Studying the Immune Synapse in HIV-1 Infection.

International audienceT cells are the main cellular targets of the human immunodeficiency virus 1 (HIV-1). HIV-1 infection induces pleiotropic effects on the infected T cell that modify the T cell capacity to respond to antigen and facilitates virus replication. HIV-1 infection subverts the formation and function of the immunological synapse altering both actin cytoskeleton remodeling and intracellular vesicle traffic. We describe here our methods to unveil how HIV-1 and in particular its protein Nef modify vesicle traffic to the immunological synapse, perturbing the synapse activation capacity

Imaging Vesicular Traffic at the Immune Synapse.

International audienceImmunological synapse formation is the result of a profound T cell polarization process that involves the coordinated action of the actin and microtubule cytoskeleton, as well as intracellular vesicle traffic. Endosomal vesicle traffic ensures the targeting of the T cell receptor (TCR) and various signaling molecules to the synapse, being necessary for the generation of signaling complexes downstream of the TCR. Here we describe the microscopy imaging methods that we currently use to unveil how TCR and signaling molecules are associated with endosomal compartments and deliver their cargo to the immunological synapse

HIV-1 Nef Hijacks Lck and Rac1 Endosomal Traffic To Dually Modulate Signaling-Mediated and Actin Cytoskeleton–Mediated T Cell Functions

International audienceEndosomal traffic of TCR and signaling molecules regulates immunological synapse formation and T cell activation. We recently showed that Rab11 endosomes regulate the subcellular localization of the tyrosine kinase Lck and of the GTPase Rac1 and control their functions in TCR signaling and actin cytoskeleton remodeling. HIV-1 infection of T cells alters their endosomal traffic, activation capacity, and actin cytoskeleton organization. The viral protein Nef is pivotal for these modifications. We hypothesized that HIV-1 Nef could jointly alter Lck and Rac1 endosomal traffic and concomitantly modulate their functions. In this study, we show that HIV-1 infection of human T cells sequesters both Lck and Rac1 in a pericentrosomal compartment in an Nef-dependent manner. Strikingly, the Nef-induced Lck compartment contains signaling-competent forms (phosphorylated on key Tyr residues) of Lck and some of its downstream effectors, TCRζ, ZAP70, SLP76, and Vav1, avoiding the proximal LAT adaptor. Importantly, Nef-induced concentration of signaling molecules was concomitant with the upregulation of several early and late T cell activation genes. Moreover, preventing the concentration of the Nef-induced Lck compartment by depleting the Rab11 effector FIP3 counteracted Nef-induced gene expression upregulation. In addition, Nef extensively sequesters Rac1 and downregulates Rac1-dependent actin cytoskeleton remodeling, thus reducing T cell spreading. Therefore, by modifying their endosomal traffic, Nef hijacks signaling and actin cytoskeleton regulators to dually modulate their functional outputs. Our data shed new light into the molecular mechanisms that modify T cell physiology during HIV-1 infection

Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation

International audienceThe immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellu-lar vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellu-lar vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intra-cellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation

Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

International audienceThe role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production

Adenomatous Polyposis Coli Defines Treg Differentiation and Anti-inflammatory Function through Microtubule-Mediated NFAT Localization

International audienceAdenomatous polyposis coli (APC) is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer development. Although extensively studied in epithelial transformation, the effect of APC on T lymphocyte activation remains poorly defined. We found that APC ensures T cell receptor-triggered activation through Nuclear Factor of Activated T cells (NFAT), since APC is necessary for NFAT’s nuclear localization in a microtubule-dependent fashion and for NFAT-driven transcription leading to cytokine gene expression. Interestingly, NFAT forms clusters juxtaposed with microtubules. Ultimately, mouse Apc deficiency reduces the presence of NFAT in the nucleus of intestinal regulatory T cells (Tregs) and impairs Treg differentiation and the acquisition of a suppressive phenotype, which is characterized by the production of the anti-inflammatory cytokine IL-10. These findings suggest a dual role for APC mutations in colorectal cancer development, where mutations drive the initiation of epithelial neoplasms and also reduce Treg-mediated suppression of the detrimental inflammation that enhances cancer growth