Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia.

The tumor microenvironment (TME) plays a central role in the pathogenesis of chronic lymphocytic leukemia (CLL), contributing to disease progression and chemoresistance. Leukemic cells shape the TME into a pro-survival and immunosuppressive niche through contact-dependent and contact-independent interactions with the cellular components of the TME. Immune synapse (IS) formation is defective in CLL. Here we asked whether soluble factors released by CLL cells contribute to their protection from cytotoxic T cell (CTL)-mediated killing by interfering with this process. We found that healthy CTLs cultured in media conditioned by leukemic cells from CLL patients or Eµ-TCL1 mice upregulate the exhaustion marker PD-1 and become unable to form functional ISs and kill target cells. These defects were more pronounced when media were conditioned by leukemic cells lacking p66Shc, a proapoptotic adapter whose deficiency has been implicated in disease aggressiveness both in CLL and in the Eµ-TCL1 mouse model. Multiplex ELISA assays showed that leukemic cells from Eµ-TCL1 mice secrete abnormally elevated amounts of CCL22, CCL24, IL-9 and IL-10, which are further upregulated in the absence of p66Shc. Among these, IL-9 and IL-10 were also overexpressed in leukemic cells from CLL patients, where they inversely correlated with residual p66Shc. Using neutralizing antibodies or the recombinant cytokines we show that IL-9, but not IL-10, mediates both the enhancement in PD-1 expression and the suppression of effector functions in healthy CTLs. Our results demonstrate that IL-9 secreted by leukemic cells negatively modulates the anti-tumor immune abilities of CTLs, highlighting a new suppressive mechanism and a novel potential therapeutical target in CLL.

End-binding protein 1 regulates the metabolic fate of CD4+ T lymphoblasts and Jurkat T cells and the organization of the mitochondrial network.

The organization of the mitochondrial network is relevant for the metabolic fate of T cells and their ability to respond to TCR stimulation. This arrangement depends on cytoskeleton dynamics in response to TCR and CD28 activation, which allows the polarization of the mitochondria through their change in shape, and their movement along the microtubules towards the immune synapse. This work focus on the role of End-binding protein 1 (EB1), a protein that regulates tubulin polymerization and has been previously identified as a regulator of intracellular transport of CD3-enriched vesicles. EB1-interferred cells showed defective intracellular organization and metabolic strength in activated T cells, pointing to a relevant connection of the cytoskeleton and metabolism in response to TCR stimulation, which leads to increased AICD. By unifying the organization of the tubulin cytoskeleton and mitochondria during CD4+ T cell activation, this work highlights the importance of this connection for critical cell asymmetry together with metabolic functions such as glycolysis, mitochondria respiration, and cell viability.

Chaperonin CCT controls extracellular vesicle production and cell metabolism through kinesin dynamics.

Cell proteostasis includes gene transcription, protein translation, folding of de novo proteins, post-translational modifications, secretion, degradation and recycling. By profiling the proteome of extracellular vesicles (EVs) from T cells, we have found the chaperonin complex CCT, involved in the correct folding of particular proteins. By limiting CCT cell-content by siRNA, cells undergo altered lipid composition and metabolic rewiring towards a lipid-dependent metabolism, with increased activity of peroxisomes and mitochondria. This is due to dysregulation of the dynamics of interorganelle contacts between lipid droplets, mitochondria, peroxisomes and the endolysosomal system. This process accelerates the biogenesis of multivesicular bodies leading to higher EV production through the dynamic regulation of microtubule-based kinesin motors. These findings connect proteostasis with lipid metabolism through an unexpected role of CCT.

Post-translational modifications and stabilization of microtubules regulate transport of viral factors during infections.

Tubulin post-translational modifications (PTMs) constitute a source of diversity for microtubule (MT) functions, in addition to the different isotypes of α and ß-tubulin acting as building blocks of MTs. Also, MT-associated proteins (MAPs) confer different characteristics to MTs. The combination of all these factors regulates the stability of these structures that act as rails to transport organelles within the cell, facilitating the association of motor complexes. All these functions are involved in crucial cellular processes in most cell types, ranging from spindle formation in mitosis to the defense against incoming cellular threats during phagocytosis mediated by immune cells. The regulation of MT dynamics through tubulin PTMs has evolved to depend on many different factors that act in a complex orchestrated manner. These tightly regulated processes are particularly relevant during the induction of effective immune responses against pathogens. Viruses have proved not only to hijack MTs and MAPs in order to favor an efficient infection, but also to induce certain PTMs that improve their cellular spread and lead to secondary consequences of viral processes. In this review, we offer a perspective on relevant MT-related elements exploited by viruses.

Rapid Visualization of Intracellular Vesicle Events During Synaptic Stimulation.

The immune synapse (IS) enables cell-cell communication between immune cells through close contacts, as well as T-cell activation and vesicle secretion. It is sustained by fine-tuned molecular interactions of receptors at both cell sides of the IS and intracellular cytoskeletal components. The resulting intracellular polarization of different organelles, through cytoskeleton-guided vesicular traffic, is a key player in IS formation and signaling. We describe herein a method to analyze rapid changes of vesicle localization through microscopy analysis upon polarization toward the IS. These vesicles are monitored using the centrosome and its associated microtubular network or the actin-based structures as spatial references during the organization of the IS.

TIRF Microscopy as a Tool to Determine Exosome Composition.

Exosomes are extracellular vesicles (EVs) containing different biomolecules with biological activity, such as proteins, miRNA, long noncoding RNA, and DNA. EVs are efficient platforms for intercellular communication, especially during immune responses, but also in some pathological contexts, such as tumor cell growth. The precise assessment of EV content is relevant for the selection of specific vesicles with specialized biological activities, whose content is hardly visualized due to their small size. We describe herein a protocol for the determination of the content of individual EVs through microscopy imaging and user-friendly analysis using TIRF microscopy.

The Swing of Lipids at Peroxisomes and Endolysosomes in T Cell Activation.

The immune synapse (IS) is a well-known intercellular communication platform, organized at the interphase between the antigen presenting cell (APC) and the T cell. After T cell receptor (TCR) stimulation, signaling from plasma membrane proteins and lipids is amplified by molecules and downstream pathways for full synapse formation and maintenance. This secondary signaling event relies on intracellular reorganization at the IS, involving the cytoskeleton and components of the secretory/recycling machinery, such as the Golgi apparatus and the endolysosomal system (ELS). T cell activation triggers a metabolic reprogramming that involves the synthesis of lipids, which act as signaling mediators, and an increase of mitochondrial activity. Then, this mitochondrial activity results in elevated reactive oxygen species (ROS) production that may lead to cytotoxicity. The regulation of ROS levels requires the concerted action of mitochondria and peroxisomes. In this review, we analyze this reprogramming and the signaling implications of endolysosomal, mitochondrial, peroxisomal, and lipidic systems in T cell activation.

Integrated miRNA and mRNA expression profiling identifies novel targets and pathological mechanisms in autoimmune thyroid diseases.

BACKGROUND: The mechanisms underlying autoimmune thyroid disease (AITD) remain elusive. Identification of such mechanisms would reveal novel and/or better therapeutic targets. Here, we use integrated analysis of miRNAs and mRNAs expression profiling to identify potential therapeutic targets involved in the mechanisms underlying AITD. METHODS: miRNA and mRNA from twenty fresh-frozen thyroid tissues (15 from AITD patients and 5 from healthy controls) were subjected to next-generation sequencing. An anti-correlated method revealed potential pathways and disease targets, including proteins involved in the formation of primary cilia. Thus, we examined the distribution and length of primary cilia in thyroid tissues from AITD and controls using immunofluorescence and scanning electron microscopy, and parsed cilia formation in thyroid cell lines in response to inflammatory stimuli in the presence of miRNA mimics. FINDINGS: We found that the expression of miR-21-5p, miR-146b-3p, miR-5571-3p and miR-6503-3p was anti-correlated with Enolase 4 (ENO4), in-turned planar cell polarity protein (INTU), kinesin family member 27 (KIF27), parkin co-regulated (PACRG) and serine/threonine kinase 36 (STK36) genes. Functional classification of these miRNA/mRNAs revealed that their differential expression was associated with cilia organization. We demonstrated that the number and length of primary cilia in thyroid tissues was significantly lower in AITD than in control (frequency of follicular ciliated cells in controls = 67.54% vs a mean of 22.74% and 21.61% in HT and GD respectively p = 0.0001, by one-way ANOVA test). In addition, pro-inflammatory cytokines (IFNγ and TNFα) and specific miRNA mimics for the newly identified target genes affected cilia appearance in thyroid cell lines. INTERPRETATION: Integrated miRNA/gene expression analysis has identified abnormal ciliogenesis as a novel susceptibility pathway that is involved in the pathogenesis of AITD. These results reflect that ciliogenesis plays a relevant role in AITD, and opens research pathways to design therapeutic targets in AITD. FUNDING: Instituto de Salud Carlos III, Comunidad de Madrid, Grupo Español de Tumores Neuroendocrinos y Endocrinos, Ministerio de Economía y Empresa and FEDER.

Phosphatase of Regenerating Liver-1 (PRL-1) Regulates Actin Dynamics During Immunological Synapse Assembly and T Cell Effector Function.

The regulatory role of most dual specific phosphatases during T cell activation remains unknown. Here, we have studied the expression and function of phosphatases of regenerating liver (PRLs: PRL-1, PRL-2, and PRL-3) during T cell activation, as well as, the dynamic delivery of PRL-1 to the Immunological Synapse (IS). We found that T cell activation downregulates the expression of PRL-2, resulting in an increased PRL-1/PRL-2 ratio. PRL-1 redistributed at the IS in two stages: Initially, it was transiently accumulated at scanning membranes enriched in CD3 and actin, and at later times, it was delivered at the contact site from pericentriolar, CD3ζ-containing, vesicles. Once at the established IS, PRL-1 distributed to LFA-1 and CD3ε sites. Remarkably, PRL-1 was found to regulate actin dynamics during IS assembly and the secretion of IL-2. Moreover, pharmacological inhibition of the catalytic activity of the three PRLs reduced the secretion of IL-2. These results provide evidence indicating a regulatory role of PRL-1 during IS assembly and highlight the involvement of PRLs in immune responses by mature T cells.

Priming of dendritic cells by DNA-containing extracellular vesicles from activated T cells through antigen-driven contacts.

Interaction of T cell with antigen-bearing dendritic cells (DC) results in T cell activation, but whether this interaction has physiological consequences on DC function is largely unexplored. Here we show that when antigen-bearing DCs contact T cells, DCs initiate anti-pathogenic programs. Signals of this interaction are transmitted from the T cell to the DC, through extracellular vesicles (EV) that contain genomic and mitochondrial DNA, to induce antiviral responses via the cGAS/STING cytosolic DNA-sensing pathway and expression of IRF3-dependent interferon regulated genes. Moreover, EV-treated DCs are more resistant to subsequent viral infections. In summary, our results show that T cells prime DCs through the transfer of exosomal DNA, supporting a specific role for antigen-dependent contacts in conferring protection to DCs against pathogen infection. The reciprocal communication between innate and adaptive immune cells thus allow efficacious responses to unknown threats.

eNOS S-nitrosylates ß-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1.

The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of ß-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated ß-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of ß-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.

Analysis of Microtubules and Microtubule-Organizing Center at the Immune Synapse.

The immune synapse (IS) is a specialized structure that enables cell-cell communication between immune cells. As such, it involves direct cell-to-cell contact. It is sustained by cytoskeletal components that allow the intracellular polarization of different organelles and the surface re-organization of signaling and adhesion receptors. The tubulin-based cytoskeleton is a key player in IS formation and signaling. We describe methods to analyze through Western blot and microscopy analysis the polarization to the IS of the centrosome, also known as microtubule-organizing center (MTOC), the dynamics of microtubule growth and polymerization from the MTOC to the IS and the activation of signaling molecules.

Aurora-A shines on T cell activation through the regulation of Lck.

Different protein kinases control signaling emanating from the T cell receptor (TCR) during antigen-specific T cell activation. Mitotic kinases, e.g. Aurora-A, have been widely studied in the context of mitosis due to their role during microtubule (MT) nucleation, becoming critical regulators of cell cycle progression. We have recently described a specific role for Aurora-A kinase in antigenic T cell activation. Blockade of Aurora-A in T cells severely disrupts the dynamics of MTs and CD3ζ-bearing signaling vesicles during T cell activation. Furthermore, Aurora-A deletion impairs the activation of signaling molecules downstream of the TCR. Targeting Aurora-A disturbs the activation of Lck, which is one of the first signals that drive T cell activation in an antigen-dependent manner. This work describes possible models of regulation of Lck by Aurora-A during T cell activation. We also discuss possible roles for Aurora-A in other systems similar to the IS, and its putative functions in cell polarization.

Aurora A drives early signalling and vesicle dynamics during T-cell activation.

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal.

CXCL12 Regulates through JAK1 and JAK2 Formation of Productive Immunological Synapses.

The adaptive immune response requires interaction between T cells and APC to form a specialized structure termed the immune synapse (IS). Although the TCR is essential for IS organization, other factors such as chemokines participate in this process. In this study, we show that the chemokine CXCL12-mediated signaling contributes to correct IS organization and therefore influences T cell activation. CXCR4 downregulation or blockade on T cells caused defective actin polymerization at the contact site with APC, altered microtubule-organizing center polarization and the IS structure, and reduced T cell/APC contact duration. T cell activation was thus inhibited, as shown by reduced expression of CD25 and CD69 markers and of IL-2 mRNA levels. The results indicate that, through Gi and JAK1 and 2 kinases activation, CXCL12 signaling cooperates to build the IS and to maintain adhesive contacts between APC and T cells, required for continuous TCR signaling.

Miro-1 links mitochondria and microtubule Dynein motors to control lymphocyte migration and polarity.

The recruitment of leukocytes to sites of inflammation is crucial for a functional immune response. In the present work, we explored the role of mitochondria in lymphocyte adhesion, polarity, and migration. We show that during adhesion to the activated endothelium under physiological flow conditions, lymphocyte mitochondria redistribute to the adhesion zone together with the microtubule-organizing center (MTOC) in an integrin-dependent manner. Mitochondrial redistribution and efficient lymphocyte adhesion to the endothelium require the function of Miro-1, an adaptor molecule that couples mitochondria to microtubules. Our data demonstrate that Miro-1 associates with the dynein complex. Moreover, mitochondria accumulate around the MTOC in response to the chemokine CXCL12/SDF-1α; this redistribution is regulated by Miro-1. CXCL12-dependent cell polarization and migration are reduced in Miro-1-silenced cells, due to impaired myosin II activation at the cell uropod and diminished actin polymerization. These data point to a key role of Miro-1 in the control of lymphocyte adhesion and migration through the regulation of mitochondrial redistribution.

End-binding protein 1 controls signal propagation from the T cell receptor.

The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unresolved. Here, we show that T cell activation requires the growth of MTs mediated by the plus-end specific protein end-binding 1 (EB1). A direct interaction of the T cell receptor (TCR) complex with EB1 provides the molecular basis for EB1 activity promoting TCR encounter with signalling vesicles at the IS. EB1 knockdown alters TCR dynamics at the IS and prevents propagation of the TCR activation signal to LAT, thus inhibiting activation of PLCγ1 and its localization to the IS. These results identify a role for EB1 interaction with the TCR in controlling TCR sorting and its connection with the LAT/PLCγ1 signalosome.

The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse.

During antigen-specific T-cell activation, mitochondria mobilize towards the vicinity of the immune synapse. We show here that the mitochondrial fission factor dynamin-related protein 1 (Drp1) docks at mitochondria, regulating their positioning and activity near the actin-rich ring of the peripheral supramolecular activation cluster (pSMAC) of the immune synapse. Mitochondrial redistribution in response to T-cell receptor engagement was abolished by Drp1 silencing, expression of the phosphomimetic mutant Drp1S637D and the Drp1-specific inhibitor mdivi-1. Moreover, Drp1 knockdown enhanced mitochondrial depolarization and T-cell receptor signal strength, but decreased myosin phosphorylation, ATP production and T-cell receptor assembly at the central supramolecular activation cluster (cSMAC). Our results indicate that Drp1-dependent mitochondrial positioning and activity controls T-cell activation by fuelling central supramolecular activation cluster assembly at the immune synapse.

Receptors, signaling networks, and disease.

Over the past years, a holistic approach has been applied to the study of the field of receptor signaling, permitting the analysis of how the interaction between receptors and their cellular environment determines receptor function and the study of the role of these receptors, under both normal and pathophysiological conditions, in whole organisms. This has been facilitated by the development of high-resolution microscopy techniques, which allow single-molecule or spatiotemporal resolution, or both, of signaling processes at the cellular and organismal levels. Concurrently, the role of these signaling pathways can be tested in increasingly sophisticated murine disease models. Finally, computational approaches aid in predicting and understanding receptor behavior. The program of the Madrid meeting reflected this integrated approach, highlighting signaling by and dynamics and regulation of immune cell receptors, the T cell receptor and B cell receptor, and signaling by and regulation of G protein-coupled receptors.