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.     

Differences in signaling molecule organization between naive and memory CD4+ T lymphocytes

The immunological synapse is a highly organized complex formed at the junction between Ag-specific T cells and APCs as a prelude to cell activation. Although its exact role in modulating T cell signaling is unknown, it is commonly believed that the immunological synapse is the site of cross-talk between the T cell and APC (or target). We have examined the synapses formed by naive and memory CD4 cells during Ag-specific cognate interactions with APCs. We show that the mature immunological synapse forms more quickly during memory T cell activation. We further show that the composition of the synapse found in naive or memory cell conjugates with APCs is distinct with the tyrosine phosphatase, CD45, being a more integral component of the mature synapses formed by memory cells. Finally, we show that signaling molecules, including CD45, are preassociated in discrete, lipid-raft microdomains in resting memory cells but not in naive cells. Thus, enhanced memory cell responses may be due to intrinsic properties of signaling molecule organization.     

Dendritic cell actin dynamics control contact duration and priming efficiency at the immunological synapse

Dendritic cells (DCs) are crucial for the priming of naive T cells and the initiation of adaptive immunity. Priming is initiated at a heterologous cell–cell contact, the immunological synapse (IS). While it is established that F-actin dynamics regulates signaling at the T cell side of the contact, little is known about the cytoskeletal contribution on the DC side. Here, we show that the DC actin cytoskeleton is decisive for the formation of a multifocal synaptic structure, which correlates with T cell priming efficiency. DC actin at the IS appears in transient foci that are dynamized by the WAVE regulatory complex (WRC). The absence of the WRC in DCs leads to stabilized contacts with T cells, caused by an increase in ICAM1-integrin–mediated cell–cell adhesion. This results in lower numbers of activated and proliferating T cells, demonstrating an important role for DC actin in the regulation of immune synapse functionality.     

Homeostatic lymphoid chemokines synergize with adhesion ligands to trigger T and B lymphocyte chemokinesis

Homeostatic chemokines such as CCL19, CCL21, and CXCL13 are known to elicit chemotaxis from naive T and B cells and play a critical role in lymphocyte homing to appropriate zones within secondary lymphoid organs (SLO). Here we tested whether CCL21 and CXCL13 modulate murine lymphocyte motility in the absence of concentration gradients, using videomicroscopy to directly observe the migration of single cells. CCL21 treatment of T cells induced rapid polarization and sustained random migration with average speeds of 5.16 +/- 2.08 microm/min; B cell migration (average velocity 4.10 +/- 1.58 microm/min) was similarly induced by CXCL13. Migration required the presence of both chemokine and adhesion ligands and was sustained for >24 h. Furthermore, in in vitro assays modeling the relative infrequency of Ag-specific T cell-dendritic cell (DC) encounters during primary immune responses, we found that CCL21 addition to T-DC cocultures accelerated the kinetics of CD69 up-regulation and enhanced by 2-fold the proliferation of Ag-specific T cells in a manner dependent on G-protein-coupled receptor signaling in T cells. These results suggest that homeostatic chemokines could substantially impact the dynamics and priming of lymphocytes within SLO even in the absence of significant concentration gradients.     

Cytoskeletal control of the secretory immune synapse

The immune synapse is a very important but often transient site for secretion between immune cells. How secretion is controlled in a coordinated fashion at the synapse is a subject of much investigation. Two key mechanisms are the polarisation of the centrosome and rapid actin dynamics across the immune synapses that form between interacting immune cells. In recent years it has become clear that different immune cells utilise a diversity of immune synapses that modify these mechanisms in order to optimise specialised modes of secretion. Here we describe some of the latest research, focusing on regulation by centrosomal and actin dynamics in a variety of immune cells.     

Immunological synapses: breaking up may be good to do

Activated T cells form stable immunological synapses with antigen-presenting cells whereas na?ve T cells initially engage in more transient interactions. Sims et al. (2007) demonstrate that these transient interactions are due to the kinase PKCtheta, which serves to destabilize the synapse thereby permitting T cells to migrate elsewhere. They also show that re-establishment of a synapse involves the actin regulator WASp.     

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.     

HIV-1 Infection of DC: Evidence for the Acquisition of Virus Particles from Infected T Cells by Antigen Uptake Mechanism

Dendritic cells (DC) play a pivotal role in transmission and dissemination of HIV-1. Earlier studies reported that DC present at the site of infection trap virus particles via DC-SIGN and transfer the virus to the interacting naïve T cells. This prompted us to ask the question whether DC could acquire virus from infected T cells during DC-T cell interaction. To address this, we investigated the likely transfer of virus from HIV-1 infected T cells to DC and the underlying mechanisms involved. Results indicate that DC acquire virus from infected T cells via antigen uptake mechanism and this results in infection of DC with expression of proteins directed by viral DNA. Further studies with HIV-1 lacking the Env protein also resulted in infection of DC. The use of antibodies against DC-SIGN and DC-SIGN-R ruled out a role for receptor in the infection of DC. Additional data show that DC infection is directly correlated with the ability of DC to take up antigen from infected T cells. Overall, these studies provide evidence to suggest that HIV-1, besides infecting immune cells, also utilizes immunological mechanism(s) to acquire and disseminate virus.     

Synapsis of Tn3 recombination sites: unpaired sites destabilize synapses by a partner exchange mechanism

Catalysis of site-specific recombination is preceded by the formation of a synapse comprising two DNA sites and multiple subunits of the recombinase, together with other "accessory" proteins in some cases. We investigated the stability of synapses of Tn3 resolvase-bound res recombination sites, in plasmids containing either two or three res sites. Although synapses are long-lived in plasmids with just two res sites, persisting for tens of minutes, a synapse of any two sites is relatively short-lived in plasmids with three res sites. The three alternative pairwise synapses that can be formed in three-res plasmids re-assort rapidly relative to the rate of recombination. We propose a "partner exchange" mechanism for this re-assortment, involving direct attack on a synapse by an unpaired res site. This mechanism reconciles studies on selective synapsis in multi-res substrates, which imply rapid interchange of synaptic pairings, with studies indicating that synapses of two Tn3res sites are stable.     

Immunological synapses and neuronal synapses

The interface between two cells from the immune system has recently been coined "immunological synapse". The authors review recent findings concerning the structure of the synapse formed between T lymphocytes and antigen-presenting cells. T cells can be part of different synapses, depending on the antigen-presenting cell (B cell hybridoma, proteo-lipid bilayer, macrophage, dendritic cell). The synapse formed with dendritic cells is discussed in more details. A comparison is made with the synapses from the nervous system. Several parallel questions are discussed: how receptors can be clustered, what is the influence of synapse functioning on the structure of the synapse. It is suggested that in both cases two modes of communication exist in parallel: direct cell-cell contacts and soluble mediators, neurotransmitters in one case, putative immunotransmitters in the other.     

T cell receptor antagonism interferes with MHC clustering and integrin patterning during immunological synapse formation

T cell activation by nonself peptide-major histocompatibility complex (MHC) antigenic complexes can be blocked by particular sequence variants in a process termed T cell receptor antagonism. The inhibition mechanism is not understood, although such variants are encountered in viral infections and may aid immune evasion. Here, we study the effect of antagonist peptides on immunological synapse formation by T cells. This cellular communication process features early integrin engagement and T cell motility arrest, referred to as the "stop signal." We find that synapses formed on membranes presenting antagonist-agonist complexes display reduced MHC density, which leads to reduced T cell proliferation that is not overcome by the costimulatory ligands CD48 and B7-1. Most T cells fail to arrest and crawl slowly with a dense ICAM-1 crescent at the leading edge. Similar aberrant patterns of LFA-1/ICAM-1 engagement in live T-B couples correlate with reduced calcium flux and IL-2 secretion. Hence, antagonist peptides selectively disable MHC clustering and the stop signal, whereas LFA-1 valency up-regulation occurs normally.     

Cytoskeletal cross-talk in the control of T cell antigen receptor signaling

T cell antigen receptor signaling is triggered and controlled in specialized cellular interfaces formed between T cells and antigen-presenting cells named immunological synapses. Both microtubules and actin cytoskeleton rearrange at the immunological synapse in response to T cell receptor triggering, ensuring in turn the accuracy of intracellular signaling. Recent reports show that the cross-talk between the cortical actin cytoskeleton and microtubule networks is key for structuring the immunological synapse and for controlling T cell receptor signaling. Immunological synapse architecture and the interaction between the signaling machinery and various cytoskeletal elements are therefore crucial for the fine-tuning of T cell signaling.     

Age-associated decline in effective immune synapse formation of CD4(+) T cells is reversed by vitamin E supplementation

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.     

Properties of a Glutamatergic Synapse Controlling Information Output from Retinal Bipolar Cells

One general categorization of retinal ganglion cells is to segregate them into tonically or phasically responding neurons, each conveying discrete aspects of the visual scene. Although best identified in the output signals of the retina, this distinction is initiated at the first synapse: between photoreceptors and the dendrites of bipolar cells. In this study we found that the output synapses of bipolar cells also contribute to separate these pathways. Both transient and sustained ganglion cells can produce maintained spike activity, but bipolar cell glutamate release exhibits a divergence that corresponds to the response characteristics of the ganglion cells. Comparing light intensity coding in the sustained and transient ON pathways revealed that they shared the intensity spectrum. The transient pathway had greater sensitivity but smaller dynamic range, and switched from intensity coding to event detection at light levels where sustained pathway sensitivity began to rise. The distinctive properties of the sustained pathway depended upon inhibition and shifted toward those of the transient pathway in the absence of inhibition. The transient system was comparatively unaffected by the loss of inhibition and this was due to the concomitant activation of perisynaptic NMDA receptors. Overall, the properties of bipolar cell dendritic and axon terminals both contribute to the formation of key aspects of the sustained/transient dichotomy normally associated with ganglion cells.     

NKT cells provide help for dendritic cell-dependent priming of MHC class I-restricted CD8+ T cells in vivo

Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires "help". We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4(+) T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4(+) T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4(+) T cell-independent priming of CTL. DC copresenting a K(b)-binding antigenic peptide and the CD1d-binding glycolipid alpha-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.     

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.     

Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation

Mast cells (MCs) produce soluble mediators such as histamine and prostaglandins that are known to influence dendritic cell (DC) function by stimulating maturation and antigen processing. Whether direct cell–cell interactions are important in modulating MC/DC function is unclear. In this paper, we show that direct contact between MCs and DCs occurs and plays an important role in modulating the immune response. Activation of MCs through FcεRI cross-linking triggers the formation of stable cell–cell interactions with immature DCs that are reminiscent of the immunological synapse. Direct cellular contact differentially regulates the secreted cytokine profile, indicating that MC modulation of DC populations is influenced by the nature of their interaction. Synapse formation requires integrin engagement and facilitates the transfer of internalized MC-specific antigen from MCs to DCs. The transferred material is ultimately processed and presented by DCs and can activate T cells. The physiological outcomes of the MC–DC synapse suggest a new role for intercellular crosstalk in defining the immune response.     

Cutting edge: cytotoxic granule polarization and cytolysis can occur without central supramolecular activation cluster formation in CD8+ effector T cells

The functional implication of molecular segregation within the immunological synapse remains uncertain. We recently reported that effector but not naive TCR transgenic murine CD8+ T cells formed immunological synapses containing a central supramolecular activation cluster (cSMAC), suggesting that execution of effector functions such as cytolytic activity might be facilitated by the cSMAC structure. We have now explored this hypothesis using two approaches. First, by simultaneously imaging cSMAC formation and mobilization of cytotoxic granules to the synapse, we observed no correlation between the presence of a cSMAC and granule reorientation. Second, we took advantage of the observation that CD28 costimulation markedly enhances cSMAC formation. Granule polarization to the contact site was indistinguishable with B7-1+ and B7-1- target cells, and cytolytic activity against B7-1+ or B7-1- targets was similar and granule-dependent. Together, our results indicate that the formation of a cSMAC is not required for cytolytic activity in CD8+ effector T cells.     

Dendritic cell maturation controls adhesion, synapse formation, and the duration of the interactions with naive T lymphocytes

The initiation of adaptive immune responses requires the direct interaction of dendritic cells (DCs) with naive T lymphocytes. It is well established that the maturation state of DCs has a critical impact on the outcome of the response. We show here that mature DCs form stable conjugates with naive T cells and induce the formation of organized immune synapses. Immature DCs, in contrast, form few stable conjugates with no organized immune synapses. A dynamic analysis revealed that mature DCs can form long-lasting interactions with naive T cells, even in the absence of Ag. Immature DCs, in contrast, established only short intermittent contacts, suggesting that the premature termination of the interaction prevents the formation of organized immune synapses and full T cell activation.     

Dendritic cells induce MUC1 expression and polarization on human T cells by an IL-7-dependent mechanism

The MUC1 transmembrane mucin is expressed on the surface of activated human T cells; however, the physiologic signals responsible for the regulation of MUC1 in T cells are not known. The present studies demonstrate that IL-7, but not IL-2 or IL-4, markedly induces MUC1 expression on CD3+ T cells. MUC1 was also up-regulated by IL-15, but to a lesser extent than that found with IL-7. The results show that IL-7 up-regulates MUC1 on CD4+, CD8+, CD25+, CD69+, naive CD45RA+, and memory CD45RO+ T cells. In concert with induction of MUC1 expression by IL-7, activated dendritic cells (DC) that produce IL-7 up-regulate MUC1 on allogeneic CD3+ T cells. DC also induce MUC1 expression on autologous CD3+ T cells in the presence of recall Ag. Moreover, DC-induced MUC1 expression on T cells is blocked by a neutralizing anti-IL-7 Ab. The results also demonstrate that DC induce polarization of MUC1 on T cells at sites opposing the DC-T cell synapse. These findings indicate that DC-mediated activation of Ag-specific T cells is associated with induction and polarization of MUC1 expression by an IL-7-dependent mechanism.