LFA-1 and kindlin-3 enable the collaborative transport of SLP-76 microclusters by myosin and dynein motors.

Integrin engagement within the immune synapse enhances T cell activation, but our understanding of this process is incomplete. In response to T cell receptor (TCR) ligation, SLP-76 (LCP2), ADAP (FYB1) and SKAP55 (SKAP1) are recruited into microclusters and activate integrins via the effectors talin-1 and kindlin-3 (FERMT3). We postulated that integrins influence the centripetal transport and signaling of SLP-76 microclusters via these linkages. We show that contractile myosin filaments surround and are co-transported with SLP-76 microclusters, and that TCR ligand density governs the centripetal movement of both structures. Centripetal transport requires formin activity, actomyosin contraction, microtubule integrity and dynein motor function. Although immobilized VLA-4 (α4ß1 integrin) and LFA-1 (αLß2 integrin) ligands arrest the centripetal movement of SLP-76 microclusters and myosin filaments, VLA-4 acts distally, while LFA-1 acts in the lamellum. Integrin ß2, kindlin-3 and zyxin are required for complete centripetal transport, while integrin ß1 and talin-1 are not. CD69 upregulation is similarly dependent on integrin ß2, kindlin-3 and zyxin, but not talin-1. These findings highlight the integration of cytoskeletal systems within the immune synapse and reveal extracellular ligand-independent roles for LFA-1 and kindlin-3. This article has an associated First Person interview with the first author of the paper.

p53 keeps bystanders at the gates.

The inappropriate expansion of self-reactive "bystander" T cells can contribute to autoimmune disease. In this issue of Immunity, Watanabe et al. (2014) demonstrate that the tumor suppressor p53 prevents the cytokine-dependent proliferation of T cells in the absence of cognate antigens.

Vav2 lacks Ca2+ entry-promoting scaffolding functions unique to Vav1 and inhibits T cell activation via Cdc42.

Vav family guanine nucleotide exchange factors (GEFs) are essential regulators of immune function. Despite their structural similarity, Vav1 promotes and Vav2 opposes T cell receptor (TCR)-induced Ca2+ entry. By using a Vav1-deficient Jurkat T cell line, we find that Vav1 facilitates Ca2+ entry via non-catalytic scaffolding functions that are encoded by the catalytic core of Vav1 and flanking linker regions. We implicate, in this scaffolding function, a previously undescribed polybasic motif that is strictly conserved in Vav1 and absent from Vav2 in tetrapods. Conversely, the catalytic activity of Vav2 contributes to the suppression of TCR-mediated Ca2+ entry. By performing an in vivo 'GEF trapping' assay in intact cells, we demonstrate that Cdc42 interacts with the catalytic surface of Vav2 but not Vav1, and that Vav1 discriminates Cdc42 from Rac1 via F56 (W56 in Rac1). Finally, the Cdc42-specific inhibitor ZCL278 and the shRNA-mediated suppression of Cdc42 each prevent the inhibition of TCR-induced Ca2+ entry by Vav2. These findings define stark differences in the functions of Vav1 and Vav2, and provide an explanation for the differential usage of these Vav isoforms by immune subpopulations.

Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis during Yersinia infection.

Cell death and inflammation are intimately linked during Yersinia infection. Pathogenic Yersinia inhibits the MAP kinase TGFß-activated kinase 1 (TAK1) via the effector YopJ, thereby silencing cytokine expression while activating caspase-8-mediated cell death. Here, using Yersinia pseudotuberculosis in corroboration with costimulation of lipopolysaccharide and (5Z)-7-Oxozeaenol, a small-molecule inhibitor of TAK1, we show that caspase-8 activation during TAK1 inhibition results in cleavage of both gasdermin D (GSDMD) and gasdermin E (GSDME) in murine macrophages, resulting in pyroptosis. Loss of GsdmD delays membrane rupture, reverting the cell-death morphology to apoptosis. We found that the Yersinia-driven IL-1 response arises from asynchrony of macrophage death during bulk infections in which two cellular populations are required to provide signal 1 and signal 2 for IL-1α/ß release. Furthermore, we found that human macrophages are resistant to YopJ-mediated pyroptosis, with dampened IL-1ß production. Our results uncover a form of caspase-8-mediated pyroptosis and suggest a hypothesis for the increased sensitivity of humans to Yersinia infection compared with the rodent reservoir.

ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters.

Antigen recognition by the T cell receptor (TCR) directs the assembly of essential signaling complexes known as SLP-76 (also known as LCP2) microclusters. Here, we show that the interaction of the adhesion and degranulation-promoting adaptor protein (ADAP; also known as FYB1) with SLP-76 enables the formation of persistent microclusters and the stabilization of T cell contacts, promotes integrin-independent adhesion and enables the upregulation of CD69. By analyzing point mutants and using a novel phospho-specific antibody, we show that Y595 is essential for normal ADAP function, that virtually all tyrosine phosphorylation of ADAP is restricted to a Y595-phosphorylated (pY595) pool, and that multivalent interactions between the SLP-76 SH2 domain and its binding sites in ADAP are required to sustain ADAP phosphorylation. Although pY595 ADAP enters SLP-76 microclusters, non-phosphorylated ADAP is enriched in protrusive actin-rich structures. The pre-positioning of ADAP at the contact sites generated by these structures favors the retention of nascent SLP-76 oligomers and their assembly into persistent microclusters. Although ADAP is frequently depicted as an effector of SLP-76, our findings reveal that ADAP acts upstream of SLP-76 to convert labile, Ca2+-competent microclusters into stable adhesive junctions with enhanced signaling potential.

A view to a kill: how ligand quality controls lethal hits.

In this issue of Immunity, Beal et al. (2009) and Jenkins et al. (2009) demonstrate that relatively weak stimuli support synapse formation and microtubule polarization, but fail to trigger efficient killing because of their inability to recruit lytic granules to the synaptic cleft.

Phagocytic Receptors Activate Syk and Src Signaling during Borrelia burgdorferi Phagocytosis.

Phagocytosis of the Lyme disease-causing pathogen Borrelia burgdorferi has been shown to be important for generating an inflammatory response to the pathogen. As a result, understanding the mechanisms of phagocytosis has been an area of great interest in the field of Lyme disease. Several cell surface receptors that participate in B. burgdorferi phagocytosis have been reported, including the scavenger receptor MARCO and integrin α3ß1. We sought to define the mechanisms by which these receptors mediate phagocytosis and to identify signaling pathways activated downstream of these receptors upon contact with B. burgdorferi We identified both Syk and Src signaling pathways as ones that participate in B. burgdorferi phagocytosis and the resulting cytokine activation. In our studies, we found that both MARCO and integrin ß1 play a role in the activation of the Src kinase pathway. However, only integrin ß1 participates in the activation of Syk. Interestingly, the integrin activates Syk without the help of the signaling adaptor Dap12 or FcRγ. Thus, we report that multiple pathways participate in B. burgdorferi internalization and that different cell surface receptors act simultaneously in cooperation and independently to mediate phagocytosis.

T cell costimulation via the integrin VLA-4 inhibits the actin-dependent centralization of signaling microclusters containing the adaptor SLP-76.

Antigen-dependent T cell activation drives the formation of signaling microclusters containing the adaptor SLP-76. Costimulatory integrins regulate SLP-76 phosphorylation and could influence SLP-76 microclusters in the integrin-rich periphery of the immune synapse. We report that costimulation by the integrin VLA-4 (alpha4beta1) required SLP-76 domains implicated in microcluster assembly. Pro-adhesive ligands enlarged the contact and increased the number of SLP-76 microclusters regardless of their costimulatory potential. Costimulatory VLA-4 ligands also prevented the centralization of SLP-76, promoted microcluster persistence, prolonged lateral interactions between SLP-76 and its upstream kinase, ZAP-70, and retained SLP-76 in tyrosine-phosphorylated peripheral structures. SLP-76 centralization was driven by dynamic actin polymerization and was correlated with inward actin flows. VLA-4 ligation retarded these flows, even in the absence of SLP-76. These data suggest a widely applicable model of costimulation, in which integrins promote sustained signaling by attenuating cytoskeletal movements that drive the centralization and inactivation of SLP-76 microclusters.

Adaptor Protein-3-Mediated Trafficking of TLR2 Ligands Controls Specificity of Inflammatory Responses but Not Adaptor Complex Assembly.

Innate immune engagement results in the activation of host defenses that produce microbe-specific inflammatory responses. A long-standing interest in the field of innate immunity is to understand how varied host responses are generated through the signaling of just a limited number of receptors. Recently, intracellular trafficking and compartmental partitioning have been identified as mechanisms that provide signaling specificity for receptors by regulating signaling platform assembly. We show that cytokine activation as a result of TLR2 stimulation occurs at different intracellular locations and is mediated by the phagosomal trafficking molecule adaptor protein-3 (AP-3). AP-3 is required for trafficking TLR2 purified ligands or the Lyme disease causing bacterium, Borrelia burgdorferi, to LAMP-1 lysosomal compartments. The presence of AP-3 is necessary for the activation of cytokines such as IL-6 but not TNF-α or type I IFNs, suggesting induction of these cytokines occurs from a different compartment. Lack of AP-3 does not interfere with the recruitment of TLR signaling adaptors TRAM and MyD88 to the phagosome, indicating that the TLR-MyD88 signaling complex is assembled at a prelysosomal stage and that IL-6 activation depends on proper localization of signaling molecules downstream of MyD88. Finally, infection of AP-3-deficient mice with B. burgdorferi resulted in altered joint inflammation during murine Lyme arthritis. Our studies further elucidate the effects of phagosomal trafficking on tailoring immune responses in vitro and in vivo.

CD209a expression on dendritic cells is critical for the development of pathogenic Th17 cell responses in murine schistosomiasis.

In murine schistosomiasis, immunopathology and cytokine production in response to parasite eggs are uneven and strain dependent. CBA/J (CBA) mice develop severe hepatic granulomatous inflammation associated with prominent Th17 cell responses driven by dendritic cell (DC)-derived IL-1ß and IL-23. Such Th17 cells fail to develop in low-pathology C57BL/6 (BL/6) mice, and the reasons for these strain-specific differences in APC reactivity to eggs remain unclear. We show by gene profiling that CBA DCs display an 18-fold higher expression of the C-type lectin receptor CD209a, a murine homolog of human DC-specific ICAM-3-grabbing nonintegrin, compared with BL/6 DCs. Higher CD209a expression was observed in CBA splenic and granuloma APC subpopulations, but only DCs induced Th17 cell differentiation in response to schistosome eggs. Gene silencing in CBA DCs and overexpression in BL/6 DCs demonstrated that CD209a is essential for egg-elicited IL-1ß and IL-23 production and subsequent Th17 cell development, which is associated with SRC, RAF-1, and ERK1/2 activation. These findings reveal a novel mechanism controlling the development of Th17 cell-mediated severe immunopathology in helminthic disease.

STING promotes homeostatic maintenance of tissues and confers longevity with aging.

Local immune processes within aging tissues are a significant driver of aging associated dysfunction, but tissue-autonomous pathways and cell types that modulate these responses remain poorly characterized. The cytosolic DNA sensing pathway, acting through cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING), is broadly expressed in tissues, and is poised to regulate local type I interferon (IFN-I)-dependent and independent inflammatory processes within tissues. Recent studies suggest that the cGAS/STING pathway may drive pathology in various in vitro and in vivo models of accelerated aging. To date, however, the role of the cGAS/STING pathway in physiological aging processes, in the absence of genetic drivers, has remained unexplored. This remains a relevant gap, as STING is ubiquitously expressed, implicated in multitudinous disorders, and loss of function polymorphisms of STING are highly prevalent in the human population (>50%). Here we reveal that, during physiological aging, STING-deficiency leads to a significant shortening of murine lifespan, increased pro-inflammatory serum cytokines and tissue infiltrates, as well as salient changes in histological composition and organization. We note that aging hearts, livers, and kidneys express distinct subsets of inflammatory, interferon-stimulated gene (ISG), and senescence genes, collectively comprising an immune fingerprint for each tissue. These distinctive patterns are largely imprinted by tissue-specific stromal and myeloid cells. Using cellular interaction network analyses, immunofluorescence, and histopathology data, we show that these immune fingerprints shape the tissue architecture and the landscape of cell-cell interactions in aging tissues. These age-associated immune fingerprints are grossly dysregulated with STING-deficiency, with key genes that define aging STING-sufficient tissues greatly diminished in the absence of STING. Changes in immune signatures are concomitant with a restructuring of the stromal and myeloid fractions, whereby cell:cell interactions are grossly altered and resulting in disorganization of tissue architecture in STING-deficient organs. This altered homeostasis in aging STING-deficient tissues is associated with a cross-tissue loss of homeostatic tissue-resident macrophage (TRM) populations in these tissues. Ex vivo analyses reveal that basal STING-signaling limits the susceptibility of TRMs to death-inducing stimuli and determines their in situ localization in tissue niches, thereby promoting tissue homeostasis. Collectively, these data upend the paradigm that cGAS/STING signaling is primarily pathological in aging and instead indicate that basal STING signaling sustains tissue function and supports organismal longevity. Critically, our study urges caution in the indiscriminate targeting of these pathways, which may result in unpredictable and pathological consequences for health during aging.

Signal initiation in T-cell receptor microclusters.

Although dynamic imaging technologies have provided important insights into the underlying processes responsible for T-cell activation, the processes that link antigen recognition to downstream signaling remain poorly defined. Converging lines of inquiry indicate that T-cell receptor (TCR) microclusters are the minimal structures capable of directing effective TCR signaling. Furthermore, imaging studies have determined that these structures trigger the assembly of oligomeric signaling scaffolds that contain the adapters and effectors required for T-cell activation. Existing models of T-cell activation accurately explain the sensitivity and selectivity of antigen recognition. However, these models do not account for important properties of microclusters, including their peripheral formation, size, and movement on the actin cytoskeleton. Here we examine how lipid rafts, galectin lattices, and protein scaffolds contribute to the assembly, function, and fate of TCR microclusters within immune synapses. Finally, we propose a 'mechanical segregation' model of signal initiation in which cytoskeletal forces contribute to the lateral segregation of molecules and cytoskeletal scaffolds provide a template for microclusters assembly.

Multiple microclusters: diverse compartments within the immune synapse.

The activation of classical alphabeta T cells is initiated when the T cell receptor (TCR) recognizes peptide antigens presented by major histocompatibility complex (pMHC) molecules. This recognition always occurs at the junction of a T cell and antigen-presenting cell (APC). Existing models of T-cell activation accurately explain the sensitivity and selectivity of antigen recognition within the immunological synapse. However, these models have not fully incorporated the diverse microcluster types revealed by current imaging technologies. It is increasingly clear that a better understanding of T-cell activation will require an appreciation of the diverse signaling assemblies arising within the immune synapse, the interrelationships between these structures, and the mechanisms by which underlying cytoskeletal systems govern their assembly and fate. Here, we will provide a brief framework for understanding these issues, review our contributions to current knowledge, and provide perspectives on the future of this rapidly advancing field.

Neutrophils require SKAP2 for reactive oxygen species production following C-type lectin and Candida stimulation.

Signaling cascades converting the recognition of pathogens to efficient inflammatory responses by neutrophils are critical for host survival. SKAP2, an adaptor protein, is required for reactive oxygen species (ROS) generation following neutrophil stimulation by integrins, formyl peptide receptors, and for host defense against the Gram-negative bacterial pathogens, Klebsiella pneumoniae and Yersinia pseudotuberculosis. Using neutrophils from murine HoxB8-immortalized progenitors, we show that SKAP2 in neutrophils is crucial for maximal ROS response to purified C-type lectin receptor agonists and to the fungal pathogens, Candida glabrata and Candida albicans, and for robust killing of C. glabrata. Inside-out signaling to integrin and Syk phosphorylation occurred independently of SKAP2 after Candida infection. However, Pyk2, ERK1/2, and p38 phosphorylation were significantly reduced after infection with C. glabrata and K. pneumoniae in Skap2-/- neutrophils. These data demonstrate the importance of SKAP2 in ROS generation and host defense beyond antibacterial immunity to include CLRs and Candida species.

Characterization of a novel interaction between transcription factor TFII-I and the inducible tyrosine kinase in T cells.

TCR signaling leads to the activation of kinases such as inducible tyrosine kinase (Itk), a key regulatory protein in T-lymphocyte activation and function. The homolog of Itk in B cells is Bruton's tyrosine kinase, previously shown to bind and phosphorylate the transcription factor TFII-I. TFII-I plays major roles in transcription and signaling. Our purpose herein was twofold: first, to identify some of the molecular determinants involved in TFII-I activation downstream of receptor crosslinking in T cells and second, to uncover the existence of Itk-TFII-I signaling in T lymphocytes. We report for the first time that TFII-I is tyrosine phosphorylated upon TCR, TCR/CD43, and TCR/CD28 co-receptor engagement in human and/or murine T cells. We show that Itk physically interacts with TFII-I and potentiates TFII-I-driven c-fos transcription. We demonstrate that TFII-I is phosphorylated upon co-expression of WT, but not kinase-dead, or kinase-dead/R29C mutant Itk, suggesting these residues are important for TFII-I phosphorylation, presumably via an Itk-dependent mechanism. Structural analysis of TFII-I-Itk interactions revealed that the first 90 residues of TFII-I are dispensable for Itk binding. Mutations within Itk's kinase, pleckstrin-homology, and proline-rich regions did not abolish TFII-I-Itk binding. Our results provide an initial step in understanding the biological role of Itk-TFII-I signaling in T-cell function.

SKAP2 is required for defense against K. pneumoniae infection and neutrophil respiratory burst.

Klebsiella pneumoniae is a respiratory, blood, liver, and bladder pathogen of significant clinical concern. We show that the adaptor protein, SKAP2, is required for protection against K. pneumoniae (ATCC 43816) pulmonary infections. Skap2-/- mice had 100-fold higher bacterial burden when compared to wild-type and burden was controlled by SKAP2 expression in innate immune cells. Skap2-/- neutrophils and monocytes were present in infected lungs, and the neutrophils degranulated normally in response to K. pneumoniae infection in mice; however, K. pneumoniae-stimulated reactive oxygen species (ROS) production in vitro was abolished. K. pneumoniae-induced neutrophil ROS response required the activity of SFKs, Syk, Btk, PLCγ2, and PKC. The loss of SKAP2 significantly hindered the K. pneumoniae-induced phosphorylation of SFKs, Syk, and Pyk2 implicating SKAP2 as proximal to their activation in pathogen-signaling pathways. In conclusion, SKAP2-dependent signaling in neutrophils is essential for K. pneumoniae-activated ROS production and for promoting bacterial clearance during infection.

Klebsiella pneumoniae is a type of bacteria that can cause life-threatening infections ­ including pneumonia, blood stream infections, and urinary tract infections ­ in hospitalized patients. These infections can be difficult to treat because some K. pneumoniae are resistant to antibiotics. The bacteria are normally found in the human intestine, and they do not usually cause infections in healthy people. This implies that healthy people's immune systems are better able to fend off K. pneumoniae infections; learning how could help scientists develop new ways to treat or prevent infections in hospitalized patients. In healthy people, a type of immune cell called neutrophils are the first line of defense against bacterial infections. Several different proteins are needed to activate neutrophils, including a protein called SKAP2. But the role of this protein in fighting K. pneumoniae infections is not clear. To find out what role SKAP2 plays in the defense against pneumonia caused by K. pneumoniae, Nguyen et al. compared infections in mice with and without the protein. Mice lacking SKAP2 in their white blood cells had more bacteria in their lungs than normal mice. The experiments showed that neutrophils from mice with SKAP2 produce a burst of chemicals called "reactive oxygen species", which can kill bacteria. But neutrophils without the protein do not. Without SKAP2, several proteins that help produce reactive oxygen species do not work. Understanding the role of SKAP2 in fighting infections may help scientists better understand the immune system. This could help clinicians to treat conditions that cause it to be hyperactive or ineffective. More studies are needed to determine if SKAP2 works the same way in human neutrophils and if it works against all types of K. pneumoniae. If it does, then scientists might be able use this information to develop therapies that help the immune system fight infections.

Role for the Abi/wave protein complex in T cell receptor-mediated proliferation and cytoskeletal remodeling.

BACKGROUND: The molecular reorganization of signaling molecules after T cell receptor (TCR) activation is accompanied by polymerization of actin at the site of contact between a T cell and an antigen-presenting cell (APC), as well as extension of actin-rich lamellipodia around the APC. Actin polymerization is critical for the fidelity and efficiency of the T cell response to antigen. The ability of T cells to polymerize actin is critical for several steps in T cell activation including TCR clustering, mature immunological synapse formation, calcium flux, IL-2 production, and proliferation. Activation of the Rac GTPase has been linked to regulation of actin polymerization after TCR stimulation. However, the molecules required for TCR-mediated actin polymerization downstream of activated Rac have remained elusive. Here we identify a novel role for the Abi/Wave protein complex, which signals downstream of activated Rac, in the regulation of actin polymerization and T cell activation in response to TCR stimulation. RESULTS: Here we show that Abi and Wave rapidly translocate from the T cell cytoplasm to the T cell:B cell contact site in the presence of antigen. Abi and Wave colocalize with actin at the T cell:B cell conjugation site. Moreover, Wave and Abi are necessary for actin polymerization after T cell activation, and loss of Abi proteins in mice impairs TCR-induced cell proliferation and IL-2 production in primary T cells. Significantly, the impairment in actin polymerization in cells lacking Abi proteins is due to the inability of Wave proteins to localize to the T cell:B cell contact site in the presence of antigen, rather than the destabilization of the components of the Wave protein complex. CONCLUSIONS: The Abi/Wave complex is a novel regulator of TCR-mediated actin dynamics, IL-2 production, and proliferation.

Vitamin E reverses impaired linker for activation of T cells activation in T cells from aged C57BL/6 mice.

Supplemental vitamin E alleviates age-related defects in interleukin (IL)-2 production, T cell proliferation, and immune synapse formation. Here, we evaluated the effect of in vitro supplementation with 46 mumol/L of vitamin E on T cell receptor-proximal signaling events of CD4(+) T cells from young (4-6 mo) and old (22-26 mo) C57BL mice. Aged murine CD4(+) T cells stimulated via CD3 and CD28, tyrosine 191 of the adaptor protein Linker for Activation of T cells (LAT), was hypo-phosphorylated. Supplementation with vitamin E eliminated this difference in the tyrosine phosphorylation of LAT. By using a flow cytometric assay, the age-related differences in the activation-induced phosphorylation of LAT were observed in both naïve and memory T cell subsets. In addition, supplementation with vitamin E eliminates the age-related differences in LAT phosphorylation in both T cell subsets. Neither age nor vitamin E supplementation altered the fraction of LAT entering the membrane compartment. Furthermore, neither age nor vitamin E influenced the phosphorylation of Lck and Zap70, indicating that associated changes in LAT phosphorylation were not caused by alterations in activation states of the upstream kinases Lck and Zap70.

T cell receptor ligation induces the formation of dynamically regulated signaling assemblies.

Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein-GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70-containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.

Persistence of cooperatively stabilized signaling clusters drives T-cell activation.

Antigen recognition triggers the recruitment of the critical adaptor protein SLP-76 to small macromolecular clusters nucleated by the T-cell receptor (TCR). These structures develop rapidly, in parallel with TCR-induced increases in tyrosine phosphorylation and cytosolic calcium, and are likely to contribute to TCR-proximal signaling. Previously, we demonstrated that these SLP-76-containing clusters segregate from the TCR and move towards the center of the contact interface. Neither the function of these clusters nor the structural requirements governing their persistence have been examined extensively. Here we demonstrate that defects in cluster assembly and persistence are associated with defects in T-cell activation in the absence of Lck, ZAP-70, or LAT. Clusters persist normally in the absence of phospholipase C-gamma1, indicating that in the absence of a critical effector, these structures are insufficient to drive T-cell activation. Furthermore, we show that the critical adaptors LAT and Gads localize with SLP-76 in persistent clusters. Mutational analyses of LAT, Gads, and SLP-76 indicated that multiple domains within each of these proteins contribute to cluster persistence. These data indicate that multivalent cooperative interactions stabilize these persistent signaling clusters, which may correspond to the functional complexes predicted by kinetic proofreading models of T-cell activation.