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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 (α4β1 integrin) and LFA-1 (αLβ2 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 β2, kindlin-3 and zyxin are required for complete centripetal transport, while integrin β1 and talin-1 are not. CD69 upregulation is similarly dependent on integrin β2, 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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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 ., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

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., Competing Interests: GN, LS, MM, KS, SB, DS, JM No competing interests declared, (© 2020, Nguyen et al.)

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 Ca 2+ entry. By using a Vav1-deficient Jurkat T cell line, we find that Vav1 facilitates Ca 2+ 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 Ca 2+ 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 Ca 2+ 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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Schistosomiasis is a major helminthic disease in which damage to the affected organs is orchestrated by a pathogenic host CD4 T helper (Th) cell-mediated immune response against parasite eggs. In the case of the species Schistosoma mansoni , the resulting granulomatous inflammation and fibrosis takes place in the liver and intestines. The magnitude of disease varies greatly from individual to individual but in a minority of patients, there is severe disease and death. S. mansoni infection in a murine model similarly results in marked strain variation of immunopathology. In the most commonly examined mouse strain, C57BL/6 (BL/6), there is relatively mild hepatic pathology arising in a Th2-dominated cytokine environment. In contrast, CBA mice develop decisively more severe lesions largely driven by proinflammatory IL-17-producing Th17 cells. Dendritic cells (DCs) from CBA mice differ sharply with those from BL/6 mice in that they vastly over-express the C-type lectin receptor (CLR) CD209a (SIGNR5), a homolog of human DC-SIGN, which senses glycans such as those produced by schistosome eggs. Silencing of CD209a, and recent studies with CD209a KO CBA mice have shown that this receptor is crucial to induce the pathogenic Th17 cell response; indeed, CD209a KO mice display markedly reduced immunopathology akin to that seen in BL/6 mice. Mechanistically, CD209a synergizes with the related CLRs Dectin-2 and Mincle to stimulate increased DC production of IL-1β and IL-23, necessary for pathogenic Th17 cell development. These findings denote key molecular underpinnings of disease variability based on selection and function of contrasting Th cell subsets.

Cell death and inflammation are intimately linked during Yersinia infection. Pathogenic Yersinia inhibits the MAP kinase TGFβ-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/β release. Furthermore, we found that human macrophages are resistant to YopJ-mediated pyroptosis, with dampened IL-1β 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., Competing Interests: The authors declare no conflict of interest.

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, Ca 2+ -competent microclusters into stable adhesive junctions with enhanced signaling potential., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The immunopathology caused by schistosome helminths varies greatly in humans and among mouse strains. A severe form of parasite egg-induced hepatic granulomatous inflammation, seen in CBA mice, is driven by Th17 cells stimulated by IL-1β and IL-23 produced by dendritic cells that express CD209a (SIGNR5), a C-type lectin receptor (CLR) related to human DC-SIGN. Here, we show that CD209a-deficient CBA mice display decreased Th17 responses and are protected from severe immunopathology. In vitro, CD209a augments the egg-induced IL-1β and IL-23 production initiated by the related CLRs Dectin-2 and Mincle. While Dectin-2 and Mincle trigger an FcRγ-dependent signaling cascade that involves the tyrosine kinase Syk and the trimolecular Card9-Bcl10-Malt1 complex, CD209a promotes the sustained activation of Raf-1. Our findings demonstrate that CD209a drives severe Th17 cell-mediated immunopathology in a helminthic disease based on synergy between DC-SIGN- and Dectin-2-related CLRs., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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 α3β1. 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 β1 play a role in the activation of the Src kinase pathway. However, only integrin β1 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., (Copyright © 2017 American Society for Microbiology.)

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., (Copyright © 2015 by The American Association of Immunologists, Inc.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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-1β 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-1β 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.

Phospholipase C-γ1 (PLC-γ1) is a key regulator of T cell receptor (TCR)-induced signaling. Activation of the TCR enhances PLC-γ1 enzymatic function, resulting in calcium influx and the activation of PKC family members and RasGRP. The current model is that phosphorylation of LAT tyrosine 132 facilitates the recruitment of PLC-γ1, leading to its activation and function at the LAT complex. In this study, we examined the phosphorylation kinetics of LAT and PLC-γ1 and the cellular localization of activated PLC-γ1. We observed that commencement of the phosphorylation of LAT tyrosine 132 and PLC-γ1 tyrosine 783 occurred simultaneously, supporting the current model. However, once begun, PLC-γ1 activation occurred more rapidly than LAT tyrosine 132. The association of LAT and PLC-γ1 was more transient than the interaction of LAT and Grb2 and a pool of activated PLC-γ1 translocated away from LAT to cellular structures containing the TCR. These studies demonstrate that LAT and PLC-γ1 form transient interactions that catalyze the activation of PLC-γ1, but that activated PLC-γ1 resides in both LAT and TCR clusters. Together, this work highlights that our current model is incomplete and the activation and function of PLC-γ1 in T cells is highly complex., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The T cell receptor (TCR) triggers the assembly of "SLP-76 microclusters," which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase-associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A "tandem dimer" containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP-interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and "inside-out" signaling to β1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and β1 integrins.

To determine whether changes in sphingolipid composition are associated with age-related immune dysfunction, we analyzed the core sphingolipidome (i.e., all of the metabolites through the first headgroup additions) of young and aged CD4(+) T cells. Since sphingolipids influence the biophysical properties of membranes, we evaluated the compositions of immune synapse (IS) and non-IS fractions prepared by magnetic immuno-isolation. Broadly, increased amounts of sphingomyelins, dihydrosphingomyelins and ceramides were found in aged CD4(+) T cells. After normalizing for total sphingolipid content, a statistically significant decrease in the molar fraction of glucosylceramides was evident in both the non-IS and IS fractions of aged T cells. This change was balanced by less dramatic increases in the molar fractions of sphingomyelins and dihydrosphingomyelins in aged CD4(+) T cells. In vitro, the direct or enzymatic enhancement of ceramide levels decreased CD4(+) T cell proliferation without regard for the age of the responding T cells. In contrast, the in vitro inhibition of glucosylceramidase preferentially increased the proliferation of aged CD4(+) T cells. These results suggest that reductions in glucosylceramide abundance contribute to age-related impairments in CD4(+) T cell function.

The guanine nucleotide exchange factor (GEF) Vav1 synergizes with the adaptor protein SLP-76 (Src homology 2 domain--containing leukocyte phosphoprotein of 76 kD) to support T cell development and activation. In response to ligation of the T cell receptor (TCR), SLP-76 is assembled into microclusters that provide an essential platform for the signaling events that drive T cell activation. We found that Vav1 selectively entered SLP-76 microclusters, rather than TCR microclusters, influencing their stability and function. The carboxyl terminus of Vav1, which consists of Src homology domains, was both necessary and sufficient for the entry of Vav1 into SLP-76 microclusters; however, this fragment of Vav1 was insufficient to stabilize the microclusters, and it potently suppressed T cell activation. This indicated that the amino terminus of Vav1, which has the GEF domain, also contributed to the integrity of SLP-76 microclusters and thereby to T cell activation. These microcluster-stabilizing functions were independent of the GEF activity in the amino terminus of Vav1 and were unaffected if the GEF function of Vav1 was either inactivated or constitutively activated by mutation. In contrast, Vav1 deletion mutants lacking either the calponin homology domain or the catalytic core of the GEF exhibited mild scaffolding defects, but they differentially affected TCR-dependent calcium ion (Ca²+) responses. We conclude that multiple GEF-independent scaffolding functions distributed throughout the amino terminus of Vav1 contribute to the activation of T cells by acting synergistically to increase the stability and function of SLP-76 microclusters.

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.

Interference reflection microscopy (IRM) is an optical technique used to study cell adhesion or cell mobility on a glass coverslip. The interference of reflected light waves generates images with high contrast and definition. IRM can be used to examine almost any cell that will rest upon a glass surface, although it is most useful in examining sites of close contact between a cell and substratum. This unit presents methods for obtaining IRM images of cells with particular emphasis on IRM imaging with a laser scanning confocal microscope (LSCM), as most LSCM are already capable of recording these images without any modification of the instrument. Techniques are presented for imaging fixed and live cells, as well as simultaneous multi-channel capture of fluorescence and reflection images., (Copyright 2009 by John Wiley & Sons, Inc.)

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.

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.

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.

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.

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.

Upon T cell receptor engagement, both the actin cytoskeleton and substrates of tyrosine phosphorylation are remodeled to create a signaling complex at the interface of the antigen-presenting cell and responding T cell. While T cell signaling has been shown to regulate actin reorganization, the mechanisms by which changes in actin dynamics affect early T cell signaling have not been fully explored. Using gelsolin, an actin-binding protein with capping and severing activities, and latrunculin, an actin-depolymerizing agent, we have further investigated the interplay between actin dynamics and the regulation of T cell signaling. Overexpression of gelsolin altered actin dynamics in Jurkat T cells, and alteration of actin dynamics correlated with dysregulation of tyrosine phosphorylation of raft-associated substrates. This perturbation of tyrosine phosphorylation was correlated with inhibition of activation-dependent signaling pathways regulating Erk-1/2 phosphorylation, NF-AT transcriptional activation and IL-2 production. Modification of actin by the depolymerizing agent latrunculin also altered the tyrosine phosphorylation patterns of proteins associated with lipid rafts, and pre-treatment with latrunculin inhibited anti-CD3 mAb-mediated NF-AT activation. Thus, our data indicate that actin cytoskeletal dynamics modulate the tyrosine phosphorylation of raft-associated proteins and subsequent downstream signal transduction.

Aging is associated with reduced IL-2 production and T cell proliferation. Vitamin E supplementation, in aged animals and humans, increases cell division and IL-2 production by naive T cells. The immune synapse forms at the site of contact between a T cell and an APC and participates in T cell activation. We evaluated whether vitamin E affects the redistribution of signaling proteins to the immune synapse. Purified CD4(+) T cells, from the spleens of young and old mice, were treated with vitamin E before stimulation with a surrogate APC expressing anti-CD3. Using confocal fluorescent microscopy, we observed that CD4(+) T cells from old mice were significantly less likely to recruit signaling proteins to the immune synapse than cells from young mice. Vitamin E increased the percentage of old CD4(+) T cells capable of forming an effective immune synapse. Similar results were found following in vivo supplementation with vitamin E. When compared with memory cells, naive T cells from aged mice were more defective in immune synapse formation and were more responsive to vitamin E supplementation. These data show, for the first time, that vitamin E significantly improves age-related early T cell signaling events in naive CD4(+) T cells.

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.

T-cell antigen receptor engagement causes the rapid assembly of signaling complexes. The adapter protein SLP-76, detected as SLP-yellow fluorescent protein, initially clustered with the TCR and other proteins, then translocated medially on microtubules. As shown by total internal reflection fluorescence microscopy and the inhibition of SLP-76 movement at 16 degrees C, this movement required endocytosis. Immunoelectron microscopy showed SLP-76 staining of smooth pits and tubules. Cholesterol depletion decreased the movement of SLP-76 clusters, as did coexpression of the ubiquitin-interacting motif domain from eps15. These data are consistent with the internalization of SLP-76 via a lipid raft-dependent pathway that requires interaction of the endocytic machinery with ubiquitinylated proteins. The endocytosed SLP-76 clusters contained phosphorylated SLP-76 and phosphorylated LAT. The raft-associated, transmembrane protein LAT likely targets SLP-76 to endocytic vesicles. The endocytosis of active SLP-76 and LAT complexes suggests a possible mechanism for downregulation of signaling complexes induced by TCR activation.

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.

T cell receptor (TCR) engagement leads to actin polymerization at the site of T cell contact with antigen-presenting cells. Here we have studied the dynamic activity of proteins involved in regulating actin polymerization in live T cells after activation. Two such adaptor proteins, Nck and the Wiskott-Aldrich syndrome protein (WASp), were recruited to the TCR during initial T cell activation, where they colocalized with the tyrosine kinase Zap70. The recruitment of Nck and WASp depended on TCR-induced tyrosine phosphorylation and the LAT and SLP-76 adaptors. Nck and WASp migrated peripherally and accumulated at an actin-rich circumferential ring. Thus, actin polymerization regulated by the TCR begins at the TCR. Molecules recruited to the TCR regulate actin polymerization and this process drives plasma membrane movement and cellular spreading.

Phosphoinositide 3-kinase (PI3K) is important in TCR signaling. PI3K generates phosphatidylinositol 3, 4, 5-trisphosphate (PI-3,4,5-P3), which regulates membrane localization and/or activity of multiple signaling proteins. PTEN (phosphatase and tensin homologue deleted on chromosome 10) opposes PI3K, reversing this reaction. Maintaining the balance between these two enzymes is important for normal T cell function. Here we use the PTEN-null Jurkat T cell line to address the role of PTEN in modulating proximal and distal TCR-signaling events. PTEN expression at levels that restored low basal Akt phosphorylation (an indicator of PI-3,4,5-P3 levels), but which were not themselves cytotoxic, had minimal effect on TCR-stimulated activation of phospholipase Cgamma1 and Ca2+ flux, but reduced the duration of extracellular signal-regulated kinase (Erk) activation. Distal signaling events, including nuclear factor of activated T cells (NFAT) activation, CD69 expression and IL-2 production, were all inhibited by PTEN expression. Notably, PTEN did not block TCR-stimulated PI-3,4,5-P3 accumulation. The effect of PTEN on distal TCR signaling events was strongly correlated with the loss of the constitutive Akt activation and glycogen synthase kinase-3 (GSK3) inhibition that is typical of Jurkat cells, and could be reversed by expression of activated Akt or pharmacologic inhibition of GSK3. These results suggest that PTEN acts in T cells primarily to control basal PI-3,4,5-P3 levels, rather than opposing PI3K acutely during TCR stimulation.

The adaptor protein Src homology (SH)2 domain-containing and leukocyte-specific phosphoprotein of 76 kDa (SLP-76) is critical for signal transduction in multiple hematopoietic lineages. It links proximal and distal T cell receptor signaling events through its function as a molecular scaffold in the assembly of multimolecular signaling complexes. Here we studied the functional roles of sub-domains within the SLP-76 proline-rich region, specifically the Gads binding domain and the recently defined P1 domain. To gain a further understanding of the functions mediated by this region, we used three complementary approaches as follows: reconstitution of SLP-76-deficient cells with functional domain deletion mutants, blocking molecular associations through the expression of a dominant negative protein fragment, and directed localization of SLP-76 to assess the role of the domains in SLP-76 recruitment. We find the Gads binding domain and the P1 domain are both necessary for optimal SLP-76 function, and in the absence of these two regions, SLP-76 is functionally inert. Furthermore, we provide direct evidence that SLP-76 localization and, in turn, function are dependent upon association with Gads.

The dynamic visualization of developing immunological synapses has been hindered by the difficulty of imaging the contact between the T cell and the antigen-presenting cell (APC). Here, we describe a technique in which T cell responses are constrained to a planar stimulatory substrate. This approach, when used in conjunction with immunofluorescent staining procedures or fluorescent protein tags, greatly facilitates detection of the dynamic molecular rearrangements that accompany the formation of contacts and the initiation of signal transduction through the T cell receptor (TCR). Using this method, we have observed signaling complexes of dynamically varying compositions that possess distinct fates.

In T lymphocytes, the "novel" protein kinase C theta (PKC theta) isoform and the transcription factor nuclear factor-kappaB (NF-kappaB) are required for cell proliferation and the production of cytokines in response to T cell activation; however, the molecular interactions that link PKCtheta and NF-kappaB have remained unknown. Two recent reports demonstrate that CARMA1 (caspase recruitment domain-containing membrane-associated guanylate kinase protein-1) bridges the gap between PKCtheta and the IkappaB kinase (IKK)-dependent activation of NF-kappaB in T cells. Excessive T lymphocyte activation and proliferation are hallmarks of T cell-derived leukemias. Given that CARMA1 is specifically expressed in lymphoid tissues, could pharmacological inhibitors be designed to inhibit CARMA1's (or PKCtheta's) ability to promote the activation of NF-kappaB?

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.

T cell activation induces functional changes in cell shape and cytoskeletal architecture. To facilitate the collection of dynamic, high-resolution images of activated T cells, we plated T cells on coverslips coated with antibodies to the T cell receptor (TCR). Using these images, we were able to quantitate the morphological responses of individual cells over time. Here, we show that TCR engagement triggers the formation and expansion of contacts bounded by continuously remodeled actin-rich rings. These processes are associated with the extension of lamellipodia and require actin polymerization, tyrosine kinase activation, cytoplasmic calcium increases, and LAT, an important hematopoietic adaptor. In addition, the maintenance of the resulting contact requires sustained calcium influxes, an intact microtubule cytoskeleton, and functional LAT.

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P(2) and PI-3,4,5-P(3) in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-gamma1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.

Itk, a Tec family tyrosine kinase, acts downstream of Lck and phosphatidylinositol 3'-kinase to facilitate T cell receptor (TCR)-dependent calcium influxes and increases in extracellular-regulated kinase activity. Here we demonstrate interactions between Itk and crucial components of TCR-dependent signaling pathways. First, the inositide-binding pocket of the Itk pleckstrin homology domain directs the constitutive association of Itk with buoyant membranes that are the primary site of TCR activation and are enriched in both Lck and LAT. This association is required for the transphosphorylation of Itk. Second, the Itk proline-rich region binds to Grb2 and LAT. Third, the Itk Src homology (SH3) 3 and SH2 domains interact cooperatively with Syk-phosphorylated SLP-76. Notably, SLP-76 contains a predicted binding motif for the Itk SH2 domain and binds to full-length Itk in vitro. Finally, we show that kinase-inactive Itk can antagonize the SLP-76-dependent activation of NF-AT. The inhibition of NF-AT activation depends on the Itk pleckstrin homology domain, proline-rich region, and SH2 domain. Together, these observations suggest that multivalent interactions recruit Itk to LAT-nucleated signaling complexes and facilitate the activation of LAT-associated phospholipase Cgamma1 by Itk.

Itk, a Tec family tyrosine kinase, plays an important but as yet undefined role in T cell receptor (TCR) signaling. Here we show that T cells from Itk-deficient mice have a TCR-proximal signaling defect, resulting in defective interleukin 2 secretion. Upon TCR stimulation, Itk-/- T cells release normal amounts of calcium from intracellular stores, but fail to open plasma membrane calcium channels. Since thapsigargin-induced store depletion triggers normal calcium entry in Itk-/- T cells, an impaired biochemical link between store depletion and channel opening is unlikely to be responsible for this defect. Biochemical studies indicate that TCR-induced inositol 1,4,5 tris-phosphate (IP3) generation and phospholipase C gamma1 tyrosine phosphorylation are substantially reduced in Itk-/- T cells. In contrast, TCR-zeta and ZAP-70 are phosphorylated normally, suggesting that Itk functions downstream of, or in parallel to, ZAP-70 to facilitate TCR-induced IP3 production. These findings support a model in which quantitative differences in cytosolic IP3 trigger distinct responses, and in which only high concentrations of IP3 trigger the influx of extracellular calcium.

The Tec family tyrosine kinase Itk has been implicated in T cell receptor (TCR) signaling, yet its precise role and mechanism of activation remain undefined. To investigate these issues, we examined the biochemical response of Itk to TCR stimulation. We found that Itk is tyrosine-phosphorylated after TCR cross-linking and that this phosphorylation depends on the presence of functional Lck. To determine if this Lck dependence results from direct phosphorylation of Itk by Lck, we generated recombinant Itk and Lck using a baculovirus expression system and used these proteins in subsequent biochemical analyses. We found that Lck phosphorylates Itk upon co-expression in insect cells and, further, that this phosphorylation of Itk results in increased Itk in vitro kinase activity. The major site of Lck phosphorylation on Itk was mapped to the conserved tyrosine (Tyr511) in the activation loop of the Itk kinase domain. Substitution of this tyrosine with phenylalanine abolishes Itk kinase activity in insect cells, indicating that phosphorylation at this site plays a critical role in regulating Itk function.

The T-cell-specific tyrosine kinase Itk is a member of the Tec family of non-receptor tyrosine kinases, and is required for signalling through the T-cell antigen receptor (TCR). The role of Itk in TCR signalling and the manner in which Itk activity is regulated are not well understood. Substrate binding and enzymatic activity of the structurally related Src kinases are regulated by an intramolecular interaction between the Src-homology-2 (SH2) domain and a phosphotyrosine. Although Itk also contains SH3, SH2 and tyrosine kinase domains, it lacks the corresponding regulatory phosphorylation site, and therefore must be regulated by an alternative mechanism. The proline-rich sequence adjacent to the SH3 domain of Tec family kinases contains an SH3 ligand, potentially allowing a different intramolecular interaction. By using multidimensional nuclear magnetic resonance we have determined the structure of a fragment of Itk, confirming that these domains interact intramolecularly. Formation of this intramolecular SH3-ligand complex prevents the Itk SH3 domain and proline-rich region from interacting with their respective protein ligands, Sam68 and Grb-2. We believe that this structure represents the first example of an intramolecular interaction between an SH3 domain and a proline-rich ligand, and has implications for the regulation of Tec family kinases.

The tyrosine kinase Itk/Tsk is a T cell specific analog of Btk, the tyrosine kinase defective in the human immunodeficiency X-linked agammaglobulinemia and in xid mice. T lymphocytes from Itk-deficient mice are refractory to mitogenic stimuli delivered through the T cell receptor (TCR). To gain insights into the biochemical role of Itk, the binding properties of the Itk SH3 domain were examined. An optimal Itk SH3 binding motif was derived by screening biased phage display libraries; peptides based on this motif bound with high affinity and selectivity to the Itk SH3 domain. Initial studies with T cell lysates indicated that the Itk SH3 domain bound Cbl, Fyn, and other tyrosine phosphoproteins from TCR-stimulated Jurkat cells. Under conditions of increased detergent stringency Sam 68, Wiskott-Aldrich Syndrome protein, and hnRNP-K, but not Cbl and Fyn, were bound to the Itk SH3 domain. By examining the ability of different SH3 domains to interact with deletion variants of Sam 68 and WASP, we demonstrated that the Itk-SH3 domain and the SH3 domains of Src family kinases bind to overlapping but distinct sets of proline-rich regions in Sam 68 and WASP.

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.