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Bernard Malissen1–6, Claude Grégoire1–3, Marie Malissen1–6 & Romain Roncagalli1–3 The activation of T cells mediated by the T cell antigen receptor (TCR) requires the interaction of dozens of proteins, and its malfunction has pathological consequences. Our major focus is on new developments in the systems-level understanding of the TCR signal-transduction network. To make sense of the formidable complexity of this network, we argue that ‘fine-grained’ methods are needed to assess the relationships among a few components that interact on a nanometric scale, and those should be integrated with high-throughput ‘-omic’ approaches that simultaneously capture large numbers of parameters. We illustrate the utility of this integrative approach with the transmembrane signaling protein Lat, which is a key signaling hub of the TCR signaltransduction network, as a connecting thread. T cell antigen receptors (TCRs) detect antigens on the surface of antigen-presenting cells (APCs) in the form of complexes of antigenic peptides bound to major histocompatibility complex (MHC) molecules. The binding of such peptide-MHC complexes to the TCR induces a cascade of events that propagate through the cytosol and the nucleus via a multilayered signal-transduction network. These events involve protein-protein and protein-lipid interactions, reversible modifications of proteins (mainly phosphorylation and ubiquitination) and the generation of second messengers such as calcium and diacylglycerol. The presence of a few foreign peptide–MHC ligands or, more probably, a single such ligand in a T cell–APC contact surface suffices to induce sustained calcium influx and cytokine production1–3. Moreover, TCR-induced signals are transmitted in a rapid manner, which results in the phosphorylation of TCR-proximal components within 4 seconds, the production of intracellular second messengers such as diacylglycerol and calcium within 6–7 seconds4,5 and submicrometric cytoskeletal reorganization within 10 seconds that leads to microvillus retraction6. Most receptors encountered in biology are encoded in the germline, and each is thus committed to bind a predetermined physiological ligand with invariant parameters. Due to the stochastic nature of the DNA recombination that directs the assembly of gene segments that encode the clonally variable domains of the TCR, a given TCR has no predetermined specificity and is able to interact with different affinities with a small spectrum of 1Centre

d’Immunologie de Marseille-Luminy, UM2 Aix-Marseille Université, Marseille, France. 2INSERM U1104, Marseille, France. 3CNRS UMR7280, Marseille, France. 4Centre d’Immunophénomique, UM2 Aix-Marseille Université, Marseille, France. 5INSERM US012, Marseille, France. 6CNRS UMS3367, Marseille, France. Correspondence should be addressed to B.M. ([email protected]). Received 18 May; accepted 10 July; published online 19 August 2014; doi:10.1038/ni.2959

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peptide-MHC ligands, a property called ‘cross-reactivity’. The phase that is known as ‘positive TCR selection’ and occurs in the thymus between recently assembled TCRs and self peptide–MHC complexes favors the development of mature T cells that express TCRs able to interact weakly with self peptide–MHC7. Therefore, the TCR signaltransduction network that operates in mature T cells needs to be highly discriminatory, since chronic, weak interactions with self peptide–MHC trigger ‘subliminal’ signals8,9 that maintain T cells in a state of heightened antigenic reactivity10, whereas slightly stronger interactions with foreign peptide–MHC lead to their full-fledged activation. A major challenge for T cell biology is thus to identify the molecules and molecular circuits in the TCR signal-transduction network that carry out antigen recognition with sensitivity, rapidity and specificity and that ‘translate’ differences in the quality and quantity of the earliest signaling events, including the seminal TCR-peptideMHC interaction, into distinct long-term functional outcomes that lead to effector and memory T cells or to tolerant T cells. It has been argued that every component of the TCR signaling machinery has been identified and mapped in terms of its place in the TCR signal-transduction network11. However, key components continue to be discovered, and rather than the tens of components previously thought to compose this network, studies now suggest the involvement of hundreds of such components. Therefore, delineating the structure of the TCR signal-transduction network will require the identification of all its components, the characterization of their respective roles, the determination of the physical interactions between them, and their organization into an integrated and dynamic ‘map’. Using as an example the scaffolding molecule Lat (‘linker for the activation of T cells’), a key signaling hub of the TCR signaltransduction network, we illustrate in this Perspective the need to integrate ‘fine-grained’ experimental methods that assess the relationships among a few interacting components of the network with high-throughput ‘-omic’ approaches that simultaneously measure large numbers of parameters. Early TCR signals The TCR consists of one α-chain and one β-chain that together directly recognize peptide-MHC ligands, and it is associated with CD3 subunits that contain one or several immunoreceptor tyrosine-based activation motifs (Fig. 1a). The coreceptors CD4 and CD8 assist the TCR in the recognition of peptide–MHC class II and peptide–MHC class I, respectively, and the cytoplasmic segment of each is associated with the tyrosine kinase Lck. A single peptide-MHC complex can simultaneously bind to a TCR and a coreceptor, trigger their juxtaposition and permit phosphorylation of the CD3 immunoreceptor tyrosine-based activation motifs by the coreceptor-associated Lck. This leads to recruitment and activation of the cytosolic VOLUME 15  NUMBER 9  SEPTEMBER 2014  nature immunology

Figure 1  Overview of the early TCR signaltransduction network. (a) The proximal TCR signal-transduction apparatus can be broken down into an antigen-recognition and triggering module made of the TCR-CD3 complex and Zap70, and a priming module made of CD4 and CD8 and their associated tyrosine kinase Lck81,82. A single peptide-MHC complex (pMHC) can simultaneously bind to a TCR and a coreceptor, which permits phosphorylation (red dots) of the CD3 subunits by Lck. This leads to the recruitment and activation of Zap70, which phosphorylates tyrosine residues present in the intracytoplasmic segments of Lat and CD6, both of which constitute signal-amplification and diversification modules. In response to signaling via the TCR, T cells quickly become responsive to cytokines such as IL-12 and IL-4, which are delivered by APCs and activate key lineage-specific transcription factors that control differentiation into various effector types. (b) Network of proteins that interact with Lat (the Lat ‘interactome’), isolated by AP-MS from CD4+ cells before and after TCR triggering14; proteins are identified by designations of the UniProt database and are classified according to function (left margin) as transmembrane receptor, tyrosine or serinethreonine protein kinase (Kinase), phospholipid or tyrosine phosphatase (Phosphatase), guanine nucleotide–exchange factor (GEF), GTPase-activating protein (GAP), ion channel, transporter or ubiquitin ligase (E3 ligase).

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tyrosine kinase Zap70 that in turn phos­ Ion channel phorylates Lat12. The intracellular domain of Lat KPNB1 TAP2 NSUN2 COX4l1 GVIN1 RPN2 NCAPD2 SF3B4 comprises approximately 200 residues and Transporter has intrinsically disordered characteristics13. CSE1L SLC25A12 MCM5 DDOST GPX1 DYN2 ECM29 PRPF40A This domain constitutes a kind of protein E3 ligase ‘fishing line’ that, upon phosphorylation of LRPPRC TMEM173 DNAJA1 TLN1 PSIP1 WIPF1 Adaptor its nine tyrosine residues by Zap70, is able p85A Itk to nucleate the assembly of a multiprotein PDCD4 ZC3HAV1 YWHAQ HSP90B1 THEMIS AT1B3 Phospholipase complex called the ‘Lat signalosome’ that involves the adaptor SLP-76 (LCP2). The MAP4K1 Fyn Unspecified CAND1 TARDBP Eif2s2 STAT1 STAT3 function Lat signalosome constitutes a device for the amplification and diversification of signals that is responsible for most of the responses that result from engage- antigen-induced proliferation of naive T cells19. Although several ment of the TCR14. For example, it couples the TCR to a network of additional components of the CD28 costimulatory pathway have been serine-threonine kinases that deliver signals to the nucleus and also identified20–22, it remains to be determined whether CD28 signals contribute, together with cytokines, to fulfillment of the stringent are qualitatively different from those induced by the TCR or amplify metabolic demands of activated T cells15. CD6, which is expressed the latter in a quantitative manner2,23–25. Various surface receptors on the surface of T cells, is also phosphorylated by Zap70 regardless in addition to CD28 have been described as having costimulatory of the presence of Lat14. CD6 nucleates the assembly of a signalosome functions26. Therapeutic antibodies to the coinhibitors CTLA-4 and that also involves SLP-76 and probably accounts for the many TCR- PD-1 present at the T cell surface have become standard treatment induced tyrosine-phosphorylated species that remain in the absence for metastatic melanoma and have led to a revival of the study of of Lat14,16–18. T cell coinhibitors27. In response to signaling via the TCR and CD28, Although T cell activation can result from signaling via the TCR naive T cells become responsive to cytokines (such as interleukin 12 alone, physiological T cell responses are ‘tuned’ by signals that result (IL-12) and IL-4) produced and delivered by APCs and activate key from the engagement of several other surface receptors that convey lineage-specific transcription factors that control their differentiation positive (costimulatory) or negative (coinhibitory) information about (‘polarization’) into various effector types. However, this ‘cytokinethe nature and state of activation of APCs. For example, after binding only’ instructive model needs to be modified, given published data to its ligands (CD80 or CD86) on APCs, the costimulator CD28 inter- showing that quantitative factors, especially the strength of antigen acts with the serine-threonine kinase PKC-θ and the adaptor CARD11 stimulation through the TCR, make important contributions to the to activate the transcription factor NF-κB and thereby sustains the T cell–differentiation ‘choice’28. Katie Vicari/Nature Publishing Group

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p e r s p e c ti v e Positive regulatory plug-ins Figure 2  Positive regulatory plug-ins of the Lat Lat interactome. Most Lat signals are funneled through phosphorylation (red dots) of the four DOCK2 Itk carboxy-terminal tyrosine residues at positions 136 136, 175, 195 and 235 in the mouse. PLC-γ1 SLP-76 p85A A palmitoylated juxtamembrane amino acid 175 GRAP2 motif targets Lat to lipid rafts (dashed arrows). Vav1 195 DYN2 The Lat interactome (Fig. 1b) can be tentatively organized into several positive regulatory Grb2 235 plug-ins. One consists of SLP-76, PLC-γ1 and Sos1 GRAP2 (Gads). Once bound to phosphorylated Lat molecules via GRAP2, SLP-76 is Activation of Erk and NFAT Activation of Erk Activation of Akt, Itk, phosphorylated by Zap70 and recruits the Increase in LFA-1 adhesiveness PLC-γ1 and Vav1 Tec-family tyrosine kinase Itk. In turn, Itk Actin polymerization Actin polymerization phosphorylates PLC-γ1 and enhances its enzymatic activity; this leads to hydrolysis of phosphatidylinositol-(4,5)-bisphosphate and to the production of diacylglycerol and inositol-(1,4,5)trisphosphate, which induces the entry of Ca2+ from outside the T cell that then stimulates the translocation of NFAT transcription factors to the nucleus. In parallel, diacylglycerol in the plasma membrane recruits RasGRP1, a guanine nucleotide-exchange factor (GEF), which acts on Ras to activate Erk1 and Erk2. Lat-bound SLP-76 also interacts with Nck and with Vav1 to promote reorganization of the actin cytoskeleton 83 and with FYB to increase the binding of the integrin CD58 (LFA-1) to its ligand ICAM-1 (ref. 84). Cooperative interactions stabilize the Lat–GRAP2–SLP-76–PLC-γ1–Itk plug-in, and loss of any one component reduces interactions among the others 85. DYN2, a member of the dynamin superfamily of large GTPases, is also recruited by phosphorylated Lat molecules14 and participates in the generation of filamentous actin 86. Sos1 binds to phosphorylated Lat via Grb2. However, full activation of Sos1 is contingent on prior activation by RasGRP1; the latter provides GTP-bound active Ras molecules that enhance Sos1 activity in an allosteric manner87. Phosphorylated Lat binds p85A, an SH2 domain–containing regulatory subunit of class IA phosphatidylinositol-3-OH kinase14, which results in the production of phosphatidylinositol-3-4,5-trisphosphate, a messenger that stabilizes recruitment to the plasma membrane of pleckstrin-homology domain–containing proteins such as PLC-γ1, Itk, Vav1, the serine-threonine kinase Akt, and the GEF DOCK2. By activating GTPases of the Rac family, DOCK2 triggers the growth and maintenance of the ring of filamentous actin present in the periphery of the IS 88.

The Lat signaling hub Affinity purification coupled with mass spectrometry (AP-MS) allows highly sensitive analysis of the dynamics of noncovalent protein complexes29,30. Early attempts to delineate the TCR signal­transduction network by AP-MS relied on the analysis of transformed T cell lines31,32. However, such cell lines lack key signaling proteins33, a feature that may preclude generalization of the conclusions of those studies to normal T cells. Mice have been developed that bear a genetic tag that permits AP-MS analysis of protein complexes containing Zap70, Lat and SLP-76 isolated from primary CD4+ T cells. A study that used this approach to monitor four time points covering 300 seconds following T cell activation has identified a Zap70–Lat–SLP-76 signaling network consisting of 90 signaling proteins linked via 112 interactions14. Proteins that have never before been observed in the context of TCR signaling were identified; these encompass transcription regulators, transporters, a central adaptor for sensing cytosolic DNA (TMEM173 (STING)), a guanine nucleotide–exchange factor for the Rho family of GTPases (ARHGEF6) and a putative GTPase-­activating protein (RASAL3). Additionally, several proteins of unknown functional relevance were found to be constitutively associated to Lat. As illustrated in the case of the Lat ‘bait’, protein ‘interactomics’ (the study of protein-protein interaction networks) can be presented as a diagram in which proteins are represented as nodes and the inter­action between them are represented as lines (Fig. 1b). This leaves much room for interpretation, since it is impossible to know on that sole basis whether the lines correspond to direct interactions or to indirect interactions. The underlying set of binary interactions can be defined through genetic or pharmacological ablation of individual proteins (nodes) or interactions (lines), a difficult exercise when cooperative interactions stabilize the macromolecular complexes under study. Causal relationships in the TCR signal-transduction network can be inferred from analysis of the kinetics of assembly-disassembly of the complexes after TCR triggering and of the kinetics of phosphorylation events that propagate in the network after TCR triggering14,17,34–36. On the basis of available structural and biochemical data, the whole set of partners that interact with Lat (the ‘Lat interactome’) can be broken 792

down into subnetworks whereby each constitutes a ‘plug-in’ that adds a specific function to the network. Congruent with its indispensable role in the development37 and function14 of T cells, Lat nucleates a large array of positive regulatory plug-ins (Fig. 2). However, Lat appears to be more than just a positive regulator of TCR signaling, in that it recruits several negative regulatory plug-ins (Fig. 3) that, as described below, help to terminate TCR-driven responses. Lat signaling pathology Malfunction of the negative regulatory plug-ins associated with Lat probably accounts for the paradoxical phenotype of mice with replacement of the tyrosine at position 136 of Lat with phenylalanine (LatY136F mice). Those mice have a severe but incomplete block in intrathymic αβ T cell development whereby only a few CD4+ or CD8+ T cells succeed in reaching the periphery. In the absence of any deliberate immunization and within 2 weeks of life, the peripheral CD4+ T cells embark on sustained proliferation and coincidently differentiate into T helper type 2 effector cells that trigger massive formation of germinal centers and the production of immunoglobulins IgG1 and IgE, which results in hypergammaglobulinemia38,39. Consequently, LatY136F mice develop autoantibodies and die of severe lupus nephritis at 4–6 months of age. Faulty thymic T cell maturation is not mandatory for development of the LatY136F pathology, since limiting expression of the mutant Lat with the Y136F substitution to the periphery or depriving mature peripheral CD4 + T cells of Lat itself leads to similar pathology16,40. Although the mutation that encodes the Y136F substitution prevents proper function of regulatory T cells41, the LatY136F pathology differs from that of mice deprived of regulatory T cells in that it results mainly from a defect intrinsic to conventional (Foxp3−) CD4+ T cells42. LatY136F T cells have low TCR expression and show ‘autistic’ responses to TCR signals. Thus, these CD4+ T cells can proliferate in hosts deprived of MHC class II molecules and can activate B cells whether or not MHC class II molecules are present43. Therefore, although the onset of the LatY136F pathology necessitates engagement of the TCR, its perpetuation involves non-TCR signals that are driven by CD28 (refs. 22,44). VOLUME 15  NUMBER 9  SEPTEMBER 2014  nature immunology

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p e r s p e c ti v e Negative regulatory plug-ins Figure 3  Negative regulatory plug-ins present in the Lat interactome. The serine-threonine kinase MAP4K1 (HPK1) is also part of the Lat interactome. It phosphorylates THEMIS the GRAP2–SLP-76 complex and induces its interaction with members of the 14-3-3 SLP-76 Cbl GRB2 GRB2 family of adaptors, which leads to its GRAP2 dissociation from Lat74,89,90. UBASH3A UBASH3A Dok1 constitutes another negative regulator SHIP-1 Cbl-b recruited by phosphorylated Lat molecules. Dok2 14-3-3T It is composed of a ubiquitin-association MAP4K1 domain that binds mono- and polyubiquitin, a central SH3 domain, and a carboxy-terminal Dismantles the Curtails early TCR signals Dampens activity of domain with weak tyrosine-phosphatase SLP-76–GRAP2–based PLC-γ1, Akt, Itk and Vav1 activity. The negative regulatory role of signaling complex UBASH3A is probably due to its ability to dephosphorylate Zap70 (ref. 91). The E3 ubiquitin ligases Cbl and Cbl-b are also part of the Lat interactome and are involved in the termination of TCR signaling92. Another addition to the list of Lat negative plug-ins is THEMIS1 (refs. 93–95), a protein that lacks any discernible catalytic activity and interacts constitutively with Grb2 and the tyrosine phosphatases PTPN6 (SHP-1) and PTPN11 (SHP-2). Upon TCR triggering, the THEMIS1nucleated complex associates with Lat via Grb2 (refs. 14,94) and may curtails TCR signaling from its inception by dephosphorylating substrates such as Lck, Lat or PLC-γ1 (ref. 93). The inositol polyphosphate-5-phosphatase SHIP-1 acts together with Grb2 to bind to phosphorylated Lat molecules. The carboxyl terminus of SHIP-1 contains two tyrosine-based motifs that are phosphorylated following TCR triggering and bind the adaptors Dok1 and Dok2, which leads to the recruitment of additional effectors such as CSK 96–98. By diminishing the amount of phosphatidylinositol-(3,4,5)trisphosphate, SHIP-1 probably dampens the activity of PLC-γ1, Akt, Itk and Vav1, whereas CSK blunts Lck activity.

Additional mutations of the gene encoding Lat have revealed that γδ T cells and CD8+ T cells can also give rise to LatY136F-like pathologies45,46. Therefore, when TCR triggering occurs in the presence of a defective Lat signalosome, T cell activation goes awry in γδ T cells, CD4+ T cells and CD8+ T cells and results in a fatal immune disorder called ‘Lat signaling pathology’47. The Lat signalosome might contribute to the termination of antigen-driven T cell responses by curtailing the activity of Lck and Zap70, and in the presence of defective Lat signalosomes, Lck and Zap70 may remain in a hyperactive state that chronically primes the CD28 signaling pathway and thereby contributes to the etiology of Lat signaling pathology16,17,22,48,49. Interestingly, the Lat mutant with the Y136F substitution is just one of several examples of such mutants that, although predicted to render T cells inert to TCR-mediated activation, result in the generation of hyperactive pathogenic T cells. For example, mutations in genes encoding CD3 immunoreceptor tyrosine-based activation motifs, Zap70, Stim1 and Stim2, NFATc2 and NFATc3, THEMIS1 and the tyrosine phosphatase PTPN22 are also associated with autoimmunity and inflammation50–55. Whether those pathological conditions result from failed central tolerance or, as demonstrated for Lat signaling pathology, result from loss of negative feedback controls on the TCRmediated activation of naive T cells remains to be determined.

possible binding of up to three Grb2 molecules by a single, multiphosphorylated Lat molecule and from the presence on the guanine nucleotide-exchange factor Sos1 of several docking sites for the SH3 domains of Grb2. A single Sos1 molecule can thus simultaneously associate with two different Grb2 molecules56,57 and act as a scaffold able to oligomerize Lat complex isoforms57 (Fig. 4a). Notably, Lat nanoclusters seem to be functionally relevant, since Sos1-induced Lat oligomerization results in optimal phosphorylation of Lat and the signaling protein PLC-γ1, enhanced Ca2+ signaling and proper development of T cells58.

Lat-based nanoclusters Lat molecules can differ in their extent of tyrosine phosphorylation, and distinct Src homology 2 (SH2) domain–containing interactors have been shown to compete for the same phosphorylated tyrosine residue of Lat. For example, the adaptors Grb2 and GRAP2 are able to bind to the same phosphorylated tyrosine of Lat. Therefore, multiple isoforms of the Lat complex probably coexist, each endowed with a specific function (Figs. 2 and 3). According to this view, the repertoire of Lat-interacting proteins identified by AP-MS corresponds to the sum of the components found in the distinct isoforms of the Lat complex, and the physical separation of these isoforms before MS analysis should permit identification of their respective composition. However, this may constitute a futile exercise, since these isoforms might not exist in isolation and instead might oligomerize into molecular assemblies of a higher order and nanometers in size, called ‘Lat nanoclusters’ here. Such oligomerization results from the

Super-resolution microscopy of Lat clusters High-resolution dual-color photoactivation localization microscopy (PALM) imaging has been used to visualize the Lat nanoclusters postulated to exist on the basis of structural and biochemical approaches. Attempts have been also made to relate them to the Lat-containing signaling microclusters that are approximately 200–500 nm in diameter and have been visualized at the immunological synapse (IS) (Fig. 4b) by diffraction-limited light microscopy59,60. PALM imaging of human Jurkat T cells activated with CD3-specific antibody immobilized on glass coverslips has shown that Lat microclusters are composed of multiple smaller nanoclusters and that Lat microclusters account for only a small portion of the Lat molecules present at the cell surface. In resting T cells, most Lat molecules reside in small nanoclusters containing only a few detectable molecules, generally fewer than three, and CD3ζ is found in small nanoclusters much like Lat. Upon activation of the T cell, CD3ζ and Zap70 show substantial colocalization, a finding consistent with the expected binding of Zap70 to phosphorylated CD3ζ. Studies using the recruitment of Grb2 as an indicator of the phosphorylation of Lat have found that Lat clusters are able to recruit Grb2 regardless of their size, which suggests that Lat molecules not confined to microclusters are also involved in the activation of T cells. Upon such activation, Zap70 has a distribution that overlaps only partially with that of Lat molecules. The zones in which Lat overlaps with CD3ζ and Zap70 may constitute transient ‘hot spots’ for the phosphorylation of Lat. A parallel study has shown that Lat nanoclusters have a short temporal persistence and peak at 3 min after engagement of the TCR, after which they disappear quickly61. That study likewise reported that upon engagement of

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Figure 4  Lat nanoclusters and microclusters. (a) Isoforms of the Lat complex can be oligomerized by Grb2-Sos1-Grb2 complexes to form nanoclusters that are thought to enhance TCR sensitivity. Interactions between SLP-76 and FYB molecules (not presented here) enhance the extent of Lat reticulation achieved with Sos1 (ref. 99). Photobleaching studies have shown that some of the nanocluster components undergo continuous exchange with the cytosol or with nearby clusters100,101. Active PLC-γ1 can also ‘hop’ to nearby docking sites36. Given that the action of tyrosine kinases is continuously antagonized by potent tyrosine phosphatases 102, Lat molecules occupying the center of those nanoclusters might be protected from tyrosine phosphatases and thereby increase their signaling output 59. Protein identification and function, Figure 1a. (b) During interaction with antigen-laden APCs, T cells undergo rapid cytoskeletal rearrangements that result in the formation of an IS located at the point of contact with APC and where TCR signaling occurs. Glass-supported planar bilayers containing mobile peptide-MHC complexes and CD80 and ICAM-1 have been developed that mimic a minimal APC surface. These allow high-resolution imaging of the IS forming at T cell–planar bilayer interface 103,104. Small microclusters (200–500 nm in diameter) of TCRs engaged by peptide-MHC complexes are immediately generated after the T cells attach to the planar bilayer (top; image obtained 1 min after T cell–planar bilayer contact). TCR microclusters trigger an actin polymerization–driven retrograde flow that pushes TCR microclusters toward the center of the interface, where they coalesce to form a central supramolecular activation comple 73 (bottom; image obtained 20 min after T cell–planar bilayer contact). Scale bar, 5 µM. DIC, differential interference contrast. Adapted from ref. 22.

the TCR, the number of molecules per nanocluster ranges from 50–80 for SLP-76 to 25–60 for Lat and 25–45 for Zap70. Therefore, PALM enables the study, with single-molecule resolution, of the TCRinduced signaling nanoclusters that develop at the interface between T cells and stimulatory coverslips. However, these approaches can be prone to caveats, since weak aggregation and activation of the TCR can even occur upon contact with a supporting glass coverslip lacking antibody to CD3 (ref. 62). Moreover, models involving interfaces of T cells and a glass-supported planar bilayer lack key features of cellular APC surfaces, such as topological deformability, stiffness, molecular complexity and the presence of glycocalyx. Accordingly, the spatiotemporal organization of early signaling events may differ substantially when a T cell is activated with an APC or a glass-supported planar bilayer63. Therefore, it remains uncertain how to ‘translate’ the findings obtained with planar bilayer interfaces to the situation in which a T cell is confronted with an APC in the environment of a secondary lymphoid organ or in body barriers where they generally exert their effector functions. Vesicular Lat molecules In addition to its localization at the plasma membrane of resting T cells, Lat also resides on intracellular vesicles64. A series of studies have suggested that triggering of the TCR induces the docking, but not the fusion, of Lat-containing intracellular vesicles below the IS65–67. Further analysis of T cells deficient in VAMP-7, a soluble N-ethylmaleimide-sensitive attachment-protein receptor involved mainly in the docking of intracellular vesicles with the presynaptic membrane of neurons, has established that the docking of Lat-containing intracellular vesicles is mandatory for proper TCR signaling66. This finding led to a model in which functionally relevant Lat signalosomes assemble at the surface of vesicles near the plasma membrane of the IS. According to this model, plasma membrane– bound, TCR-activated Zap70 molecules phosphorylate vesicleassociated Lat molecules, which permits their docking to SLP76–Gads complexes that are already associated with the plasma membrane, possibly through phosphorylated CD6 molecules68. Given 794

that Lat molecules localized to the plasma membrane are efficiently phosphorylated in the seconds that follow engagement of the TCR, it has been argued that vesicular Lat molecules are required to sustain signaling only at late time points69. It has been also proposed that Lat-containing intracellular vesicles fuse with the plasma membrane at the IS70. Such fusion events, triggered by engagement of the TCR, are thought to contribute to the functional nanoscale organization of the IS. Therefore, the assembly of signaling complexes may not rely solely on the lateral reorganization of molecules embedded in the plane of the T cell plasma membrane71 but may also rely on preformed vesicular ‘packets’ that are released on demand to the IS membrane, as is also observed at the neuronal synapse72. Conversely, endocytosis of vesicles from the plasma membrane contributes to the dismantling of TCR signaling complexes via internalization73,74, together with the shedding of TCRs in microvesicles that also occurs at the center of the IS75. Therefore, although indisputable experimental evidence supports the proposal of the existence of a pool of vesicular Lat molecules, contrasting views remain as to its exact function. T cell activation at the single-cell level Biochemical and imaging approaches suggest the existence of TCRinduced signaling nanoclusters. Determining the signaling output of these dynamic and probably heterogeneous macromolecular structures constitutes a formidable challenge, and ‘optogenetics’, a combination of techniques from optics and genetics, whereby light is used to control cells or proteins that have been genetically sensitized to light, may help meet such a challenge. By manipulating at will the activity of a given signaling protein at the single-cell level, optogenetics allows researchers to monitor quantitatively and qualitatively how information flows through a given section of a signal-transduction network. Moreover, cellular optogenetics allows researchers to cope with the normal random variability between genetically identical cells in their expression of many molecular components76. As an example, a study based on cellular optogenetics has highlighted how the dynamic activity of an intracellular effector is ‘decoded’ by fibroblastic cells77. VOLUME 15  NUMBER 9  SEPTEMBER 2014  nature immunology

p e r s p e c ti v e This study revealed that the signaling pathway of the GTPase Ras and the kinase Erk rejects Ras-generated inputs with a duration of less than 4 minutes and converts them in an ‘analog’ manner into phosphorylated Erk species if they last from 4 minutes to multiple hours. Interestingly, a signaling module using the enzymatically active Erk species present in the cytoplasm as the input and phosphorylation of the transcription factor c-Fos as the output functions as an ‘analog-to-digital’ converter that dictates cell-fate ‘decisions’ and cell-cycle progression 78. Therefore, it is anticipated that the application of optogenetics to T cells will provide invaluable information about the signaling properties of the building blocks that constitute the TCR signaltransduction network.

Acknowledgments This paper is dedicated to the memory of François Kourilsky. We thank R. Germain and P. Bongrand for discussions. Supported by the Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Aix-Marseille Université, French National Infrastructure for Mouse Phenogenomics (PHENOMIN), Agence Nationale de Recherche (Basilic project to M.M.) and European Research Council (“Integrate” grant to B.M.). COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html.

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Perspectives The ever-increasing capacity of ‘-omics’ approaches is not commensurate with the ability to delineate the dynamic relationships between signaling ‘protagonists’ in the T cell interior. Moreover, a general model of T cell activation will be difficult to achieve because the requirements for the activation of a T lymphocyte vary according to the functional subset to which it belongs, its state of differentiation and the cells with which it interacts. Such interactions may occur in the thymus during T cell development or in secondary lymphoid organs at the onset of T cell responses. There has been a surge of interest in effector T cells and effector-memory T cells, since unlike naive T cells, they can pass through body barriers and interact with professional and nonprofessional APCs to elicit local T cell immunity. Such interactions further emphasize the need to understand the rules of T cell activation beyond secondary lymphoid organs, including those in tumor microenvironments 79, and to take into account the possibility that some T cell functions are reactivated in a TCRindependent manner80. It may therefore be hazardous to interpret in the same way results relevant to different circumstances of T cell stimulation. Ultimately, several ‘submodels’ of T cell activation will probably be required. Given the considerable challenges that exist, humility is a more appropriate reaction than hubris. However, as illustrated here, the prospects of integrating in vitro, in vivo and in silico approaches, each with unique advantages and drawbacks, in modeling T cell activation are excellent. A major goal is to predict how differences in the quality and quantity of the earliest T cell signaling events affect long-term functional outcomes. Identifying redundancy (the duplication of critical components) constitutes a critical issue in predicting whether the TCR signal-transduction network will be particularly vulnerable to a given drug or instead will be able to use an alternative route for signal propagation to bypass the component targeted by the drug. A current expectation is that incorporating the mathematical tools that have often been highly successful in physics and chemistry biology should allow prediction of the unfolding of T cell activation from the initial conditions. However, despite substantial attempts, such modeling may not be achievable yet because all the initial conditions are still not yet known with sufficient accuracy to warrant reliable predictions. For example, in addition to APCs, other factors extrinsic to a T cell can influence its activation. Neighboring T cells can compete for peptideMHC ligands and influence the response of a given T cell clone. However, although the understanding of TCR signaling remains incomplete, it is rewarding to see that such knowledge has already found application in the treatment of diseases, in that autologous T cells expressing recombinant chimeric antigen receptors and therapeutic antibodies directed at various T cell coinhibitors represent promising reagents for cancer immunotherapy. nature immunology  VOLUME 15  NUMBER 9  SEPTEMBER 2014

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Huang, J. et al. A single peptide-major histocompatibility complex ligand triggers digital cytokine secretion in CD4+ T cells. Immunity 39, 846–857 (2013). Two key papers (refs. 1 and 3) showing that a single peptide-MHC suffices to trigger T cell activation. Manz, B.N., Jackson, B.L., Petit, R.S., Dustin, M.L. & Groves, J. T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters. Proc. Natl. Acad. Sci. USA 108, 9089–9094 (2011). O’Donoghue, G.P., Pielak, R.M., Smoligovets, A.A., Lin, J.J. & Groves, J.T. Direct single molecule measurement of TCR triggering by agonist pMHC in living primary T cells. eLife 2, e00778 (2013). Houtman, J.C., Houghtling, R.A., Barda-Saad, M., Toda, Y. & Samelson, L.E. Early phosphorylation kinetics of proteins involved in proximal TCR-mediated signaling pathways. J. Immunol. 175, 2449–2458 (2005). Huse, M. et al. Spatial and temporal dynamics of T cell receptor signaling with a photoactivatable agonist. Immunity 27, 76–88 (2007). Two key papers (refs. 5 and 6) showing the high speed at which TCR signaling proceeds. Brodovitch, A., Bongrand, P. & Pierres, A. T lymphocytes sense antigens within seconds and make a decision within one minute. J. Immunol. 191, 2064–2071 (2013). Hogquist, K.A.J. & Jameson, S.C. The self-obsession of T cells: how TCR signaling thresholds affect fate ‘decisions’ and effector function. Nat. Immunol. 15, XXX–XXX (2014). Pitcher, L.A. et al. The formation and functions of the 21- and 23-kDa tyrosinephosphorylated TCR zeta subunits. Immunol. Rev. 191, 47–61 (2003). Moran, A.E. et al. T cell receptor signal strength in Treg and iNKT cell development demonstrated by a novel fluorescent reporter mouse. J. Exp. Med. 208, 1279–1289 (2011). Hochweller, K. et al. Dendritic cells control T cell tonic signaling required for responsiveness to foreign antigen. Proc. Natl. Acad. Sci. USA 107, 5931–5936 (2010). Huppa, J.B. & Davis, M.M. The interdisciplinary science of T-cell recognition. Adv. Immunol. 119, 1–50 (2013). Chakraborty, A.K.W. & Weiss, A. Insights into the initiation of TCR signaling. Nat. Immunol. 15, 815–823 (2014). Sangani, D., Venien-Bryan, C. & Harder, T. Phosphotyrosine-dependent in vitro reconstitution of recombinant LAT-nucleated multiprotein signalling complexes on liposomes. Mol. Membr. Biol. 26, 159–170 (2009). Roncagalli, R. et al. Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor-independent TCR signaling hub. Nat. Immunol. 15, 384–392 (2014). Key paper describing the Zap70–Lat–SLP-76 interactome in primary CD4+ T cells. Navarro, M.N.C. & Cantrell, D. Serine-threonine kinases in TCR signaling. Nat. Immunol. 15, XXX–XXX (2014). Mingueneau, M. et al. Loss of the LAT adaptor converts antigen-responsive T cells into pathogenic effectors that function independently of the T cell receptor. Immunity 31, 197–208 (2009). Salek, M. et al. Quantitative phosphoproteome analysis unveils LAT as a modulator of CD3zeta and ZAP-70 tyrosine phosphorylation. PLoS ONE 8, e77423 (2013). Ou-Yang, C.W. et al. Role of LAT in the granule-mediated cytotoxicity of CD8 T cells. Mol. Cell. Biol. 32, 2674–2684 (2012). Pagán, A.J., Pepper, M., Chu, H.H., Green, J.M. & Jenkins, M.K. CD28 promotes CD4+ T cell clonal expansion during infection independently of its YMNM and PYAP motifs. J. Immunol. 189, 2909–2917 (2012). Kong, K.F. et al. A motif in the V3 domain of the kinase PKC-θ determines its localization in the immunological synapse and functions in T cells via association with CD28. Nat. Immunol. 12, 1105–1112 (2011). Chuang, H.C. et al. The kinase GLK controls autoimmunity and NF-κB signaling by activating the kinase PKC-θ in T cells. Nat. Immunol. 12, 1113–1118 (2011). Liang, Y. et al. The lymphoid lineage-specific actin-uncapping protein Rltpr is essential for costimulation via CD28 and the development of regulatory T cells. Nat. Immunol. 14, 858–866 (2013). Michel, F. & Acuto, O. CD28 costimulation: a source of Vav-1 for TCR signaling with the help of SLP-76? Sci. STKE 2002, pe35 (2002).

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p e r s p e c ti v e 24. Kim, J.E. & White, F.M. Quantitative analysis of phosphotyrosine signaling networks triggered by CD3 and CD28 costimulation in Jurkat cells. J. Immunol. 176, 2833–2843 (2006). 25. Iezzi, G., Karjalainen, K. & Lanzavecchia, A. The duration of antigenic stimulation determines the fate of naive and effector T cells. Immunity 8, 89–95 (1998). 26. Chen, L. & Flies, D.B. Molecular mechanisms of T cell co-stimulation and coinhibition. Nat. Rev. Immunol. 13, 227–242 (2013). 27. Simpson, T.R. et al. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J. Exp. Med. 210, 1695–1710 (2013). 28. van Panhuys, N., Klauschen, F. & Germain, R.N. T cell receptor-dependent signal intensity dominantly controls CD4+ T cell polarization in vivo. Immunity 41, 63–74 (2014). 29. Varjosalo, M. et al. Interlaboratory reproducibility of large-scale human proteincomplex analysis by standardized AP-MS. Nat. Methods 10, 307–314 (2013). 30. Zheng, Y. et al. Temporal regulation of EGF signalling networks by the scaffold protein Shc1. Nature 499, 166–171 (2013). Key paper analyzing the causal relationships within a complex signal-transduction network. 31. Mayya, V. et al. Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions. Sci. Signal. 2, ra46 (2009). 32. Mbonye, U.R. et al. Phosphorylation of CDK9 at Ser175 enhances HIV transcription and is a marker of activated P-TEFb in CD4+ T lymphocytes. PLoS Pathog. 9, e1003338 (2013). 33. Astoul, E., Edmunds, C., Cantrell, D.A. & Ward, S.G. PI 3-K and T-cell activation: limitations of T-leukemic cell lines as signaling models. Trends Immunol. 22, 490–496 (2001). 34. Brockmeyer, C. et al. T cell receptor (TCR)-induced tyrosine phosphorylation dynamics identifies THEMIS as a new TCR signalosome component. J. Biol. Chem. 286, 7535–7547 (2011). 35. Navarro, M.N., Goebel, J., Feijoo-Carnero, C., Morrice, N. & Cantrell, D.A. Phosphoproteomic analysis reveals an intrinsic pathway for the regulation of histone deacetylase 7 that controls the function of cytotoxic T lymphocytes. Nat. Immunol. 12, 352–361 (2011). 36. Cruz-Orcutt, N., Vacaflores, A., Connolly, S.F., Bunnell, S.C. & Houtman, J.C. Activated PLC-γ1 is catalytically induced at LAT but activated PLC-γ1 is localized at both LAT- and TCR-containing complexes. Cell. Signal. 26, 797–805 (2014). 37. Zhang, W. et al. Essential role of LAT in T cell development. Immunity 10, 323–332 (1999). 38. Aguado, E. et al. Induction of T helper type 2 immunity by a point mutation in the LAT adaptor. Science 296, 2036–2040 (2002). 39. Sommers, C.L. et al. A LAT mutation that inhibits T cell development yet induces lymphoproliferation. Science 296, 2040–2043 (2002). 40. Chuck, M.I., Zhu, M., Shen, S. & Zhang, W. The role of the LAT-PLC-γ1 interaction in T regulatory cell function. J. Immunol. 184, 2476–2486 (2010). 41. Koonpaew, S., Shen, S., Flowers, L. & Zhang, W. LAT-mediated signaling in CD4+CD25+ regulatory T cell development. J. Exp. Med. 203, 119–129 (2006). 42. Wang, Y. et al. Th2 lymphoproliferative disorder of LatY136F mutant mice unfolds independently of TCR-MHC engagement and is insensitive to the action of Foxp3+ regulatory T cells. J. Immunol. 180, 1565–1575 (2008). 43. Genton, C. et al. The Th2 lymphoproliferation developing in LatY136F mutant mice triggers polyclonal B cell activation and systemic autoimmunity. J. Immunol. 177, 2285–2293 (2006). 44. Chevrier, S., Genton, C., Malissen, B., Malissen, M. & Acha-Orbea, H. Dominant role of CD80–CD86 Over CD40 and ICOSL in the massive polyclonal B cell activation mediated by LAT(Y136F) CD4+ T cells. Front Immunol 3, 27 (2012). 45. Nuñez-Cruz, S. et al. LAT regulates gammadelta T cell homeostasis and differentiation. Nat. Immunol. 4, 999–1008 (2003). 46. Archambaud, C. et al. STAT6 deletion converts the Th2 inflammatory pathology afflicting Lat(Y136F) mice into a lymphoproliferative disorder involving Th1 and CD8 effector T cells. J. Immunol. 182, 2680–2689 (2009). 47. Roncagalli, R., Mingueneau, M., Gregoire, C., Malissen, M. & Malissen, B. LAT signaling pathology: an “autoimmune” condition without T cell self-reactivity. Trends Immunol. 31, 253–259 (2010). 48. Kortum, R.L. et al. A phospholipase C-γ1-independent, RasGRP1-ERK-dependent pathway drives lymphoproliferative disease in linker for activation of T cells-Y136F mutant mice. J. Immunol. 190, 147–158 (2013). 49. Cao, L. et al. Quantitative phosphoproteomics reveals SLP-76 dependent regulation of PAG and Src family kinases in T cells. PLoS ONE 7, e46725 (2012). 50. Siggs, O.M. et al. Opposing functions of the T cell receptor kinase ZAP-70 in immunity and tolerance differentially titrate in response to nucleotide substitutions. Immunity 27, 912–926 (2007). 51. Hirota, K. et al. T cell self-reactivity forms a cytokine milieu for spontaneous development of IL-17+ Th cells that cause autoimmune arthritis. J. Exp. Med. 204, 41–47 (2007). 52. Hsu, L.Y., Tan, Y.X., Xiao, Z., Malissen, M. & Weiss, A. A hypomorphic allele of ZAP-70 reveals a distinct thymic threshold for autoimmune disease versus autoimmune reactivity. J. Exp. Med. 206, 2527–2541 (2009). 53. Vang, T. et al. Autoimmune-associated lymphoid tyrosine phosphatase is a gainof-function variant. Nat. Genet. 37, 1317–1319 (2005). 54. Oh-Hora, M. et al. Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T cell activation and tolerance. Nat. Immunol. 9, 432–443 (2008).

796

55. Chabod, M. et al. A spontaneous mutation of the rat Themis gene leads to impaired function of regulatory T cells linked to inflammatory bowel disease. PLoS Genet. 8, e1002461 (2012). 56. McDonald, C.B. et al. Multivalent binding and facilitated diffusion account for the formation of the Grb2-Sos1 signaling complex in a cooperative manner. Biochemistry 51, 2122–2135 (2012). 57. Houtman, J.C. et al. Oligomerization of signaling complexes by the multipoint binding of GRB2 to both LAT and SOS1. Nat. Struct. Mol. Biol. 13, 798–805 (2006). One of three key papers (refs. 57, 58 and 99) demonstrating the role of cooperative interactions in the assembly of the TCR signal-transduction network. 58. Kortum, R.L. et al. The ability of Sos1 to oligomerize the adaptor protein LAT is separable from its guanine nucleotide exchange activity in vivo. Sci. Signal. 6, ra99 (2013). 59. Sherman, E. et al. Functional nanoscale organization of signaling molecules downstream of the T cell antigen receptor. Immunity 35, 705–720 (2011). One of two key papers (refs. 59 and 61) describing the use of super-resolution microscopy to determine the nanoscale organisation of signaling molecules in T cells. 60. Sherman, E., Barr, V.A. & Samelson, L.E. Resolving multi-molecular protein interactions by photoactivated localization microscopy. Methods 59, 261–269 (2013). 61. Hsu, C.J. et al. Ligand mobility modulates immunological synapse formation and T cell activation. PLoS ONE 7, e32398 (2012). 62. James, J.R. et al. The T cell receptor triggering apparatus is composed of monovalent or monomeric proteins. J. Biol. Chem. 286, 31993–32001 (2011). 63. Roybal, K.T., Sinai, P., Verkade, P., Murphy, R.F. & Wulfing, C. The actin-driven spatiotemporal organization of T-cell signaling at the system scale. Immunol. Rev. 256, 133–147 (2013). 64. Bonello, G. et al. Dynamic recruitment of the adaptor protein LAT: LAT exists in two distinct intracellular pools and controls its own recruitment. J. Cell Sci. 117, 1009–1016 (2004). 65. Williamson, D.J. et al. Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat. Immunol. 12, 655–662 (2011). One of three key papers (refs. 65–67) suggesting a role for intracellular vesicular Lat molecules in the TCR signal-transduction network. 66. Larghi, P. et al. VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites. Nat. Immunol. 14, 723–731 (2013). 67. Purbhoo, M.A. et al. Dynamics of subsynaptic vesicles and surface microclusters at the immunological synapse. Sci. Signal. 3, ra36 (2010). 68. Malissen, B. & Marguet, D. La(s)t but not least. Nat. Immunol. 12, 592–593 (2011). 69. Balagopalan, L., Barr, V.A., Kortum, R.L., Park, A.K. & Samelson, L.E. Cutting edge: cell surface linker for activation of T cells is recruited to microclusters and is active in signaling. J. Immunol. 190, 3849–3853 (2013). 70. Soares, H. et al. Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse. J. Exp. Med. 210, 2415–2433 (2013). Key paper suggesting that intracellular vesicles fuse with the plasma membrane beyond the IS to generate signaling ‘nanoterritories’. 71. Lillemeier, B.F. et al. TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation. Nat. Immunol. 11, 90–96 (2010). 72. Choquet, D. & Triller, A. The dynamic synapse. Neuron 80, 691–703 (2013). 73. Vardhana, S., Choudhuri, K., Varma, R. & Dustin, M.L. Essential role of ubiquitin and TSG101 protein in formation and function of the central supramolecular activation cluster. Immunity 32, 531–540 (2010). 74. Lasserre, R. et al. Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation. J. Cell Biol. 195, 839–853 (2011). 75. Choudhuri, K. et al. Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse. Nature 507, 118–123 (2014). 76. Feinerman, O., Veiga, J., Dorfman, J.R., Germain, R.N. & Altan-Bonnet, G. Variability and robustness in T cell activation from regulated heterogeneity in protein levels. Science 321, 1081–1084 (2008). 77. Toettcher, J.E., Weiner, O.D. & Lim, W.A. Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155, 1422–1434 (2013). Key paper demonstrating the power of using cellular optogenetics to delineate a complex signal-transduction network. 78. Nakakuki, T. et al. Ligand-specific c-Fos expression emerges from the spatiotemporal control of ErbB network dynamics. Cell 141, 884–896 (2010). 79. Breart, B., Lemaitre, F., Celli, S. & Bousso, P. Two-photon imaging of intratumoral CD8+ T cell cytotoxic activity during adoptive T cell therapy in mice. J. Clin. Invest. 118, 1390–1397 (2008). 80. Soudja, S.M., Ruiz, A.L., Marie, J.C. & Lauvau, G. Inflammatory monocytes activate memory CD8+ T and innate NK lymphocytes independent of cognate antigen during microbial pathogen invasion. Immunity 37, 549–562 (2012). 81. Acuto, O., Di Bartolo, V. & Michel, F. Tailoring T-cell receptor signals by proximal negative feedback mechanisms. Nat. Rev. Immunol. 8, 699–712 (2008). 82. Malissen, B. An evolutionary and structural perspective on T cell antigen receptor function. Immunol. Rev. 191, 7–27 (2003).

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83. Pauker, M.H., Reicher, B., Fried, S., Perl, O. & Barda-Saad, M. Functional cooperation between the proteins Nck and ADAP is fundamental for actin reorganization. Mol. Cell. Biol. 31, 2653–2666 (2011). 84. Ophir, M.J., Liu, B.C. & Bunnell, S.C. The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms. J. Cell Biol. 203, 1021–1041 (2013). 85. Balagopalan, L., Coussens, N.P., Sherman, E., Samelson, L.E. & Sommers, C.L. The LAT story: a tale of cooperativity, coordination, and choreography. Cold Spring Harb. Perspect. Biol. 2, a005512 (2010). 86. Gomez, T.S. et al. Dynamin 2 regulates T cell activation by controlling actin polymerization at the immunological synapse. Nat. Immunol. 6, 261–270 (2005). 87. Jun, J.E., Rubio, I. & Roose, J.P. Regulation of Ras exchange factors and cellular localization of Ras activation by lipid messengers in T cells. Front Immunol 4, 239 (2013). 88. Le Floc’h, A. et al. Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse. J. Exp. Med. 210, 2721–2737 (2013). 89. Shui, J.W. et al. Hematopoietic progenitor kinase 1 negatively regulates T cell receptor signaling and T cell-mediated immune responses. Nat. Immunol. 8, 84–91 (2007). 90. Di Bartolo, V. et al. A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14–3-3 proteins on SLP-76. J. Exp. Med. 204, 681–691 (2007). 91. San Luis, B., Sondgeroth, B., Nassar, N. & Carpino, N. Sts-2 is a phosphatase that negatively regulates ζ-associated protein (ZAP)-70 and T cell receptor signaling pathways. J. Biol. Chem. 286, 15943–15954 (2011). 92. Balagopalan, L. et al. Enhanced T-cell signaling in cells bearing linker for activation of T-cell (LAT) molecules resistant to ubiquitylation. Proc. Natl. Acad. Sci. USA 108, 2885–2890 (2011).

93. Fu, G. et al. Themis sets the signal threshold for positive and negative selection in T-cell development. Nature 504, 441–445 (2013). 94. Paster, W. et al. GRB2-mediated recruitment of THEMIS to LAT is essential for thymocyte development. J. Immunol. 190, 3749–3756 (2013). 95. Lesourne, R. et al. Themis, a T cell-specific protein important for late thymocyte development. Nat. Immunol. 10, 840–847 (2009). 96. Dong, S. et al. T cell receptor for antigen induces linker for activation of T celldependent activation of a negative signaling complex involving Dok-2, SHIP-1, and Grb-2. J. Exp. Med. 203, 2509–2518 (2006). 97. Yasuda, T. et al. Dok-1 and Dok-2 are negative regulators of T cell receptor signaling. Int. Immunol. 19, 487–495 (2007). 98. Schoenborn, J.R., Tan, Y.X., Zhang, C., Shokat, K.M. & Weiss, A. Feedback circuits monitor and adjust basal Lck-dependent events in T cell receptor signaling. Sci. Signal. 4, ra59 (2011). 99. Coussens, N.P. et al. Multipoint binding of the SLP-76 SH2 domain to ADAP is critical for oligomerization of SLP-76 signaling complexes in stimulated T cells. Mol. Cell. Biol. 33, 4140–4151 (2013). 100. Bunnell, S.C. et al. T cell receptor ligation induces the formation of dynamically regulated signaling assemblies. J. Cell Biol. 158, 1263–1275 (2002). 101. Douglass, A.D. & Vale, R.D. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 121, 937–950 (2005). 102. Kleiman, L.B., Maiwald, T., Conzelmann, H., Lauffenburger, D.A. & Sorger, P.K. Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding. Mol. Cell 43, 723–737 (2011). 103. Yokosuka, T. et al. Spatiotemporal regulation of T cell costimulation by TCR-CD28 microclusters and protein kinase C theta translocation. Immunity 29, 589–601 (2008). 104. Dustin, M.L. The cellular context of T cell signaling. Immunity 30, 482–492 (2009).

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