Altered Actin Centripetal Retrograde Flow in Physically Restricted Immunological Synapses

Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3ε on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network.     

Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells

Actin retrograde flow and actomyosin II contraction have both been implicated in the inward movement of T cell receptor (TCR) microclusters and immunological synapse formation, but no study has integrated and quantified their relative contributions. Using Jurkat T cells expressing fluorescent myosin IIA heavy chain and F-tractin-a novel reporter for F-actin-we now provide direct evidence that the distal supramolecular activation cluster (dSMAC) and peripheral supramolecular activation cluster (pSMAC) correspond to lamellipodial (LP) and lamellar (LM) actin networks, respectively, as hypothesized previously. Our images reveal concentric and contracting actomyosin II arcs/rings at the LM/pSMAC. Moreover, the speeds of centripetally moving TCR microclusters correspond very closely to the rates of actin retrograde flow in the LP/dSMAC and actomyosin II arc contraction in the LM/pSMAC. Using cytochalasin D and jasplakinolide to selectively inhibit actin retrograde flow in the LP/dSMAC and blebbistatin to selectively inhibit actomyosin II arc contraction in the LM/pSMAC, we demonstrate that both forces are required for centripetal TCR microcluster transport. Finally, we show that leukocyte function-associated antigen 1 clusters accumulate over time at the inner aspect of the LM/pSMAC and that this accumulation depends on actomyosin II contraction. Thus actin retrograde flow and actomyosin II arc contraction coordinately drive receptor cluster dynamics at the immunological synapse.     

Myosin IIA modulates T cell receptor transport and CasL phosphorylation during early immunological synapse formation

Activation of T cell receptor (TCR) by antigens occurs in concert with an elaborate multi-scale spatial reorganization of proteins at the immunological synapse, the junction between a T cell and an antigen-presenting cell (APC). The directed movement of molecules, which intrinsically requires physical forces, is known to modulate biochemical signaling. It remains unclear, however, if mechanical forces exert any direct influence on the signaling cascades. We use T cells from AND transgenic mice expressing TCRs specific to the moth cytochrome c 88-103 peptide, and replace the APC with a synthetic supported lipid membrane. Through a series of high spatiotemporal molecular tracking studies in live T cells, we demonstrate that the molecular motor, non-muscle myosin IIA, transiently drives TCR transport during the first one to two minutes of immunological synapse formation. Myosin inhibition reduces calcium influx and colocalization of active ZAP-70 (zeta-chain associated protein kinase 70) with TCR, revealing an influence on signaling activity. More tellingly, its inhibition also significantly reduces phosphorylation of the mechanosensing protein CasL (Crk-associated substrate the lymphocyte type), raising the possibility of a direct mechanical mechanism of signal modulation involving CasL.     

Effects of Intracellular Calcium and Actin Cytoskeleton on TCR Mobility Measured by Fluorescence Recovery

Background

The activation of T lymphocytes by specific antigen is accompanied by the formation of a specialized signaling region termed the immunological synapse, characterized by the clustering and segregation of surface molecules and, in particular, by T cell receptor (TCR) clustering.

Methodology/Principal Findings

To better understand TCR motion during cellular activation, we used confocal microscopy and photo-bleaching recovery techniques to investigate the lateral mobility of TCR on the surface of human T lymphocytes under various pharmacological treatments. Using drugs that cause an increase in intracellular calcium, we observed a decrease in TCR mobility that was dependent on a functional actin cytoskeleton. In parallel experiments measurement of filamentous actin by FACS analysis showed that raising intracellular calcium also causes increased polymerization of the actin cytoskeleton. These in vitro results were analyzed using a mathematical model that revealed effective binding parameters between TCR and the actin cytoskeleton.

Conclusion/Significance

We propose, based on our results, that increase in intracellular calcium levels leads to actin polymerization and increases TCR/cytoskeleton interactions that reduce the overall mobility of the TCR. In a physiological setting, this may contribute to TCR re-positioning at the immunological synapse.     

Tec kinases: shaping T-cell activation through actin

Following stimulation, T cells undergo marked actin-dependent changes in shape that are required for productive cellular interactions and movement during immune responses. Reorganization of the actin cytoskeletal is also necessary for the formation of an immunological synapse - the convergence of several signaling molecules at the plasma membrane that occurs after effective T-cell receptor (TCR) signaling. Much emerging evidence indicates that the Tec family of tyrosine kinases has a role in actin cytoskeleton reorganization. Specifically, T cells that lack or express mutant versions of the Tec kinase Itk show impaired TCR-induced actin polymerization, cell polarization and regulation of the signaling events involved in cytoskeletal reorganization. These data, as well as other findings, support roles for Tec kinases in actin cytoskeleton regulation.     

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.     

Dynamic regulation of T cell activation and co-stimulation through TCR-microclusters

TCR-microclusters (MC) are generated upon TCR stimulation prior to the immune synapse formation independently of lipid rafts. TCR-MCs contain receptors, kinases and adaptors, and function as the signaling unit for T cell activation. The TCR complex, but not the signaling molecules, is transported to the center to form cSMAC. The co-stimulation receptor CD28 joins the signaling region of cSMAC and recruits PKCθ and Carma1. CTLA-4 accumulates in the same region and competes with CD28 for negative regulation of T cell activation. T cell activation is therefore mediated by two spatially distinct signaling compartments: TCR signaling by the peripheral TCR-MC and co-stimulation signal by the central signaling cSMAC.     

Lateral compartmentalization of T cell receptor versus CD45 by galectin-N-glycan binding and microfilaments coordinate basal and activation signaling

Lateral compartmentalization of membrane proteins into microdomains regulates signal transduction; however, structural determinants are incompletely understood. Membrane glycoproteins bind galectins in proportion to the number (i.e. NX(S/T) sites) and degree of GlcNAc branching within attached N-glycans, forming a molecular lattice that negatively regulates T cell function and autoimmunity. We find that in resting T cells, partition of CD45 inside and T cell receptor (TCR)/CD4-Lck/Zap-70 outside microdomains is positively and negatively regulated by the galectin lattice and actin cytoskeleton, respectively. In the absence of TCR ligands, the galectin lattice counteracts F-actin to retain CD45 in microdomains while concurrently blocking TCR/CD4-Lck/Zap-70 partition to microdomains by preventing a conformational change in the TCR that recruits Nck/Wiscott Aldrich Syndrome (WASp)/SLP76/F-actin/CD4 to TCR. The counterbalancing activities of the galectin lattice and actin cytoskeleton negatively and positively regulate Lck activity in resting cells and CD45 versus TCR clustering and signaling at the early immune synapse, respectively. Microdomain-localized CD45 inactivates Lck and inhibits TCR signaling at the early immune synapse. Thus, the galectin lattice and actin cytoskeleton interact on opposing sides of the plasma membrane to control microdomain structure and function, coupling basal growth signaling with thresholds to activation.     

Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP

Actin polymerization at the immune synapse is required for T cell activation and effector function; however, the relevant regulatory pathways remain poorly understood. We showed previously that binding to antigen presenting cells (APCs) induces localized activation of Cdc42 and Wiskott-Aldrich Syndrome protein (WASP) at the immune synapse. Several lines of evidence suggest that Tec kinases could interact with WASP-dependent actin regulatory processes. Since T cells from Rlk-/-, Itk-/-, and Rlk-/- x Itk-/- mice have defects in signaling and development, we asked whether Itk or Rlk function in actin polymerization at the immune synapse. We find that Itk-/- and Rlk-/- x Itk-/- T cells are defective in actin polymerization and conjugate formation in response to antigen-pulsed APCs. Itk functions downstream of the TCR, since similar defects were observed upon TCR engagement alone. Using conformation-specific probes, we show that although the recruitment of WASP and Arp2/3 complex to the immune synapse proceeds normally, the localized activation of Cdc42 and WASP is defective. Finally, we find that the defect in Cdc42 activation likely stems from a requirement for Itk in the recruitment of Vav to the immune synapse. Our results identify Itk as a key element of the pathway leading to localized actin polymerization at the immune synapse.     

Regulation of Vesicular Traffic at the T Cell Immune Synapse: Lessons from the Primary Cilium

The signals that orchestrate the process of T cell activation are coordinated at the specialized interface that forms upon contact with an antigen presenting cell displaying a specific MHC‐associated peptide ligand, known as the immune synapse. The central role of vesicular traffic in the assembly of the immune synapse has emerged only in recent years with the finding that sustained T‐cell receptor (TCR) signaling involves delivery of TCR/CD3 complexes from an intracellular pool associated with recycling endosomes. A number of receptors as well as membrane‐associated signaling mediators have since been demonstrated to exploit this process to localize to the immune synapse. Here, we will review our current understanding of the mechanisms responsible for TCR recycling, with a focus on the intraflagellar transport system, a multimolecular complex that is responsible for the assembly and function of the primary cilium which we have recently implicated in polarized endosome recycling to the immune synapse.      

Tec kinases regulate TCR-mediated recruitment of signaling molecules and integrin-dependent cell adhesion

T cells deficient in the Tec kinases Itk or Itk and Rlk exhibit defective TCR-stimulated proliferation, IL-2 production, and activation of phospholipase C-gamma. Evidence also implicates Tec kinases in actin cytoskeleton regulation, which is necessary for cell adhesion and formation of the immune synapse in T lymphocytes. In this study we show that Tec kinases are required for TCR-mediated up-regulation of adhesion via the LFA-1 integrin. We also demonstrate that the defect in adhesion is associated with defective clustering of LFA-1 and talin at the site of interaction of Rlk-/-Itk-/- and Itk-/- T cells with anti-TCR-coated beads. Defective recruitment of Vav1, protein kinase Ctheta, and Pyk2 was also observed in Rlk-/-Itk-/- and Itk-/- T cells. Stimulation with ICAM-2 in conjunction with anti-TCR-coated beads enhanced polarization of Vav1, protein kinase Ctheta, and Pyk2 in wild-type cells, demonstrating a role for integrins in potentiating the recruitment of signaling molecules in T cells. Increased recruitment of signaling molecules was most pronounced under conditions of low TCR stimulation. Under these suboptimal TCR stimulation conditions, ICAM-2 could also enhance the recruitment of signaling molecules in Itk-/-, but not Rlk-/-Itk-/- T cells. Thus, Tec kinases play key roles in regulating TCR-mediated polarization of integrins and signaling molecules to the site of TCR stimulation as well as the up-regulation of integrin adhesion.     

The immunological synapse: the more you look the less you know..

Before T cells of the immune system can recognize pathogens, antigen presenting cells (APCs) must process pathogen-derived peptides and present them together with major histocompatibility complex molecules (MHC) to T lymphocytes. T lymphocytes then scan the surface of APCs and antigen-specific activation of the T cell will happen after interaction of T cell antigen receptor (TCR) with MHC-peptide complexes expressed at the membrane of APCs. This interaction takes place in a nanometer scale gap between the two cells, referred to as an immunological synapse. Recent three-dimensional fluorescence analysis of this synapse revealed a dynamic spatial organization of membrane receptors, cytoskeleton and intracellular signaling complexes on the T cell side showing specific patterns, which depend on the nature of the T cell:APC pair. Although it is obvious that establishment of an intimate contact between T cells and APCs will facilitate cell:cell communication it is not clear what is the role, if any, of this receptors patterning. This molecular reorganization has long been thought to enhance and/or sustain TCR signaling and thus T cell activation, but this is now a matter of controversy. Moreover, mechanisms controlling immunological synapse formation are still unraveled. Segregation of proteins may occur spontaneously as proposed by mathematical modeling taking into account membrane fluidity, protein size and receptor/ligand affinity. Alternatively patterning of the molecules at the cell:cell interface could be driven by active processes involving T cell signaling and/or specific features of the APC. These different questions will be discussed herein.     

Cross Talk between CD3 and CD28 Is Spatially Modulated by Protein Lateral Mobility

Functional convergence of CD28 costimulation and TCR signaling is critical to T-cell activation and adaptive immunity. These receptors form complex microscale patterns within the immune synapse, although the impact of this spatial organization on cell signaling remains unclear. We investigate this cross talk using micropatterned surfaces that present ligands to these membrane proteins in order to control the organization of signaling molecules within the cell-substrate interface. While primary human CD4⁺ T cells were activated by features containing ligands to both CD3 and CD28, this functional convergence was curtailed on surfaces in which engagement of these two systems was separated by micrometer-scale distances. Moreover, phosphorylated Lck was concentrated to regions of CD3 engagement and exhibited a low diffusion rate, suggesting that costimulation is controlled by a balance between the transport of active Lck to CD28 and its deactivation. In support of this model, disruption of the actin cytoskeleton increased Lck mobility and allowed functional T-cell costimulation by spatially separated CD3 and CD28. In primary mouse CD4⁺ T cells, a complementary system, reducing the membrane mobility increased the sensitivity to CD3-CD28 separation. These results demonstrate a subcellular reaction-diffusion system that allows cells to sense the microscale organization of the extracellular environment.     

LFA-1-mediated T cell costimulation through increased localization of TCR/class II complexes to the central supramolecular activation cluster and exclusion of CD45 from the immunological synapse

T cell activation is associated with a dramatic reorganization of cell surface proteins and associated signaling components into discrete subdomains within the immunological synapse in T cell:APC conjugates. However, the signals that direct the localization of these proteins and the functional significance of this organization have not been established. In this study, we have used wild-type and LFA-1-deficient, DO11.10 TCR transgenic T cells to examine the role of LFA-1 in the formation of the immunological synapse. We found that coengagement of LFA-1 is not required for the formation of the central supramolecular activation cluster (cSMAC) region, but does increase the accumulation of TCR/class II complexes within the cSMAC. In addition, LFA-1 is required for the recruitment and localization of talin into the peripheral supramolecular activation cluster region and exclusion of CD45 from the synapse. The ability of LFA-1 to increase the amount of TCR engaged during synapse formation and segregate the phosphatase, CD45, from the synapse suggests that LFA-1 might enhance proximal TCR signaling. To test this, we combined flow cytometry-based cell adhesion and calcium-signaling assays and found that coengagement of LFA-1 significantly increased the magnitude of the intracellular calcium response following Ag presentation. These data support the idea that in addition to its important role on regulating T cell:APC adhesion, coengagement of LFA-1 can enhance T cell signaling, and suggest that this may be accomplished in part through the organization of proteins within the immunological synapse.     

Synaptic transfer by human gamma delta T cells stimulated with soluble or cellular antigens

B, alpha beta T, and NK lymphocytes establish immunological synapses (IS) with their targets to enable recognition. Transfer of target cell-derived Ags together with proximal molecules onto the effector cell appears also to occur through synapses. Little is known about the molecular basis of this transfer, but it is assumed to result from Ag receptor internalization. Because human gamma delta T cells recognize soluble nonpeptidic phosphoantigens as well as tumor cells such as Daudi, it is unknown whether they establish IS with, and extract molecules from, target cells. Using flow cytometry and confocal microscopy, we show in this work that Ag-stimulated human V gamma 9/V delta 2 T cells conjugate to, and perform molecular transfer from, various tumor cell targets. The molecular transfer appears to be linked to IS establishment, evolves in a dose-dependent manner in the presence of either soluble or cellular Ag, and requires gamma delta TCR ligation, Src family kinase signaling, and participation of the actin cytoskeleton. Although CD45 exclusion characterized the IS performed by gamma delta T cells, no obvious capping of the gamma delta TCR was detected. The synaptic transfer mediated by gamma delta T cells involved target molecules unrelated to the cognate Ag and occurred independently of MHC class I expression by target cells. From these observations, we conclude that despite the particular features of gamma delta T cell activation, both synapse formation and molecular transfer of determinants belonging to target cell characterize gamma delta T cell recognition of Ags.     

Recruitment of the actin-binding protein HIP-55 to the immunological synapse regulates T cell receptor signaling and endocytosis

Actin cytoskeleton dynamics critically regulate T cell activation. We found that the cytoplasmic adaptor HIP-55, a Src/Syk-kinases substrate and member of the drebrin/Abp1 family of actin-binding proteins, localized to the T cell-antigen-presenting cell (APC) contact site in an antigen-dependent manner. Using green fluorescent protein fusion proteins, both Src homology 3 (SH3) and actin binding domains were found necessary for recruitment at the T cell-APC interface. HIP-55 was not implicated in conjugate formation and actin polymerization but regulated distal signaling events through binding and activation of hematopoietic progenitor kinase 1 (HPK1), a germinal center kinase (GCK) family kinase involved in negative signaling in T cells. Using RNA interference and overexpression experiments, the HIP-55-HPK1 complex was found to negatively regulate nuclear factor of activated T cell (NFAT) activation by the T cell antigen receptor. Moreover, we show that HIP-55, which partly co-localized with early endocytic compartments, promoted both basal and ligand-dependent T cell receptor (TCR) down-modulation, resulting in a decreased TCR expression. SH3 and actin-depolymerizing factor homology domains were required for this function. As controls, the expression of CD28 and the glycosylphosphatidylinositol-linked protein CD59 was not affected by HIP-55 overexpression. These results suggest that, in addition to binding to HPK1, HIP-55 might negatively regulate TCR signaling through down-regulation of TCR expression. Our findings show that HIP-55 is a key novel component of the immunological synapse that modulates T cell activation by connecting actin cytoskeleton and TCRs to gene activation and endocytic processes.     

Peptide-specific intercellular transfer of MHC class II to CD4+ T cells directly from the immunological synapse upon cellular dissociation

The transfer of membrane proteins from APC to T cells was initially described in the 1970s, and subsequent work has described two mechanisms of transfer: APC-derived exosomes and direct transfer of small packets, while cells remain conjugated. Using fibroblast APC expressing a GFP-tagged I-E(k) molecule with covalently attached antigenic peptide, we observed a third mechanism in live cell imaging: T cells spontaneously dissociating from APC often capture MHC:peptide complexes directly from the immunological synapse. Using two I-E(k)-restricted murine TCR transgenic T cells with different peptide specificity, we show in this study that the MHC transfer is peptide specific. Using blocking Abs, we found that MHC:peptide transfer in this system requires direct TCR-MHC:peptide interactions and is augmented by costimulation through CD28-CD80 interactions. Capture of the GFP-tagged MHC:peptide complexes correlates with an activated phenotype of the T cell, elevated CD69 with down-modulated TCR. The transferred MHC:peptide molecules transferred to the T cell are associated with molecules that imply continued TCR signaling; p56(lck), phosphotyrosine, and polarization of the actin cytoskeleton.     

Immunological synapse formation licenses CD40-CD40L accumulations at T-APC contact sites

The maintenance of tolerance is likely to rely on the ability of a T cell to polarize surface molecules providing "help" to only specific APCs. The formation of a mature immunological synapse leads to concentration of the TCR at the APC interface. In this study, we show that the CD40-CD154 receptor-ligand pair is also highly concentrated into a central region of the synapse on mouse lymphocytes only after the formation of the TCR/CD3 c-SMAC. Concentration of this ligand was strictly dependent on TCR recognition, the binding of ICAM-1 to T cell integrins and the presence of an intact cytoskeleton in the T cells. This may provide a novel explanation for the specificity of T cell help directing the help signal to the site of Ag receptor signal. It may also serve as a site for these molecular aggregates to coassociate and/or internalize alongside other signaling receptors.     

Lipid rafts mediate association of LFA-1 and CD3 and formation of the immunological synapse of CTL

Lipid rafts accumulate in the immunological synapse formed by an organized assembly of the TCR/CD3, LFA-1, and signaling molecules. However, the precise role of lipid rafts in the formation of the immunological synapse is unclear. In this study, we show that LFA-1 on CTL is constitutively active and mediates Ag-independent binding of CTL to target cells expressing its ligands. LFA-1 and CD3 on CTL, but not resting T cells, colocalize in lipid rafts. Binding of LFA-1 on CTL to targets initiates the formation of the immunological synapse, which is formed by LFA-1, CD3, and ganglioside GM1 distributed in the periphery of the cell contact site and cholesterol is more widely distributed. The formation of this synapse is Ag independent, but the recognition of Ag by the TCR induces accumulation of tyrosine phosphorylated proteins in the synapse as well as redistribution of the microtubule organization center toward the cell contact site. Our results suggest that LFA-1 recruits lipid rafts and the TCR/CD3 to the synapse, and facilitates efficient and rapid activation of CTL.