Andrés Alcover

Vav proteins, masters of the world of cytoskeleton organization

Vav proteins are evolutionarily conserved from nematodes to mammals and play a pivotal role in many aspects of cellular signaling, coupling cell surface receptors to various effectors functions. In mammals, there are three family members; Vav1 is specifically expressed in the hematopoietic system, whereas Vav2 and Vav3 are more ubiquitously expressed. Vav proteins contain multiple domains that enable their function in various fashions. The participation of the Vav proteins in several processes that require cytoskeletal reorganization, such as the formation of the immunological synapse (IS), phagocytosis, platelet aggregation, spreading, and transformation will be discussed in this review. We will also cover how the Vav proteins succeed in controlling these processes by their function as guanine nucleotide exchange factors (GEFs) for the Rho/Rac family of GTPases. The contribution of the Vav proteins in a GEF-independent manner to the organization of the cytoskeleton will also be deliberated. The scope of this review is to highlight the numerous roles of the Vav signal transducer proteins in actin organization.

Role of ICAM-3 in the initial interaction of T lymphocytes and APCs

Antigen-independent adhesive interactions between T lymphocytes and antigen-presenting cells (APCs) are essential for scanning for specific antigens on the APC surface and for initiating the immune response. Here we show, through time-lapse imaging of live cells, that the intercellular adhesion molecule 3 (ICAM-3, also known as CD50) is clustered specifically at the region of the T lymphocyte surface that initiates contact with APCs. We describe the role of ICAM-3 in T cell–APC conjugate formation before antigen recognition, in early intracellular signaling and in cytoskeletal rearrangement. Our data indicate that ICAM-3 is important in the initial scanning of the APC surface by T cells and, therefore, in generating the immune response.

ZAP‐70 kinase regulates HIV cell‐to‐cell spread and virological synapse formation

HIV efficiently spreads in lymphocytes, likely through virological synapses (VSs). These cell–cell junctions share some characteristics with immunological synapses, but cellular proteins required for their constitution remain poorly characterized. We have examined here the role of ZAP‐70, a key kinase regulating T‐cell activation and immunological synapse formation, in HIV replication. In lymphocytes deficient for ZAP‐70, or expressing a kinase‐dead mutant of the protein, HIV replication was strikingly delayed. We have characterized further this replication defect. ZAP‐70 was dispensable for the early steps of viral cycle, from entry to expression of viral proteins. However, in the absence of ZAP‐70, intracellular Gag localization was impaired. ZAP‐70 was required in infected donor cells for efficient cell‐to‐cell HIV transmission to recipients and for formation of VSs. These results bring novel insights into the links that exist between T‐cell activation and HIV spread, and suggest that HIV usurps components of the immunological synapse machinery to ensure its own spread through cell‐to‐cell contacts.

Modulation of the immunological synapse: a key to HIV-1 pathogenesis?

AIDS is the result of a constant struggle between the lentivirus HIV and the immune system. Infection with HIV interferes directly with the function of CD4+ T cells and manipulates the host immune response to the virus. Recent studies indicate that the viral protein Nef, a central player in HIV pathogenesis, impairs the ability of infected lymphocytes to form immunological synapses with antigen-presenting cells and affects T-cell-receptor-mediated stimulation. An integrative picture of the abnormal behaviour of HIV-infected lymphocytes is therefore emerging. We propose that modulating lymphocyte signalling, apoptosis and intracellular trafficking ensures efficient spread of the virus in the hostile environment of the immune system.

Triggering the TCR Complex Causes the Downregulation of Nonengaged Receptors by a Signal Transduction-Dependent Mechanism

Downregulation of the TCR complex is believed to be intimately tied to T cell activation, allowing serial triggering of receptors and desensitization of stimulated cells. We studied transfected and transgenic T cells expressing CD3zeta chimeras to demonstrate that ligand engagement of the TCR or chimeras causes comodulation of nonengaged receptors. Comodulation required protein tyrosine kinase activity but not trans-phosphorylation of nonengaged receptors. The TCR appears to be downregulated by at least two mechanisms. One mechanism requires direct engagement, independent of signaling. The second requires signaling and downregulates nontriggered receptors. These results shed new light on the process of TCR downregulation and indicate that the number of downregulated TCRs cannot be assumed to equal the number of engaged receptors.

CD28 Utilizes Vav-1 to Enhance TCR-Proximal Signaling and NF-AT Activation

The mechanism through which CD28 costimulation potentiates TCR-driven gene expression is still not clearly defined. Vav-1, an exchange factor for Rho GTPases thought to regulate, mainly through Rac-1, various signaling components leading to cytokine gene expression, is tyrosine phosphorylated upon CD28 engagement. Here, we provide evidence for a key role of Vav-1 in CD28-mediated signaling. Overexpression of Vav-1 in Jurkat cells in combination with CD28 ligation strongly reduced the concentration of staphylococcus enterotoxin E/MHC required for TCR-induced NF-AT activation. Surprisingly, upon Vav-1 overexpression CD28 ligation sufficed to activate NF-AT in the absence of TCR engagement. This effect was not mediated by overexpression of ZAP-70 nor of SLP-76 but necessitated the intracellular tail of CD28, the intactness of the TCR-proximal signaling cascade, the Src-homology domain 2 (SH2) domain of Vav-1, and SLP-76 phosphorylation, an event which was favored by Vav-1 itself. Cells overexpressing Vav-1 formed lamellipodia and microspikes reminiscent of Rac-1 and Cdc42 activation, respectively, for which the SH2 domain of Vav-1 was dispensable. Together, these data suggest that CD28 engagement activates Vav-1 to boost TCR signals through a synergistic cooperation between Vav-1 and SLP-76 and probably via cortical actin changes to facilitate the organization of a signaling zone.

Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse

T‐cell receptor (TCR) signalling is triggered and tuned at immunological synapses by the generation of signalling complexes that associate into dynamic microclusters. Microcluster movement is necessary to tune TCR signalling, but the molecular mechanism involved remains poorly known. We show here that the membrane‐microfilament linker ezrin has an important function in microcluster dynamics and in TCR signalling through its ability to set the microtubule network organization at the immunological synapse. Importantly, ezrin and microtubules are important to down‐regulate signalling events leading to Erk1/2 activation. In addition, ezrin is required for appropriate NF‐AT activation through p38 MAP kinase. Our data strongly support the notion that ezrin regulates immune synapse architecture and T‐cell activation through its interaction with the scaffold protein Dlg1. These results uncover a crucial function for ezrin, Dlg1 and microtubules in the organization of the immune synapse and TCR signal down‐regulation. Moreover, they underscore the importance of ezrin and Dlg1 in the regulation of NF‐AT activation through p38.

Functional Analysis via Standardized Whole-Blood Stimulation Systems Defines the Boundaries of a Healthy Immune Response to Complex Stimuli

Standardization of immunophenotyping procedures has become a high priority. We have developed a suite of whole-blood, syringe-based assay systems that can be used to reproducibly assess induced innate or adaptive immune responses. By eliminating preanalytical errors associated with immune monitoring, we have defined the protein signatures induced by (1) medically relevant bacteria, fungi, and viruses; (2) agonists specific for defined host sensors; (3) clinically employed cytokines; and (4) activators of T cell immunity. Our results provide an initial assessment of healthy donor reference values for induced cytokines and chemokines and we report the failure to release interleukin-1α as a common immunological phenotype. The observed naturally occurring variation of the immune response may help to explain differential susceptibility to disease or response to therapeutic intervention. The implementation of a general solution for assessment of functional immune responses will help support harmonization of clinical studies and data sharing.

ARF6 GTPase controls bacterial invasion by actin remodelling

The obligate intracellular bacterium Chlamydia penetrates the host epithelial cell by inducing cytoskeleton and membrane rearrangements reminiscent of phagocytosis. Here we report that Chlamydia induces a sharp and transient activation of the endogenous small GTP-binding protein ARF6, which is required for efficient uptake. We also show that a downstream effector of ARF6, phosphatidylinositol 4-phosphate 5-kinase and its product, phosphatidylinositol 4,5-bisphosphate were instrumental for bacterial entry. By contrast, ARF6 activation of phospholipase D was not required for Chlamydia uptake. ARF6 activation was necessary for extensive actin reorganization at the invasion sites. Remarkably, these signalling players gathered with F-actin in a highly organized three-dimensional concentric calyx-like protrusion around invasive bacteria. These results indicate that ARF6, which controls membrane delivery during phagocytosis of red blood cells in macrophages, has a different role in the entry of this small bacterium, controlling cytoskeletal reorganization.

Group Selection and Contribution of Minority Variants during Virus Adaptation Determines Virus Fitness and Phenotype.

Understanding how a pathogen colonizes and adapts to a new host environment is a primary aim in studying emerging infectious diseases. Adaptive mutations arise among the thousands of variants generated during RNA virus infection, and identifying these variants will shed light onto how changes in tropism and species jumps can occur. Here, we adapted Coxsackie virus B3 to a highly permissive and less permissive environment. Using deep sequencing and bioinformatics, we identified a multi-step adaptive process to adaptation involving residues in the receptor footprints that correlated with receptor availability and with increase in virus fitness in an environment-specific manner. We show that adaptation occurs by selection of a dominant mutation followed by group selection of minority variants that together, confer the fitness increase observed in the population, rather than selection of a single dominant genotype.