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. Here, we use a cancer-on-a-chip platform to evaluate the impact of adenomatous polyposis coli (APC) mutation on CTL migration and cytotoxicity against 3D tumor spheroids. The APC mutated CTLs are found to have a reduced ability to destroy tumor spheroids compared with control cells, even though APC mutants migrate in the extra-tumoral space and accumulate on the spheroids as efficiently as control cells. Once in contact with the tumor however, mutated CTLs display reduced engagement with the cancer cells, as measured by a metric that distinguishes different modes of CTL migration. Realigning the CTL trajectories around localized killing cascades reveals that all CTLs transition to high engagement in the 2 h preceding the cascades, which confirms that the low engagement is the cause of reduced cytotoxicity. Beyond the study of APC mutations, this platform offers a robust way to compare cytotoxic cell efficiency of even closely related cell types, by relying on a multiscale cytometry approach to disentangle complex interactions and to identify the steps that limit the tumor destruction.

T cell migration and effector function differences in familial adenomatous polyposis patients with APC 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 Adenomatous polyposis coli (APC) gene is often at the origin of this disease, as well as of a high percentage of spontaneous colorectal tumors. APC is therefore considered a tumor suppressor gene. While the role of APC in intestinal epithelium homeostasis is well characterized, its importance in immune responses remains ill defined. Our recent work indicates that the APC protein is involved in various phases of both CD4 and CD8 T cells responses. This prompted us to investigate an array of immune cell features in FAP subjects carrying APC mutations. A group of 12 FAP subjects and age and sex-matched healthy controls were studied. We characterized the immune cell repertoire in peripheral blood and the capacity of immune cells to respond ex vivo to different stimuli either in whole blood or in purified T cells. A variety of experimental approaches were used, including, pultiparamater flow cytometry, NanosString gene expression profiling, Multiplex and regular ELISA, confocal microscopy and computer-based image analyis methods. We found that the percentage of several T and natural killer (NK) cell populations, the expression of several genes induced upon innate or adaptive immune stimulation and the production of several cytokines and chemokines was different. Moreover, the capacity of T cells to migrate in response to chemokine was consistently altered. Finally, immunological synapses between FAP cytotoxic T cells and tumor target cells were more poorly structured. Our findings of this pilot study suggest that mild but multiple immune cell dysfunctions, together with intestinal epithelial dysplasia in FAP subjects, may facilitate the long-term polyposis and colorectal tumor development. Although at an initial discovery phase due to the limited sample size of this rare disease cohort, our findings open new perspectives to consider immune cell abnormalities into polyposis pathology.

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. Thus, for example, we have shown that the T-cell receptor and several of its proximal signaling proteins reside on microvilli, while others are excluded from these projections. These results have indicated that microvilli can function as key signaling hubs for the initiation of the immune response. MC has facilitated our observations of particular surface proteins and their specialized distribution on microvillar and non-microvillar compartments. MC combines membrane topography imaging, using variable-angle total internal microscopy, with stochastic localization nanoscopy, which generates deep sub-diffraction maps of protein distribution. Since the method is based on light microscopy, it avoids some of the pitfalls inherent to electron-microscopy-based techniques, such as dehydration, the need for carbon coating, and immunogold clustering, and is amenable to future developments involving, for example, live-cell imaging. This protocol details the procedures we developed for MC, which can be readily adopted to study a broad range of cell-surface molecules and dissect their distribution within distinct surface assemblies under multiple cell activation states.

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. Understanding how lytic granule delivery is regulated and quantifying its efficiency under physiological and pathological conditions may help to improve immune responses against infection and cancer.

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. Here, we address whether APC plays a role in T lymphocyte migration. Using a series of cell biology tools, we unveiled that T cells from FAP patients carrying APC mutations display impaired adhesion and motility in constrained environments. We further dissected the cellular mechanisms underpinning these defects in APC-depleted CEM T cell line that recapitulate the phenotype observed in FAP T cells. We found that APC affects T cell motility by modulating integrin-dependent adhesion and cytoskeleton reorganization. Hence, APC mutations in FAP patients not only drive intestinal neoplasms but also impair T cell migration, potentially contributing to inefficient antitumor immunity.

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. Hence some of these proteins are tumor suppressors. These cellular processes rely on the establishment of cell polarity characterized by the asymmetric localization of proteins, RNAs, membrane domains, or organelles that together condition cell shape and function. Whether apparently stable, as in epithelia or neurons, or very dynamic, as in immune cells, cell polarity is an active process. It involves cytoskeleton reorganization and targeted intracellular traffic, and results in cellular events such as protein synthesis, secretion and assembly taking place at defined cell poles. Multiple polarity regulators orchestrate these processes. Immune cells are particularly versatile in rapidly polarizing and assuming different shapes, so to swiftly adopt specialized behaviors and functions. Polarity regulators act in various ways in different immune cell types and at their distinct differentiation states. Here we review how cell polarity regulators control different processes and functions along T lymphocyte physiology, including cell migration through different tissues, immunological synapse formation and effector functions.

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. In T lymphocytes, SNX27 was found localized at recycling endosomal compartments that polarized to the IS, suggesting a function in polarized traffic to this structure. Proteomic analysis of PDZ-SNX27 interactors during IS formation identify proteins with known functions in cytoskeletal reorganization and lipid regulation, such as diacylglycerol (DAG) kinase (DGK) ζ, as well as components of the retromer and WASH complex. In this study, we investigated the consequences of SNX27 deficiency in cytoskeletal reorganization during IS formation. Our analyses demonstrate that SNX27 controls the polarization towards the cell-cell interface of the PDZ-interacting cargoes DGKζ and the retromer subunit vacuolar protein sorting protein 26, among others. SNX27 silencing abolishes the formation of a DAG gradient at the IS and prevents re-localization of the dynactin complex component dynactin-1/p150Glued, two events that correlate with impaired microtubule organizing center translocation (MTOC). SNX27 silenced cells show marked alteration in cytoskeleton organization including a failure in the organization of the microtubule network and defects in actin clearance at the IS. Reduced SNX27 expression was also found to hinder the arrangement of signaling microclusters at the IS, as well as the polarization of the secretory machinery towards the antigen presenting cells. Our results broaden the knowledge of SNX27 function in T lymphocytes by showing a function in modulating IS organization through regulated trafficking of cargoes.

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. Cytoskeletal and vesicle traffic dynamics and their interplay are coordinated by a variety of regulatory molecules. Among them, polarity regulators and membrane-cytoskeleton linkers are master controllers of this interplay. Here, we review the various ways the T cell plasma membrane, receptors, and their signaling machinery interplay with the actin and microtubule cytoskeleton and with intracellular vesicular compartments. We highlight the importance of this fine-tuned crosstalk in three key stages of T cell biology involving cell polarization: T cell migration in response to chemokines, immunological synapse formation in response to antigen cues, and effector functions. Finally, we discuss two examples of perturbation of this interplay in pathological settings, such as HIV-1 infection and mutation of the polarity regulator and tumor suppressor adenomatous polyposis coli (Apc) that leads to familial polyposis and colorectal cancer.

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 ApcMin/+ 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. Ultimately, Apc tunes cytotoxic T cell activity, leading to tumor cell killing. Furthermore, Apc modulates early TCR signaling and nuclear translocation of the NFAT transcription factor with mild consequences on the expression of some differentiation markers. In contrast, no differences in the production of effector cytokines were observed. These results, together with our previous findings on Apc function in regulatory T cells, indicate that Apc mutations may cause a dual damage, first unbalancing epithelial cell differentiation and growth driving epithelial neoplasms and, second, impairing T cell-mediated antitumor immunity at several levels.

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 TCRαß 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. Notably, phosphorylated proteins of the ERM (ezrin, radixin, and moesin) family colocalize with TCRαß as well as with actin filaments, implying a role for one or more ERMs in linking the TCR complex to the actin cytoskeleton within microvilli. Our results establish microvilli as key signaling hubs, in which the TCR complex and its proximal signaling molecules and adaptors are preassembled prior to activation in an ERM-dependent manner, facilitating initial antigen sensing.

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. 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.

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. Moreover, Rho family GTPases are pivotal for the control of the actin and microtubule cytoskeleton. However, the interplay between these 2 types of GTPases has been rarely reported. We discuss here our recent findings showing that Rab11, a key regulator of endosomal recycling, and Rac1, a central actin cytoskeleton regulator involved in lamellipodium formation and cell migration, interplay on endosomes through the Rab11 effector FIP3. In the context of the rapidly reactive T lymphocytes, Rab11-Rac1 endosomal functional interplay is important to control cell shape changes and cell symmetry during lymphocyte spreading and immunological synapse formation and ultimately modulate T cell activation.

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 αß subunits displaying immunoglobulin-like variable domains that recognize peptide antigens associated with major histocompatibility complex molecules expressed on the surface of antigen-presenting cells. TCRαß subunits are associated with the CD3 complex formed by the γ, δ, ε, and ζ subunits, which are invariable and ensure signal transduction. Here, we review how the expression and function of TCR complexes are orchestrated by several fine-tuned cellular processes that encompass (a) synthesis of the subunits and their correct assembly and expression at the plasma membrane as a single functional complex, (b) TCR membrane localization and dynamics at the plasma membrane and in endosomal compartments, (c) TCR signal transduction leading to T cell activation, and (d) TCR degradation. These processes balance each other to ensure efficient T cell responses to a variety of antigenic stimuli while preventing autoimmunity.

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. 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.

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.

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. 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.

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). 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.

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. To address this question, we generated a SLP76-S376A-expressing knock-in mouse strain and investigated the effects of Ser376 mutation on T cell development and function. We report here that SLP76-S376A-expressing mice exhibit normal thymocyte development and no detectable phenotypic alterations in mature T cell subsets or other lymphoid and myeloid cell lineages. Biochemical analyses revealed that mutant T cells were hypersensitive to TCR stimulation. Indeed, phosphorylation of several signaling proteins, including SLP76 itself, phospholipase Cγ1 and the protein kinases AKT and ERK1/2, was increased. These modifications correlated with increased Th1-type and decreased Th2-type cytokine production by SLP76-S376A T cells, but did not result in significant changes of proliferative capacity nor activation-induced cell death susceptibility. Hence, our results reveal that SLP76-Ser376 phosphorylation does not mediate all HPK1-dependent regulatory effects in T cells but it fine-tunes helper T cell responses.

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. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular 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.

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. Thus, the tight and dynamic interplay between receptor signaling, actin and microtubule cytoskeleton, and targeted vesicle traffic are all critical features to build functional phagosomes and immunological synapses. Interestingly, both phagocytic cups and immunological synapses display particular spatial and temporal patterns of receptors and signaling molecules, leading to the notion of "phagocytic synapse." Here, we discuss both types of structures, their organization, and the mechanisms by which they are generated and regulated.