*Copyright © 2016 Onnis, Finetti and Baldari. 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.*

# Comparative Anatomy of Phagocytic and Immunological Synapses

### *Florence Niedergang1,2,3\*† , Vincenzo Di Bartolo4,5\*† and Andrés Alcover4,5\*†*

*1U1016, Institut Cochin, INSERM, Paris, France, 2UMR 8104, CNRS, Paris, France, 3Université Paris Descartes, Sorbonne Paris Cité, Paris, France, 4 Lymphocyte Cell Biology Unit, Department of Immunology, Institut Pasteur, Paris, France, 5U1221, INSERM, Paris, France*

The generation of phagocytic cups and immunological synapses are crucial events of the innate and adaptive immune responses, respectively. They are triggered by distinct immune receptors and performed by different cell types. However, growing experimental evidence shows that a very close series of molecular and cellular events control these two processes. Thus, the tight and dynamic interplay between receptor signaling, actin and microtubule cytoskeleton, and targeted vesicle traffic are all critical features to build functional phagosomes and immunological synapses. Interestingly, both phagocytic cups and immunological synapses display particular spatial and temporal patterns of receptors and signaling molecules, leading to the notion of "phagocytic synapse." Here, we discuss both types of structures, their organization, and the mechanisms by which they are generated and regulated.

Keywords: phagocytosis, immunological synapse, immune receptor, signal transduction, actin, microtubules, exocytosis, endocytosis

# INTRODUCTION

Immunological synapses are organized cell–cell contacts shaped at the interface between T cells and antigen-presenting cells (APCs) (**Figure 1**). They are triggered by the binding of T cell antigen receptors (TCR) to their ligands, peptide antigens associated with major histocompatibility complex molecules (pMHC) expressed on the surface of APCs. TCR engagement induces the polarization of the T cell toward the APC and a coordinated reorganization of various T cell components, including receptors, signaling and adhesion molecules, the actin and microtubule cytoskeleton, and intracellular vesicle traffic. Thus, the TCR and its proximal signaling molecules (e.g., protein kinases and phosphatases, signaling adapters, and effectors molecules) form dynamic signaling complexes at the immunological synapse that drive T cell activation. Moreover, TCR signaling triggers the fine reorganization of the actin and microtubule cytoskeleton that ensures synapse architecture and signaling complex dynamics, critical for TCR signal regulation. Finally, various intracellular compartments polarize toward the immunological synapse, including the Golgi apparatus, early and late endosomes, and mitochondria. Importantly, the TCR signaling machinery, actin and microtubule cytoskeleton, and intracellular vesicle traffic interplay at the synapse to sustain and regulate T cell activation (1).

Phagocytic cup formation mirrors a large number of events occurring during immunological synapse formation, before leading to a productive engulfment of the target (**Figure 1**). First, clustering of phagocytic receptors induced by particle-associated ligands triggers signal transduction pathways similar to those engaged by the TCR. In particular, a similar spatial and temporal segregation of tyrosine kinases and phosphatases was observed at both immunological synapses and phagocytic cups, leading to the notion of "phagocytic synapse" (2). Second, phagocytic receptor signaling triggers

### *Edited by:*

*Nick Gascoigne, National University of Singapore, Singapore*

### *Reviewed by:*

*Daniel Denis Billadeau, Mayo Clinic, USA Cosima T. Baldari, University of Siena, Italy*

### *\*Correspondence:*

*Florence Niedergang<EMAIL_ADDRESS>Vincenzo Di Bartolo<EMAIL_ADDRESS>Andrés Alcover<EMAIL_ADDRESS>
*† All three authors contributed equally to this review.*

### *Specialty section:*

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

*Received: 19 November 2015 Accepted: 14 January 2016 Published: 28 January 2016*

### *Citation:*

*Niedergang F, Di Bartolo V and Alcover A (2016) Comparative Anatomy of Phagocytic and Immunological Synapses. Front. Immunol. 7:18. doi: 10.3389/fimmu.2016.00018*

and their focal recycling participate in the regulation of T cell activation. Finally, in either system, triggering of multiple signaling pathways downstream of the surface

a profound reorganization of the actin cytoskeleton that is similar to the one induced by the TCR, generating large membrane extensions rich in filamentous (F)-actin. Third, microtubule dynamics are also important for some receptor-mediated phagocytosis. Fourth, intracellular traffic involving several vesicular compartments reorients toward the phagocytic cup. Fifth, internalization of the triggered receptors together with their ligands occurs and may lead to their degradation or recycling back to the plasma membrane. Interestingly, the TCRs may be phagocytosed from the immunological synapse internalizing with them their pMHC ligands together with portions of the APC membrane (3). Finally, a series of downstream signaling events lead to cytokine gene activation in both cases.

receptors leads to *de novo* transcriptional programs controlling cell survival, activation, and cytokine production.

We review here the molecular and cellular events taking place in both phagocytic and immunological synapses, highlighting their mechanisms of regulation.

### RECEPTOR SIGNAL TRANSDUCTION IN T CELLS AND PHAGOCYTES

T cell receptor engagement induces a series of molecular reorganization events that stabilize T cell–APC interactions and optimize signal transduction. Several other receptors are recruited to the immunological synapse and contribute to the activation process. These include the co-receptors CD4 and CD8, co-stimulatory receptors such as CD28, or adhesion proteins such as the integrins αLβ2 (LFA1) or α4β1 (VLA4) [reviewed in Ref. (4)].

One of the earliest events elicited by antigen recognition is the sequential activation of protein tyrosine kinases belonging to the Src and Syk families. The Src-family kinases Lck and/or Fyn, phosphorylate several TCR complex subunits, namely CD3 (ɛ, γ, and δ) and ζ (5). These subunits are all endowed with one or more consensus sequences called immunoreceptor tyrosine-based activation motif (ITAM), each containing two phosphorylatable tyrosine residues. Doubly phosphorylated ITAMs then recruit Syk-family kinases, either ZAP-70 or Syk (6), whose tandem SH2 domains provide specific, high-affinity binding to ITAM phosphotyrosines. Src kinases may be further required to phosphorylate and activate Syk kinases, in particular ZAP-70. The interplay between these two families of tyrosine kinases is crucial for transmitting downstream signals. Thus, Syk family kinases phosphorylate adaptor proteins, such as LAT and SLP-76 that in turn gather signaling effectors within multiprotein complexes, or signalosomes (6). Moreover, both Src- and Syk-family kinases activate several enzymes recruited in these signalosomes that are responsible for the generation of intracellular second messengers, such as Ca2<sup>+</sup> or phosphoinositides. Collectively, these early steps, induced within seconds after TCR engagement, initiate a cascade of downstream events leading to cytoskeletal rearrangement and cellular polarization. Concomitantly, various serine–threonine kinases, including MAP kinases, are activated, regulating the activation of several transcription factors that will drive in turn T cell growth and differentiation and the production of effector cytokines (7).

Detection and engulfment of bacteria or fungi by phagocytic cells are triggered by a similar sequence of early events. However, multiple unrelated ligands trigger phagocytosis by engaging distinct receptors. Indeed, phagocytic receptors can recognize their target by binding either to specific molecules expressed on the target's surface or to opsonizing antibodies or complement subunits previously bound to the target. For instance, phagocytosis of IgG-coated pathogens is triggered upon antibody recognition by Fcγ receptor (FcγR), whereas integrins, such as αMβ2 (also known as Mac-1 or CR3), can recognize complement-coated particles. Finally, phagocytosis of fungi expressing β-glucans on their cell wall is triggered by Dectin-1 receptor (8).

Phagocytic Fc receptors (FcγRII and FcγRIII) belong to the immunoreceptor family and are structurally related to antigen receptors. Importantly, they transmit activating signals using ITAM motifs that are either built in the receptor intracellular tail or in the associated common γ-chain (9). Hence, early signaling events involve Src- and Syk-family kinases, similarly to what explained above for the TCR. In macrophages, the Src kinases Lyn, Hck, and Fgr are involved in FcR-induced phagocytosis. However, phagocytosis was significantly reduced but not abolished in cells of triple knockout mice, suggesting the existence of further redundancy or alternative triggering mechanisms (10). In contrast, Syk knockout resulted in a complete block of phagocytosis, indicating the indispensable role of this kinase (11). Since Syk, but not ZAP-70, has been shown to phosphorylate ITAM motifs (12), it can be envisaged that Syk can trigger some phagocytic activity in the absence of Src kinases.

The β-glucan receptor Dectin-1, a member of the C-type lectin receptor (CLR) family, also induces sequential activation of Src and Syk kinases. Dectin-1 displays in its cytoplasmic domain ITAM-like sequences named hem-ITAM, each containing a single tyrosine-based motif. Once phosphorylated by Src kinases, they are able to bind Syk and trigger downstream activation (13). Since Dectin-1 is a dimer, it has been proposed that Syk binds in trans to two phosphorylated hem-ITAMs on adjacent subunits in order to be recruited to the activated receptor (13). However, this model has not been validated experimentally. Furthermore, a potential alternative mechanism for Syk recruitment has been revealed recently, implying a scaffolding role of the protein tyrosine phosphatase SHP-2 in bridging Syk to Dectin-1 and other CLRs (14).

The molecular mechanisms underlying integrin-dependent phagocytosis, such as that elicited by complement-coated particles binding to CR3, are more complex than those described for FcRs and Dectin-1. Importantly, integrin binding to their ligand requires prior activation *via* a conformational change induced by "inside-out signaling." This priming phase is induced by inflammatory or pathogen-specific signals, such as those triggered by G-protein-coupled (GPCRs) or toll-like receptors (TLRs). These proteins initiate different signaling cascades converging on a common effector, the GTPase Rap1 (15). Active Rap1 induces the recruitment of RapL, RIAM, and talin to integrin cytoplasmic tails, thus promoting the switch of integrins to their extended conformation that can bind ligands with high affinity (16). Then, ligand-bound integrins transmit "outside-in" signals that drive actin polymerization and downstream activation. These steps involve several effectors including the protein kinases FAK (or Pyk2) and ILK, non-muscle myosin II, and Rho GTPases (17). Nonetheless, the fact that Syk inhibition impairs CR3-mediated phagocytosis demonstrates the existence of some crosstalk between integrin activation and ITAM-bearing receptors or adaptors (18). Interestingly, FcRs have confined mobility in the plasma membrane, in fenestrated cortical actin structures that depend on the activity of Src- and Syk-family kinases (19). Integrins or pattern recognition receptors, such as the scavenger receptor CD36, are potentially initiating Syk activation, leading to FcR increased mobility and engagement (8). However, further work is needed to define the molecular basis of integrin interplay with ITAM-dependent signaling.

# SPATIOTEMPORAL ORGANIZATION OF IMMUNE AND PHAGOCYTIC RECEPTORS AND THEIR SIGNALING MACHINERIES

How early signals are elicited by antigen or phagocytic receptors engagement is still a matter of debate. One model proposed for TCR activation postulates that initial triggering is achieved when key inhibitory proteins, such as the tyrosine phosphatase CD45, are segregated away from the engaged TCR and the proximal tyrosine kinase Lck. This segregation is mainly driven by the size of membrane protein ectodomains. Indeed, the length of the TCR–pMHC pairs is relatively small (7 nm) compared to that of CD45 ectodomain (28–50 nm); hence, TCR engagement by pMHC induces the formation of areas of close juxtaposition of T cell and APC membranes from which phosphatases are excluded (20, 21). As a consequence, local activity of tyrosine kinases would be favored, leading to an increase in net phosphorylation of TCR downstream effectors and T cell activation. Interestingly, a similar mechanism was observed during Dectin-1-dependent phagocytosis, leading to the "phagocytic synapse" model. Indeed, Dectin-1 engagement by β-glucan-bearing particles results in local exclusion of phosphotyrosine phosphatases CD45 and CD148 from receptor-enriched areas containing phosphotyrosine, thus triggering downstream signaling (e.g., Syk phosphorylation) and phagocytic cup formation (2). Importantly, several results suggest that this mechanism also concerns FcRs (22, 23); hence, it may be relevant for all phagocytic receptors.

Concomitantly to initial kinase and phosphatase segregation, T cell receptor subunits, the tyrosine kinases Lck and ZAP70, and the adapters LAT and SLP76 associate into dynamic signaling complexes that nucleate at the periphery of immunological synapses and then migrate toward its center, where they concentrate or vanish (24–26). Interestingly, centripetal dynamics of signaling complexes at the immunological synapse and their concentration in the center is a regulatory mechanism that depends on actin and microtubule cytoskeleton and is meant to downregulate proximal TCR signaling (27–29). Various mechanisms have been proposed for TCR signal downregulation at the synapse. These include relocalization to membrane regions containing the tyrosine phosphatase CD45 (28), internalization and degradation of TCR and signaling complexes (30–32), post-translational modification of signaling adapters leading to signalosome disassembly (33), or the extracellular release of vesicles containing TCR (34). Of note, in FcR-mediated phagocytosis, receptors are engaged sequentially in a receptor-guided, zipper-like advance of the membrane over the particle surface, and there is no evidence for a movement of the receptors toward the base of the phagocytic cup. Receptors are downregulated from the surface with the engulfment of the particle. Thus, the late events in the mature immunological synapse differ from those observed in phagosome completion and closure.

### ACTIN AND MICROTUBULE CYTOSKELETON INTERPLAY

Signaling downstream of the TCR and phagocytic receptors leads to intense and transient actin polymerization that relies on the activation of Rho family GTPases (35). In T cells and phagocytes, Rho GTPase activation occurs to a large extent *via* tyrosine phosphorylation and activation of the Rac1 and Cdc42 guanine exchange factor (GEF) Vav (36, 37). In addition, Rac1 can be activated by other GEFs, including DOCK2, DOCK8, Tiam1, and Trio. DOCK2 is involved in Rac1 activation downstream of the TCR and in lymphocyte migration in response to chemokines. DOCK2 and DOCK8 physiological relevance has been underscored by the discovery of human-inherited immunodeficiencies caused by *DOCK2* or *DOCK8* gene mutations. B and T cells from these patients display impaired actin polymerization and migration in response to chemokines, as well as impaired lytic granule release by NK cells (38, 39). DOCK family proteins are also involved in phagocytosis as regulators of Rac1 (40).

In phagocytes, the pioneering description of the involvement of Rho family proteins initially led to the classification of type I phagocytosis implicating Rac1 and Cdc42 downstream of FcR and type II phagocytosis relying on RhoA downstream of CR3 (41). More recently, RhoG has been shown to act as regulator for both FcR and CR3-mediated phagocytosis (42). As RhoG is also critical for phagocytosis of apoptotic bodies (43), and for the nibbling of MHC-associated portions of APC membrane by T cells (3), it could well act as a still ill-defined "master regulator" in immunological synapse and phagosome formation. Dynamic studies by fluorescence resonance energy transfer (FRET) revealed different patterns of activation for Rac and Cdc42 downstream of FcR. Active GTP-Cdc42 is present at the tip of the advancing pseudopod where it colocalizes with polymerizing actin, while Rac1 activation is biphasic. GTP-Rac1 is induced at a low level early after particle binding and peaked at the time of pseudopod fusion (44). Cdc42 activation and phosphatidylinositol-4,5-bisphosphate PI(4,5)P2 accumulation in the nascent phagocytic cup activate effectors among which the actin nucleation promoting factor (NPF) N-WASP that acts on the Arp2/3 actin nucleation complex. Rac1 is then essential for F-actin polymerization to complete extension and closure, through activation of another NPF, the WAVE complex. In CR3-mediated phagocytosis, RhoA is critical for the signaling to actin polymerization as it activates the Rho-Kinase (ROCK), the formin mDia1, and myosin II that are implicated in polymerization and contraction of F-actin around the particles (41, 45–47). The microtubules are important for this pathway, and CLIP1 (CLIP-170), a microtubule plus-end protein, is especially required for efficient recruitment of mDia1 downstream of CR3 and therefore for efficient phagocytosis (48, 49), showing crosstalk between microtubules and actin.

Immunological synapse formation and function require the coordinated activation of RhoA after initial LFA-1 clustering and Rac1 and Cdc42 activation downstream of the TCR (35). Active Cdc42 and its effector WASP are independently recruited to the synapse. WASP seems not to be necessary for broad actin polymerization at the synapse, but rather for the generation of dynamically polymerizing actin foci that facilitate PLCγ activation and calcium flux (50). Consistently, WASP is necessary for efficient IL2 production (51, 52). In contrast, WAVE2, Arp2/3, and the cortactin homolog HS1 are required for T cells to regulate actin polymerization at the synapse (53–55). In turn, actin dynamics is necessary for triggering and sustaining T cell activation (56). This occurs in various concomitant ways, including the regulation of T cell–APC conjugate formation *via* integrin clustering (57), the interplay between actin cytoskeleton regulators and the calcium second messenger (58), or the regulation of immunological synapse architecture and its interplay with the TCR signaling machinery (59). Finally, the formation of signaling microclusters around the synapse periphery and their convergence toward the center depends on actin dynamics and F-actin inward flows (24, 60).

Cortical actin-associated proteins, such as ezrin and moesin, play important roles in building an activation competent immunological synapse. These proteins connect the cortical cytoskeleton with membrane components. Thus, moesin supports CD43 exclusion from the center of the synapse, a mechanism proposed to remove the CD43-dependent steric inhibition and to facilitate synapse formation (61–63). Moreover, ezrin and moesin contribute to the architecture of the immunological synapse, cell cortex rigidity, and T cell activation as well as differentially regulate early and late activation events (64–67).

Microtubules are finely reorganized at the immunological synapse bringing the microtubule-organizing center (MTOC) close to T cell–APC contact (67–69). Microtubule polarization depends on TCR-induced signaling (70, 71) and the microtubule-driven molecular motor dynein (72). Interestingly, ezrin plays a critical role in driving the MTOC close to the synapse, in controlling microtubule network organization, and in signaling microcluster dynamics at the synapse. Ezrin does so *via* its association with the polarity regulator Dlg1 (67). Moreover, the actin-nucleating proteins Diaphanous 1 (mDia1) and formin-like 1 (FMNL1) are also necessary to polarize MTOC to the synapse (53). The involvement of ezrin and formins in MTOC polarization highlights that actin and microtubule network organization at the synapse are tightly connected. Microtubule stability modulated by the HDAC6 deacetylase is also regulated during immunological synapse formation and necessary for synapse formation and T cell activation (73). Actin and microtubule interplay is also critical for T cell effector function, such as polarized secretion of helper cytokines, since it is necessary for Golgi complex polarization toward the APC (74).

As mentioned above, microtubule–actin interplay is also necessary for efficient phagocytosis (48). Of note, the MTOC has also been reported to be relocated at the site of phagosome formation (75), but given that multiple targets are often phagocytosed at the same time, how this applies to uptake in physiological situations is uncertain. Similarly, when a cytotoxic T cell is engaged in multiple contacts, the antigen-specific delivery of lytic granules occurs independently of centrosome positioning (76).

Microtubule dynamics and organization ensure the delivery of TCRs and signaling molecules to the synapse *via* recycling endosomes (77–79). Moreover, microtubules, together with actin flows, drive signaling microcluster centripetal movement at the synapse (67, 80). Therefore, microtubules drive the arrival and removal of TCRs and signaling molecules in a way to sustain and regulate TCR signaling at the synapse.

### ACTIN DYNAMICS AND CLEARANCE

Actin polymerization is crucial to achieve efficient pseudopod extension and phagosome formation, but actin turnover and depolymerization is as important. This turnover, which occurs at the base of the phagocytic cup (81), is directly dependent on the hydrolysis of PI(4,5)P2 (82), which is mediated by several effectors including phosphatases that hydrolyze PI(4,5)P2, such as phospholipase C, PI3 kinase, and 5′ phosphatases, such as Inpp5b or oculocerebrorenal syndrome of Lowe (OCRL) (81, 83–85). In addition, the severing protein cofilin is recruited to the site of phagocytosis and its activity is regulated by LIM kinase (86). Interestingly, the presence of OCRL at sites of phagocytosis was shown to depend on vesicular recruitment of AP1 and EpsinR adaptors, which is under the unexpected control of the NF-kB signaling protein Bcl10 (81), showing how interconnected the signaling and trafficking events are. Inactivation of Rho GTPases is also achieved by several Rho GAP proteins, such as ARHGAP12, ARHGAP25, and SH3BP1, that are recruited under the dependence of PI3K and synergistically inactivate Rac and Cdc42 (87). Actin clearance from the base of the phagocytic cup, which is required for large but not small particle internalization (87), is then necessary for vesicles to make their way to the plasma membrane (81).

Actin clearance is also observed in immunological synapses, and it is thought to be important to facilitate vesicle fusion at the synapse, particularly in cytotoxic T cells, which destroy target cells by the polarized secretion of lytic granules (88). F-actin relocalization at the immunological synapse depends on PI(3,4,5)P3 (89) and modulates cytotoxicity. Actin and PI(4,5)P2 are cleared from the site of secretion, indicating a tight interplay between actin cytoskeleton reorganization and phospholipid second messenger at the synapse (68, 90).

Therefore, the reorganization of the actin and microtubule cytoskeleton is triggered by TCR and phagocytic receptors and is the key to maintain the structure and function of phagocytic cups and immunological synapses.

# VESICLE TRAFFIC DURING PHAGOCYTIC CUP AND IMMUNOLOGICAL SYNAPSE FORMATION

Phagocytic cup formation generates membrane protrusions capable of engulfing particles of different sizes. Instead of a decrease in membrane surfaces after internalization of the phagosomes, an increase in cell surface was reported during phagosome formation using capacitance measurements (91). This is in agreement with the concept of membrane remodeling and "focal delivery" of intracellular compartments at the site of phagosome formation (92, 93). The requirement for focal vesicle fusion in optimal phagocytosis of large targets came from studies interfering with the fusion machineries composed of soluble *N*-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). These are membrane fusion regulatory proteins that form a tri-party complex composed of one vesicle (v)-SNARE and two target membrane (t)-SNAREs. SNARE complex formation helps bringing together the two membranes to facilitate their fusion. SNAREs act with various regulatory proteins, such as Rab GTPases, Munc proteins, and the calcium sensors synaptotagmins to bring together, dock, tether, and fuse vesicles with target membranes, either the plasma membrane or other vesicles (94). Several intracellular vesicular compartments have been implicated in focal recruitment and fusion concomitant with phagosome formation (95–97). These include recycling endosomes bearing the v-SNARE VAMP3 on their surface (98–100) and late endocytic compartments displaying the v-SNARE VAMP7 or lysosomes (101, 102). The endocytic compartments also harbor the adaptor proteins AP1 and EpsinR, both implicated in efficient phagosome formation, while the AP2 complexes and the clathrin-related endocytic machinery are not involved (81, 100). Interestingly, VAMP3<sup>+</sup>/AP1<sup>+</sup> endosomes also partially colocalize with TNFα, a cytokine that is delivered at the site of forming phagosomes (103).

Similarly, different endosomal compartments and vesicle traffic regulators are involved in immunological synapse formation. These compartments differentially transport TCRs, the tyrosine kinase Lck, and the adapter LAT to the synapse by recycling these proteins back and forth between their plasma membrane location and endosomes. These endosomal compartments display different traffic regulators, such as Rab GTPases (i.e., Rab4, Rab8, Rab11, Rab27, and Rab35), transport proteins (i.e., MAL, intraflagellar transport proteins), or vesicle fusion regulators (i.e., VAMP3, VAMP7, Synaptotagmin-7, and Munc13) (77, 78, 104–108). The immunological synapse clusters the t-SNAREs SNAP23 and syntaxin 4 preparing the zone for active vesicle fusion activity. It is still a matter of debate whether vesicles transporting the signaling adapter LAT only dock and stay as subsynaptic vesicles (106, 109, 110) or fuse with the plasma membrane driving LAT clustering at the synapse (77, 78, 111, 112). The regulated exocytosis of vesicular compartments in T cells might also be important during the early stages of synapse formation when a large lamellipodium-like membrane structure is formed over the APC. Finally, vesicle traffic is important for T cell effector functions, such as polarized secretion of cytokines or cytotoxic granules in helper and cytotoxic cells, respectively (88, 113).

During phagosome formation, the recruitment of compartments and their fusion are regulated by small GTPases of the Rab and ARF families. Rab11, localized on the recycling compartments, is implicated in efficient phagocytosis (114–116). ARF6 was shown to be activated during phagosome formation and to control the delivery of VAMP3<sup>+</sup> recycling endosomes (99, 117, 118). Rab35 regulates actin-dependent phagosome formation by recruiting ACAP2, an ARF6 GTPase-activating protein (119), or by regulating the localization of Rac1 and Cdc42 (120). In addition, Rab11 and ARF6 activities might be coordinated *via* common effectors, such as the Rab11-FIP3/4/ RIP/RCP (Rab-coupling proteins), also named arfophilins, which were implicated in phagosome formation and maturation (115). Rab31 (Rab22b) recruits the adaptor APPL2 that participates in PI3K/Akt signaling and phagosome completion (121). As Rab35 recruits the OCRL phosphatase during cytokinesis (122), it could also be implicated together with Rab5 (85) in OCRL recruitment during phagocytosis, although this has not been demonstrated. There are therefore multiple levels of regulation that implicate tight coordination between the signaling platforms and their subcellular localization, and further investigations are required to dissect them both in the context of the immunological synapse and the phagocytic cup.

### CONCLUSION

Although we have largely progressed in our understanding of the mechanisms underlying the membrane and cytoskeletal reorganization that support phagosome and immunological synapse formation, there are still a number of issues that need further indepth investigation. These issues may be different in the phagocytosis and the immunological synapse fields, but a comparison of the two systems may help solve these different questions faster. These include how some phagocytic receptors get engaged and the type of signals they generate? What is the phospholipid

### REFERENCES

chemistry of each of the systems and its influence on cytoskeleton organization? What is the precise time and space organization of signaling complexes and vesicular compartments? Interestingly, we have recently described several examples of "*ménage à trois*" between receptor signals, vesicle traffic, and cytoskeletal structures in both processes; for instance, the involvement of the proinflammatory signaling pathway NFκB in the control of vesicle trafficking and actin clearance in nascent phagosomes *via* the signaling protein Bcl10 (81), or the orchestrated action of the TCR signaling machinery, the actin and microtubule cytoskeleton, and intracellular vesicle traffic in ensuring immunological synapse architecture and function in T cell activation and effector functions, such as polarized secretion of cytokines or cytotoxic granules (1). Collectively, the vast majority of data presented here emphasize the similarities between immunological and phagocytic synapses formation and suggest a possible evolutionary link between these two structures, whereby the phagocytic synapse of innate immune cells would be an ancestor of the immunological synapse in the adaptive immune system (123).

### AUTHOR CONTRIBUTIONS

FN, VDB, and AA contributed equally to this review.

# FUNDING

Work in the laboratory of FN was supported by CNRS, INSERM, and Université Paris Descartes, and grants from Agence Nationale de la Recherche, ANR (2011 BSV3 025 02), Fondation pour la Recherche Médicale (FRM, INE20041102865), and l'Agence Nationale pour la Recherche sur le SIDA et les Hépatites Virales (ANRS). Work in the laboratory of AA and VDB was supported by grants from Institut Pasteur, CNRS, ANR (2011 BSV3 025 02), Fondation ARC pour la Recherche sur le Cancer, La Ligue Contre le Cancer, and ANRS.

calcium entry during T cell activation. *Curr Biol* (2006) **16**(1):24–34. doi:10.1016/j.cub.2005.11.036

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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 Niedergang, Di Bartolo and Alcover. 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.*

# The Dendritic Cell Synapse: A Life Dedicated to T Cell Activation

### *Federica Benvenuti\**

*Cellular Immunology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy*

T-cell activation within immunological synapses is a complex process whereby different types of signals are transmitted from antigen-presenting cells to T cells. The molecular strategies developed by T cells to interpret and integrate these signals have been systematically dissected in recent years and are now in large part understood. On the other side of the immune synapse, dendritic cells (DCs) participate actively in synapse formation and maintenance by remodeling of membrane receptors and intracellular content. However, the details of such changes have been only partially characterized. The DCs actin cytoskeleton has been one of the first systems to be identified as playing an important role in T-cell priming and some of the underlying mechanisms have been elucidated. Similarly, the DCs microtubule cytoskeleton undergoes major spatial changes during synapse formation that favor polarization of the DCs subcellular space toward the interacting T cell. Recently, we have begun to investigate the trafficking machinery that controls polarized delivery of endosomal vesicles at the DC–T immune synapse with the aim of understanding the functional relevance of polarized secretion of soluble factors during T-cell priming. Here, we will review the current knowledge of events occurring in DCs during synapse formation and discuss the open questions that still remain unanswered.

### *Edited by:*

*Andres Alcover, Institut Pasteur, France*

### *Reviewed by:*

*José Luis Rodríguez-Fernández, Consejo Superior de Investigaciones Científicas, Spain Anne Hosmalin, Cochin Institute, France*

*\*Correspondence:*

*Federica Benvenuti<EMAIL_ADDRESS>
### *Specialty section:*

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

*Received: 17 December 2015 Accepted: 15 February 2016 Published: 07 March 2016*

### *Citation:*

*Benvenuti F (2016) The Dendritic Cell Synapse: A Life Dedicated to T Cell Activation. Front. Immunol. 7:70. doi: 10.3389/fimmu.2016.00070*

Keywords: immune synapse, dendritic cells, actin cytoskeleton, polarized secretion, antigen presentation

# INTRODUCTION

Once in the lymph node, dendritic cells (DCs) have few hours to transfer the information gathered in the periphery to T cells. Live imaging of the lymph node unveiled that, depending on the inflammatory context and antigen density, T-cell activation can ensue from long-lasting interactions or by collection of signals from subsequent repetitive contacts (1). Recent technologies to visualize recruitment and activation of signaling molecules *in vivo* within individual DC–T contacts is beginning to shed light on the actual functional meaning of various types of intercellular encounters (2, 3). Nevertheless, our present understanding of the subcellular events underlying informational transfer at the synapse and signal decoding by T cells mostly comes from *in vitro* studies. As largely discussed in this topic, sophisticated analysis unveiled the mechanism by which the antigen-presenting cell (APC)-derived flow of information is turned into fine tuned T-cell activation. Less is known, instead, about the events that control efficient delivery of antigen-derived signals to T cells. Among APCs, DCs are uniquely potent in their ability to launch adaptive responses. They are composed of a complex network of different subsets that differentially control T-cell functions. The classical CD8α+ with the ontogenetically related tissue resident CD103<sup>+</sup> cells and the Cd11b subsets share some general principles, i.e., the ability to migrate from tissue to regional lymph nodes charged with antigens, but are specialized in the activation of CD8<sup>+</sup> and CD4<sup>+</sup> T lymphocytes, respectively (4). Yet, this paradigm is still evolving and these distinctions may turn out to be less strict.

In the following paragraphs, the events that takes place on the DCs side of the immune synapse (DC-IS) refer mostly to studies conducted using DCs models, i.e., cells differentiated from monocytes in the human system or from bone marrow precursors in the mouse system and unfortunately do not yet take into account such complexity. Still, common themes have emerged that will certainly be translated into more physiological cell types in the near future.

# SURFACE RECEPTORS

To simplify and categorize events occurring at the DC-IS, it is useful to proceed from the surface inward to the cytoplasm. Remodeling of the plasma membrane architecture and redistribution of the surface receptors have been documented in DCs from the earliest instance upon T-cell contact and even before the two cell bodies become adherent. Some conserved mechanisms to support intercellular interactions described in the neuronal synapse operate in DCs and are essential for contact formation, including semaphorins, plexins, and neuropilins (5). Plexin-1A, a receptor for class-3 semaphorins is highly expressed in mature DCs, cluster at the IS, and controls T-cell priming by regulating cytoskeletal remodeling, possibly *via* Rho activation (6, 7). A second receptor for semaphorins, neuropilin-1, is expressed on both sides of the synapse and can control DC–T encounter during priming and during instruction of regulatory T cells (8, 9). The way DCs interact with T cells is peculiar in terms of global geometry and microdomains organization, as compared with the synapse between B cells and T cells. Unlike B cells, DCs possess a highly dynamic membrane with projections such as veils and ruffles that are increased during the process of maturation (10, 11) and further modify when an approaching T cell is sensed by chemokine receptors (12, 13). In human DCs, a peculiar microvilli-like structure was shown to be the preferential site of association with T cells leading to multiple aggregates of TCR/CD28 signaling complexes in T cells, as opposed to the concentrically structured immune synapse formed by B cells (14). A multifocal synapse is frequently formed also in murine T cells interacting with DCs, further strengthening that antigen presentation by DCs is more dynamic than by B cell (14, 15). Mirroring TCR clustering, MHC class-II and costimulatory molecules such as CD86 are recruited at the DC contact area very early during initial DC–T scanning. This recruitment is driven by ICAM-1/3 interactions with LFA-1 *via*, a mechanism that depends on an intact actin cytoskeleton and the Vav1/Rac1 effectors (16). Delivery of MHC class-II at the plasma membrane depends on long tubules of late endosomal origin that contain MHC–peptide complexes and move directionally toward the contact zone. This process is conserved in murine and human DCs both during antigen presentation and cross-presentation (17, 18) and ensures continuous fueling of TCR ligands in the IS region. Other costimulatory molecules undergo spatial reorganization on the APC. CD40 clusters at the synapse in B cells during delivery of T-cell help and it is stored in intracellular vesicles and rapidly released upon contact with an allogeneic CD4 T cells in rat lymph DCs (19, 20). Mirroring CD40 clustering, CD40L in T cells is recruited at the IS with human DCs and this polarization is important for DCs–T cross-talk and T-cell induced DCs maturation as it triggers IL-12 production by DCs (21). T-cell-induced DC maturation provides an important demonstration of the mutual exchange that occurs between the two cells [reviewed in Ref. (22)]. A further costimulatory molecule CD70, a TNF-family member receptor with critical roles in T-cell priming, is concentrated at the DC-IS. Interestingly, CD70 is found in intracellular compartments that overlap with class-II positive vesicles, and this localization depends on the Invariant chain (Ii), suggesting a shared control of antigen peptides and costimulatory molecules transport to the synaptic area (23, 24). Similarly, ICAM-1 is recycled at the interaction zone through recycling compartments where it intersects MHC class-II molecules (25). ICAM-1 mobility on the DCs membrane is a critical factor controlling activation of T cells. Besides being the main adhesive force to stabilize the interaction, LFA-1 is important to modulate signaling from the TCR. Binding of LFA-1 to its ligand, in fact, enhances signaling for key TCR signal transduction molecules, such as PI3K, PLC-γ, MAP kinases, and SLP-76, reinforcing IL-2 production and T-cell proliferation (26–29). A recent study revealed that cytoskeletal remodeling during DC maturation causes a decrease in the lateral mobility of ICAM-1 in the membrane. Laterally confined ICAM-1 imposes forces on the interacting LFA-1 that promote ligand-dependent activation and increase T-cell priming (30).

### SIGNALING MOLECULES

Underneath the plasma membrane and concomitantly to redistribution of membrane receptors, several events take place in the cytoplasm of DCs, suggesting that the DC-IS is an active signaling zone. Spinophilin is a PDZ domain-containing protein that is highly expressed in the dendritic spines of neurons where it controls interactions with the underlying cytoskeleton and with membrane trafficking proteins. Spinophilin was shown to be expressed in DCs and to cluster at the DC membrane in antigen-specific conjugates, and its depletion caused a strong inhibition of T-cell priming (31). This study did not address the consequences of spinophilin loss in terms of cytoskeletal proteins distribution and overall cell symmetry. It is likely that spinophilin participates in the network of PDZ domain-containing proteins regulating cell polarity, indicating the importance of asymmetric distribution of functional subdomains for antigen presentation.

Durable presentation of antigens to T cells, either *via* longlasting contacts or cumulative interactions, is essential for efficient priming of T cells in lymph nodes (1). The life span of DCs is short, and it is therefore essential to control their fitness when they come to accomplish their ultimate task in the lymph node. Intriguingly, it was demonstrated that synapse formation provides antiapoptotic signals to DCs. Formation of the synapse induces recruitment and activation of the antiapoptotic protein Akt at the DC-IS followed by activation of prosurvival signals, including inhibition of FOXO and activation of NF-κB [(32) and reviewed in Ref. (33)]. Given the extensive remodeling of actin and microtubule cytoskeleton in DCs engaged in synapses (see below), it is likely to expect that other large signaling complexes become selectively recruited and activated at the site of interaction. However, the membrane receptors primarily involved, the composition of these signaling platforms, and the hierarchy of their assembly still remains to be elucidated.

### THE ACTIN CYTOSKELETON

All synaptic events, from intercellular contact formation and adhesion to signal transduction, are orchestrated by the actin and microtubules cytoskeleton, their upstream regulators and downstream effectors in a continuous feedback loop.

Soon after the discovery of the supramolecular activation cluster in T cells, researchers discovered that DCs actively participate in synapse formation by polarizing the actin cytoskeleton, unlike B cells. The causal link was strong as F-actin pharmacological inhibitors inhibited the T-cell priming capacity of DCs (34). Actin remodeling is controlled by a set of proteins that promote actin nucleation and polymerization, in turn controlled by Rho GTPases. Studies to decipher the molecular pathways involved in the control of actin remodeling in DCs showed, at first, an involvement of the actin-bundling protein, fascin. This protein is highly expressed selectively in DCs, upregulated during maturation, and necessary for DCs full antigen-presenting function (35). Few years later, we found that one essential mediators of actin remodeling during synapse formation in DCs is the small GTPases, Rac. Genetic deletion of Rac1 and 2 rendered DCs unable to establish productive interaction with T cells, leading to extremely inefficient T-cell activation (36). Two main classes of actin nucleating factors, formins and WASp family, involved, respectively, in the formation of long actin polymer elongation and branched actin networks were both shown to play a role in DCs migration and interaction with T cells. Deletion of mDia, a formin family member, inhibits DCs migration to LNs and decreases the capacity to establish long contacts with T cells and to induce their activation (37). WASp, expressed selectively in hematopoietic cells and target of mutations in Wiskott–Aldrich syndrome (38), controls various DCs functions, including the capacity to form stable interactions with T cells and the stability and structure of the synapse. *In vivo* two-photon microscopy analysis in the 3D environment of the lymph node showed decreased contact time between WASp null DCs and CD8<sup>+</sup> T cells. Closer inspection *in vitro* revealed a defect in accomplishing the complete cycle of movements required to establish a full contact. As a consequence, WASp null conventional DCs often undergo repetitive contacts of short duration, resulting in decreased T-cell proliferation and IFN-γ production (39). Similarly, CD4 T cells are inefficiently primed by WASp null DCs *in vivo* and *in vitro* (40). In this last work, Ca2<sup>+</sup> signaling and clustering of signaling molecules were shown to be decreased in T cells interacting with mutant cells, indicating that failure to sustain proximal events represents an important contribution to the overall immunodeficiency in WAS. Recently, a novel cell-free system to mimic the T-cell surface was used to address the role of WASp at the DC-IS with increased resolution. The results show that a spatially organized structure containing MHC-II, ICAM-1, and actin forms at the DC-IS, and it is stabilized in a WASp- and Arp2/3-dependent manner (41). It would be interesting to investigate whether WASp is involved in controlling the lateral mobility of ICAM-1, a parameter recently emerged to be critically during antigen presentation by DCs (30). Interestingly, the function of WASp goes beyond the control of motility and signaling at the IS as it also acts in tuning innate responses by toll-like receptors in plasmacytoid DCs (42).

The role of actin and actin regulatory proteins involved in synapse formation in DCs is summarized in **Figure 1**. Despite these insights, the triggers and the players of actin remodeling during synapse formation in DCs are not yet understood in sufficient details. As shown in a recent elegant study, DCs switch between different actin nucleating machineries during the process of migration and antigen uptake (43). What type of actin machinery prevails during antigen presentation at the IS and how the switch to this third DCs' specific function is achieved remains an interesting open question.

### POLARITY AND SECRETION ON THE APC SIDE OF THE IMMUNE SYNAPSE

Polarity is a highly conserved mechanism to spatially organize different functions within a cell, common to many processes, including the regulation of immune cells functions. The importance of polarity is well characterized in T cells where centrosome polarization in CTLs and T-helper cells is a fairly well-understood mechanism to optimize release of cytotoxic granules and cytokines, respectively (44–46). In B cells, centrosome polarization at the synapse is a mechanism to orient the secretion of

polarized and cluster enriched at the junction with an antigen-specific T cells. Fascin, an actin-bundling protein implicated in extension of membrane protrusion and development of dendrites is enriched in this area and controls the capacity of DCs to activate T cells. The Rho GTPase Rac is required for synapse formation and T-cell activation, likely *via* WAVE-mediated actin remodeling. WASp is a further activator of the Arp2/3 complex that is essential to support the antigen-presenting activities of DCs by promoting actin branching, thereby stabilizing the synaptic structure. WASp activation is mediated by cdc42 that is also found enriched at the DC-IS. The formin family mDia, involved in elongation of long actin filaments is important to establish and stabilize DC–T contacts and to support T-cell activation.

lysosomes and to promote the extraction of immobilized BCR ligands (47). B cells were shown to polarize also in the context of a B–T cell synapse with both the centrosome and the Golgi apparatus reoriented toward the interaction zone (48).

Many evidences exist that cell polarity and polarized membrane trafficking establishes also in DCs upon formation of antigen-specific conjugates. For instance, tubules of MHC class-II emanating in the direction of the interacting T cell indicate that remodeling of membrane trafficking is induced by antigen-specific contacts (17). The ability to polarize the tubules is acquired upon signals from toll-like receptors and depends on microtubules integrity (49, 50). DCs contacting NK cells were shown to polarize cytokines in the synaptic cleft, further suggesting directional vesicular transport (51, 52). Indeed, it was formally demonstrated that DC, following induction by a microbial maturation stimuli, polarizes the microtubule-organizing center (MTOC) toward the DC–T interface (53). This process depends on cdc42 as genetic deletion of this key polarity protein hampers the ability of DCs to reposition the centrosome underneath the synaptic membrane. Cdc42 is indeed highly enriched at the contact zone in DCs indicating its possible role as a WASp activator as well. Vesicles carrying newly synthesized interleukin 12, a key mediator of T-cell priming, clustered around the Golgi and were rapidly repositioned at the DC-IS, resulting in polarized secretion and local induction of IL-12-dependent signaling in T cells (53) (**Figure 2**). Thus, similarly to what have been shown in T cells, polarized transport of soluble mediators is a fundamental process that contributes to the antigen-presenting properties of DCs as ablation of cdc42-mediated polarization inhibits T-cell proliferation (53). The notion that the so-called signal 3 (proinflammatory cytokines) can be locally coupled to antigenic and costimulatory signals, and it can be delivered in *cis* may have important consequences for the fate imprinted to different T cells that interact sequentially with the same APC.

These findings raise the question of the link between microtubules and membrane trafficking proteins that support the directional transport of mediators en route to exocytosis. In macrophages and in T cells, members of the SNARE and Rab family were shown to control selectively directional secretion of various cytokines (44, 54). In DCs, we have recently discovered that the SNARE VAMP-7 plays a key role in the intracellular trafficking and secretion of newly synthesized IL-12. VAMP-7 not only controls the multidirectional transport of the cytokine toward the plasma membrane but, most importantly, it is absolutely required for clustering and secretion of IL-12 at the DC-IS (55). These findings reveal an usual transport route *via* late endosomes and represent the first insight into the molecular mechanism that orchestrate trafficking and secretion of soluble factors in DCs.

### REFERENCES

# CONCLUSION

and triggering of IFN-γ in the interacting T cell.

Both synaptic partners contribute to the successful outcome of the intercellular interaction between an activated DCs and a naive T cell, i.e., T-cell activation.

It is becoming obvious that a complex cross-talk between the two cells exists and that understanding T-cell activation cannot do without a deep understanding of the DCs synapse. Further efforts are envisaged in at least two directions. The first is the systematic dissection of the receptors and the downstream effectors operating sequentially in DCs and their functional significance. This goal will be achieved, thanks to the recent development of cell-free system to reconstitute the T-cell membrane. Second, to increase physiological relevance, future studies should take into account the complexity of the DCs system, which is composed of various DCs subsets that are most likely equipped with different tools to transmit information to their partners.

### AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and approved it for publication.

### ACKNOWLEDGMENTS

The author wishes to thank Italian Telethon and Associazione Italiana Ricerca Cancro grants IG9076 to the PI for the support on the work referenced in this review.

compartments in dendritic cells. *J Cell Sci* (2010) **123**:3817–27. doi:10.1242/ jcs.068510

and release of the DC maturation factor HMGB1. *Blood* (2005) **106**:609–16. doi:10.1182/blood-2004-10-3906

**Conflict of Interest Statement:** The author declares 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 Benvenuti. 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.*

# Dynamic Regulation of TCR–Microclusters and the Microsynapse for T Cell Activation

*Akiko Hashimoto-Tane and Takashi Saito\**

*Laboratory for Cell Signaling, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan*

The interaction between a T cell and an antigen-presenting cell is the initiating event in T cell-mediated adaptive immunity. The Immunological Synapse (IS) is formed at the interface between these two cell types, and is the site where antigen (Ag)-specific recognition and activation are induced through the T cell receptor (TCR). This occurs at the center of the IS, and cell adhesion is supported through integrins in the area surrounding the TCR. Recently, this model has been revised based on data indicating that the initial Ag-specific activation signal is triggered prior to IS formation at TCR–microclusters (MCs), sites where TCR, kinases and adaptors of TCR proximal downstream signaling molecules accumulate as an activation signaling cluster. TCR–MCs then move into the center of the cell–cell interface to generate the cSMAC. This translocation of TCR–MCs is mediated initially by the actin cytoskeleton and then by dynein-induced movement along microtubules. The translocation of TCR–MCs and cSMAC formation is induced upon strong TCR stimulation through the assembly of a TCR–dynein super complex with microtubules. The Ag-specific activation signal is induced at TCR–MCs, but the adhesion signal is now shown to be induced by generating a "microsynapse," which is composed of a core of TCR–MCs and the surrounding adhesion ring of integrin and focal adhesion molecules. Since the microsynapse is critical for activation, particularly under weak TCR stimulation, this structure supports a weak TCR signal through a cell–cell adhesion signal. The microsynapse has a structure similar to the IS but on a micro-scale and regulates Ag-specific activation as well as cell–cell adhesion. We describe here the dynamic regulation of TCR–MCs, responsible for inducing Ag-specific activation signals, and the microsynapse, responsible for adhesion signals critical for cell–cell interactions, and their interrelationship.

Keywords: immunological synapse, microcluster, microsynapse, dynein, cSMAC, cytoskeleton, F-actin, LFA1

### TCR–MICROCLUSTERS AND THE IMMUNE SYNAPSE

### T Cell Activation Signaling through TCR–Microclusters

Acquired immunity is exemplified by antigen (Ag)-specific responses, which are initiated by specific recognition of Ag by T or B cells. In the case of T cells, an Ag-specific cell encounters and interacts with Ag-bearing dendritic cell (DC) in the draining lymph node and uses its T cell receptor (TCR) to recognize the Ag peptide–MHC complex on the DC. This interaction induces an Immunological Synapse (IS) at the interface between the T cell and DC. The initial finding of this structure by

### *Edited by:*

*Pedro Roda-Navarro, University Complutense of Madrid (UCM), Spain*

### *Reviewed by:*

*José Luis Rodríguez-Fernández, Consejo Superior de Investigaciones Científicas, Spain Janis K. Burkhardt, Children's Hospital of Philadelphia, USA*

> *\*Correspondence: Takashi Saito<EMAIL_ADDRESS>
### *Specialty section:*

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

*Received: 15 December 2015 Accepted: 15 June 2016 Published: 29 June 2016*

### *Citation:*

*Hashimoto-Tane A and Saito T (2016) Dynamic Regulation of TCR–Microclusters and the Microsynapse for T Cell Activation. Front. Immunol. 7:255. doi: 10.3389/fimmu.2016.00255*

Kupfer's group was mainly based on microscopic visualization of the *z*-axis of the interface using advanced microscopy and deconvolution. They observed that the interface had a specifically segregated bulls-eye structure with a centralized TCR (with PKCθ) surrounded by the integrin LFA-1 (with Talin) (1). This segregation was achieved by accumulation of TCRs and adhesion molecules together with certain signaling molecules. Thus, the structure appeared to be related to T cell activation, and they termed the structure, the Supra Molecular Activation Cluster (SMAC), the central area for TCR accumulation as the cSMAC, and the peripheral LFA-1 accumulated area as the pSMAC. The initial analysis already noted the size difference of molecules in the c- and p-SMAC, i.e., that smaller molecules with one or two Ig domains in their extracellular region tended to accumulate in the cSMAC, while larger molecules such as integrins or CD45 accumulated in the pSMAC or distal dSMAC surrounding the cSMAC (2). These observations led to the segregation kinetics model of T cell activation (3), which proposes that the segregation in the periphery of large sized phosphatases such as CD45 from the central region of TCR engagement allows for activation of Lck kinase, followed by induction of the initial downstream signals for T cell activation (4).

The mature IS, supported by cellular adhesion through LFA-1, was thought to be an ideal structure for inducing an activation signal upon Ag recognition through the TCR. However, it was noticed that IS formation can be variable, depending on the cell types and stimulation conditions; some T cells do not form IS, nor do T cells with B cells rather than DC as antigen-presenting cell (APC) (5, 6). It was also proposed that only strong stimulation induced cSMAC formation (these situations are discussed later in terms of their relationship with the microsynapse). Furthermore, since the generation of the cSMAC, even on a supported planar bilayer, took about 10 min after interaction of the TCR and pMHC, it was noted that this would be too late for triggering the initial TCR signals (7). Analysis of very early activation after the interaction of Ag-specific T cells and a planar bilayer containing specific Ag peptide–MHC revealed that the TCR begins clustering immediately after T cells recognize the peptide–MHC on the planar bilayer, prior to mature IS formation. We stated to call these initial clusters TCR–microclusters (TCR–MC) as a minimal unit of clusters mediating both initial and sustained TCR signaling (8–12). MCs were described by Krummel and Davis as small clusters of CD3ζ accumulating at the center of the interface upon stimulation, and which were synchronized with the calcium response (13, 14). Quantification analysis of the TCR–MCs revealed that each cluster contains approximately one hundred (50–300) TCR molecules. This TCR accumulation immediately upon peptide/MHC stimulation was found to be associated with the simultaneous accumulation of the kinase ZAP70 and adaptor proteins LAT and SLP76 in the same TCR–MC. Upon stimulation, every TCR–MC is stained by Abs against phospho-ZAP70, phospho-SLP-76, and phospho-tyrosine. Thus, a TCR–MC is generated by accumulation of a hundred TCR–CD3 complexes, kinases and adaptors and induces immediate phosphorylation of these molecules. TCR–MCs also contain a substantial quantity of the known proximal signaling intermediates including ZAP70, LAT, SLP-76, PLCγ, and cytoskeleton-related molecules Nck and Vav (15, 16), which further induce triggering of a Ca2<sup>+</sup> flux and activation of downstream effector molecules. TCR–MCs are generated first at the center of the interface between the T cell and the planar bilayer or APC, and then are increased over the entire interface as the T cells spread. The initial activation signal is therefore induced in the newly generated TCR–MCs on the cell surface. Regarding the relationship of TCR–MC and the IS, TCR– MCs move toward the center of the interface after maximum cellular spreading, and the accumulated TCRs generate the cSMAC of the IS. It was noticed that only the TCR–CD3 complexes move to the center to form the cSMAC, while other signaling molecules such as ZAP70 and SLP-76 move only a short distance toward the center but do not accumulate in the cSMAC. These molecules disappear during their transport toward the center, probably by endocytosis. It has been noticed that TCR–MCs do not generate a cSMAC in some T cells, such as thymocytes and hybridomas, or under certain conditions, including weak Ag stimulation. However, even under conditions without cSMAC formation, T cells generate TCR–MCs to induce activation signals.

Signaling clusters induced upon TCR stimulation had been previously demonstrated when Jurkat cells were stimulated by immobilized anti-CD3 Ab (15). In this situation, TCR-CD3 appeared to be fixed and immobilized on the cover slip, but clusters of LAT, SLP76, and PLCγ, which induce the phosphorylation and activation of downstream signaling molecules, were generated. In this system, distal signaling intermediate molecules become dissociated from the immobilized TCR and move to intracellular compartments; SLP76 moves to a perinuclear structure and Nck and WASP to an actin-rich compartment and the immobilized TCRs do not move to the center or make the cSMAC (15, 16). Although there are some differences between pMHC-induced TCR–MC in normal T cells and Ab-stimulated signaling clusters in Jurkat cells, a general common feature is that, prior to the IS formation, TCR–MCs composed of TCR-CD3, kinases and adaptors are generated at the interface upon Ag recognition, which induces the initial signal for T cell activation. Later, though depending on stimulation conditions, the TCR–MCs move to the center of the interface to generate the cSMAC of the mature IS.

Recent imaging analysis using super-resolution microscopy as well as EM analysis revealed that several molecules of TCR or LAT are pre-clustered prior to Ag stimulation as "nanoclusters," which are then assembled together upon stimulation to form a MC (17, 18). In this regard, it is noted that the dynamics of signaling molecules within TCR–MCs are not uniformly regulated, and the signaling components within the cluster are variable and dynamic in their behavior.

A transient initial activation signal is not sufficient for full activation of T cells to induce cytokine production and cell proliferation, instead sustained activation for several hours is at a minimum required (19). Not only initial activation but also sustained continuous activation is induced through TCR–MCs at the peripheral region of the interface (9). TCR–MCs are continuously generated at the cellular edge with lamellipodial structures and move inward to the cSMAC. When the generation of the peripheral MCs is interrupted by the addition of anti-pMHC Ab, the formation of peripheral TCR–MCs is immediately halted, but the cSMAC is maintained (10). Moreover, the blockade of newly Hashimoto-Tane and Saito TCR–Microcluster and Microsynapse

generated TCR–MCs functionally inhibited activation signals. Therefore, the continuous generation of TCR–MCs is critical for inducing sustained activation signals.

# Compartmentalization of TCR Signaling at the IS

The cSMAC, as the representative structure of the original description of the IS, has several specific functions: (a) increasing the cell adhesion between the T cell and APC. Since the affinity of the individual TCR and pMHC interaction is so low, on the order of 10<sup>−</sup><sup>4</sup> M (20), the accumulation of thousands of TCRs increases the avidity for pMHC for cellular adhesion between the T cell and APC, although the adhesion is mainly supported by integrin binding in the pSMAC. (b) directing the targeted secretion of cytotoxic granules and cytokines toward APC (21, 22). (c) serving as the site for endocytosis and/or exocytosis (23, 24) of the TCR complex, which functions to negatively regulate T cell activation. (d) inducing co-stimulation signals as described below.

In the case of the IS formed between cytotoxic T cells and target cells, the cSMAC area is further segregated into two functional domains, a signaling domain through the TCR and a secretory domain, through which cytotoxic granules are secreted onto target cells (23, 25). These functional domains are present even in CD4<sup>+</sup> T cells.

Because the TCR/CD3 complex accumulates in the cSMAC, whereas upon Ag recognition, the majority of the downstream kinases and adaptors do not (9, 10), and little phosphorylation of these signaling molecules was observed in the cSMAC, it is thought to be inactive in signaling. Rather, the cSMAC is thought to be responsible for endocytosis and degradation of the TCR, which consequently contributes to negative regulation of T cell activation by decreasing the TCR complex. Indeed, a large invagination of the TCR was observed (26), and endocytic/ degradation machinery such as TSG101 (27) and a lipid for multivesicular body formation for degradation, lysobisphosphatidic acid (LBPA), is assembled in the cSMAC (10), indicating that the TCR complex is endocytosed at the cSMAC and targeted for degradation. In contrast to its function in TCR endocytosis, it was recently reported that vesicles containing the TCR are secreted from the cSMAC (24). Thus, the contribution of endocytosis vs. exocytosis of TCR-containing vesicles in the cSMAC has to be better understood. When the cSMAC area was precisely analyzed by microscopy, two distinct areas were found – CD3hi and CD3lo (**Figure 1**) Bleaching experiments revealed that the CD3hi area is rigid whereas the CD3lo area is very mobile and dynamically regulated. Using planar membranes containing MHC class II with covalently linked peptide, the CD3lo area but not the CD3hi region was found to be associated with pMHC, suggesting that the CD3lo region is actively participating in binding to pMHC, but the CD3hi region may contain TCR complexes that are either in the process of dissociation from pMHC or have already done so and are on the path to endocytosis and/ or exocytosis (28, 29).

In contrast to these data showing that the cSMAC is in general a negative regulatory site through TCR endocytosis/degradation, imaging analysis of co-stimulation signals indicated that a sustained co-stimulation signal is induced through a part of the cSMAC (30, 31). We demonstrated that upon Ag stimulation, the positive co-stimulatory receptor CD28 is first co-localized in the peripheral TCR–MC (recall that a co-stimulation signal is induced through the TCR–MC in the initial phase of activation) and then moves to and later accumulates in the cSMAC region, particularly in the CD3lo area (we call this area the "signaling cSMAC") (**Figure 1**). A search for the molecules whose behavior is similar to CD28 identified PKCθ and CARMA1, which also accumulated in a similar region of the cSMAC. A CTLA4–Ig fusion protein is used to inhibit CD28 co-stimulation since it binds more strongly than CD28 to the common ligand CD80/86. Addition of CTLA4–Ig blocks the association between CD28 and its ligands and results in no accumulation of CD28 in the signaling cSMAC. At the same time, PKCθ was also no longer found in the cSMAC, indicating that CD28 recruits PKCθ to the signaling cSMAC, probably to mediate co-stimulation. Using a biochemical approach, CD28 was found to be physically associated with PKCθ through association with Lck. The V3 region of PKCθ binds to the SH3 region of Lck and the SH2 region of Lck binds to the proline-rich region of CD28 (32). The accumulated CD28 recruits PKCθ and then CARMA1 into the CD3lo signaling cSMAC region, where sustained co-stimulation signals are induced, including NF-κB activation.

The analysis of TCR–MC and the cSMAC has revealed spatially distinct signaling compartments within a single T cell. These might be structural correlates corresponding to the old idea that both signal 1 and signal 2 are required for full T cell activation, i.e., TCR-induced Ag-recognition signal as signal 1 is mediated through the TCR–MC whereas the CD28-induced sustained co-stimulation signal as signal 2 is mediated through the signaling cSMAC (**Figure 1**). Recently, an actin-uncapping protein Rltpr was shown to be essential for CD28-mediated costimulation, a finding that connects CD28 and PKCθ/CARMA1 (33). Rltpr is also localized in the same signaling cSMAC upon TCR stimulation, where it may mediate the co-stimulation signaling function.

Negative regulation of T cell activation by the inhibitory costimulation receptor CTLA-4 is also induced at the same cSMAC region. Because CD28 and CTLA4 share the same ligands CD80/ CD86 and CTLA4 has a much higher (20-fold) affinity for these ligands, even low expression of CTLA4 on the T cell surface can compete with CD28 for ligand binding, which is the major mechanism for CTLA4-mediated inhibition (34, 35). CTLA-4 mostly accumulates in the intracellular secretory lysosomes and, upon TCR stimulation (36), it moves toward the plasma membrane at the cSMAC, particularly to the CD3lo signaling cSMAC, the same region where CD28 accumulates. Accumulated CTLA4 at the signaling cSMAC locally competes with CD28 for ligand binding. Therefore, CTLA-4-mediated inhibition is induced by ejecting the CD28-PKCθ–CARMA1 signaling machinery from the signaling cSMAC (11).

Thus, the current view of the cSMAC has evolved. It is not merely a site for negative regulation through TCR endocytosis/ degradation, but instead a particular region within the cSMAC is the site for inducing activation signals and is also a regulatory site by virtue of its inhibitory co-stimulation.

Figure 1 | Compartmentalization of TCR activation signals: TCR–microcluster and cSMAC. [(A), *x*–*y* axis; (B), *z*-axis; (C), a model for the assembly on the membrane] Upon Ag recognition, T cells form a conjugate with the APC (left) and generate TCR–microclusters (MCs) at the interface with the APC. TCR–MCs contain TCRs (red) and the proximal signaling molecules as well as the CD28 co-stimulation receptor (blue), and induce the initial activation signal (middle). After maximum spreading, TCR–MCs begin to move toward the center of the interface to form the cSMAC (right). It was noted that there is a CD3hi region (red) and a CD3lo region (mixture of red and blue) within the cSMAC; the CD3hi region is rigid and may represent the site for TCR endocytosis, whereas the CD3lo region is dynamically regulated and various costimulation molecules as CD28 and CTLA-4 are co-localized. Thus, we named this CD3lo region the "signaling cSMAC." In the cSMAC, the TCR complex is subjected to endocytosis/degradation for negative regulation, whereas CD28 recruits PKCθ and CARMA1 to induce a sustained co-stimulation signal leading to downstream events such as of NF-κB activation. The inhibitory co-stimulation receptor CTLA4 is translocated to the same cSMAC area as CD28 and competes with CD28 to eject CD28-PKCθ from the cSMAC, resulting in inhibition of activation. Thus, the TCR activation signal is regulated by spatially distinct signals: The Ag recognition signal as "Signal 1" is mediated by TCR–MCs and a sustained co-stimulation signal as "Signal 2" is mediated by the signaling region of the cSMAC.

# CYTOSKELETAL REGULATION OF TCR–MICROCLUSTER MOVEMENT

When T cells are stimulated with different concentrations of peptide/MHC on a planar bilayer, the cSMAC is formed with relatively high concentrations of antigen (>1 μM) but cannot be formed with low concentrations (<10 nM). Stimulation with low doses of Ag induces TCR–MCs, but they do not move toward the center of the interface and do not form the cSMAC. Considering that the cSMAC negatively regulates T cell activation through TCR endocytosis/degradation, weak stimulation to trigger weak signals may not require such an inhibitory mechanism. In contrast, upon strong stimulation with high doses of Ag, TCR–MCs move to and accumulate in the center, generating a cSMAC. Therefore, the movement of TCR–MCs is regulated by activation signal strength. At the steady state before stimulation, the TCR forms small clusters on the cell surface consisting of a few to ten molecules as "nanoclusters" (17, 37), as described above. Some signaling molecules such as LAT have been shown to be in nano-clusters distinct from the TCR. However, upon TCR stimulation, these distinct nano-clusters begin to form larger clusters by coalescing with signaling molecules such as LAT (17). These coalesced clusters are likely to be equivalent to TCR–MCs as a signaling unit. The size of TCR–MCs to be translocated centripetally into the cSMAC should be minimum (38).

The translocation and function of TCR–MCs is dependent on the actin cytoskeleton. Upon TCR stimulation, the actin cytoskeleton dynamically changes the cell morphology to promote centripetal flow at the periphery (39, 40). Inhibition of actin polymerization at the initial stage of T cell activation resulted in blockade of T cell adhesion, generation of TCR–MCs and activation. Addition of the actin inhibitor during the initial formation of TCR–MCs inhibits the generation of additional TCR–MCs, and consequently inhibits activation signals (8, 10). F-actin is initially generated upon TCR activation as a distal ring at the peripheral edge of the cell along with cell spreading, and then forms a large peripheral ring. In addition to this distal lamellipodial ring, small foci of F-actin have been found in co-localization with TCR–MCs (41). The peripheral actin exhibits retrograde flow toward the center of the interface. The new TCR–MCs, which are generated at the lamellipodial edge in a random manner upon interaction with peptide–MHC, then start to move toward the center, coincident with the actin retrograde flow (42). Since the interaction of TCR–MCs and actin appears to be weak and the actin centripetal flow is faster than the movement of TCR–MCs, TCR–MCs may be propelled by transient linkage to the actin retrograde flow (42, 43). However, the actin retrograde flow can only reach to about the middle of the path to the center, and the central/peripheral areas are free of actin (44). This raises the question of how are TCR–MCs translocated further to the cSMAC? We found that TCR–MCs translocate further into the central region along microtubules by assembly with the microtubule-associated motor protein dynein (45) (**Figure 2**). Dynein generally transports various cellular cargos by walking along cytoskeletal microtubules toward the minus-end of the microtubule. Indeed, we could co-immunoprecipitate the dynein–dynactin complex with the TCR complex upon TCR stimulation. When T cells were treated to (a) down-modulate dynein expression by siRNA-mediated knockdown, (b) inhibit dynein kinase activity, or (c) inhibit microtubule formation, TCR–MCs failed to move to the center and did not form the cSMAC. Consequently, these treatments resulted in augmented activation signals, resulting in enhanced phosphorylation of downstream signal molecules, such as SLP76, Vav and Erk, and elevated cytokine production. The finding that inhibition of cSMAC formation resulted in augmented activation indicates that the cSMAC functions as negative regulator, as previously shown similarly in CD2AP-deficient mice (46).

T cell receptor stimulation induces two events in relation to dynein-mediated translocation of TCR–MCs; one is the assembly of the TCR complex with the dynein/dynactin complex, and the other is the translocation of the MTOC (microtubule organization center, or centrosome) to the vicinity to the engagement site on the membrane at the interface. Kinetic studies revealed that MTOC translocation takes place first, followed by the translocation of TCR–MCs (45). Thus, TCR–MCs move along the microtubules, which are localized close to the plasma membrane after the MTOC moves to the site of TCR engagement. The TCR complex is assembled with the dynein complex and associates with microtubules after the MTOC and microtubules become

Thereafter, TCR–MCs are translocated to the cSMAC along the microtubules (blue line), which are translocated together with the MTOC into close proximity to the membrane in a dynein-mediated manner. The TCR/CD3 complex associates with the dynein–dynactin complex upon TCR stimulation, and then further assembles with microtubules. The dynein-mediated translocation of TCR–MCs regulates T cell activation because blockade of microtubules or dynein function prevents cSMAC formation and enhances T cell activation.

localized to the membrane. This interplay leads the movement of the TCR–MCs toward the center along microtubules in a dynein-mediated fashion, thus generating the cSMAC. Therefore, the translocation of TCR–MCs is regulated cooperatively both through F-actin retrograde flow initially, and then later by dynein-mediated movement along microtubules, ultimately leading to formation of the cSMAC (**Figure 2**).

The translocation of the MTOC to the interface of the TCR engagement site is regulated by TCR signals upon pMHC stimulation (22, 47, 48). TCR engagement upon triggering with weak stimulation induces neither MTOC translocation nor the translocation of the TCR–MCs to generate the cSMAC. Such a weak stimulatory signal, which is induced at the TCR–MCs, may not need negative regulation at the cSMAC.

### MICROSYNAPSES SUPPORT ADHESION AND SIGNALING

Ag recognition and subsequent activation of T cells requires strong contact and adhesion with APC for a certain extended time period to induce full activation. Because the affinity of the TCR–pMHC interaction is very low, Ag recognition by the TCR is supported by strong cellular adhesion through specific adhesion molecules, particularly the integrin LFA-1 binding to its ligand ICAM-1/ICAM-2. The TCR-induced activation signal and the LFA1-mediated adhesion signal are mutually regulated. The TCR signal induces a specific LFA-1 conformational change that results in high affinity binding to the ligand, a process known as "inside-out signaling" (49, 50). This inside-out signal involves the activation of SLP76, ADAP, RIAM, and Rap1/RapL. Furthermore, the high affinity configuration of LFA-1 is acquired through an LFA-1-mediated downstream signal (51, 52), known as "outside-in signaling." This outside-in signal induces activation of kinases and clustering of SLP76/ADAP (53, 54).

In the mature IS, the cSMAC as the TCR-enriched central region is surrounded by LFA-1 at the peripheral region as the pSMAC, which forms a "bulls-eye" shaped structure. During the course of IS formation, the cSMAC is generated by the translocation of peripherally induced TCR–MCs into the center of the interface. Then how is LFA-1 accumulated into the pSMAC? We found that LFA-1, as well as focal adhesion molecules represented by Pyk2, Paxillin, and vinculin, accumulate just around the TCR– MC and form a kind of "adhesion-ring" in micro scale during the very initial stage of T cell activation (55). The formation of the micro adhesion ring is dependent on LFA1, because no adhesion ring is formed in the absence of the LFA1-ICAM1 interaction on a planar bilayer lacking ICAM-1. The micro adhesion-ring is induced transiently after the initial formation of TCR–MCs, and disappears before the TCR–MCs move to the center to form a cSMAC (**Figure 3**) (55). The bulls-eye shaped structure with the central TCR–MCs surrounded by the micro adhesion-ring of LFA1, Pyk2, and Paxillin resembles the structure of the mature IS, represented by the central TCR surrounded by LFA1, therefore we suggest naming this structure the "microsynapse" (**Figure 3**). In addition to LFA1 signals, microsynapse formation is totally dependent on F-actin, since inhibitors of both F-actin and Arp2/3 block the formation of the adhesion ring, but this treatment had

Figure 3 | A microsynapse composed of a core TCR–MC and a surrounding micro adhesion-ring. (A) time course of microsynapse generation, (B) molecular assembly in the microsynapse. Immediately after TCR–MCs are formed, an adhesion-ring composed of integrin LFA-1 and focal adhesion molecules such as Paxillin and Pyk2 is generated around the TCR–MC. Because the structure resembles the mature Immunological Synapse in a micro scale, this structure was designated the microsynapse. The adhesion-ring is dependent on LFA-1 outside-in signaling and is supported by F-actin and Myosin II. Cluster formation by LAT and SLP76, but not the TCR or ZAP70, is supported by the microsynapse. The adhesion-ring is a transient structure and disappears before cSMAC formation. The microsynapse is sustained upon weak TCR stimulation, whereas it disappears quickly upon strong stimulation, suggesting that it functions to augment the TCR activation signal upon weak stimulation.

no effect on TCR–MC formation. The involvement of Myosin II as an F actin-related effector molecule in microsynapse formation was also analyzed. Treatment with a Myosin II inhibitor reduced microsynapse formation while retaining TCR–MC formation. These observations all indicate that F-actin supports the formation and function of the microsynapse. The functional importance of the microsynapse was revealed by the observation that it is maintained for a longer period when T cells are stimulated with low doses of Ag or weak stimulation, whereas it exists only transiently upon strong stimulation. This was similarly observed upon T cell stimulation with low affinity Ag peptide, such as altered peptide ligand (APL) or with T cells whose TCR had low affinity for the Ag–MHC. These data suggest that the microsynapse structure functions to enhance cellular adhesion to support TCR–MCs, which generate initial activation signals particularly upon weak stimulation. Weak interaction between the TCR and pMHC may require stronger cell adhesion mediated by the microsynapse to induce Ag recognition, followed by triggering initial activation signals. On the other hand, strong TCR engagement can induce sufficient signals for activation by itself in the relative absence of such strong cellular adhesion or co-stimulation.

The requirement for F-actin in microsynapse formation is consistent with the formation of F-actin clusters in TCR–MCs, which are localized at the center of the microsynapse. The F-actin at the TCR–MC is clearly distinct from the peripheral large actin ring and is specialized to support the microsynapse. Whereas the clustering of TCR, CD3, and ZAP70 is relatively independent of F-actin, clustering of LAT and SLP76, as well as molecules in the adhesion-ring such as LFA1, Pyk2 and paxillin, is strongly dependent on F-actin. Therefore, the components of the microsynapse induce two different types of clusters; F-actin dependent clusters (LAT, SLP76, adhesion-ring) and relatively F-actin independent clusters (TCR, ZAP70, etc.). Dependency of F-actin was found to parallel the dependency on TCR signal strength. Whereas TCR and ZAP70 cluster formation is dependent on stimulation signal strength, LAT and SLP76 clusters are relatively independent of signal strength. Such differences in the molecular dynamics of LAT and SLP76 from TCR and ZAP70 are evidence that LAT and SLP76 clustering are dependent on the microsynapse, which is supported by F-actin. Recently it was reported that the phospho-PLCγ cluster is formed in an F-actin- and WASP-dependent manner (41). Since the "actin foci" supporting phospho-PLCγ are quite similar to the F-actin cluster at the center of the microsynapse, the PLCγ cluster may also be supported by the microsynapse.

### CONCLUDING REMARKS

Initial contact of a T cell with a cognate Ag-bearing APC induces T cell activation. This critical interaction creates the IS to deliver signals into T cells. The activation unit leading to T cell activation

### REFERENCES

is the TCR–MC, which recruits downstream signaling molecules and mediates the activation signal. The TCR–MC is supported by a ring of adhesion molecules as the microsynapse. Since the cSMAC is formed mainly upon strong stimulation and under limited circumstances *in vivo*, microsynapses generated even upon weak stimulation may play more general and critical roles for early T cell activation in physiological situations. Although at present, the fine analyses described here can be achieved only by using a planar bilayer system, the technique should be extended to the analysis of cell–cell interactions *in vivo* by using *in vivo* imaging microscopy with better resolution. To get a clear picture of the signal events at the IS, first, the cooperative signaling between the TCR–MC signal and other signals such as co-stimulation, adhesion, cytokine, and innate signals should be clarified, and second, individual signaling pathways downstream of the TCR–MC, e.g., Ras-MAPK and PI3K-mTOR, should be analyzed in a spatial-temporal manner, i.e., both the timing and cellular compartmentalization of positive vs. negative signaling molecules need to be studied. The dynamics of these opposing signals may fine tune the activation signals, which controls the direction of cell fate.

### AUTHOR CONTRIBUTIONS

AH-T performed experiments, and TS and AH-T wrote the review article.

### FUNDING

Supported by Grant-in-Aid for Scientific Research (S) 24229004 for TS and (C) 10415226 for AH-T from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

**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 Hashimoto-Tane and Saito. 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.*

# Cell Type-Specific Regulation of Immunological Synapse Dynamics by B7 Ligand Recognition

*Joanna Brzostek1 \*, Nicholas R. J. Gascoigne1 and Vasily Rybakin1,2*

*1Department of Microbiology and Immunology, Yong Loo Lin School of Medicine and Immunology Programme, National University of Singapore, Singapore, Singapore, 2 Laboratory of Immunobiology, Rega Institute for Medical Research, KU Leuven, Leuven, Belgium*

B7 proteins CD80 (B7-1) and CD86 (B7-2) are expressed on most antigen-presenting cells and provide critical co-stimulatory or inhibitory input to T cells via their T-cell-expressed receptors: CD28 and CTLA-4. CD28 is expressed on effector T cells and regulatory T cells (Tregs), and CD28-dependent signals are required for optimum activation of effector T cell functions. CD28 ligation on effector T cells leads to formation of distinct molecular patterns and induction of cytoskeletal rearrangements at the immunological synapse (IS). CD28 plays a critical role in recruitment of protein kinase C (PKC)-θ to the effector T cell IS. CTLA-4 is constitutively expressed on the surface of Tregs, but it is expressed on effector T cells only after activation. As CTLA-4 binds to B7 proteins with significantly higher affinity than CD28, B7 ligand recognition by cells expressing both receptors leads to displacement of CD28 and PKC-θ from the IS. In Tregs, B7 ligand recognition leads to recruitment of CTLA-4 and PKC-η to the IS. CTLA-4 plays a role in regulation of T effector and Treg IS stability and cell motility. Due to their important roles in regulating T-cell-mediated responses, B7 receptors are emerging as important drug targets in oncology. In this review, we present an integrated summary of current knowledge about the role of B7 family receptor–ligand interactions in the regulation of spatial and temporal IS dynamics in effector and Tregs.

Keywords: Treg, PKC-theta, PKC-eta, CTLA-4, costimulation, immunological synapse, co-inhibition

# INTRODUCTION

The adaptive immune system must distinguish between self and non-self in order to provide protection from pathogenic challenges while sparing the organism's own tissues. Recognition of B7 ligands (CD80 and CD86, also known as B7-1 and B7-2, respectively) by co-stimulatory CD28 and co-inhibitory CTLA-4 (cytotoxic T-lymphocyte-associated protein 4, also known as CD152) receptors plays a critical role in regulation of effective self versus non-self discrimination. CD28 signaling is required for optimum proliferation and function of effector T cells, whereas CTLA-4 plays a critical role in negative regulation of immune responses, as it is required for turning off effector T cell signaling and regulatory T cell (Treg) development and suppressive functions. These opposing immunomodulatory roles of CTLA-4 and CD28 are of considerable clinical significance. CTLA-4 was the first immune checkpoint receptor targeted for cancer immunotherapy, and the anti-CTLA-4 antibody ipilimumab is used in the clinic for treatment of advanced melanoma (1).

### *Edited by:*

*Andres Alcover, Institut Pasteur, France*

### *Reviewed by:*

*Janis K. Burkhardt, Children's Hospital of Philadelphia, USA Bernard Malissen, Centre d'Immunologie de Marseille Luminy, France*

> *\*Correspondence: Joanna Brzostek<EMAIL_ADDRESS>
### *Specialty section:*

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

*Received: 03 November 2015 Accepted: 18 January 2016 Published: 04 February 2016*

### *Citation:*

*Brzostek J, Gascoigne NRJ and Rybakin V (2016) Cell Type-Specific Regulation of Immunological Synapse Dynamics by B7 Ligand Recognition. Front. Immunol. 7:24. doi: 10.3389/fimmu.2016.00024*

CD28 co-stimulatory function is also relevant for cancer immunotherapy, as chimeric antigen receptors (CARs) containing CD28 cytoplasmic regions have been shown to induce efficient T cell effector functions (2). However, targeting CD28 with the superagonistic monoclonal antibody TGN1412 was a tragic failure, when administration of the antibody during a phase I clinical trial induced severe systemic inflammatory responses in healthy volunteers (3). Therefore, a comprehensive understanding of expression patterns, signaling pathways, and functional roles of CD28 and CTLA-4 on effector and Treg subsets can have significant medical impact.

CD28 and CTLA-4 recognize their B7 ligands in the context of the cell-to-cell interface, termed the immunological synapse (IS), formed between a T cell and an antigen-presenting cell (APC). Receptor ligation at the IS leads to accumulation of interacting molecules at different regions of the synapse, forming distinct molecular patterns known as supramolecular activation clusters (SMAC) (4–6). The canonical mature T cell IS consists of a central SMAC (cSMAC) containing TCR (on the T cell) and pMHC (on the APC) molecules, surrounded by the peripheral SMAC (pSMAC) containing LFA-1 (on T cell) and ICAM-1 (on APC) adhesion molecules as well as F-actin. The outer ring of the IS, known as the distal SMAC (dSMAC) contains molecules with large ectodomains, such as CD45 and CD43. The SMAC regions contain smaller microdomains, known as microclusters (7). The IS is highly dynamic, with movement of TCR microclusters toward the center of the synapse, where they undergo endocytosis. Antigen recognition under physiological conditions does not always result in formation of this canonical IS structure; nevertheless this model provides a useful framework for understanding spatial dynamics of molecular interactions at the interface between T cell and APC membranes. The IS is the main site of immune receptor triggering and recruitment of signaling intermediates, leading to signal initiation and integration. B7 ligand recognition leads to distinct localization of CD28 and CTLA-4 receptors at the SMAC, modulation of cytoskeletal dynamics as well as recruitment of protein kinase C (PKC) isoforms to the IS. The effect of B7 ligand recognition on the IS dynamics is cell type specific, with effector T cells and Tregs displaying different CD28 and CTLA-4 localization, leading to differential recruitment of PKC-θ and PKC-η to the effector T cell and Treg synapses. This review presents a brief outline of the roles of CD28 and CTLA-4 in the immune system, followed by a more detailed discussion of CD28 and CTLA-4 localization patterns in the IS, and the consequences of B7 ligand recognition on IS structure and stability in T effector and Tregs.

# B7 LIGAND RECOGNITION: STRUCTURAL FEATURES AND EXPRESSION PATTERNS

B7-1 and B7-2 (CD80 and CD86) molecules share a similar structure, consisting of one membrane-distal variable domain-like and one membrane-proximal constant domain-like immunoglobulin superfamily (IgSF) domain. Purified CD80 crystallizes in a dimeric form, and undergoes spontaneous homodimerization in solution (8), whereas CD86 crystalizes as a monomer (9). The two different oligomeric states of B7 were also observed using Forster resonance energy transfer (FRET) analysis on the surface of APCs, with CD80 present on the cell surface mainly in the form of dimers, and CD86 being monomeric (10, 11). CD80 and CD86 are expressed on dendritic cells (DCs), macrophages, and B cells, with CD86 displaying higher constitutive expression and more rapid upregulation after activation. B7 molecules are also expressed on activated mouse and human effector T cells (12–14). CD80 and CD86 bind to CTLA-4 with significantly higher affinity than to CD28. CD80 is a stronger ligand, with KD 0.2 μM for CTLA-4 and 4 μM for CD28 interaction, whereas the KD for CD86 binding to CTLA-4 is 2 and 20 μM for CD28 (15).

CD28 monomers consist of a V-like IgSF extracellular domain, transmembrane regions, and a short cytoplasmic tail with no enzymatic activity. CD28 is expressed on the cell surface as a glycosylated, disulfide-linked homodimer of 44 kDa chains. In adult humans, CD28 is constitutively expressed on approximately 80% of CD4+ and 50% of CD8+ T lymphocytes. Loss of CD28 expression, most marked in the CD8 compartment, has been observed in humans during aging and autoimmune diseases (16–18). CD28 is expressed on all mouse T cells, and it is not downmodulated during aging (19). Repeated *in vitro* antigenic stimulation (20, 21) and exposure to common-γ chain cytokines or type I interferons (22) leads to downregulation of CD28 expression on human T cells. However, *in vivo* antigenic stimulation has been reported to increase CD28 surface levels on mouse T cells (23).

CTLA-4 shares structural similarity with CD28, forming homodimers of V-like IgSF monomers. CTLA-4 contains a 36-amino-acid-long cytoplasmic tail with no enzymatic activity. CTLA-4 is not expressed on the surface of resting effector T cells (24, 25), but is expressed constitutively in Tregs (26) under control of Foxp3 and NFAT (27–29). In both conventional T cells and Tregs, surface CTLA-4 is continuously endocytosed via a clathrin- and dynamin-mediated pathway, and recycled to the plasma membrane (30–34). Activation of effector and Tregs leads to upregulated levels of CTLA-4 on the cell surface. CTLA-4 internalization is mediated by the heterotrimeric adapter protein AP-2 (30, 34, 35) [regulation of CTLA-4 trafficking is the subject of an excellent recent review in Ref. (36)], whereas CTLA-4 trafficking from the trans-Golgi network to the cell surface involves formation of a multimeric complex consisting of transmembrane adapters TRIM and LAX, as well as small GTPase Rab8 (37, 38). CTLA-4 present in recycling endosomes is protected from lysosomal targeting through interaction between LRBA protein (lipopolysaccharide-responsive and beige-like anchor protein) and CTLA-4's tail region (39). Since its lysosomal degradation involves interaction with another clathrin adaptor complex AP-1 that binds to the same tyrosine-based motif (Y201) of CTLA-4 as LRBA (35) (the interaction motifs in CTLA-4 cytoplasmic region are summarized in **Figure 1**), it has been suggested that the binding of LRBA may prevent interaction with AP-1 and thereby protect the protein from degradation (39).

Both CTLA-4 and CD28 rely on the amino acid motif MYPPPY in the vicinity of Y139 in human CTLA-4 and Y123 in CD28 for binding to the B7 proteins (46–48). Importantly, despite the identical amino acid sequence of the interaction site, CTLA-4 and CD28 are capable of effectively discriminating between B7 proteins. A key study from the Allison lab (48) reported that

the binding of a B7 ligand was critical for the concentration of CTLA-4 at the IS and contributed to the concentration of CD28, and that CD86 was a preferred ligand for CD28 and CD80 for CTLA-4. Antigen-pulsed B cells expressing CD80 effectively concentrated CTLA-4 at the synapse. Furthermore, in synapses formed by B cells expressing only CD80, there was evidence for competition between CTLA-4 and CD28 for ligand binding, as CD28 accumulation was reduced even further when CTLA-4 was present at the IS. Conversely, peptide-pulsed B cells expressing only CD86 strongly increased the accumulation of CD28 at the synapse, but failed to recruit CTLA-4 (48).

### CD28 IN REGULATION OF THE IMMUNE RESPONSE

CD28 is the prototypic co-stimulatory molecule, and CD28 ligation leads to enhanced cytokine production, cell survival, and proliferation of effector T cells. The critical role of CD28 mediated signaling in optimum T cell responses is demonstrated by the T cell effector functions afforded to second-generation CARs containing cytoplasmic regions of CD28 and CD3ζ, but not by first-generation CARs lacking CD28 sequences (2). The cytoplasmic region of CD28 contains two main signaling motifs (summarized in **Figure 1**): a proximal YMNM motif and a distal proline-rich PYAP motif (49). The YMNM motif mediates phosphatidylinositol 3-kinase (PI3K) binding (50–52), leading to Akt activation; YMNM can also bind to GRB2/GADS adaptor proteins (51, 53) and the PYAP motif binds to Lck (54), filamin A, and GRB2/GADS (53, 55). The YMNM motif is followed immediately by another poly-proline motif PRRP, reported to bind the kinase Ikt (56). Analysis of knock-in mutant mice revealed that the PYAP motif is critical for IL-2 production and proliferation *in vitro*, as well as for *in vivo* antibody production and germinal center formation (57), whereas YMNM plays a role in augmenting T cell proliferation (58). Interestingly, knock-in T cells with both their YMNM and PYAP motifs mutated display less severe activation defects than CD28-deficient T cells, suggesting some functional role for the PRRP motif and/or yet unidentified cytoplasmic sequences.

CD28 is required for the thymic generation and peripheral maintenance of a functional Treg population. CD4+ Foxp3+ Tregs are key negative regulators of T cell-mediated immunity and are required for the control of spontaneous responses to self through several mechanisms (59, 60). Contact-mediated suppression relies on CTLA-4 interactions with its ligands and is discussed in detail below. Bystander suppression is mediated by suppressive cytokines, mainly IL-10 (61) and TGF-β (62) produced by activated Tregs, and by induction of cytokine starvation in target cells by IL-2 clearance (63). B7 ligand recognition plays an important role in Treg development and function, summarized in **Table 1**. In CD28-deficient NOD mice, the percentage of peripheral Tregs is strongly reduced (64). Similar reductions are observed in NOD mice lacking both CD80 and CD86, leading to the conclusion that the B7–CD28 interaction is required for the formation of the full Treg repertoire. The reduction in the percentage of Tregs in NOD mice treated with B7-blocking CTLA-4-Ig correlates with a higher incidence of spontaneous autoimmune diabetes (64). Subsequent analysis revealed that Treg deficiency in CD28<sup>−</sup>/<sup>−</sup> mice can be traced back to thymic development. The percentage of Treg precursors among thymic CD4 single-positive cells is significantly reduced in CD28<sup>−</sup>/<sup>−</sup> mice as well as in NOD mice injected with anti-CD80 and CD86 antibodies (65), and in B7 double knockout mice (66).

### Table 1 | B7 ligand recognition in Treg synapse formation and suppressive functions.

Peripheral homeostatic expansion of Tregs – but not effector T cells – in normal syngeneic hosts is also strongly suppressed by anti-CD80 and CD86 antibodies (66). A mechanistic explanation for the thymic requirement for CD28 was proposed by Tai et al. (67) who examined the consequences of CD28 deletion in a TCR-transgenic model. Mice expressing the AND TCR and its agonist ligand, a pigeon cytochrome *c* peptide, were found to effectively induce thymic Tregs only in the presence of CD28. This means that while a strong selection signal through TCR is indeed required (68), it is not sufficient for the full initiation of the agonist selection program leading to the generation of Tregs, and that a co-stimulatory signal from mTEC-expressed B7 molecules through CD28 is also required. It is noteworthy that a small proportion of regulatory phenotype T cells were still generated in the absence of CD28 but these cells lacked suppressive capacity (67). Earlier data from the same group indicated that CD28 is also required for the deletion of thymocytes by negative selection (69, 70).

A study using mice in which CD28 was selectively deleted in cells expressing Foxp3 (Cd28-ΔTreg), reported only a minor decrease in the percentage of thymic Treg precursors (71). This is in line with previous observations that CD28 is involved in the generation of early, Foxp3-negative Treg precursors (72). However, in stark contrast to earlier studies, homeostatic expansion of Tregs in the periphery was reported to occur independently of CD28 (73). Tregs from CD28-ΔTreg mice displayed reduced suppressive capacity, and consequently CD28-ΔTreg animals developed spontaneous autoimmunity (71). Costimulation through CD28 is required for *in vivo* expansion of Tregs in the presence of TCR stimulation and IL-2 (74). CD28 stimulation is also required for the conversion of naïve CD4 T cells into Tregs *in vivo* (75, 76) and *in vitro* (77).

# CTLA-4 IN REGULATION OF THE IMMUNE RESPONSE

CTLA-4 is a critical negative regulator of the immune response. Germline CTLA-4 knockout in mice results in massive lymphoproliferation (78), and is lethal at 3–4 weeks of age (78, 79). The peripheral T cell profile in these animals is strongly skewed toward CD4 cells that rapidly proliferate in a CTLA-4-Ig-sensitive manner – indicating the dependence on B7–CD28 interaction – and infiltrate non-lymphoid tissues (78, 80). Introduction of CTLA-4-sufficient Tregs reverts the lymphoproliferative disorder and prevents early lethality in CTLA-4 knockout mice (81), whereas blocking of CTLA-4 on Tregs completely abrogates their suppressive function (62, 66, 82). CTLA-4-deficient Tregs are unable to control lymphopenia-driven homeostatic expansion of conventional CD4 cells (83). Importantly, interaction between CTLA-4 and B7 expressed on effector T cells was found to be dispensable for the control of the latter in mixed bone marrow chimera experiments as both B7<sup>−</sup>/<sup>−</sup>CTLA-4<sup>−</sup>/<sup>−</sup> and B7<sup>+</sup>/<sup>+</sup>CTLA-4<sup>−</sup>/<sup>−</sup> effector T cells were efficiently suppressed by CTLA-4-sufficient Tregs (66). B7 expression is also not required on Tregs themselves (66). These data indicate that B7 expressed on a cell subset distinct from effector and Tregs mediates interactions with Treg-expressed CTLA-4 and immune suppression. CTLA-4-deficient Tregs are characterized by similar expression of CD25, PD1, GITR, and of suppressive cytokines IL-10 and IL-35 (83). Foxp3 promoter-controlled deletion of CTLA-4 in Tregs resulted in lymphoproliferative disease and tissue infiltration, and was lethal at ~7–8 weeks of age [i.e., somewhat delayed compared to germline knockout Ref. (84)]. Similarly to Foxp3-driven CD28 deficiency, thymic development of Tregs was normal, as was their survival in the periphery. However, cells lacking CTLA-4 were unable to control proliferation of target cells stimulated by anti-CD3 antibody and DC, and to induce tumor rejection (84).

Unlike CD28, CTLA-4 is not required for Treg development in the thymus. CTLA-4 is expressed by a subset of thymocytes predominantly residing at the corticomedullary junction (85) and is strongly upregulated upon induction of negative selection (86). There is no requirement for CTLA-4 expression to initiate central Treg development and peripheral expansion, as CTLA-4 knockout mice exhibit elevated percentage of Tregs and increased Ki67 expression, indicative of their active proliferation (87). Moreover, deletion of CTLA-4 in TCR-transgenic mice increases the frequency of Foxp3-positive Treg precursors in the thymus and leads to the formation of a specific population of Foxp3-positive DP thymocyte subsets in the thymic cortex (85). However, CTLA-4 can play a role in formation of the induced Treg population, as CTLA-4 has been shown to induce expression of Foxp3 and Treg conversion in the intestine (88).

# CD28 AND IMMUNOLOGICAL SYNAPSE ARCHITECTURE IN EFFECTOR T CELLS

CD28 shows a unique cSMAC localization pattern that is important for its efficient co-stimulatory functions. CD28 co-localizes with TCR microclusters at the earliest observable time-point after agonist pMHC recognition (89, 90), and the early accumulation of CD28 at the IS shows similar kinetics and localization as the TCR complex. In a mature IS, CD28 is present at the cSMAC, but segregates away from TCR (90, 91). This segregation of CD28 from TCR at the IS is required for optimum T cell activation, as shown in a study comparing different anti-CD3 and CD28 micropatterns on planar stimulatory surfaces (92). The spatial separation of TCR and CD28 at the mature IS is regulated by localization of CD28 ligands, as full length CD80 separates from TCR at the IS, but CD80 with deleted cytoplasmic region localizes with TCR (93). Moreover, the tailless CD80 molecule does not provide an optimum co-stimulatory signal and does not show efficient accumulation at APC: T cell contact interface (94, 95). This suggests a role for B7 interactions with cytoskeleton and/or other cytoplasmic components in regulation of IS architecture. CD28 recruitment and maintenance at the synapse requires both CD28 and TCR ligand binding (90, 96). CD28 accumulation at the synapse has been shown to be independent of antigenic pMHC affinity to TCR, with weak and strong agonist pMHC complexes inducing similar levels of CD28 recruitment (97). The role of CD28-mediated signaling and interactions in regulation of CD28 localization at the synapse is somewhat controversial, with a report indicating unperturbed IS localization of CD28 with mutated or deleted cytoplasmic region (90), whereas another study observed impaired IS localization of CD28 with deleted cytoplasmic domain or with a mutation at Y188 within the CD28 PYAP motif (96).

CD28 ligation has been shown to induce rapid internalization of the receptor, with half of the endocytosed fraction degraded in lysosomes and half recycled back to the cell surface (98). CD28 downregulation depends on PI3K (73), with preferential endocytosis of CD28 molecules associated with PI3K (98). CD28 is endocytosed via clathrin-coated pits, and this process requires coupling of WASP to PI3K and CD28 via sorting nexin 9 (73). CD28 downregulation from the synapse can also be influenced by stoichiometry of its B7 ligands (11). FRET analysis of B7 fluorescent protein fusions demonstrated that CD80 is present at the cell surface as a mixed population of dimers and monomers, with CD86 predominantly present in monomeric form (10). Experimental increase in CD80 dimerization resulted in enhanced T cell: APC conjugate formation and more sustained accumulation of Lck and PKC-θ at the IS (11).

Co-stimulatory signals play a critical role in regulation of cytoskeleton dynamics at the IS during T cell interaction with APC (summarized in **Table 2**). CD28 ligation induces movement of actin cytoskeleton toward the IS (99), and CD28 engagement is required for sustained actin accumulation at the IS (100). CD28 stimulation alone leads to actin polymerization and recruitment of actin at the IS (101). CD28 signaling is important in multiple pathways involved in actin filament nucleation, elongation, and depolymerization. The guanine nucleotide exchange factor Vav1 controls the activity of small Rho GTPases Cdc42 and Rac1 that regulate actin polymerization activity of WASP and WAVE2, respectively. WASP and WAVE2 are actin nucleation-promoting factors that, together with the Arp2/3 complex, facilitate formation of new actin filaments.

CD28 ligation induces tyrosine phosphorylation of Vav1 (102), and CD28-dependent actin remodeling requires Cdc42 (103) and Rac1 (104). The molecular interactions linking CD28 to Vav1 phosphorylation are not yet fully elucidated. CD28-dependent Vav1 phosphorylation has been shown to require binding of the adaptor protein GRB2 to CD28 (105, 106), but a recent report provided evidence for GRB2-independent Vav1 binding to CD28 and a role of PIP5K1A (phosphatidylinositol 4-Phosphate 5-Kinase α) and Vav1 cooperation in regulation of actin, downstream of CD28 (107). Jurkat cells expressing CD28 with mutated C-terminal PYAP motif, important for GRB2 binding, failed to recruit Vav1 to the IS or to rearrange actin after CD28 ligation (107); however, Vav1 phosphorylation in response to CD28 ligation was not assessed in this study, and in another report the PYAP motif was shown to be dispensable for CD28-dependent Vav1 phosphorylation (108). The Arp2/3 actin nucleation complex cooperates with filamins, large multidomain proteins with a role in actin crosslinking (109), to establish actin structure. Filamin A is phosphorylated (110) and recruited to the IS (108) after CD28 ligation in T cells, with the PYAP motif on the cytoplasmic region of CD28 mediating the interaction with filamin A (108, 111). Knockdown of filamin A expression did not affect CD28 mediated Vav1 phosphorylation, but reduced Cdc42 activity and impaired CD28-mediated costimulation (108). However, changes

in actin structure or dynamics at the IS of filamin A knockdown cells have not been reported. Moreover, knocking down filamin A did not impair ezrin accumulation at the IS (108).

Actin filaments contain a fast growing barbed end, which can be bound to actin capping protein. Capping protein binding to the barbed end prevents addition of new actin subunits, limiting the filament elongation. Actin capping and subsequent actin polymerization can be regulated by actin-uncapping proteins (112). An actin-uncapping protein Rltpr is required for CD28 dependent costimulation (113), and Rltpr colocalizes with CD28 in CD80-dependent signaling microclusters (113), suggesting a role of Rltpr in CD28-mediated actin rearrangement at the synapse. However, a direct role of Rltpr for CD28-dependent actin modification is unclear. Rltpr does not immunoprecipitate with CD28 (113) and is not phosphorylated after CD28 ligation (110). Moreover, an Rltpr mutation that abolishes CD28-mediated costimulation does not impair Rltpr's actin-uncapping ability or CD28-dependent actin rearrangements at the synapse (113). The Rltpr mutation that reduced CD28-dependent costimulation abrogates CD28-dependent recruitment of PKC-θ and Carma1 to the IS (113) through a yet unidentified molecular mechanism, suggesting that Rltpr acts as an adaptor at the IS independently of its actin-uncapping functions. A recent phosphoproteomic screen identified actin-uncapping CapZIP as part CD28 signaling network (110). Importantly, CapZIP is required for CD28 dependent costimulation of cytokine production (110). However, it has not yet been reported if CapZIP can directly interact with CD28 and if it is required for CD28-dependent changes in actin dynamics. In summary, the current evidence suggests that CD28-dependent signaling may regulate actin capping through actin-uncapping proteins CapZIP and potentially Rltpr.

CD28 signaling regulates actin dynamics through control of activity of the actin-severing protein cofilin. Cofilin is a ubiquitously expressed 19 kDa protein that cleaves actin filaments, thus, promoting actin depolymerization, but also creating new barbed ends for filament elongation (114). Cofilin's actin binding capacity is negatively regulated by its phosphorylation at serine 3 (115, 116), and binding to phospholipids (117). Blocking cofilin interaction with actin reduces T cell proliferation and cytokine production, as well as conjugation with APCs (118). In resting human T cells, cofilin is present mainly in the inactive phosphorylated form, and CD28 or CD2 signal together with TCR, but not TCR signal alone, induces cofilin dephosphorylation and actin binding (119, 120). The precise sequence of signaling events linking CD28 ligation to cofilin activation is unknown. Cofilin is dephosphorylated by serine phosphatases PP1 and PP2A (121), and CD3/CD28-induced cofilin dephosphorylation requires Ras (120). Additionally, CD28 may regulate cofilin activity through control of levels of membrane phospholipids (114).

There is strong evidence that CD28-dependent regulation of actin dynamics is important for the effector T cell functions. CD28 enhances T cell:APC conjugate formation *in vitro* (122, 123). Knock-in mice with mutated PYAP motif show reduced IL-2 production and proliferation *in vitro*, and impaired *in vivo* antibody production and germinal center formation (57). This could be a result of impaired cytoskeletal rearrangement, as the PYAP motif is implicated in Vav1 and filamin A recruitment. However, the effects of PYAP mutations on cytoskeletal dynamics and synapse stability have not yet been reported for primary T cells, and this motif is also important for binding to Lck (54), GRB2/GADS (53, 55), and PKC-θ (124), as discussed below. Analysis of a mouse mutant with inducible inhibition of Csk, a negative regulator of Src family kinases, strongly suggested that CD28-dependent actin remodeling is critical for initiation of full TCR signal in thymocytes (125). However, thymocytes from PYAP mutant knock-in mice do not show obvious phenotypic defects (57), suggesting that CD28-independent pathways can regulate actin cytoskeleton dynamics during thymocyte development.

### CD28 AND REGULATION OF PKC-**θ** LOCALIZATION AT THE EFFECTOR T CELL IS

CD28 plays a critical role in regulation of the IS localization of the novel protein kinase C (nPKC) isoform PKC-θ (summarized in **Table 3**). The PKC family consists of 10 serine/threonine kinase isoforms, with important roles in regulation of multiple cellular processes in different cell types. All nPKC isoforms (PKC-θ, PKC-δ, PKC-ϵ, and PKC-η) require diacylglycerol (DAG), but

not Ca2<sup>+</sup>, for activation, and are expressed in T cells and play multiple roles in regulation of T cell signaling and effector functions (126). Central localization of PKC-θ is one of the hallmarks of the mature effector T cell IS. A seminal study by Monks at al. identified PKC-θ as the only PKC isoform recruited to effector T cell IS (127). However, more recent studies show that PKC-η and PKC-ϵ are also recruited (128–130), with some evidence that their recruitment precedes that of PKC-θ (129). PKC-ϵ and PKC-η display homogeneous distribution over the entire synapse, whereas PKC-θ displays discrete cSMAC localization contained within the peripheral actin ring (128–132).

An important study using lipid bilayers presenting antigen and co-stimulatory signal, and TIRF microscopy to examine PKC-θ localization at the effector T cell IS, revealed initial colocalization of PKC-θ with TCR/CD28 microclusters (90). This was followed by PKC-θ recruitment to the cSMAC, where it segregated, together with CD28, to TCRlow regions in the periphery of cSMAC (90). The initial stages of PKC-θ recruitment to the effector T cell IS do not depend on CD28 ligand binding, but CD28 ligation is required for sustained PKC-θ localization at the synapse and colocalization of PKC-θ with CD28 (90, 133). PKC-θ interacts with CD28 after PMA treatment (90) (which induces PKC activation) and TCR/CD28 stimulation (124).

The molecular determinants of PKC-θ synapse localization have been mapped to the V3 hinge region and C1 domains (132). nPKCs share a conserved structure, with an amino-terminal C2 domain, tandem C1 domains, and V3 hinge linked to a carboxyl-terminal kinase domain. PKC-θ interaction with CD28 and cSMAC localization requires a polyproline motif within the V3 hinge region (124), and V3 hinge regions from PKC-ϵ and PKC-η mediate their diffuse accumulation at the synapse (134). A carboxyl-terminal poly-proline motif (PYAP) in the CD28 cytoplasmic tail is required for its interaction with PKC-θ, with strong evidence suggesting that this is an indirect interaction mediated through Lck, with the Lck SH3 domain binding to the polyproline motif in PKC-θ V3 and the Lck SH2 domain binding a phosphorylated tyrosine within the CD28 PYAP motif (124). Tyrosine 188 within the PYAP motif was also identified as critical for CD28 and PKC-θ central synapse localization in an earlier study (96).

Additionally, C1 domains of PKC-θ also play a role in its synapse localization, through interaction with DAG at the synapse membrane (134, 135). C1 domains can mediate initial PKC-θ recruitment to the synaptic membrane (135), but they do not support PKC-θ central accumulation and retention and the membrane (136), and nPKC C1 domains are not sufficient to determine the respective synapse localizations of PKC-θ versus PKC-ϵ and PKC-η (134). Phosphorylation of PKC-θ threonine 219 (T219), in a hinge region between the tandem C1 domains, is required for PKC-θ localization at the IS (137). Moreover, sustained synapse localization is dependent on PKCθ kinase activity (137, 138), most likely through a requirement for PKC-θ autophosphorylation at T219 (132, 137). PKC-θ recruitment to the IS also requires expression of wild-type Rltpr actin-uncapping protein (113). T cells from mice expressing an Rltpr mutant could not recruit PKC-θ to the IS (113). The precise role of Rltpr in the regulation of PKC-θ synapse localization is unknown but seems to be independent of Rltpr actin-uncapping function, and no direct interactions between Rltpr and PKC-θ have been observed (113).

A recent report identified a novel activation-dependent posttranslational modification of PKC-θ that modulates CD28–PKCθ interactions and IS architecture (139). TCR stimulation of resting murine and human T cells leads to conjugation of SUMO1 (small ubiquitin-like modifier) to PKC-θ lysine (K) 325 and K506 by SUMO E3 ligase PIASxβ (139). Importantly, TCR and CD28 costimulation resulted in stronger PKC-θ sumoylation than TCR stimulation alone. Sumoylation-resistant PKC-θ with mutated K325 and K506 residues showed reduced interaction with CD28 and filamin A, and diffuse localization at the membrane in the IS (139). Inhibiting PKC-θ sumoylation through PIASxβ knockdown or overexpression of a desumoylating enzyme also abrogated PKC-θ localization at the IS, and reduced its colocalization with CD28 (139). Wild-type PKC-θ segregated from filamin A at the IS, with mainly pSMAC localization of filamin A. Inhibition of PKC-θ sumoylation altered the IS architecture, inducing colocalization of PKC-θ and filamin A at the periphery of the synapse (139).

The localization of PKC-θ to the center of the IS is critical for its functions in effector T cells. Mutations of the poly-proline motif in the V3 region of PKC-θ reduced activation of primary effector CD4+ T cells (124). Critically, overexpression of murine V3 domain sequesters PKC-θ from CD28 and cSMAC in mouse CD4+ T cells, and reduces PKC-θ-dependent gene expression *in vitro*, as well as CD4+ Th2 and Th17 immune responses *in vivo* (124). Similarly, expression of sumoylation-resistant PKC-θ mutants, with impaired synapse localization, does not rescue defects in cytokine production, activation of PKC-θ dependent transcription factors, and Th2 differentiation of human T cells with downregulated expression levels of endogenous PKC-θ (139). Additionally, mutations in the CD28 PYAP motif, required for PKC-θ interaction with CD28 and for IS localization, severely impaired effector T cell functions *in vivo* (57). However, it must be noted that PKC-θ synapse localization seems to be inseparable from its interaction with CD28, and the observed functional effects of impaired PKC-θ synapse recruitment could also be caused by reduced CD28 interactions with PKC-θ, Lck, and/or filamin A.

# CTLA-4 DYNAMICS AT THE EFFECTOR AND Treg IS

Recognition of B7 ligands by CD28 and CTLA-4 at the effector T cell IS leads to competitive displacement of CD28 and PKC-θ from its central region. In the absence of stimulation, regulatory and conventional T cells express similar levels of CD28, but CTLA-4 expression is significantly higher in unstimulated Tregs (71, 140, 141). TCR signaling induces polarization of both intracellular (142) and membrane pools of CTLA-4 toward the IS of effector T cells, and TCR signal strength determines CTLA-4 localization at the IS (97). CTLA-4 is recruited to the effector T cell cSMAC with delayed kinetics relative to that of TCR and CD28, segregates away from CD3high regions and forms a ring-like structure (141). CTLA-4 recruitment to and stabilization at the IS depends on its ligand binding, but occurs at both high and low B7 ligand densities (141). Critically, recruitment of CTLA-4 to the IS influences CD28 localization, due to competition for ligand binding. At high ligand densities, CTLA-4 recruitment leads to exclusion of CD28 from the cSMAC and its accumulation outside the pSMAC (141). At low ligand densities, CTLA-4 prevents formation of CD28 clusters at the T effector IS (141). Importantly, CTLA-4-mediated displacement of CD28 from the cSMAC leads to impaired synaptic localization of PKC-θ (141). CTLA-4 ligation has also been reported to reduce the size of T cell: APC contact interface and to reduce ZAP70 microcluster formation (143).

The localization of CTLA-4 in the T effector synapse depends on the molecular dimensions of the extracellular region of the protein, as CTLA-4 molecules with elongated ectodomains failed to accumulate at cSMAC despite unimpaired ligand binding (141). However, it has not been reported if CTLA-4 with elongated ectodomains affected CD28 clustering at the synapse, and it is not known whether similar dimensions of CD28 and CTLA-4 receptor–ligand complexes are important for efficient regulation of co-stimulatory signal and/or competition for ligand binding at the synapse. The matching sizes of activating and inhibitory receptor–ligand complexes are critical for signal integration and regulation of NK-cell functions (144, 145), and it is plausible that a similar requirement exists for co-stimulatory and co-inhibitory signaling in effector T cells.

A molecular mechanism for CTLA-4 involvement in the downregulation of CD80/CD86 has been established in the seminal work by Qureshi et al. (146). Using co-cultured CHO cells expressing either human CTLA-4 or GFP-tagged human CD86, they observed transfer of GFP signal into CTLA-4 expressing cells, and its accumulation in the endolysosomal system, indicative of CD86 trans-endocytosis. Endocytosis-deficient CTLA-4 failed to induce trans-endocytosis of CD86-GFP and resulted in the accumulation of CD86 at cell contacts. These findings were confirmed using purified human Tregs incubated with DC where CD86 expression on the surface of the DC was reduced in the presence of Tregs but not effector T cells, and TCR stimulation increased the rate of trans-endocytosis (146). The most direct consequence of reduction of B7 proteins on the surface of APC is manifested in fewer and less prolonged interactions between APC and effector T cells (147, 148) reduced PKC-θ recruitment and activation in these cells (149) and, consequently, repression of IL-2 production by effector T cells (150). Recently, it has been shown that surface expression of CTLA-4 on effector T cells is sufficient for downregulation of CD86 expression from APCs (40).

Tregs display radically different synapse localization of CD28 and PKC-θ than effector T cells (summarized in **Figure 2**). In a stimulating planar lipid bilayer system, the recruitment of CD28 to the Treg IS is barely detectable, whereas CTLA-4 is recruited robustly, in stark contrast to conventional CD4 T cells (141). Displacement of CD28 from Treg synapses by CTLA-4 coincides with the absence of PKC-θ clusters in the cSMAC zone of Treg synapses. Similarly, the switch of developmental program during the *in vitro* conversion of naïve CD4 T cells into Tregs results

in a loss of PKC-θ signal at the synapse. Correct localization of CTLA-4 to the IS is functionally important, as elongation of the extracellular domain of CTLA-4 resulted in a loss of its concentration in the synapse and reduction of suppressive activity of Tregs (141). In a lipid bilayer system, addition of CD80 or ICAM-1 to the bilayer increases the recruitment of PKC-θ to the synapse in both effector and Tregs, but stimulation through TCR strongly decreases the recruitment in Tregs (151). Reduction of PKC-θ activity results in increased Treg proliferation and elevated suppressive capacity (151).