CD28 engagement promotes actin polymerization through the activation of the small Rho GTPase Cdc42 in human T cells

Engagement of the costimulatory molecule CD28 is an important step in the optimal activation of T cells. Nevertheless, the specific role of CD28 in the formation of the immunological synapse and cytoskeletal changes that occur upon TCR/CD3 complex engagement is still poorly understood. Using Ab-coated surfaces, we show that CD28 engagement in the absence of any other signal induced the formation of cytoplasmic elongations enriched in filamentous actin (F-actin), in this work called filopodia or microspikes. Such structures were specific for engagement of CD28 on mAb-coated surfaces because they could not be observed in surfaces coated with either poly(L-lysine) or anti-CD3 mAb. The signaling pathway coupling CD28 to cytoskeletal rearrangements required Src-related kinase activity and promoted Vav phosphorylation and Cdc42 activation independently of the zeta-chain-associated kinase (ZAP-70). CD28-induced filopodia required Cdc42 GTPase activity, but not the related Rho GTPase Rac1. Moreover, Cdc42 colocalized to areas of increased F-actin. Our results support a specific role for the activation of the small Rho GTPase Cdc42 in the actin reorganization mediated by CD28 in human T cells.     

Induction of Membrane Ceramides: A Novel Strategy to Interfere with T Lymphocyte Cytoskeletal Reorganisation in Viral Immunosuppression

Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase–dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to β1 integrin ligation or αCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics.     

CD28 signals in the immature immunological synapse

T cell recognition of peptide-MHC complexes on APCs results in the aggregation of TCRs at a central supramolecular activation complex (c-SMAC) within a mature immunological synapse. T cells require a second "costimulatory" signal for activation, the most important of which, for naive T cells, is from CD28. However the time at which CD28-derived signals are induced relative to c-SMAC formation is not well understood. In this study, we have assessed the kinetics of CD28 localization and function relative to well-established aspects of c-SMAC formation. CD28 accumulates at the immature synapse alongside the TCR and is likewise enriched at the synapse at the onset of the calcium signal. In addition, using CD28 deficient or reconstituted murine cells in a single-cell recording approach shows that CD28 regulates this signal within seconds of a TCR-mediated rise in intracellular calcium levels. Finally, CD28 exerts effects on both the initiation and stabilization of the synapse in parallel with its effects on the downstream proliferation of T cells. Together, the data show that CD28 functions in the immunological synapse before the formation of the c-SMAC.     

Silencing OCILRP2 leads to intrinsic defects in T cells in response to antigenic stimulation

We have previously demonstrated that OCILRP2 interaction with its ligand NKRP1f provides a co-stimulatory signal for optimal T cell proliferation and IL-2 production. Here, using RNA interference technology, we will demonstrate that silencing OCILRP2 in vivo leads to intrinsic impairment in T cell response to CD3- and CD28-cross-linking as well as antigenic stimulation. OCILRP2-silenced T cells have reduced cell proliferation and IL-2 production, which can be bypassed by PMA and ionomycin treatment. OCILRP2-silenced T cells also failed to undergo TCR capping and had impaired cytoskeleton reorganization. Moreover, in OCILRP2-silenced T cells, tyrosine phosphorylation of Lck was diminished, while tyrosine phosphorylation of linkers for activation of T cells was unchanged. Interestingly, NF-kappaB activation was also impaired as the result of OCILRP2 silencing. Together, our data strongly support a novel role for OCILRP2 C-type lectin in TCR-mediated signal transduction. The observation that OCILRP2 is involved in TCR capping and cytoskeletal organization suggests that OCILRP2-NKRP1f may facilitate lipid rafts and immunological synapse formation during T cell interaction with antigen presenting cells.     

CD3/CD28 costimulation-induced NF-kappaB activation is mediated by recruitment of protein kinase C-theta, Bcl10, and IkappaB kinase beta to the immunological synapse through CARMA1

CARMA1 (also known as CARD11) is a scaffold molecule and contains a caspase-recruitment domain (CARD) and a membrane-associated guanylate kinase-like (MAGUK) domain. It plays an essential role in mediating CD3/CD28 costimulation-induced NF-kappaB activation. However, the molecular mechanism by which CARMA1 mediates costimulatory signals remains to be determined. Here, we show that CARMA1 is constitutively associated with the cytoplasmic membrane. This membrane association is essential for the function of CARMA1, since a mutant of CARMA1, CARMA1(L808P), that is defective in the membrane association cannot rescue CD3/CD28 costimulation-induced NF-kappaB activation in JPM50.6 CARMA1-deficient T cells. Although CD3/CD28 costimulation effectively induces the formation of the immunological synapse in CARMA1-deficient T cells, the recruitment of protein kinase C-theta (PKC-theta), Bcl10, and IkappaB kinase beta (IKKbeta) into lipid rafts of the immunological synapse is defective. Moreover, expression of wild-type CARMA1, but not CARMA1(L808P), restores the recruitment of PKC-theta, Bcl10, and IKKbeta into lipid rafts in CARMA1-deficient T cells. Consistently, expression of a mutant CARMA1, CARMA1(DeltaCD), that cannot associate with Bcl10 failed to restore CD3/CD28 costimulation-induced NF-kappaB activation in JPM50.6 cells, whereas expression of Bcl10-CARMA(DeltaCD) fusion protein effectively restored this NF-kappaB activation. Together, these results indicate that CARMA1 mediates CD3/CD28 costimulation-induced NF-kappaB activation by recruiting downstream signaling components into the immunological synapse.     

Age-related defects in moesin/ezrin cytoskeletal signals in mouse CD4 T cells

Cytoskeletal proteins of the ezrin-radixin-moesin (ERM) family contribute to T cell activation in response to Ag, and also to T cell polarization in response to connective tissue matrix proteins and chemokine gradients. Previous work has shown that T cells from aged mice are defective in their ability to develop molecular linkages between surface macromolecules and the underlying cytoskeletal framework, both for proteins that move to the synapse and those that are excluded from the site of T cell-APC interaction. T cells from aged mice also show defective cytoskeletal rearrangements and lamellipodia formation when placed in contact with slides coated with Abs to the TCR/CD3 complex. In this study, we show that old CD4 T cells differ from young CD4 T cells in several aspects of ERM biochemistry, including ERM phosphorylation and ERM associations with CD44, CD43, and EBP50. In addition, CD4 T cells from aged mice show defects in the Rho GTPase activities known to control ERM function.     

Immune synapses formed with measles virus-infected dendritic cells are unstable and fail to sustain T cell activation

Interaction with dendritic cells (DCs) is considered as central to immunosuppression induced by viruses, including measles virus (MV). Commonly, viral infection of DCs abrogates their ability to promote T cell expansion, yet underlying mechanisms at a cellular level are undefined. We found that MV-infected DCs only subtly differed from LPS-matured with regard to integrin activation, acquisition of a migratory phenotype and motility. Similarly, the organization of MV-DC/T cell interfaces was consistent with that of functional immune synapses with regard to CD3 clustering and MHC class II surface recruitment. The majority of MV-DC/T cell conjugates was, however, unstable and only promoted abortive T cell activation. Thus, MV-infected DCs retain activities required for initiating, but not sustaining T cell conjugation and activation. This is partially rescued if surface expression of the MV glycoproteins on DCs is abolished by infection with a recombinant MV encoding VSV G protein instead, indicating that these contribute directly to synapse destabilization and thereby act as effectors of T cell inhibition.     

The CD3-zeta chimeric antigen receptor overcomes TCR Hypo-responsiveness of human terminal late-stage T cells

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1(+) CD57(+) CD7(-) phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8(+) T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter.     

Targeting CD28, CTLA-4 and PD-L1 Costimulation Differentially Controls Immune Synapses and Function of Human Regulatory and Conventional T-Cells

CD28, CTLA-4 and PD-L1, the three identified ligands for CD80/86, are pivotal positive and negative costimulatory molecules that, among other functions, control T cell motility and formation of immune synapse between T cells and antigen-presenting cells (APCs). What remains incompletely understood is how CD28 leads to the activation of effector T cells (Teff) but inhibition of suppression by regulatory T cells (Tregs), while CTLA-4 and PD-L1 inhibit Teff function but are crucial for the suppressive function of Tregs. Using alloreactive human T cells and blocking antibodies, we show here by live cell dynamic microscopy that CD28, CTLA-4, and PD-L1 differentially control velocity, motility and immune synapse formation in activated Teff versus Tregs. Selectively antagonizing CD28 costimulation increased Treg dwell time with APCs and induced calcium mobilization which translated in increased Treg suppressive activity, in contrast with the dampening effect on Teff responses. The increase in Treg suppressive activity after CD28 blockade was also confirmed with polyclonal Tregs. Whereas CTLA-4 played a critical role in Teff by reversing TCR-induced STOP signals, it failed to affect motility in Tregs but was essential for formation of the Treg immune synapse. Furthermore, we identified a novel role for PD-L1-CD80 interactions in suppressing motility specifically in Tregs. Thus, our findings reveal that the three identified ligands of CD80/86, CD28, CTLA-4 and PD-L1, differentially control immune synapse formation and function of the human Teff and Treg cells analyzed here. Individually targeting CD28, CTLA-4 and PD-L1 might therefore represent a valuable therapeutic strategy to treat immune disorders where effector and regulatory T cell functions need to be differentially targeted.     

Cutting edge: dendritic cell actin cytoskeletal polarization during immunological synapse formation is highly antigen-dependent

Dendritic cells (DC) actively rearrange their actin cytoskeleton to participate in formation of the immunological synapse (IS). In this study, we evaluated the requirements for DC participation in the IS. DC rearrange their actin cytoskeleton toward naive CD4(+) T cells only in the presence of specific MHC-peptide complexes. In contrast, naive CD4(+) T cells polarized their cytoskeletal proteins in the absence of Ag. DC cytoskeletal rearrangement occurred at the same threshold of peptide-MHC complexes as that required for T cell activation. Furthermore, T cell activation was inhibited by specific blockade of DC cytoskeletal rearrangement. When TCR-MHC interaction was bypassed by using Con A-activated T cells, DC polarization was abrogated. In addition, directional ligation of MHC class II resulted in DC cytoskeletal polarization. Our findings suggest that a high Ag specificity is required for DC IS formation and that MHC class II signaling plays a central role in this process.     

Vitamin E, signalosomes and gene expression in T cells

CD4+T cells from aged humans or mice show significant reductions in IL-2 production upon activation. The resulting decreased proliferation is linked to higher risks of infection in the elderly. Several lines of evidence indicate that intrinsic defects preferentially affecting the naïve subset of CD4+T cells contribute to this reduced IL-2 production. Comparison of the biochemical pathways that transduce activation signals from the T cell receptor to the IL-2 promoter in young and old CD4+T cells has demonstrated age-related impairments at initial molecular events, in particular the phosphorylation of kinases and adapter proteins involved in the formation of signalosomes - complex multiprotein assemblies that provide the framework for effective signal transduction. Confocal microscopy has demonstrated a series of age-related impairments in effective immune synapse formation. Vitamin E can reverse many of these CD4+T cell age-associated defects, including reduced levels of phosphorylation of critical signaling/adapter proteins as well as defective immune synapse formation. Vitamin E also enhances IL-2 production, expression of several cell cycle control proteins, and proliferation. Although the precise mechanisms underlying this effect are not understood, it is possible that this antioxidant lipophilic vitamin can prevent the propagation of polyunsaturated fatty acid peroxidation in the cell membrane, influence the biochemical characteristics of specific lipid bilayer microdomains involved in signal transduction, modulate the activity of kinases/phosphatases, or interact with intracellular receptors.     

The dendritic cell cytoskeleton is critical for the formation of the immunological synapse

The binding of a T cell to an APC results in T cell actin cytoskeletal rearrangement leading to the formation of an immunological synapse. The APC cytoskeleton has been thought to play a passive role in this process. In this study, we demonstrate that dendritic cells (DC), unlike other APC, actively polarize their actin cytoskeleton during interaction with T cells. DC cytoskeletal rearrangement was critical for both the clustering and the activation of resting T cells. This study provides compelling evidence that the APC cytoskeleton plays an active role in the immunological synapse and may explain the unique ability of DC to activate resting T cells.     

CD28 interaction with filamin-A controls lipid raft accumulation at the T-cell immunological synapse

During physiological T-cell stimulation by antigen presenting cells (APCs), a major T-cell membrane rearrangement is known to occur leading to the organization of 'supramolecular activation clusters' at the immunological synapse. A possible role for the synapse is the generation of membrane compartments where signalling may be organized and propagated. Thus, engagement of the costimulatory molecule CD28 at the immunological synapse promotes the organization of a signalling compartment by inducing cytoskeletal changes and lipid raft accumulation. We identified the actin-binding protein Filamin-A (FLNa) as a novel molecular partner of CD28. We found that, after physiological stimulation, CD28 associated with and recruited FLNa into the immunological synapse, where FLNa organized CD28 signalling. FLNa knockdown by short interfering RNA (siRNA) inhibited CD28-mediated raft accumulation at the immunological synapse and T-cell costimulation. Together, our data indicate that CD28 binding to FLNa is required to induce the T-cell cytoskeletal rearrangements leading to recruitment of lipid microdomains and signalling mediators into the immunological synapse.     

Human immunodeficiency virus type 1 envelope gp120 induces a stop signal and virological synapse formation in noninfected CD4+ T cells

Human immunodeficiency virus type 1 (HIV-1)-infected T cells form a virological synapse with noninfected CD4⁺ T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T-cell activation and effector functions mediated by the T-cell antigen receptor. The immunological synapse stops T-cell migration to allow a sustained interaction between T-cells and antigen-presenting cells. Here, we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1-infected cell also delivers a stop signal and if this is sufficient to induce a virological synapse. We demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4 T cells that occurs spontaneously in the presence of ICAM-1 and induced the formation of a virological synapse, which was characterized by segregated supramolecular structures with a central cluster of envelope surrounded by a ring of ICAM-1. The virological synapse was formed transiently, with the initiation of migration within 30 min. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in noninfected CD4⁺ T cells.     

Cutting edge: A role for inside-out signaling in TCR regulation of CD28 ligand binding

Efficient T cell activation depends on the engagement of both TCR and CD28, although the molecular mechanisms that control this signal integration are not fully understood. Using fluorescence resonance energy transfer, we show that T cell activation can drive a reorientation of the cytosolic tails of the CD28 dimer. However, this is not mediated through CD28 ligand binding. Rather, TCR signaling itself mediates this conformation change in CD28. We also show that TCR signaling can induce CD28-ligand interactions. Although the CD28 dimer appears to bind ligand monovalently in solution, we show that both ligand binding sites are required to efficiently recruit CD28 to the immunological synapse. These results suggest, that analogous to the cross-talk from TCR that regulates integrin activation, TCR-initiated inside-out signaling may induce a conformational change to the extracellular domains of CD28, enabling ligand binding and initiating CD28 signaling.     

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.     

A molecular framework for two-step T cell signaling: Lck Src homology 3 mutations discriminate distinctly regulated lipid raft reorganization events

Costimulation by CD28 or lipid-raft-associated CD48 potentiate TCR-induced signals, cytoskeletal reorganization, and IL-2 production. We and others have proposed that costimulators function to construct a raft-based platform(s) especially suited for TCR engagement and sustained and processive signal transduction. Here, we characterize TCR/CD48 and TCR/CD28 costimulation in T cells expressing Lck Src homology 3 (SH3) mutants. We demonstrate that Lck SH3 functions after initiation of TCR-induced tyrosine phosphorylation and concentration of transducers within rafts, to regulate the costimulation-dependent migration of rafts to the TCR contact site. Expression of kinase-active/SH3-impaired Lck mutants disrupts costimulation-dependent raft recruitment, sustained TCR protein tyrosine phosphorylation, and IL-2 production. However, TCR-induced apoptosis, shown only to require "partial" TCR signals, is unaffected by expression of kinase-active/SH3-impaired Lck mutants. Therefore, two distinctly regulated raft reorganization events are required for processive and sustained "complete" TCR signal transduction and T cell activation. Together with recent characterization of CD28 and CD48 costimulatory activities, these findings provide a molecular framework for two signal models of T cell activation.     

The lymphocyte receptor CD6 interacts with syntenin-1, a scaffolding protein containing PDZ domains

CD6 is a type I membrane glycoprotein expressed on thymocytes, mature T and B1a lymphocytes, and CNS cells. CD6 binds to activated leukocyte cell adhesion molecule (CD166), and is considered as a costimulatory molecule involved in lymphocyte activation and thymocyte development. Accordingly, CD6 partially associates with the TCR/CD3 complex and colocalizes with it at the center of the mature immunological synapse (IS) on T lymphocytes. However, the signaling pathway used by CD6 is still mostly unknown. The yeast two-hybrid system has allowed us the identification of syntenin-1 as an interacting protein with the cytoplasmic tail of CD6. Syntenin-1 is a PDZ (postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1) domain-containing protein, which functions as an adaptor protein able to bind cytoskeletal proteins and signal transduction effectors. Mutational analyses showed that certain amino acids of the most C-terminal sequence of CD6 (-YDDISAA) and the two postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1 domains of syntenin-1 are relevant to the interaction. Further confirmation of the CD6-syntenin-1 interaction was obtained from pull-down and co-immunoprecipitation assays in mammalian cells. Image analyses also showed that syntenin-1 accumulates at CD6 caps and at the IS. Therefore, we propose that syntenin-1 may function as a scaffolding protein coupling CD6 and most likely other lymphocyte receptors to cytoskeleton and/or signaling effectors during IS maturation.