Cancer-on-a-chip model shows that the adenomatous polyposis coli mutation impairs T cell engagement and killing of cancer spheroids.

Evaluating the ability of cytotoxic T lymphocytes (CTLs) to eliminate tumor cells is crucial, for instance, to predict the efficiency of cell therapy in personalized medicine. However, the destruction of a tumor by CTLs involves CTL migration in the extra-tumoral environment, accumulation on the tumor, antigen recognition, and cooperation in killing the cancer cells. Therefore, identifying the limiting steps in this complex process requires spatio-temporal measurements of different cellular events over long periods.

T cell migration and effector function differences in familial adenomatous polyposis patients with gene mutations.

Familial adenomatous polyposis (FAP) is an inherited disease characterized by the development of large number of colorectal adenomas with high risk of evolving into colorectal tumors. Mutations of the gene is often at the origin of this disease, as well as of a high percentage of spontaneous colorectal tumors. is therefore considered a tumor suppressor gene.

Microvillar Cartography: A Super-Resolution Single-Molecule Imaging Method to Map the Positions of Membrane Proteins with Respect to Cellular Surface Topography.

We describe microvillar cartography (MC), a method to map proteins on cellular surfaces with respect to the membrane topography. The surfaces of many cells are not smooth, but are rather covered with various protrusions such as microvilli. These protrusions may play key roles in multiple cellular functions, due to their ability to control the distribution of specific protein assemblies on the cell surface.

Imaging polarized granule release at the cytotoxic T cell immunological synapse using TIRF microscopy: Control by polarity regulators.

Immunological synapse formation results from a profound T cell polarization process that involves the coordinated action of the actin and microtubule cytoskeleton, and the intracellular traffic of several vesicular organelles. T cell polarization is key for both T cell activation leading to T cell proliferation and differentiation, and for T cell effector functions such as polarized secretion of cytokines by helper T cells, or polarized delivery of lytic granules by cytotoxic T cells. Efficient targeting of lytic granules by cytotoxic T cells is a crucial event for the control and elimination of infected or tumor cells.

The tumor suppressor adenomatous polyposis coli regulates T lymphocyte migration.

Adenomatous polyposis coli (APC) is a tumor suppressor whose mutations underlie familial adenomatous polyposis (FAP) and colorectal cancer. Although its role in intestinal epithelial cells is well characterized, APC importance in T cell biology is ill defined. APC regulates cytoskeleton organization, cell polarity, and migration in various cell types.

Sorting Nexin 27 Enables MTOC and Secretory Machinery Translocation to the Immune Synapse.

Sorting nexin 27 (SNX27) association to the retromer complex mediates intracellular trafficking of cargoes containing PSD95/Dlg1/ZO-1 (PDZ)-binding C-terminal sequences from endosomes to the cell surface, preventing their lysosomal degradation. Antigen recognition by T lymphocyte leads to the formation of a highly organized structure named the immune synapse (IS), which ensures cell-cell communication and sustained T cell activation. At the neuronal synapse, SNX27 recycles PDZ-binding receptors and its defective expression is associated with synaptic dysfunction and cognitive impairment.

Cell polarity regulators, multifunctional organizers of lymphocyte activation and function.

Cell polarity regulators are ubiquitous, evolutionary conserved multifunctional proteins. They contain a variety of protein-protein interaction domains endowing them the capacity to interact with cytoskeleton structures, membrane components and multiple regulatory proteins. In this way, they act in complexes and are pivotal for cell growth and differentiation, tissue formation, stability and turnover, cell migration, wound healing, and others.

Coordinating Cytoskeleton and Molecular Traffic in T Cell Migration, Activation, and Effector Functions.

Dynamic localization of receptors and signaling molecules at the plasma membrane and within intracellular vesicular compartments is crucial for T lymphocyte sensing environmental cues, triggering membrane receptors, recruiting signaling molecules, and fine-tuning of intracellular signals. The orchestrated action of actin and microtubule cytoskeleton and intracellular vesicle traffic plays a key role in all these events that together ensure important steps in T cell physiology. These include extravasation and migration through lymphoid and peripheral tissues, T cell interactions with antigen-presenting cells, T cell receptor (TCR) triggering by cognate antigen-major histocompatibility complex (MHC) complexes, immunological synapse formation, cell activation, and effector functions.

Adenomatous Polyposis Coli Modulates Actin and Microtubule Cytoskeleton at the Immunological Synapse to Tune CTL Functions.

Adenomatous polyposis coli (Apc) is a cell polarity regulator and a tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Apc involvement in T lymphocyte functions and antitumor immunity remains poorly understood. Investigating Apc-depleted human CD8 T cells and CD8 T cells from mutant mice, we found that Apc regulates actin and microtubule cytoskeleton remodeling at the immunological synapse, controlling synapse morphology and stability and lytic granule dynamics, including targeting and fusion at the synapse.

ERM-Dependent Assembly of T Cell Receptor Signaling and Co-stimulatory Molecules on Microvilli prior to Activation.

T cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that ≥90% of T cell receptor (TCR) complex molecules TCRαβ and TCRζ, as well as the co-receptor CD4 (cluster of differentiation 4) and the co-stimulatory molecule CD2, reside on microvilli of resting human T cells. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, including the protein tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase) and the key adaptor LAT (linker for activation of T cells), are also enriched on microvilli.

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

Endosomal 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.

Cell Biology of T Cell Receptor Expression and Regulation.

T cell receptors (TCRs) are protein complexes formed by six different polypeptides. In most T cells, TCRs are composed of αβ subunits displaying immunoglobulin-like variable domains that recognize peptide antigens associated with major histocompatibility complex molecules expressed on the surface of antigen-presenting cells. TCRαβ subunits are associated with the CD3 complex formed by the γ, δ, ε, and ζ subunits, which are invariable and ensure signal transduction.

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

Adenomatous 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.

Studying the Immune Synapse in HIV-1 Infection.

T 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.

Imaging Vesicular Traffic at the Immune Synapse.

Immunological 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.

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

The 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).

Serine Phosphorylation of SLP76 Is Dispensable for T Cell Development but Modulates Helper T Cell Function.

The adapter protein SLP76 is a key orchestrator of T cell receptor (TCR) signal transduction. We previously identified a negative feedback loop that modulates T cell activation, involving phosphorylation of Ser376 of SLP76 by the hematopoietic progenitor kinase 1 (HPK1). However, the physiological relevance of this regulatory mechanism was still unknown.

The functional interplay of Rab11, FIP3 and Rho proteins on the endosomal recycling pathway controls cell shape and symmetry.

Several families of small GTPases regulate a variety of fundamental cellular processes, encompassing growth factor signal transduction, vesicular trafficking and control of the cytoskeleton. Frequently, their action is hierarchical and complementary, but much of the detail of their functional interactions remains to be clarified. It is well established that Rab family members regulate a variety of intracellular vesicle trafficking pathways.

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

The 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 intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay.

Comparative Anatomy of Phagocytic and Immunological Synapses.

The generation of phagocytic cups and immunological synapses are crucial events of the innate and adaptive immune responses, respectively. They are triggered by distinct immune receptors and performed by different cell types. However, growing experimental evidence shows that a very close series of molecular and cellular events control these two processes.

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.

[Immunological synapse is a dynamic signaling platform for T cell activation].

Adaptive immune responses are initiated by the recognition of antigens by T lymphocytes. Antigen recognition triggers the generation of immunological synapses. These are dynamic and finely organized cell-cell contacts formed between T lymphocytes and antigen presenting cells.

Orchestrating cytoskeleton and intracellular vesicle traffic to build functional immunological synapses.

Immunological synapses are specialized cell-cell contacts formed between T lymphocytes and antigen-presenting cells. They are induced upon antigen recognition and are crucial for T-cell activation and effector functions. The generation and function of immunological synapses depend on an active T-cell polarization process, which results from a finely orchestrated crosstalk between the antigen receptor signal transduction machinery, the actin and microtubule cytoskeletons, and controlled vesicle traffic.

Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse.

How the vesicular traffic of signaling molecules contributes to T cell receptor (TCR) signal transduction at the immunological synapse remains poorly understood. In this study, we show that the protein tyrosine kinase Lck, the TCRζ subunit, and the adapter LAT traffic through distinct exocytic compartments, which are released at the immunological synapse in a differentially regulated manner. Lck vesicular release depends on MAL protein.