays a key role in T cell conjugate formation and T cell activation, how changes in the DC cytoskeleton are regulated, and their role in IS-mediated signal transduction are open questions in the field.

# Maturation-Associated Changes in the DC Actin Network

Following recognition of danger signals through pattern recognition receptors, DCs undergo a maturation process that increases their stimulatory potential as APCs. Maturation is associated with an increase in F-actin content and increased plasma membrane ruffling, as well as major changes in actinregulatory proteins. The resulting cytoskeletal changes alter antigen uptake and migratory behavior and increase the stimulatory potential of DCs by modifying the F-actin network at the DC–IS. Among the actin-regulatory proteins that are upregulated during maturation are the actin bundling protein fascin, which is greatly increased in expression (237, 238), the actin-severing protein cofilin, which is activated by dephosphorylation (239), and the actin-binding protein moesin, which is increased both by enhanced expression and phosphorylation-dependent activation (240). Interestingly, there is evidence that fascin and cofilin can work together to remodel the F-actin network (241). Moreover, fascin polarizes to the site of T cell engagement on DCs (231), and we have observed that T cells preferentially bind to pre-formed moesin-rich regions (240). It is important to point out that in addition to changes in proteins such as fascin, cofilin, and moesin that directly bind to actin filaments, DC maturation is associated with changes in regulatory molecules that control actin dynamics. For example, the activation of the Rho-family GTPase CDC42 is diminished during DC maturation (242). Similarly, the semaphorin receptor plexin-A1 is upregulated during maturation and is recruited to the DC–IS, where it mediates Rho activation, F-actin polarization, and T cell activation (234, 243). Going forward, it will be important to define how these changes in cytoskeletal proteins and their regulators impact DC–IS function. Likely mechanisms include physical reorganization of the DC membrane and generation of cytoskeletal tethers or corrals that limit the lateral mobility of T cell ligands.

# Regulated Mobility of T Cell Ligands on the DC Surface

Consistent with the idea that activation of T cell surface receptors is force dependent, *in vitro* analysis shows that modulation of ligand mobility can influence T cell activation (244). Though comparatively few studies have addressed ligand mobility on the DC surface, there is already evidence that ligand mobility is regulated in ways that are important for T cell priming.

### Control of MHC Lateral Mobility

The strongest evidence that ligand mobility is important for TCR triggering comes from studies in which diffusion of an αCD3 antibody in stimulatory bilayers is limited by a physical barrier. Under these conditions, TCR microclusters are trapped in the periphery of the IS, resulting in increased microcluster phosphorylation and cellular activation (77). Interestingly, limiting the forward mobility of TCR microclusters causes local deformation of the F-actin network (6, 74, 245), suggesting that molecular forces between the TCR and the viscoelastic actin network are increased.

Though MHC class II lateral mobility is not constrained by the F-actin network in B cells or DCs (57, 240), the lateral mobility of MHC class I is restricted by the actin cytoskeleton (246–248). It is worth noting that many of the studies documenting TCR catch-bonds were done using the OTI or 2C CD8 TCR transgenic models, suggesting the possibility that TCR mechanotransduction and control of MHC lateral mobility may be more important in MHCI/TCR than MHCII/TCR interactions. It will be interesting to examine the lateral mobility of MHCI in professional APCs and to ask if the control of MHCI lateral mobility correlates with TCR triggering.

### Control of Integrin Ligand Lateral Mobility

As mechanosenstive proteins, integrins respond to the physical properties of their ligand-binding environment. In fact, integrin-mediated cell spreading does not occur unless the ligand can withstand roughly 40 pN of applied force (223). In line with this idea, stiffness of the extracellular matrix correlates with outside in signaling (249), and surface immobilization of ICAM-1 is required for TCR-induced LFA-1 conformational change (193, 250). Importantly, the lateral mobility of ICAM-1 can have dramatic effects on immune cell function. In particular, NK cells adhere firmly to target cells in which ICAM-1 lateral mobility is low, and increasing ICAM-1 mobility decreases the efficiency of conjugate formation and lytic granule polarization (251). This suggests that restriction of the lateral mobility of integrin ligands increases tension on the ICAM-1/LFA-1 bond. In endothelial cells, members of the actinin and ERM family of actin-binding proteins limit the lateral mobility of ICAM-1 through interactions with a concerted polybasic region on the ICAM-1 cytoplasmic tail. Importantly, the constrained lateral mobility of ICAM-1 greatly increases the efficiency of T cell diapedesis, suggesting that this is a critical determining factor for LFA-1 adhesiveness (252, 253). We have recently shown that during DC maturation, ICAM-1 undergoes a specific decrease in lateral mobility mediated by interactions with moesin and α-actinin, which are upregulated and activated in response to inflammatory stimuli (240). This decrease in ICAM-1 lateral mobility enhances conjugate formation and LFA-1 affinity maturation, and ultimately contributes to T cell priming. This evidence indicates that DC maturation is associated with biophysical changes that constrain ligand mobility and promote mechanical integrin activation.

### Control of CD80/86 Lateral Mobility

Constrained lateral mobility of the CD28 ligands CD80 and CD86 on the APC surface may also be important for APC function. Consistent with this idea, the cytoplasmic tails of CD80 and CD86 are essential for their costimulatory activity and mediate the separation of CD28 microclusters from TCR microclusters (254). These tails contain a highly conserved poly-basic motif that mediates protein clustering and cytoskeletal interactions (255, 256). This motif resembles known ERM-binding sites in other proteins, including ICAM-1 (253, 257). Given the importance of F-actin linkage and reorganization to CD28 function, it will be interesting to see if DCs can modulate costimulatory signals by regulating the lateral mobility of CD80 and 86.

## T Cells and DCs Coordinately Regulate the Activation of LFA-1 at the IS

Although there is increasing evidence that the TCR, CD28, and integrins can be activated by application of external forces on individual receptors, it is critically important to know if the molecular forces generated internally at the IS can drive these same activation pathways. We have recently shown that T cell actin retrograde flow drives conformational change of the integrin LFA-1 into the high affinity form, as well as its accumulation and organization at the IS (193). In a reciprocal process, DCs actively limit ICAM-1 lateral mobility on the plasma membrane through upregulation of the proteins moesin and α-actinin-1 (240). The limitation on ICAM-1 lateral mobility opposes the forces applied by the T cell actin cytoskeleton, thus enhancing tension and promoting LFA-1 affinity maturation, T cell adhesion, and priming. Thus, the F-actin networks in the T cell and the interacting DC work in concert to efficiently activate the integrin LFA-1. Interestingly, coordinated regulation of force application on LFA-1 and constrained ICAM-1/ligand lateral mobility is also at play during T cell migration on and diapedesis through inflamed endothelium (253, 258, 259). The theme that appears is that T cell adhesion to either APCs or endothelial cells, whether that is brought on by antigen recognition or exposure to chemokine, involves both a concerted change in the T cell and a reciprocal change in the interacting partner. Thus, for mechanosensitive molecules involved in T cell function, the physical properties of the ligand on the surface of interacting cells must be considered along with the forces applied to the receptor itself.

# FUTURE DIRECTIONS FOR BASIC AND TRANSLATIONAL RESEARCH

Our understanding of the mechanobiology associated with signaling events at the IS is in its infancy. In particular, we lack concrete information on how the relevant forces are generated at the IS, which molecules act as true mechanosensors, and how the function of these molecules is coordinated to tune the immune response. Pioneering studies using biophysical approaches, such as modulation of substrate stiffness, physical manipulation using micropipettes, and traction force measurements, have all been informative (81, 138, 260), and there is much work yet to do in this arena.

To relate these biophysical measurements to T cell signaling, we also need to develop additional probes for protein conformation and activation state. For example, antibodies specific for integrin activation intermediates have been quite valuable (175, 193), as have antibodies that detect stretching of Cas (150, 222). Conformational probes for integrins and Cas family members have been successful because these proteins undergo large scale, concerted, changes as part of their regulated function. Given the paucity of robust conformational changes that are detectible in the ectodomains of the TCR, it is unsurprising that similar reagents do not exist for the study of TCR mechanosensing. One conformation-specific antibody has been reported for the TCR, though this epitope is exposed upon pMHC binding even in force-free conditions (131). Building upon work showing that agonist mAbs bind to the membrane-distal CD3ϵ lobe and exert torque on the CD3 complex (136), it may be possible to ask if cytoskeletal forces on TCRs engaged to pMHC have a similar effect by measuring changes in binding of anti-CD3ϵ Fabs in the presence or absence of F-actin dynamics. In any case, it seems likely that analysis of TCR force sensing will continue to be challenging, and that novel tools that detect changes transmitted across the cell membrane will be needed.

One caveat to the use of conformation-specific antibodies is that they often induce or stabilize the conformations they detect. This considerably limits their use, especially in live cell experiments. Thus, future studies are likely to require fluorescent tension biosensors for live cell microscopy similar to those that have been used to detect force on vinculin (261). This technology is evolving rapidly (262, 263). FRET-based biosensors have already been used to detect activation of VLA-4 in migrating T cells (263), and similar biosensors have been used to create integrin ligands that register force applied by spreading cells (264, 265). Another ligand-based approach involves a tension gauge tether consisting of a piece of double stranded DNA attached to an integrin ligand on one strand and to the substratum on the other such that the DNA strands separate with applied force, which can be varied over a range of 12–56 pN (223). Related ligand-based force sensors could be extremely useful in measuring the forces applied to specific receptor–ligand pairs, and the effects of cytoskeletal perturbations on these forces.

As a closing note, it is worth considering how new knowledge about the mechanobiology of T cell signaling could influence immunotherapy. Recent clinical successes have generated great interest in T cell-based immunotherapies for cancer and infection (266, 267). Although many approaches exist for adoptive T cell therapy, a common first step involves the *ex vivo* expansion of T cell subsets, typically through the use of stimulatory beads.

### REFERENCES

Much thought has gone into optimizing the antibody cocktails used for bead coating, but relatively little attention has been paid to their physical properties. Yet, these properties are likely to be highly significant. For example, most stimulatory magnetic beads commercially available for T cell activation are ~4 μm in diameter; yet, human peripheral blood T cells engaging APCs readily spread to a diameter of 12 μm and resting blasts can spread to over 20 μm. A bead that is not large enough to induce robust spreading is not likely to induce the symmetric actin flow associated with mechanical signaling. Thus, beads with a larger surface area may enhance T cell activation and expansion. Another key parameter is the rigidity of the stimulatory surface. Human T cells reportedly respond better on softer surfaces, with increased IL-2 secretion, proliferation, and effector function increasing upon decreased cell surface rigidity (81). Interestingly, the substrate rigidity range used in this study was 100–1000 kPa, several orders of magnitude above physiological values for soft lymphoid tissues. Further research aimed at optimizing surface area and rigidity may prove valuable in creating artificial APCs for clinical applications.

### AUTHOR CONTRIBUTIONS

WC and JB reviewed and interpreted the literature, drafted and revised the manuscript, and approved the final version for publication. They both agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

### ACKNOWLEDGMENTS

The authors thank members of the Burkhardt lab for helpful conversations, and Drs. Nathan Roy and Daniel Blumenthal for critical reading of the manuscript.

### FUNDING

This work was supported by NIH grants R01AI065644, R01 HL128551, and R01GM104867.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Comrie and Burkhardt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Biophysical Aspects of T Lymphocyte Activation at the Immune Synapse

*Claire Hivroz1,2,3\* and Michael Saitakis1,2,3†*

*<sup>1</sup> Institut Curie Section Recherche, Paris, France, 2 INSERM U932, Paris, France, 3PSL Research University, Paris, France*

T lymphocyte activation is a pivotal step of the adaptive immune response. It requires the recognition by T-cell receptors (TCR) of peptides presented in the context of major histocompatibility complex molecules (pMHC) present at the surface of antigen-presenting cells (APCs). T lymphocyte activation also involves engagement of costimulatory receptors and adhesion molecules recognizing ligands on the APC. Integration of these different signals requires the formation of a specialized dynamic structure: the immune synapse. While the biochemical and molecular aspects of this cell–cell communication have been extensively studied, its mechanical features have only recently been addressed. Yet, the immune synapse is also the place of exchange of mechanical signals. Receptors engaged on the T lymphocyte surface are submitted to many tensile and traction forces. These forces are generated by various phenomena: membrane undulation/protrusion/ retraction, cell mobility or spreading, and dynamic remodeling of the actomyosin cytoskeleton inside the T lymphocyte. Moreover, the TCR can both induce force development, following triggering, and sense and convert forces into biochemical signals, as a *bona fide* mechanotransducer. Other costimulatory molecules, such as LFA-1, engaged during immune synapse formation, also display these features. Moreover, T lymphocytes themselves are mechanosensitive, since substrate stiffness can modulate their response. In this review, we will summarize recent studies from a biophysical perspective to explain how mechanical cues can affect T lymphocyte activation. We will particularly discuss how forces are generated during immune synapse formation; how these forces affect various aspects of T lymphocyte biology; and what are the key features of T lymphocyte response to stiffness.

Keywords: T lymphocytes, immune synapse, force control, TCR, LFA-1, biomechanics, stiffness

### INTRODUCTION

T lymphocytes are motile small cells, which play a key role in adaptive immune responses against pathogens and tumor cells. T lymphocyte activation is triggered by the recognition *via* the T-cell receptor (TCR) expressed at the surface of T lymphocytes, of antigenic peptides, derived from pathogens or tumors and associated with major histocompatibility complex (MHC) molecules exposed at the surface of antigen-presenting cells (APCs). Numerous costimulatory or co-inhibitory receptor/ ligand pairs present at the plasma membrane of both cells can also modulate T lymphocyte activation (1). Thus, T lymphocyte activation is crucially dependent on the close interaction between both plasma membranes. This interaction is organized in time and space by the formation of structures,

### *Edited by:*

*Vincenzo Di Bartolo, Institut Pasteur, France*

### *Reviewed by:*

*Salvatore Valitutti, INSERM, France Michael Loran Dustin, Harvard University, USA*

> *\*Correspondence: Claire Hivroz<EMAIL_ADDRESS>
*† Michael Saitakis is the recipient of a grant from the Fondation pour la Recherche Médicale.*

### *Specialty section:*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology*

*Received: 02 November 2015 Accepted: 31 January 2016 Published: 15 February 2016*

### *Citation:*

*Hivroz C and Saitakis M (2016) Biophysical Aspects of T Lymphocyte Activation at the Immune Synapse. Front. Immunol. 7:46. doi: 10.3389/fimmu.2016.00046*

termed immune synapses, in which molecules are unevenly distributed and segregated while remaining mobile (2–4).

Thanks to increasingly sophisticated visualization techniques, more and more information is accumulated on the organization of both plasma membrane receptors and signaling molecules at the immune synapses. Visualization of T lymphocyte interactions with APCs showed that these cellular partners were submitted to pulling, pushing, and shearing forces due to cell motility relative to each other (5); continuous spontaneous motion of plasma membrane (6); and cytoskeletal remodeling (7–9). A specific function of mechanical forces in T lymphocyte activation was even proposed in the first study showing the dynamic formation of immune synapse (10). Forces exerted by T lymphocytes during these contacts have only been quantified recently (11–13). The TCR itself was shown to be a mechanosensor, i.e., able to convert the mechanical forces exerted during TCR binding to peptide–MHC complexes into a biochemical signal (14–16). Finally, at the resting T lymphocyte membrane, there are organized complexes of receptors and signaling molecules, maintained in a state of basal activity where the membrane receptors are readily available to interact with their ligands on an APC surface and to induce a signaling cascade. This dynamic organization resembles a buffer condition that is able to respond to a minute amount of agonist pMHC in a sea of endogenous pMHCs and is optimized not only for the identification of antigen but also for the initiation and amplification of signals following successful antigen recognition (17–19).

Although formation of the immune synapse has been extensively studied, information on the mechanical properties of the microenvironment and on how these properties affect T lymphocyte functions has only recently become available. We will thus review herein recent advances on the knowledge of how T lymphocytes generate or respond to forces during antigen recognition and immune synapse formation.

# FORCES IN T CELLS

When interacting with an APC, T lymphocyte morphology changes drastically: the cell moves on the APC surface, develops invadosome-like structures which push into the cortex of the APC (20–22), spreads on the APC, and eventually stops. During each of these steps, T lymphocytes exert and/or are submitted to forces, which can affect receptor/ligand bonds. We will discuss the pathways involved in the generation of these forces.

# Spontaneous Membrane Oscillations and Formation of Protrusions

Lymphocytes, such as other cell types, display membrane undulations with amplitude of several tens of nanometer and frequency ranging between 0.2 and 30 Hz (23). Moreover, when interacting with a surface, T lymphocytes rapidly develop protrusions and retractions that are organized in lateral waves along the cell membrane (24). Filopodia or microvilli are protrusive structures with a length between 0.1 and several micrometers that display receptors at their tips and present cycles of protrusions/retractions, which allow them to sense both the mechanical and biochemical environments (25). In order to grow, filopodia have to develop protrusion forces against the membrane that are mainly produced by actin polymerization at the filopodial tip (26–28). Filopodial diameter is in the same range as the diameter of membrane tubes, the generation of which requires forces ranging from 5 to 30 pN (29). Filopodia not only exert protrusive/pushing forces but also retracting/pulling ones, which have been measured using traction force microscopy, i.e., by recording the local deformation of a soft substrate of known stiffness in which fluorescent beads are embedded (29, 30). In neuron cells, the pulling forces developed by filopodia have been shown to be in the order of 1 nN (31). During their migration on endothelial cells, T cells can form F-actin-based protrusions, termed invadosome-like protrusions (ILPs) (32). These structures are small (diameter of ~0.2 μm) and transient (half-life of ~20 s) and physically push against the endothelial cell surface (20, 33). It has been postulated that ILPs can sense the stiffness of endothelial cells by "tiptoing" their surface (32, 34, 35). More recently, Yang et al. (36) described the forces developed by chemotactic T lymphocytes. A laser trap was used to position two beads, one as source of chemokine gradient and the other to measure the forces exerted by the migrating T lymphocytes (in their case, the Jurkat leukemic T cell line). The protrusion forces measured at the leading edge of Jurkat cells migrating in a gradient of SDF-1 were as high as 1000 pN and increased in parallel to the chemoattractant gradient. Moreover, the forces required to stop the migrating cells ranged from 100 to 300 pN. Finally, tensile forces may also be present at the membrane when short molecule bridges at the T lymphocyte/APC interface (i.e., TCR/pMHC or CD2/CD58 pairs) exclude other larger molecules (LFA-1/ICAM-1 pairs or CD45) (37). These results (see **Figure 1** for summary) demonstrate that spontaneous undulations of the T lymphocyte membrane and formation/ retraction of filopodia and other cellular protrusions can generate forces that facilitate probing of the biomechanical microenvironment (38). Meanwhile, receptor/ligand bonds are submitted to a wide range of forces during cell migration. This will result in the modulation of signaling cascades induced by mechanosensitive molecules.

# TCR Engagement Induces Force Generation

Despite the increasing knowledge of signaling pathways engaged after recognition of pMHC by the TCR, the triggering mechanism of the signaling cascade still remains controversial (39). Several mechanisms have been proposed, which involve aggregation, conformational changes, and segregation (40). This matter has been reviewed extensively and will not be addressed further. Yet, studies aiming to investigate if and how receptor engagement generates forces that might then be converted in biochemical signal are sparse.

In one study, Hosseini et al. used atomic force microscopy (AFM) to measure the adhesion forces between a T cell hybridoma and a B cell line used as APC (41). Results showed that in the presence of antigen, adhesion forces built up with time of conjugate formation, starting from 1 to 2 nN at the beginning of the interaction to 14 nN after 30 min. The adhesion forces were mainly due

to lymphocyte function-associated antigen-1 (LFA-1)-mediated adhesion, since the integrin inhibitor BIRT377 almost completely abolished forces in the conjugates (41). Similar experiments were performed on conjugates formed between mouse primary T lymphocytes expressing the OT1 transgenic TCR and a mouse dendritic cell line presenting OVA peptides of different affinities (42). In this experimental model, adhesion forces between cellular partners were smaller (up to 1.5 nN) and correlated to the ability of the different peptides to activate the T lymphocytes; better agonist peptides induced stronger adhesion forces (42).

Even though the above studies provided values for interaction forces between T lymphocytes and APCs, they could neither address the question of the relative contribution of different molecules to the forces measured nor the question of the contribution of each cell partner in force generation. Therefore, we adapted the biomembrane force probe (BFP) technique, which was developed to probe molecular adhesion (43), in order to assay the generation of forces by T lymphocytes. The BFP consisted of a red blood cell (RBC), which was on one side coupled to a bead coated with antibodies and held on the other side by a pipette. A human primary CD4<sup>+</sup> T lymphocyte held by a second pipette was brought into contact with the BFP. Activation of the T lymphocyte was monitored by imaging increases in the intracellular Ca2+ concentration, [Ca2<sup>+</sup>]i, and forces exerted by the T lymphocyte on the BFP were measured on time lapse stacks of images by determining the elongation of the RBC with respect to the position of the fixed micropipette (11). When the bead was coated with anti-CD3 antibodies, three consecutive phases were observed following T lymphocyte contact with the BFP: a latency phase, which lasted less than a minute during which no force and no [Ca2<sup>+</sup>]i increase were observed; a pushing phase consisting of the growth of a directional cell protrusion characterized by an initial axial compression of the RBC and a peak in [Ca2<sup>+</sup>]i increase; and, in most cases, a pulling phase characterized by protrusion/ retraction and generation of pulling forces, as witnessed by the elongation of the BFP. The initial forces exerted by T lymphocytes on the RBC were around 25 pN for a probe stiffness of 50 pN/μm. Measurement of elongations showed that CD3 engagement on T lymphocytes triggered a constant pulling loading rate of ~2 pN/s. These characteristic three phases were not observed when the bead was coated with anti-MHC-I antibodies, showing that the mere binding of the bead to the T lymphocyte membrane is not sufficient to induce forces (11). Engagement of LFA-1 together with CD3 modified the forces exerted compared to CD3 alone: when the bead coupled to the BFP was coated with an anti-CD18 antibody (specific against the β2 chain of LFA-1), a clear decrease in growth velocity and protrusion length during the pushing phase was observed. Moreover, the pulling phase started earlier and the protrusion morphology was changed from a "tube-like" to a "cup-like" structure resembling the phagocytic cup. Engagement of LFA-1 alone on resting primary T lymphocytes did not generate any pushing phase. It also generated 100-fold lower pulling loading rates (0.2 pN/s for a probe stiffness of 50 pN/μm) than the pulling loading rates induced by CD3 engagement alone (25 pN/s for the same probe stiffness). Absence of force generation in response to just LFA-1 triggering can be attributed to the fact that T lymphocytes were not pretreated for inside-out signaling induction (i.e., pretreatment with chemokines, anti-CD3, or phorbol-ester). Thus, integrins can generate traction forces and modify the forces induced upon CD3/TCR triggering. Indeed, force measurements performed on human neutrophils submitted to chemotactic gradients on hydrogel substrates revealed that neutrophils also generated traction forces, which were dependent on β2 integrin engagement and signaling (44). This was not specific to LFA-1 engagement since binding of α5β1 integrins to fibronectin and activation of these integrins by addition of Mn2<sup>+</sup> were also been shown to induce traction forces (45).

Two more studies confirmed that T lymphocytes generate significant forces upon CD3 engagement. In the first one, Bashour et al. used elastomer pillar arrays of known spring constant coated with activating antibodies (12). In this experimental setting, each pillar tip deflection caused by cell attachment and spreading is monitored using live cell videomicroscopy (46, 47). Human primary CD4<sup>+</sup> T lymphocytes were put on micropillars coated with anti-CD3 antibodies and several phases were observed (12). In the first phase, cell spreading generated only minor forces. After this phase, cells ceased to spread and started to exert significant traction forces, which were essentially centripetal and exerted mostly at the cell periphery. The forces generated per pillar were around 50 pN. In the same study, forces exerted by mouse primary CD4<sup>+</sup> T lymphocytes on the same pillars were fourfold higher (200 pN/pillar). No forces were measured when pillars were coated with an antibody against the costimulatory molecule CD28 alone. However, the dual presence of anti-CD3 and anti-CD28 antibodies resulted in doubling the traction forces exerted by T lymphocytes on the micropillars. This was observed when the anti-CD28 Ab was present on the pillar together with anti-CD3 or when added in solution (12). These results suggest that the traction forces induced by CD28 engagement are not directly generated through the CD28 receptor. They are rather due to signaling-dependent amplification of the forces triggered by TCR engagement.

In another study, Hui et al. used traction force microscopy to measure the forces exerted by Jurkat cells during TCR activation (13). Jurkat cells were put on polyacrylamide gels coated with anti-CD3 antibodies and embedded with fluorescent beads at the top surface. The traction forces exerted by the cells were measured by tracking fluorescent bead displacement. In the presence of anti-CD3 and for a substrate stiffness rigidity of 1–2 kPa, traction forces were in the order of 2 nN, whereas forces exerted on substrates coated with a non-activating antibody were below 1 nN (13).

From the above results, it is evident that TCR–CD3 engagement can generate forces in T lymphocytes. These forces can be modified by the engagement of costimulators, such as LFA-1 and CD28. We will now discuss the potential outcomes of forces on T cell activation.

### EFFECT OF FORCES ON T CELL ACTIVATION

We have seen in the previous paragraphs that membrane undulations, protrusions and retractions, cell migration, and TCR triggering can generate forces that can be exerted on receptor/ ligand bonds. In the next section, we will discuss the effect of these forces on specific receptor/ligand pairs at the T lymphocyte/ APC interface, i.e., TCR/pMHC and LFA-1/intercellular adhesion molecule-1 (ICAM-1), and on overall T lymphocyte activation.

### Forces Exerted on TCR/pMHC Bonds

T lymphocytes typically recognize peptides of 8–11 amino acids presented by MHC molecules. The TCR can "sense" a single amino acid substitution and translate it in a different functional response. Moreover, T lymphocytes can precisely discriminate a small number (2–10) of pMHC complexes for which they are specific within a sea of self or foreign peptide-MHC molecules (17–19). How this exquisite specificity and sensitivity is achieved is still a matter of investigation. Forces exerted on the TCR–pMHC bonds may have a key role in these processes. Indeed, it has been shown that the TCR functions as a mechanosensor, i.e., it can convert mechanical cues into biochemical signals (16). The first direct evidence was obtained by E. Reinherz's group, who used optically trapped beads coated with non-activating anti-CD3ϵ antibodies or pMHC to apply forces on the TCR and monitored T lymphocyte activation by measuring [Ca2<sup>+</sup>]i increase (14). They showed that T cells were triggered mechanically, since application of a tangential force (50 pN) to the coated bead induced calcium signaling. Force application on beads coated with pMHC complexes that did not bind TCR had no effect on calcium flux. In another study, Li et al. used a fibroblast cell line expressing a single chain Fv anti-CD3ϵ antibody elongated by a tether (15). Binding to CD3 did not induce calcium signaling unless a mild perpendicular shear stress or a normal pulling force on the T lymphocyte bound to the surrogate APC was applied (15). By contrast, pulling forces applied on CD28 or CD62L did not increase intracellular calcium levels. These studies demonstrated that the TCR could transform a mechanical signal (force) into a biochemical one ([Ca2<sup>+</sup>]i increase). Yet, several questions still remain unresolved and particularly whether forces applied on TCR/pMHC bonds can affect T cell antigen recognition and discrimination. Work from the group of C. Zhu elegantly demonstrated that mechanical forces applied using a BFP on TCR/ pMHC-I (48) and TCR/pMHC-II (49, 50) affected dissociation kinetics in a peptide-specific way. Forces applied to the bonds prolonged the lifetimes of single TCR–pMHC bonds for agonists (catch bonds) but shortened those for antagonists (slip bonds). When forces of 10 pN were applied by BFP on OT1 TCR/pMHC bonds, the ratio of OT1 TCR–pMHC bond lifetimes for the agonist peptide versus a weaker altered peptide grew 57-fold compared to when no force was applied (49, 50), demonstrating that forces can increase the power of antigen discrimination. The functional outcome of different peptides recognized by the same TCR was also shown to be coupled with the cumulative lifetime of the TCR–pMHC bonds (49).

A TCR deformation model where mechanical stress could induce conformational changes that would unmask sites of phosphorylation and allow TCR signaling was also proposed (51). Application of forces on the TCR would expose the immunoreceptor tyrosine-based activation motifs present in the CD3ϵ and ζ chains, otherwise buried into the hydrophobic core of the membrane lipid bilayer (52, 53). More recently, it was proposed that the structural features of TCR–CD3 complexes are adapted to permit sensing and discrimination of the forces to which TCR/ pMHC bonds are submitted (54). Das et al. used optical tweezers and DNA tether spacer technology, which allow for application of forces in the order of piconewton with a spatial precision of nanometer, in order to address the mechanisms involved in the control of strength and lifetime of the TCR–pMHC-I bonds (55). They confirmed that forces applied on TCR–pMHC-I bonds increased the lifetime of the bond and showed that the state of the CβFG loop region, a 12-amino acid peptide present in the constant region of the β chain of the TCR of all mammalian αβTCRs (56), is involved in the increased lifetime of TCR–pMHC-I bonds submitted to tensile forces (55). This study suggests that forces physically modify the αβTCR by switching it from an "extended form" that binds weakly to a "compact form" that binds more robustly. The conformational changes of the TCR would then be transmitted to the CD3 signaling complexes associated with the TCR through mechanisms that have yet to be discovered. Finally, a recent study showed that the juxtamembrane region of ζ–ζ homodimers are divaricated within the TCR–CD3ζ complexes and that TCR engagement drives the intra-complex juxtaposition of the ζ–ζ juxtamembrane regions (57). This mechanical switch might thus couple TCR engagement with CD3ζ-dependent signaling.

### Forces Exerted on Integrin/Ligand Bonds

Integrins are heterodimeric transmembrane proteins that mediate interactions in-between cells and interactions between cells and the extracellular matrix. Avidity of integrins is regulated by changing their valency, i.e., by changing their density at the cell/ cell interface and/or changing their affinity for ligands (58). The LFA-1 integrin plays an essential role for T lymphocyte trafficking, immune synapse formation, and T lymphocyte activation (59). In resting T lymphocytes, LFA-1 is in an inactive bent conformation state, which binds with low avidity to its ligand ICAM-1. TCR stimulation induces a change in LFA-1 conformation, resulting in a more extended conformation of the integrin with an intermediate affinity (60). Finally, binding of LFA-1 to its ligand modifies further its conformation with further increase in its affinity (61, 62). Forces have been shown to play a role in affinity maturation of integrins. Indeed, application of tensile forces on integrin/ligand bonds increases bond strength and longevity (63). This was also reported for LFA-1/ICAM-1 interactions, indicating that, as for TCR/pMHC, these molecules form catch bonds (64). Moreover, it has been shown that the integrin bonds "remember" the history of the forces they have been submitted to. This phenomenon was called "cyclic mechanical reinforcement," as the bond strength accumulates over repeated cycles of forces and is maintained after force removal (65). For instance, fibronectin/α5β1 integrin bonds dissociate within 1 s at a force of 5 pN, while upon cyclic mechanical reinforcement, the bond lifetimes can be extended to 14 s. Similar mechanisms apply to LFA-1/ICAM-1-specific bonds (65). Although head rearrangements of integrins are induced by ligand binding, this might take seconds to happen in the absence of force (66). Application of forces on the bonds would thus shorten the time required for conformational change. Moreover, cyclic mechanical reinforcement would strengthen the bonds by easing and accumulating the reversible conformational change of integrins with multiple force cycles. Therefore, during immune synapse formation, dynamic cyclic traction forces are exerted on LFA-1/ICAM-1 bonds by cycles of membrane undulations, protrusions, and retractions or by direct LFA-1 engagement, since, as described above, this can lead to force generation in T lymphocytes. By inducing conformational changes of integrins, forces during immune synapse formation can facilitate adhesion between T lymphocytes and APCs and probably participate to the costimulatory activity of LFA-1, although this remains to be tested.

### THE ACTOMYOSIN CYTOSKELETON: A FORCE GENERATOR AT THE IMMUNE SYNAPSE

Forces experienced by T lymphocytes during synapse formation can come from the exterior but can also come from the interior generated by the cell's own cytoskeleton. Many reviews have described and discussed remodeling of the cytoskeleton at the immune synapse and its potential role. We will herein concentrate on the role of the actomyosin cytoskeleton on the generation of forces. In the first dynamic study of immune synapse formation on artificial lipid bilayer, Grakoui et al. proposed a model of synapse formation in three stages (10): in the first stage, LFA-1 binding in the center of nascent synapse would provide "a fulcrum for cytoskeletal protrusive mechanisms that force an outermost ring of T cell membrane into close apposition with the substrate"; in the second stage, the transport of TCR–pMHC pairs to the center of the synapse would be actin driven; and in the last stage, the forces exerted would equilibrate, leading to stabilization (10). This model already proposed that forces generated by the T lymphocyte cytoskeleton would play a key role in immune synapse formation. It is remarkable to note that this model fitted so well to the experimental data obtained later on. Actin cytoskeleton has long been known to control T lymphocyte activation at different levels, such as adhesion to APC, early signaling through the TCR, and release of cytolytic granules or cytokines (67–71). T lymphocytes, when activated by the TCR–CD3, spread rapidly (in 2–4 min) on the activating substrate or cell they interact with, they stabilize (for 15–20 min), and then retract (10, 21, 72–74). These phases are reminiscent of the phases observed when adherent cells spread on their substrate (75). Indeed, the different zones of the immune synapse or supramolecular activation clusters (SMACs) have been compared to the lamellipodium (for the distal SMAC), the adhesive lamella (for the peripheral SMAC), and the uropod (for the non-adhesive central SMAC) of a mobile adherent cell (76). During synapse formation, microclusters of receptors form in the periphery and then move toward the center of the synapse (77). LFA-1 clusters stop in the pSMAC lamella zone, whereas TCR microclusters follow their path toward the cSMAC where they are endocytosed (78, 79) or secreted (80).

In the context of spreading described earlier, the centripetal movement of receptor clusters has been proposed to be driven by a combination of pushing forces originating from actin retrograde flow in the lamellipodium and pulling forces generated in the lamella by myosin-based contraction. Indeed, the inward flow of cortical F-actin at the immune synapse has been shown to be the major driving force behind microcluster movement (67, 81–84). The role of myosin II-based contractions at the lamella in microcluster movement, although more controversial (85), has also been shown to control the centripetal movement of both TCR and LFA-1 microclusters (86–88). One can speculate that the resistance of TCR, LFA-1, and other receptors to this mobilization would generate traction forces on the receptor/ligand bonds. Thus, coupling of receptors with the actin cytoskeleton together with mobility of the ligands at the membrane of the APC would be key elements in force generation on receptors. Adaptor molecules, such as talin, mediate interaction of LFA-1 with the actin cytoskeleton (89). The generation of localized traction forces by actin retrograde flow has indeed been shown to regulate adhesion (90, 91) in many cell types, including T lymphocytes forming immune synapses (92). In contrast, coupling of TCR to the actin cytoskeleton remains elusive. Yet, interactions of TCR clusters with actin have been revealed in experiments that introduced selective barriers, which altered TCR microcluster transport to the central SMAC (82, 93). Association of signalosomes with tyrosine-phosphorylated CD3 complexes may contribute to dynamic coupling of TCR–CD3 complexes with actin flow. The mobility of ligands on the surface of APC is another parameter to take into account into the generation of forces on receptor/ligand bonds (92, 94, 95) (**Figure 2** and see later discussion in Section "T Lymphocytes Interact with Cells That Have Different Mechanical Properties"). More studies and modeling analysis are required to address these specific aspects.

### EFFECT OF SUBSTRATE STIFFNESS ON FORCE DEVELOPMENT AND T CELL ACTIVATION

The mechanical behavior of solid materials, such as plastic and glass, can be described as purely elastic. This means that their stiffness can be expressed as the ratio of the applied stress and the resulting deformation, which is termed elastic (or Young's) modulus. On the other hand, cells and tissues display viscous properties in addition to their elastic ones and are, hence, viscoelastic materials. Two components can describe the mechanical properties of viscoelastic materials, one elastic and the other viscous, referred to as storage and loss moduli. In viscoelastic materials, the duration of force/stress application is also important, resulting in time-dependent deformations. Storage and loss moduli are different from the elastic or Young's modulus that is more often reported in literature, since calculation of the latter is not taking into account the duration of force application. Henceforth, we refer to elastic modulus as a measure of stiffness, unless otherwise mentioned.

The effects of substrate stiffness can be as diverse as growth, differentiation, migration, and survival (96–99). Particularly, it was demonstrated that cells display differential spreading (100), velocities (101), traction forces (102), and physiological behavior (103) in response to variations in stiffness. In a seminal study (104), Discher and co-workers showed that stem cell fate could be influenced just by the stiffness of culture substrates. Another

Hivroz and Saitakis Biophysics of T Lymphocyte Activation

important observation was that cells match their stiffness to that of the environment by regulating their actin cytoskeleton (105). Moreover, several cell types have been reported to display durotaxis, i.e., migration from soft toward stiff substrates (101, 106).

In this section, we will report on recent studies that have begun to shed light on the mechanical properties of T lymphocyte environment and on T cell responses to these properties.

# T Lymphocytes Interact with Cells That Have Different Mechanical Properties

T lymphocytes are mobile cells that are exposed to different chemical and mechanical environments. Inside lymph nodes, T lymphocytes interact transiently with a number of different APCs, each of them potentially activated by different stimuli and presenting a varying repertoire of agonist/non-agonist peptides on MHC molecules (5, 107). In blood vessels, T lymphocytes interact with endothelial cells, and inside tissues, effector T lymphocytes interact with their targets, i.e., infected or tumor cells. Much of the studies on immune synapse formation and T cell activation have been performed on plastic or glass surfaces or on planar lipid bilayers supported on glass. Even though these surfaces provide an ideal substrate to follow receptor/ligand interactions and rearrangements, they are flat and rigid with no topological variation. Moreover, plastic and glass display stiffness in the ranges of gigapascal. In contrast, cells in the body generally display stiffness in the range of 50 Pa–40 kPa (97) with primary human T lymphocytes and Jurkat cells being at the soft end of this range (108, 109) with their stiffness ranging from 50 to 90 Pa. Therefore, in order to really study the effect of mechanical properties on T lymphocyte biology, it is vital to know the mechanical landscape that the cells encounter *in vivo* and use substrates with stiffness values inside this physiological range. Using a singlecell rheometer (110), we recently showed that different human myeloid APCs have different viscoelastic properties and that their Young's modulus values vary from 500 Pa for monocytes and DCs to 900 Pa for macrophages (109). Moreover, inflammatory conditions modified the viscoelastic properties of myeloid cells, which were halved or doubled when cells were treated with a TNFα/ PGE2 cocktail or IFNγ, respectively (109). These results suggest that viscoelastic properties of myeloid cells are additional parameters of inflammation that can be integrated with biochemical factors to generate an adapted T lymphocyte response. Other studies have also reported variations in myeloid cell mechanical properties (111–113). Finally, it is worth noting that endothelial cells have also been shown to change their viscoelastic properties in response to inflammation (35), suggesting that this might be a more general process.

In our study, the viscoelastic properties of human myeloid cells were dependent on myosin IIA activity and correlated to the F-actin content in each type of cells (109). These results suggest that the actomyosin cytoskeleton of myeloid cells is responsible for their mechanical properties. Interestingly, older reports have shown that DC cytoskeleton was indispensable for priming of T cells since following DC treatment with actin depolymerizing drugs, naïve CD4<sup>+</sup> T cells were unable to proliferate (114). DCs were shown to polarize their cytoskeleton toward the immune synapse only upon successful antigen recognition by the T cell, and this was critical for TCR triggering and IL-2 production (115, 116). Maturation of DCs has also been associated with remodeling of their cytoskeleton, leading to development of projections directed toward T lymphocytes to optimize cell/cell interactions (117, 118). More recently, it has been shown that the cortical actin network of DCs regulated ICAM-1 lateral mobility at the cell surface and that DC maturation regulated mobility and clustering of ICAM-1 (95). The constrained ICAM-1 mobility associated with DC maturation was shown to promote formation of T lymphocyte/DC conjugates as well as T lymphocyte proliferation. On the T lymphocyte side, it was shown that LFA-1 affinity maturation correlated to ICAM-1 lateral mobility on the DC surface, i.e., low mobility of ICAM-1 induced high-affinity conformational changes of LFA-1 (95). The same group showed that actin flow in T lymphocytes was indispensable to maintain LFA-1 in the highaffinity conformation at the immune synapse and that ICAM-1 mobility directly affected distribution of high-affinity LFA-1 on the surface of engaged T lymphocytes (92). These results suggest a model in which ICAM-1 mobility on APC surface modulates resistance to tensile forces applied by the T lymphocyte actin cytoskeleton on LFA-1/ICAM-1 bonds, highlighting the role of mechanotransduction in cell conjugate formation and T lymphocyte activation (**Figure 2**). These mechanisms may apply to other receptor/ligand pairs, such as CD28/CD80-CD86, which are also coupled to the actin cytoskeleton (119–122). They may also apply to other cell types, such as endothelial cells. Therefore, these studies show that APCs can contribute both biochemical and mechanical cues to T cell activation. Overall, the above findings are (1) highlighting the requirements for APC/T lymphocyte crosstalk (123) for immune synapse formation and T lymphocyte activation and the need for more studies focusing on the mechanical properties of both sides of the immune synapse and (2) stressing the importance for T lymphocytes to sense the mechanical and topological properties of their environment in order to locate a specific target and respond.

It is worth noting that the mechanical properties of tissues and organs can be also modified in normal and pathological conditions. For example, it was recently shown that the contractility of fibroblastic reticular cells is regulated upon inflammation by the expression of CLEC-2 on mature dendritic cells (124, 125). CLEC-2, by interacting with podoplanin expressed on fibroblastic reticular cells, induces the relaxation of these cells that leads to a decrease of the lymph node stiffness that is probably important for its expansion (125). Moreover, tumor mechanics, and in particular the rigidity of tumoral tissues, has been shown to play a role in tumor development (126). These changes in mechanical properties of tissues and organs might also affect overall T lymphocyte activity.

Finally, it is possible that viscoelastic properties of T lymphocytes themselves are also modified by activation. The strength of TCR signaling may induce changes in T lymphocyte stiffness, which in turn may affect their interactions with APC and target cells as well as their migratory properties. Along this line, it is worth noting that T lymphocytes can adopt two types of migratory behavior (5, 127). Strong TCR stimulation can lead to complete arrest of T lymphocyte migration and stable conjugation with an APC, which can last several hours (128), while when interacting with TCR ligands of low potency or low affinity, T lymphocytes do not completely stop migrating and establish brief dynamic contacts with the APC (129), termed kinapses (127). TCR signaling strength modifies the actomyosin cytoskeleton of T lymphocytes (130, 131), which may lead to an alteration of their mechanical properties. It would thus be interesting to measure the effect of TCR signaling strength and also cytokine environment on T lymphocyte viscoelastic properties.

# T Lymphocytes Sense and Adapt to Substrate Stiffness

As discussed above, forces exerted by T lymphocytes may be important to probe their environment and particularly to test the stiffness, as we do when exerting pressure with our finger on a substrate. For example, it was proposed that "T lymphocytes are guided by the mechanical 'path of least resistance' as they transverse the endothelium" (34). In fact, T lymphocytes develop ILPs that physically push against the endothelial cell surface (20, 33, 34), suggesting that the role of these protrusions is to test the stiffness of endothelial cells in order to find "soft" areas to cross through (35). It is worth noting that these protrusions have also been proposed to facilitate the activation of memory/effector T cells to pMHC exposed on endothelial cells (22). Thus, T lymphocytes can sense the stiffness of the substrate they interact with. We have shown that not only T lymphocytes sense stiffness but also adapt to it. The pulling forces exerted by T lymphocytes upon TCR–CD3 triggering increased with the stiffness of the BFP used (11). This adaptation of forces to stiffness was not found in another study (12). Yet, the stiffness range used in each study might be very different.

Recent studies have addressed the effect of substrate stiffness on T lymphocyte activation. Using polyacrylamide gels with varying stiffness (range from 2 to 200 kPa) coated with an activating anti-CD3ϵ antibody, it was shown that mouse naïve CD4<sup>+</sup> T lymphocytes modulated their response according to the stiffness of gel substrates (132). Production of IL-2 and early phosphorylation of Zap70 and Src family kinases was higher on "stiff " (100–200 kPa) substrates. This response to substrate stiffness was observed only when the anti-CD3ϵ antibody was attached to the gel and was abrogated in the presence of the myosin inhibitor blebbistatin (132). These results suggest that the mechanotransduction involved in T cell activation requires coupling of the TCR–CD3 to the substrate and intact myosin II activity. In another study, human naïve CD4<sup>+</sup> and CD8<sup>+</sup> T lymphocytes were cultured on poly-dimethoxysilane (PDMS) substrates with stiffness ranging from 100 to 10,000 kPa. The "soft" substrates (~100 kPa) induced higher IL-2 and IFNγ production as well as more T lymphocyte proliferation (133). These results seem inconsistent with the previous study (132). Yet, for poly-acrylamide gel substrates, immobilization was performed by coupling of biotinylated activating antibodies on acrylamideconjugated streptavidin. In contrast, coating of PDMS substrates was performed by passive adsorption of antibodies on the hydrophobic surface (133), possibly resulting in both loss of immobilized material over time and passive adsorption of proteins from the culture medium. In a third study, human CD4<sup>+</sup> T lymphoblasts were activated on PDMS substrates of varying stiffness, which presented anti-CD3 antibodies either alone or together with ICAM-1 molecules (134). In this study, "soft" (5 kPa) substrates induced less tyrosine phosphorylation than the "rigid" (2000 kPa) ones, and ICAM-1 increased the response to "stiff " substrates (134).

Even though the aforementioned reports provide very interesting results, their focus is on a stiffness range (2–10,000 kPa) that is non-physiological for T cells in the body, since APCs were shown to display stiffness ranging from 0.19 to 1.45 kPa (109). A recent study (13) looked at the response of Jurkat cells to substrates of a more physiologically relevant stiffness range (0.2–6 kPa). The authors used polyacrylamide gels, treated with hydrazine hydrate and coated with poly-l-lysine and an activating anti-CD3 antibody. They quantified the effect of substrate stiffness on CD3-induced signaling by following tyrosine phosphorylation by immunoblotting and microscopy (13). They showed that tyrosine phosphorylation peaked higher and more rapidly on "stiff " gels (5 kPa) but decreased more rapidly than on "soft" gels (1 kPa).

Although these studies (summarized in **Table 1**) are difficult to directly compare because they use different cell types, substrate chemistry, antibody immobilization, and stiffness ranges, they overall reveal that T lymphocytes are indeed mechanosensitive. It is not entirely clear what is the mechanosensing mechanism, yet, as summarized above, it requires TCR-dependent actomyosin remodeling. One explanation for the effects on TCR triggering and subsequent activation could be the local spreading and

deformation of the T cell membrane in contact with substrate of different stiffness. In the kinetic-segregation model (37), local membrane deformation and segregation of large glycoproteins have to occur before TCR and pMHC can come close to one another and interact (40). It was proposed that this process required cytoskeleton-derived force (135). One could postulate that this could occur at the tips of ILPs or other small protrusions of the cell, i.e., short filopodia. Regarding the deformability of the substrate, "stiff " substrates would allow more deformation of the T lymphocyte membrane and better molecular segregation at the T cell protrusions compared to "soft" substrates and this, in turn, would result in an increased number of successful interactions between TCR and ligands (anti-CD3 antibodies or pMHC complexes). The increased number of TCR engagements would induce increased intracellular signaling that would then activate the actin cytoskeleton to produce larger cell protrusions and generate forces (11–13). This process can eventually result in increasing bond lifetimes of TCR and LFA-1 for their ligands. By inducing more conformational changes, it would lead to increased T lymphocyte activation. Thus, "stiff " substrates would display a kind of mechanical signal amplification. This mechanism has already been proposed for fibroblast adhesion on substrates of varying stiffness (100). Further work will be required to test this hypothesis for T lymphocytes.

### CONCLUSION AND PERSPECTIVES

Recent evidence has shown that TCR signaling and T lymphocyte activation are not solely regulated by chemical signals of the environment but also by mechanical cues. Forces exerted by the exterior or the T lymphocyte itself regulate the lifetime of receptor/ligand bonds. This, in turn, increases adhesion of T

### REFERENCES

lymphocytes to APCs and allows for better discrimination of agonist pMHC. Forces exerted by T lymphocytes also help the cells probe the substrates they interact with by testing their stiffness, which might be a key parameter of T cell activation. We now need to explore further the viscoelastic properties of cells and tissues in physiological and pathological conditions in order to develop experimental models that better mimic the mechanical landscape of T lymphocytes. At the molecular level, we need to study the role of known costimulators or co-inhibitors of T lymphocyte activation in force development and force sensing and find out potential mechanical crosstalk between receptors. Finally, at the cell level, it would be interesting to study if and how mechanical cues can modulate the functions of different T lymphocyte subsets. It would be particularly important to see if mechanics can modulate naïve T lymphocyte priming or effector T lymphocyte functions (cytotoxicity and cytokine secretion). Elucidating these issues will provide further insight into T lymphocyte activation under normal and pathological conditions that could be translated in novel therapeutic strategies.

# AUTHOR CONTRIBUTIONS

CH wrote the review, edited the manuscript, and designed table and figures. MS wrote part of the review and edited the manuscript and figures and table.

# FUNDING

Supported by funds from ANR (ANR-12-BSV5-0007-01, ImmunoMeca; ANR-13-BSV2-0018 "NeuroImmunoSynapse"), DC-Biol Labex, and Fondation pour la Recherche Médicale (FRM, FRM DEQ20140329513).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Hivroz and Saitakis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Vesicular Trafficking to the Immune Synapse: How to Assemble Receptor-Tailored Pathways from a Basic Building Set

### *Anna Onnis† , Francesca Finetti† and Cosima T. Baldari\**

*Department of Life Sciences, University of Siena, Siena, Italy*

The signals that orchestrate T-cell activation are coordinated within a highly organized interface with the antigen-presenting cell (APC), known as the immune synapse (IS). IS assembly depends on T-cell antigen receptor engagement by a specific peptide antigen-major histocompatibility complex ligand. This primary event leads to polarized trafficking of receptors and signaling mediators associated with recycling endosomes to the cellular interface, which contributes to IS assembly as well as signal termination and favors information transfer from T cells to APCs. Here, we will review recent advances on the vesicular pathways implicated in IS assembly and maintenance, focusing on the spatiotemporal regulation of the traffic of specific receptors by Rab GTPases. Based on accumulating evidence that the IS is a functional homolog of the primary cilium, which coordinates several central signaling pathways in ciliated cells, we will also discuss the similarities in the mechanisms regulating vesicular trafficking to these specialized membrane domains.

### *Edited by:*

*Pedro Roda-Navarro, University Complutense of Madrid, Spain*

### *Reviewed by:*

*David Escors, University College London, UK Hyun Park, National Cancer Institute, USA*

### *\*Correspondence:*

*Cosima T. Baldari<EMAIL_ADDRESS>
*† Anna Onnis and Francesca Finetti contributed equally.*

### *Specialty section:*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology*

*Received: 26 October 2015 Accepted: 01 February 2016 Published: 15 February 2016*

### *Citation:*

*Onnis A, Finetti F and Baldari CT (2016) Vesicular Trafficking to the Immune Synapse: How to Assemble Receptor-Tailored Pathways from a Basic Building Set. Front. Immunol. 7:50. doi: 10.3389/fimmu.2016.00050*

Keywords: immune synapse, receptor trafficking, Rab GTPases, IFT, primary cilium

# INTRODUCTION

Adaptive immunity relies on the presentation of major histocompatibility complex-associated peptide ligand (pMHC) by an antigen-presenting cell (APC) to a cognate T cell to allow for its activation. This process is coordinated by a highly specialized membrane domain that forms at the interface between T cell and APC, known as the immune synapse (IS), which ensures the long-lasting signaling required for T cell activation (1, 2).

T cell responses are finely regulated by the dynamic modulation of the levels of surface T cell receptor (TCR) (3). In quiescent T cells, TCR expression is dependent on a balance of *de novo* synthesis, endocytosis, recycling, and degradation, recycling between the plasma membrane and the cytoplasmatic pool being a major determinant (4). Constitutive endocytosis of the TCR requires protein kinase C (PKC)-dependent phosphorylation of a di-leucine motif on the CD3γ chain, which enables the CD3 complex to interact with the clathrin adaptor protein 2 (AP-2) and to be directed to recycling endosomes for returning to the plasma membrane (5). Constitutive TCR recycling subserves a dual function. First, it acts as a quality control mechanism allowing for the identification and degradation of TCR complexes that have lost their integrity. Second, it permits the formation of an intracellular pool of functional TCRs that can be rapidly polarized to the IS in response to engagement of plasma membrane-associated TCRs.

T-cell antigen receptor triggering induces receptor internalization, which is followed by either polarized recycling to the plasma membrane or receptor degradation (6). The pathway of ligand-dependent TCR internalization is mediated by the lymphocyte-specific protein tyrosine kinase (Lck) (7), the membrane-remodeling GTPase dynamin 2 (8), and PKCregulated activation of CD3γ, which routes the internalized TCRs to the recycling compartment (9). Alternatively, activated TCRs may undergo degradation to allow for signal termination. The E3 ligase casitas B-lineage lymphoma (Cbl-b), which is upregulated in response to the interaction of programed death 1 ligand 1 (PD-L1) on APC and programed death 1 (PD-1) on CD8<sup>+</sup> T cells, plays a key role in this process (10). TCR triggering induces CD3ζ ubiquitination by Cbl-b, which is recruited to the engaged TCRs by the protein tyrosine kinases Lck and ζ-chain-associated protein kinase 70 (ZAP-70) (10–12). Ubiquitinated TCRs are recognized by tumor susceptibility gene 101 (Tsg101) and sorted to multivesicular bodies (MVBs) for degradation (13), thereby making space for incoming TCRs and turning off signaling. Interestingly, recent evidence indicates that internalized TCRs continue to signal, thereby contributing to sustained signaling from their endosomal localization (8, 14). Moreover, internalized TCRs are in part delivered to the APC as microvesicles (15), highlighting a role for TCR endocytosis beyond the canonical function of signal termination.

Here, we will summarize of our current understanding of the recycling pathways that regulate the traffic of endosomal TCRs as well as of other receptors, including the C-X-C chemokine receptor type 4 (CXCR4) and the transferrin receptor (TfR), and of membrane-associated signaling mediators, such as Lck and linker for activation of T cells (LAT), which participate as key players in IS assembly and function. We will also discuss the emerging role of the IS as a platform for vesicular traffic-mediated transcellular communication beyond its established role in the secretion of soluble effectors.

### POLARIZED TCR RECYCLING AT THE IS: SEEING THE TIP OF THE ICEBERG

### Rab GTPases and Their Effectors in TCR Trafficking to the IS

Recycling receptors traffic through at least two temporally and spatially distinct highly conserved pathways orchestrated by members of the Rab GTPase family: a short-loop and a longloop. Following internalization, receptors are delivered to early endosomes, marked by Rab5, and rapidly returned to the plasma membrane under the control of Rab4 (short-loop). Alternatively, recycling receptors may transit from early endosomes to the pericentrosomal endocytic recycling compartment and return to the plasma membrane *via* a Rab11-dependent route, thus completing the long-loop recycling (16–18). Intracellular TCRs have been found associated with both Rab4<sup>+</sup> and Rab11<sup>+</sup> endosomes, with the Rab11<sup>+</sup> compartment centrally implicated in endosome recycling to the IS (19, 20). In addition to these universally used recycling Rabs, more specific Rab GTPases and traffic regulators have been mapped to the TCR recycling pathway. One example is Rab35 and its GTPase-activating protein (GAP) EPI64C (21). Rab35 is a Rab GTPase implicated in cytokinesis in *Drosophila* (22) and in the regulation of endosomal trafficking as well as actin polymerization in several insect and mammalian cell lines (23). In T lymphocytes, Rab35 colocalizes with the TCR at the pericentrosomal compartment, wherefrom it is recruited at the IS, thus regulating polarized TCR recycling, and IS formation in concert with EPI64C (21). We have moreover recently identified Rab29, an as yet poorly characterized Rab GTPase, as a new component of the TCR recycling pathway. The Rab29 subfamily, which also includes Rab32 and Rab38, has been implicated in the traffic of melanosomes (24), as well as of the mannose-6-phosphate receptor (MPR) in epithelial and neuronal cells (25, 26). We found that in T cells, Rab29 acts as a complex with Rab11 to control TCR delivery to the IS membrane through microtubule-dependent polarized recycling. In Rab29-depleted cells, recycling TCRs accumulate indeed in Rab11<sup>+</sup> endosomes that fail to polarize to the IS notwithstanding a correct positioning of the centrosome due to defective recruitment of the dynein microtubule motor (27).

Recycling endosomes are associated not only with microtubules but also with actin that generates force for vesicle movement along the microtubule tracks. Both early and recycling endosomes polarizing to the IS during T cell activation have been shown to colocalize with the nucleation promoting factor WASP and SCAR homolog (WASH) (28), which mediates local actin polymerization by recruitment of the Arp2/3 actin adapter complex. WASH is required for TCR trafficking following T cell stimulation (29). Accordingly, activated WASH-deficient T cells express reduced TCR levels, which is likely to lead to a defect in sustained signaling, accounting for their proliferation defect (29). WASH also contributes to maintain the levels of the costimulatory receptor CD28 and the integrin lymphocyte function-associated antigen 1 (LFA-1), as well as of the glucose transporter 1 (GLUT1), at the surface of activated T cells (29), which indicates that the role of this adaptor in receptor trafficking is not restricted to the TCR.

A previously uncharacterized role in T cell activation has been recently ascribed to the sorting nexins (SNX), which regulate several steps in vesicular trafficking. Both SNX17 and SNX27 have been shown to accumulate at the IS, where they play opposite roles in T cell activation. SNX17 colocalizes with endosomal TCRs and is required for their recycling to the plasma membrane (30). At variance, SNX27 interacts with diacyl glycerol kinase ζ (DGKζ), which negatively controls TCR signaling, at early and recycling endosomes, and traffics to the IS to blunt the Ras–Erk pathway (31).

# Control of TCR Trafficking to the IS by Regulators of Primary Cilium Assembly

Further insights into the pathway of TCR recycling have emerged with the unexpected identification in this pathway of components of the intraflagellar transport (IFT) system, which regulates the assembly of the primary cilium, an organelle of which T cells are normally devoid (32, 33). IFT particles are responsible for cargo movement into the cilium and back to the cell body through their interaction with the microtubule motors kinesin-2 and cytoplasmic dynein-2, respectively (34, 35). We have shown that in T cells, IFT20 cooperates with the IFT proteins IFT88, IFT57, and IFT52 to promote TCR recycling to the IS downstream of centrosome polarization (32, 36). IFT20 participates early in the trafficking pathway by forming a complex with Rab5 and the TCR on early endosomes. In IFT20-depleted cells, recycling TCRs accumulate in Rab5<sup>+</sup> endosomes, which fail to cluster at the IS despite a normal polarization of the centrosome, indicating that IFT20 controls TCR traffic from early to recycling endosomes (36).

The finding that proteins implicated in ciliogenesis are exploited by T cells to assemble the IS has provided support to the emerging notion that these specialized structures are functional homologs. In addition to their morphological similarities, underscored by the polarized arrangement of the centriole and Golgi apparatus beneath the respective membrane domains, both the primary cilium and the IS act as signaling platforms as well as sites of intense vesicular trafficking and polarized exocytosis (37–40). These similarities can be exploited to identify new components of the pathways governing IS assembly, as witnessed by our recent implication of the small GTPase Rab8 in polarized TCR recycling. Growth of the ciliary membrane and targeting of specific receptors to this location is orchestrated by a Rab11– Rab8 cascade, which interfaces with a multimolecular complex known as the Bardet–Biedl syndrome complex (BBSome) (41). In this cascade, serum starvation represents the signal that promotes the centrosomal trafficking of the Rab8 guanine nucleotide exchange factor Rabin8 through its association with Rab11 and the transport protein particle II (TRAPPII) complex. At the centrosome, the BBSome associates with Rabin8 and activates Rab8 to allow ciliary membrane biogenesis (42). We have recently demonstrated that in T cells, Rab8 colocalizes with IFT20, Rab11, and Rab29 and acts downstream of these trafficking mediators to regulate polarized TCR recycling and T cell activation (27, 43). Of note, Rab8, at variance with IFT20 and Rab29, is not required for the polarization of TCR<sup>+</sup> endosomes to the IS (**Figure 1**). Rather, Rab8 controls the docking/fusion step at the plasma membrane of TCR<sup>+</sup> endosomes that have clustered beneath the IS membrane by recruiting vesicle-associated membrane protein-3 (VAMP-3) (43), a vesicular soluble NSF attachment protein (v-SNARE), which had been previously reported to regulate docking and fusion of the TCR<sup>+</sup> endosomes with the IS membrane (44). Independent evidence for a role for Rab8 in TCR trafficking has been provided by Soares et al., who showed that vesicular TCRζ colocalizes with both Rab8b and Rab3d (45).

It is noteworthy that the elucidation of the pathways controlling receptor traffic to the IS has helped furthering our understanding of the mechanisms that orchestrate ciliogenesis. We have recently found that ciliated cells express Rab29 which, similar to T cells, participates in a complex that includes Rab8, Rab11, and IFT20, as well as the molecular motors kinesin and dynein. Rab29-depleted cells show defects in ciliogenesis, with a reduction in the number of cells forming a cilium and, where present, a reduced ciliary length. Ultrastructural analysis reveal that these cells have no alterations in the structure of the cilium but a significant vesicle enrichment around the ciliary base indicating that Rab29 controls ciliary assembly by favoring cargo trafficking to the cilium, a central one being the Hedgehog-associated transmembrane receptor Smoothened (Smo) (46). Additionally, we demonstrated that, similar to T cells, VAMP-3 interacts with Rab8 in ciliated cells, promoting the delivery of Smo to the ciliary membrane (43). VAMP-7, another v-SNARE implicated in traffic to the IS, has also been recently implicated in ciliogenesis (47). Hence, studying IS assembly and ciliogenesis provides a unique opportunity of cross-feeding, as recently highlighted by the implication of the Hedgehog pathway, one of the major signaling pathways orchestrated by the primary cilium, in the release of cytotoxic granules at the synapse of cytotoxic T cell effectors (48).

# A COMBINATORIAL STRATEGY FOR THE DELIVERY OF SPECIFIC RECYCLING RECEPTORS TO THE IS

Signaling at the IS to promote T cell activation, while triggered by the TCR, requires the coengagement of several other receptors as well the recruitment of key membrane-bound signaling mediators. Emerging evidence highlights the existence of individual trafficking modules, which ensure cargo specificity within the universal recycling pathways orchestrated by Rab4 and Rab11. In this section, we will present some examples that illustrate the versatility of the vesicular pathways that control endosomal trafficking to the IS.

### C-X-C Chemokine Receptor Type 4

Among the receptors known to become enriched at the IS is CXCR4, a ubiquitously expressed heterotrimeric G proteincoupled receptor, which regulates T cell development, migration, and activation (49, 50). CXCR4 participates in IS assembly, signaling through Gi and the janus-activated kinases 1/2 (JAK1/2) to maintain the T cell:APC contact (51) and promoting local actin polymerization and centrosome polarization (51). Ligand binding induces CXCR4 internalization through clathrin-coated pits *via* a PKC-mediated, β-arrestin-dependent pathway leading to CXCR4 sorting either to lysosomes (52–54) or to Rab11<sup>+</sup> recycling endosomes containing TCR cargo (19, 55). The colocalization of CXCR4 with TCR<sup>+</sup> endosomes, which depends on actin polymerization mediated by Gα13 and Rho (19), may reflect the ability of CXCR4 to heterodimerize with the TCR both at the plasma membrane and in endosomes (49). The pathway that controls CXCR4 targeting for lysosomal degradation, which involves its ubiquitination by the E3 ligase atrophin interacting protein 4 (AIP4) (56) and its interaction with the endosomal-sorting complex required for transport (ESCRT) (57), has been extensively characterized. Conversely, while CXCR4 deubiquitination has been identified as one of the factors that favor CXCR4 recycling (57), relatively little is known about the molecular mechanisms mediating this process. In T cells, CXCR4 surface expression is dependent on IQ motif-containing GTPase-activating protein 1 (IQGAP1), a cytoskeleton-interacting scaffold protein that is required for tethering CXCR4<sup>+</sup> early endosomes to microtubules to redirect their receptor cargo to the plasma membrane (58). We have recently provided further insights into the pathway that controls the traffic of endosomal CXCR4 by identifying Rab29

and Rab8 as new regulators of both constitutive and polarized recycling of this receptor to the IS. It is noteworthy that the IFT proteins do not participate in this pathway, notwithstanding their functional interplay with both Rab29 and Rab8 in TCR recycling (**Figure 1**) (27, 36, 43).

# Transferrin Receptor

Interestingly, these same traffic regulators are used in a different combination for the traffic of yet another receptor, the TfR, to the IS. The TfR, which plays a central role in iron homeostasis, is one of the best characterized recycling receptors. Upon binding iron-loaded transferrin, the TfR enters the cell through clathrin-mediated endocytosis, which is regulated by the clathrin adaptor AP-2, the phosphoinositide PtdIns(4,5) P2, the membrane-remodeling GTPase dynamin 2, Rab5, the cortical actin regulator cortactin, and the kinase Src (59, 60). It subsequently accumulates in recycling endosomes, wherefrom it returns to the plasma membrane both through the short-loop (Rab4-dependent) and the long-loop (Rab11-dependent) pathways (61, 62). In T cells, the function of the TfR goes beyond controlling iron uptake. It has indeed been demonstrated that the TfR interacts with CD3ζ and promotes its tyrosine phosphorylation following binding of holotransferrin (63), suggesting that it might participate in lymphocyte activation by modulating TCR signaling. In support of this notion, surface expression of the TfR increases upon TCR stimulation and the receptor polarizes to the IS. Blocking the TfR using a neutralizing antibody results in defective T-cell:APC conjugate formation and TCR clustering at the IS, underscoring a function for this receptor in IS assembly (44, 64). Although several Rabs and trafficking mediators other than Rab4 and Rab11 (e.g., Rab12, Rab22, Rab8, and Arl13b) have been implicated in the regulation of TfR recycling in other cells types (65–68), the pathway controlling polarized recycling of this receptor to the T cell IS is only beginning to be elucidated. Similar to the TCR, the TfR requires Rab35 and its GAP EPI64C as well as the actin adaptor WASH to recycle to the T cell plasma membrane (21, 28). Moreover, we have recently demonstrated that IFT20 interacts with the TfR and is implicated in its recycling to the IS. Remarkably, IFT20, while regulating TCR and TfR recycling, is not involved in CXCR4 recycling. At variance, Rab8 and Rab29, while participating in TCR and CXCR4 recycling, are dispensable for TfR recycling. This suggests a scenario where different receptors (TCR, CXCR4, and TfR), while sharing some components of the universal short-loop and long-loop pathways, adopt personalized routes by combining individual traffic regulators, such as IFT20, Rab8, and Rab29, allowing specificity to be achieved during polarized recycling to the IS (**Figure 1**).

# Lck and LAT

The combinatorial strategy used by T cells to coordinate the traffic of specific receptors or membrane-associated signaling mediators to the IS has been recently shown to be also exploited to generate signaling nanodomains at the TCR activation sites (45, 69). Similar to the TCR, the initiating protein tyrosine kinase Lck and the transmembrane adaptor LAT are present in T cells as two cellular pools, of which one is associated with the plasma membrane and the other with recycling endosomes, the latter becoming polarized to the IS upon TCR triggering (70–72). Sorting of Lck and LAT to the central domain of the IS, known as central supramolecular activation cluster (cSMAC), is regulated by the lipid raft-associated myelin and lymphocyte (MAL) protein, whose rapid accumulation at the raft-enriched IS promotes the recruitment of the microtubule and transport vesicle docking machinery (73). Moreover, both LAT phosphorylation and the recruitment of LAT<sup>+</sup> vesicles to TCR activation sites critically depend on the v-SNARE VAMP7 (74). In their recent report, Soares et al. (45) provided evidence for the existence of traffic modules specifically tailored to promote the synaptic transport of Lck and LAT versus TCRζ in response to TCR engagement. They found that Rab11<sup>+</sup> vesicles containing Lck rapidly localize to the IS. Subsequently, in response to increased calcium levels, Rab27a<sup>+</sup>Rab37<sup>+</sup> vesicles containing LAT and Rab3d<sup>+</sup> Rab8b<sup>+</sup> vesicles containing TCRζ are delivered to the IS. This is achieved through the interaction of VAMP-7 associated to both LAT<sup>+</sup> and TCRζ+ vesicles with the calcium sensor synaptotagmin-7. This report not only provides new insights into the modularity of the traffic pathways exploited by T cells to target specific molecules to the IS but also supports the notion that the synaptic membrane is a mosaic of nanodomains generated with the central contribution of vesicular traffic that coordinate signaling to promote the assembly of a fully competent IS.

The combinatorial assembly of unique trafficking modules within the common basic recycling pathways orchestrated by Rab4 and Rab11 provides an explanation to emerging evidence generated in several different cell types that the endosome pools marked by these GTPases are actually mosaics of Rab4<sup>+</sup> or Rab11<sup>+</sup> endosome subpopulations characterized by specific arrays of traffic regulators and carrying distinct receptor cargoes. For instance, in epithelial-like CHO cells, the TfR and the glucose transporter GLUT4 transit through distinct pools of Rab4<sup>+</sup> endosomes (75). Similarly, while the majority of ciliary proteins share the Rab8- Rab11 cascade, the traffic of specific receptors to the ciliary membrane is controlled by unique mediators. For example, Rab23 is specifically required for the ciliary traffic of Smo and the dopamine receptor but is dispensable for ciliary targeting of the receptor protein Kim1 and of the microtubular tip end-binding protein 1 (EB1), notwithstanding their common requirement for Rab8 (76, 77). IFT25 and IFT27 have been specifically implicated in ciliary trafficking of Smo, without affecting either the formation of the ciliary axoneme or the localization of other ciliary membraneassociated proteins, such as ADP-ribosylation factor-like protein 13B (ARL13B) and adenylate cyclase 3 (ADCY3) (78–81). We have moreover provided evidence that while required for Smo trafficking, Rab29 is dispensable for the ciliary localization of β1 integrin (27). Hence, the trafficking machinery is emerging as a combinatorial system of dynamic modules that ensure the specificity of receptor/cargo transport (81). This system, of which we are only beginning to fathom the complexity, is further complicated by indications that the traffic modules that have been identified may be tissue specific. For example, at variance with epithelial cells, the TfR is associated in neuronal cells with a distinct subpopulation of Rab11+ endosomes marked by ADPribosylation factor 6 (Arf6) (82). Unraveling this complexity is a major future challenge.

### VESICULAR TRAFFICKING AS A MEANS OF TRANSCELLULAR COMMUNICATION

The role of the IS as a platform for focalized exocytosis of cytokines and/or lytic granules by effector T cells is well established and has been extensively reviewed (40). Emerging evidence indicates however that vesicular traffic at the IS subserves important regulatory functions during the interactions that occur between T cell and cognate APC. In this context, the IS is exploited as a means of cell-to-cell communication to fine tune both the T cell and the APC (83).

T cells are able to extract surface molecules from other cells with which they establish contacts upon dissociation (84). This process is known as trogocytosis and leads to intercellular exchange of membrane patches. T cells take up into their plasma membranes costimulatory molecules, adhesion molecules, and pMHC expressed on APCs probably as a consequence of coincidental T cell phagocytosis of APC membrane during TCR downmodulation (85). It has been proposed that phagocytosed APC membrane fragments fuse with the endosomal compartment and recycle to the T cell plasma membrane, conferring to T cells the capacity to directly activate other CD4<sup>+</sup> T cells, which allows for an increase in the number of APCs presenting cognate antigen and facilitates the activation of effector T cells (86). In addition, trogocytosis has been linked to sustained T cell signaling since the pMHCs extracted from the APC remain associated with the engaged TCRs, resulting in elevated levels of ZAP-70 and phosphorylated proteins and thus prolonging the presentation step (87, 88).

The second example of transcellular communication involving vesicular traffic at the IS is the release by T cells of exosomes that are taken up by the APC (89). Exosomes are formed by inward budding of the limiting membrane of MVBs which, upon TCR triggering, are polarized toward the APC and fuse with the plasma membrane to release the vesicles. The polarization of MVBs is regulated by phospholipids (diacylglycerol and PIP3), the lipid kinase DGKα, and the serine/threonine protein kinases 1/2 (PKD1/2) (89, 90). Among the proteins that are involved in exosome biogenesis and release, an important role is played by the ESCRT complex as well as by several Rab GTPases (e.g., Rab4, Rab11, Rab27a, Rab27b, and Rab35) (91). It will be interesting to address the potential implication of the recently identified regulators of vesicular trafficking to the T cell IS in exosome secretion. Important insights into the function of the exosomes released at the IS have emerged from the analysis of their contents. Mittelbrunn et al. showed indeed that these exosomes are loaded with microRNAs that are able to regulate gene expression following their uptake by the APC, one being the Sry-box transcription factor 4 (Sox-4) (92). This may affect the ability of the APC to shape the differentiation program of the engaged T cell. Of note, since the MVBs in the APC do not polarize to the IS (92), the current model posits a unidirectional transfer of exosomes from the T lymphocyte to the APC.

It has been recently shown that the ESCRT-I protein Tsg101 is involved in targeting internalized ubiquitinated TCRs to microvesicles that are subsequently released at the IS through a mechanism regulated by vacuolar protein sorting 4 (VPS4). These synaptic microvesicles are delivered to the APC bearing cognate pMHC where they initiate intracellular signals, thereby acting as a means of transcellular communication (15). Interestingly, synaptically released TCR-enriched microvesicles are able to activate signaling in B cells-presenting specific pMHC, suggesting a novel mechanism of T cell help where the amount of help is adjusted to the density of pMHC at the B cell surface (15).

The role of extracellular vesicles as a means of cell-to-cell communication is now well established, particularly in immune cells, where they act as vehicles for the transfer of immunomodulatory molecules (89, 93). The focused release of extracellular vesicles at the IS ensures a cellular confinement to allow for their uptake by the APC with minimal diffusion. Of note, similar to the IS, the primary cilium has been identified as a site of focused release of membrane vesicles. Vesicle secretion from the distal ends of the cilium has been reported in vertebrate retinal photoreceptors as well as in epithelial cells lining the urinary lumen (94). In the model organism, *Chlamydomonas reinhardtii*, these ciliary ectosomes carry a protease implicated in the liberation of the daughter cell following mitosis (95), underscoring the shared informative role of the IS and the cilium achieved through the release of extracellular vesicles.

### FUTURE PERSPECTIVES

Emerging evidence of a role for post-translational modifications other than phosphorylation in the regulation of the molecular machinery that orchestrates vesicular traffic is adding a further layer of complexity to this biological process (96, 97). While receptor ubiquitination has long been known to act in concert with kinases and β-arrestins to regulate the trafficking of G protein-coupled receptors (GPCRs) (57), specific components of the pathways that control receptor traffic have turned out to be regulated by ubiquitination. For example, the GPCR β2 adrenoceptor (β2AR) is able to regulate its own trafficking by ubiquitination and activation of Rab11a (98). Moreover, the activity of the F-actin-nucleating protein WASH is fine tuned through K63-linked ubiquitination by the MAGE-L2- TRIM27 ubiquitin ligase and by the ubiquitin-specific peptidase 7 (USP7) deubiquitinating enzyme to prevent its overactivation, thus ensuring a proper WASH-mediated endosomal actin assembly and protein recycling (99, 100). In T cells, ubiquitination has recently been implicated in the traffic not only of receptors, such as the TCR and CXCR4, but also of signaling mediators that traffic to the IS, as recently exemplified by LAT. This adaptor has been shown to recruit to the IS the E3 ligase TNF receptor-associated factor 6 (TRAF6), which is essential for its ubiquitin-dependent phosphorylation (101), highlighting this cooperation between LAT and TRAF6 as a new regulatory mechanism in T-cell activation. Sumoylation, a reversible post-translational process mediated by small ubiquitinlike modifier (SUMO), has also been recently implicated in IS assembly and function, as documented for PKC-θ, which has been shown to be sumoylated is response to TCR engagement (102). These new findings underscore post-translational modifications as a new important area of study for the dissection of the traffic-related mechanisms that regulate IS assembly and T cell activation. Again, the primary cilium, where recent evidence has been provided for the regulation of ciliary trafficking by ubiquitination or sumoylation (103, 104), may provide interesting candidates to further our understanding of the post-translational control of traffic to the IS.

The identification of vesicular traffic as a central regulator of IS assembly and function highlights another important emerging area of investigation, namely the causative implication of traffic defects in T cell-mediated diseases. Abnormalities in TCR and CD4 trafficking have been associated to enhanced signaling in systemic lupus erythematosus (SLE) (105, 106). SLE T cells show enhanced endocytic trafficking due to overexpression of Rab5 and HRES-1/Rab4, a small GTPase encoded by the HRES-1 human endogenous retrovirus, which is essential for the targeting of TfR, CD4, and TCRζ for lysosomal degradation in T cells (105, 107). Paradoxically, these trafficking abnormalities, which are likely to account for the reduced expression of TCRζ at the surface of SLE T cells, have been associated with enhanced signaling. Alterations in the intracellular localization and degradation of signaling mediators, such as LAT, have also been associated to the signaling abnormalities observed in SLE T cells (108). While implicating trafficking defects in the hypersensivitity of SLE T cells, these data underscore the importance of elucidating the underlying mechanisms.

Interestingly, HRES-1/Rab4 appears to play a role also in the context of human immunodeficiency virus (HIV) pathogenesis. Overexpression of HRES-1/Rab4 in T cells has been shown to abrogate HIV infection by inhibiting surface expression of CD4 and targeting it for lysosomal degradation (107). Since expression of the HIV coreceptor CXCR4 is crucial to mediate viral entry (109), modulation of CXCR4 internalization and recycling may also contribute to HIV infection (110). In addition, mutations in CXCR4 that impair its intracellular traffic, resulting in impaired receptor recruitment to the IS (111), have been associated to the development of the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) immunodeficiency syndrome.

Collectively, these results point to abnormalities in vesicular traffic as an important determinant in T cell-related diseases. We expect that furthering our understanding of the pathways that control this process may be exploited to identify relevant molecular targets for which existing approved drugs might already be available or new drugs might be designed.

### AUTHOR CONTRIBUTIONS

AO, FF, and CB wrote the manuscript. AO and FF prepared the figure.

### FUNDING

Part of the results described in this review were obtained with the financial support of Telethon-Italy (Grant GGP1102).

# REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.