Balbino Alarcón - Academia.edu (original) (raw)

Papers by Balbino Alarcón

ABSTRACTIn CD4+ T cells, CCR5 is not only a coreceptor for HIV-1 infection, but also contributes ... more ABSTRACTIn CD4+ T cells, CCR5 is not only a coreceptor for HIV-1 infection, but also contributes to their functional fitness. Here we show that by limiting GATA-1-induced transcription of specific ceramide synthases, CCR5 signaling reduces ceramide levels and thereby increases T cell antigen receptor (TCR) nanoclustering in antigen-experienced mouse and human CD4+ T cells. This activity is CCR5-specific and independent of CCR5 costimulatory activity. CCR5-deficient mice showed reduced production of high affinity class-switched antibodies, but only after antigen rechallenge, which implies an impaired memory CD4+ T cell response. This study identifies a CCR5 function in the generation of CD4+ T cell memory responses, and establishes an antigen-independent mechanism that regulates TCR nanoclustering by altering specific lipid species.

Journal of Experimental Medicine, 2019

Signal strength controls the outcome of αβ T cell selection in the thymus, resulting in death if ... more Signal strength controls the outcome of αβ T cell selection in the thymus, resulting in death if the affinity of the rearranged TCR is below the threshold for positive selection, or if the affinity of the TCR is above the threshold for negative selection. Here we show that deletion of the GTPase RRAS2 results in exacerbated negative selection and above-normal expression of positive selection markers. Furthermore, Rras2−/− mice are resistant to autoimmunity both in a model of inflammatory bowel disease (IBD) and in a model of myelin oligodendrocyte glycoprotein (MOG)–induced experimental autoimmune encephalomyelitis (EAE). We show that MOG-specific T cells in Rras2−/− mice have reduced affinity for MOG/I-Ab tetramers, suggesting that enhanced negative selection leads to selection of TCRs with lower affinity for the self-MOG peptide. An analysis of the TCR repertoire shows alterations that mostly affect the TCRα variable (TRAV) locus with specific VJ combinations and CDR3α sequences t...

Nature communications, Jan 5, 2018

The T-cell antigen receptor (TCR) is pre-organised in oligomers, known as nanoclusters. Nanoclust... more The T-cell antigen receptor (TCR) is pre-organised in oligomers, known as nanoclusters. Nanoclusters could provide a framework for inter-TCR cooperativity upon peptide antigen-major histocompatibility complex (pMHC) binding. Here we have used soluble pMHC oligomers in search for cooperativity effects along the plasma membrane plane. We find that initial binding events favour subsequent pMHC binding to additional TCRs, during a narrow temporal window. This behaviour can be explained by a 3-state model of TCR transition from Resting to Active, to a final Inhibited state. By disrupting nanoclusters and hampering the Active conformation, we show that TCR cooperativity is consistent with TCR nanoclusters adopting the Active state in a coordinated manner. Preferential binding of pMHC to the Active TCR at the immunological synapse suggests that there is a transient time frame for signal amplification in the TCR, allowing the T cells to keep track of antigen quantity and binding time.

Nature Communications, 2016

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing ... more 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.

Journal of Leukocyte Biology, 2015

Discussion on how the adaptor Nck plays a fundamental role for survival of folicular helper T cells.

Proceedings of the National Academy of Sciences of the United States of America, Jan 20, 2006

T cell antigen receptor (TCR) triggering determines the fate of immature thymocytes. The affinity... more T cell antigen receptor (TCR) triggering determines the fate of immature thymocytes. The affinity of the TCR for its endogenous peptide/MHC ligands serves as a signal for positive or negative selection through mechanisms that are still little understood. We have used a conformation-specific antibody to demonstrate that recognition of TCR ligands that lead to negative selection induces a conformational change in the TCR in situ. In contrast, this conformational change is elicited in only a small percentage of immature thymocytes during positive selection. Using a TUNEL assay, we demonstrate that the conformational change in the TCR is strongly linked to activation of programmed cell death in conditions leading to negative selection. Furthermore, the few conformational change-positive thymocytes detected in conditions that preferably lead to positive selection are also TUNEL-positive. Thus, the conformational change in the TCR may underlie the discrimination of ligands leading to posi...

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2015

In the last decade an increasing number of plasma membrane (PM) proteins have been shown to be no... more In the last decade an increasing number of plasma membrane (PM) proteins have been shown to be nonrandomly distributed but instead forming submicron-sized oligomers called nanoclusters. Nanoclusters exist independently of the ligand-bound state of the receptors and their existence implies a high degree of lateral organisation of the PM and its proteins. The mechanisms that drive receptor nanoclustering are largely unknown. One well-defined example of a transmembrane receptor that forms nanoclusters is the T cell antigen receptor (TCR), a multisubunit protein complex whose nanoclustering influences its activity. Membrane lipids, namely cholesterol and sphingomyelin, have been shown to contribute to TCR nanoclustering. However, the identity of the membrane microdomain in which the TCR resides remains controversial. Using a GFP-labeled TCR we show here that the resting TCR localized in the disordered domain of giant PM vesicles (GPMVs) and PM spheres (PMSs) and that single and nanoclustered TCRs are found in the high-density fractions in sucrose gradients. Both findings are indicative of non-raft localization. We discuss possible mechanisms of TCR nanoclustering in T cells. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

Frontiers in Immunology, 2011

T cells reorganize their actin and tubulin-based cytoskeletons to provide a physical basis to the... more T cells reorganize their actin and tubulin-based cytoskeletons to provide a physical basis to the immune synapse. However, growing evidence shows that their roles on T cell activation are more dynamic than merely serving as tracks or scaffold for different molecules. The crosstalk between both skeletons may be important for the formation and movement of the lamella at the immunological synapse by increasing the adhesion of the T cell to the antigen-presenting cells (APC), thus favoring the transport of components toward the plasma membrane and in turn regulating theT-APC intercellular communication. Microtubules and F-actin appear to be essential for the transport of the different signaling microclusters along the membrane, therefore facilitating the propagation of the signal. Finally, they can also be important for regulating the endocytosis, recycling, and degradation of the T cell receptor signaling machinery, thus helping both to sustain the activated state and to switch it off.

Cell Reports, 2014

Activation of the T cell receptor (TCR) by antigen is the key step in adaptive immunity. In the a... more Activation of the T cell receptor (TCR) by antigen is the key step in adaptive immunity. In the abTCR, antigen induces a conformational change at the CD3 subunits (CD3 CC) that is absolutely required for abTCR activation. Here, we demonstrate that the CD3 CC is not induced by antigen stimulation of the mouse G8 or the human Vg9Vd2 gdTCR. We find that there is a fundamental difference between the activation mechanisms of the abTCR and gdTCR that map to the constant regions of the TCRab/gd heterodimers. Enforced induction of CD3 CC with a less commonly used monoclonal anti-CD3 promoted proximal gdTCR signaling but inhibited cytokine secretion. Utilizing this knowledge, we could dramatically improve in vitro tumor cell lysis by activated human gd T cells. Thus, manipulation of the CD3 CC might be exploited to improve clinical gd T cellbased immunotherapies.

Frontiers in Immunology, 2012

Increasing evidence favors the notion that, before triggering, the T cell antigen receptor (TCR) ... more Increasing evidence favors the notion that, before triggering, the T cell antigen receptor (TCR) forms nanometer-scale oligomers that are called nanoclusters. The organization of the TCR in pre-existing oligomers cannot be ignored when analyzing the properties of ligand (pMHC) recognition and signal transduction. As with other membrane receptors, the existence of TCR oligomers points out to cooperativity phenomena. We review the data in support of conformational changes in the TCR as the basic principle to transduce the activation signal to the cytoplasm and the incipient data suggesting cooperativity within nanoclusters.

Trends in Immunology, 2006

During antigen recognition, T cells show high sensitivity and specificity, and a wide dynamic ran... more During antigen recognition, T cells show high sensitivity and specificity, and a wide dynamic range. Paradoxically, these characteristics are based on low-affinity receptor-ligand interactions [between the T-cell antigen receptor (TCR-CD3) complex and the antigen peptide bound to MHC]. Recent evidence indicates that the TCR-CD3 is expressed as multivalent complexes in the membrane of non-stimulated T cells and that conformational changes in the TCR-CD3 can be induced by strong but not weak agonists. Here, we propose a thermodynamic model whereby the specificity of the TCR-CD3-pMHC interaction is explained by its multivalent nature. We also propose that the free energy barriers involved in the change in conformation of the receptor impose a response threshold and determine the kinetic properties of recognition. Finally, we suggest that multivalent TCR-CD3s can amplify signals by spreading them from pMHC-engaged TCR-CD3s to unengaged complexes as a consequence of the cooperativity in the system.

The EMBO Journal, 2012

The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unr... more 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 PLCc1 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/PLCc1 signalosome.

The EMBO Journal, 2014

T-cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antige... more T-cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen-presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified b-arrestin-1 as a ligand of non-phosphorylated resting TCRs. Using dominant-negative and knockdown approaches we demonstrate that b-arrestin-1 is required for the internalization and downregulation of non-engaged bystander TCRs. Furthermore, TCR triggering provokes the b-arrestin-1-mediated downregulation of the G-protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that b-arrestin-1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCRtriggered PKC-mediated phosphorylation of b-arrestin-1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal.

Proceedings of the National Academy of Sciences, 1999

Whether there is one or multiple αβT cell antigen receptor (TCR) recognition modules in a given T... more Whether there is one or multiple αβT cell antigen receptor (TCR) recognition modules in a given TCR/CD3 complex is a long-standing controversy in immunology. We show that T cells from transgenic mice that coexpress comparable amounts of two distinct TCRβ chains incorporate at least two αβTCRs in a single TCR/CD3 complex. Evidence for bispecific αβTCRs was obtained by immunoprecipitation and immunoblotting and confirmed on the surface of living cells both by fluorescence resonance energy transfer and comodulation assays by using antibodies specific for TCRβ-variable regions. Such (αβ) 2 TCR/CD3 or higher-order complexes were evident in T cells studied either ex vivo or after expansion in vitro . T cell activation is thought by many, but not all, to require TCR cross-linking by its antigen/major histocompatibility complex ligand. The implications of a multivalent (αβ) 2 TCR/CD3 complex stoichiometry for the ordered docking of specific antigen/major histocompatibility complex, CD4, or ...

PLoS ONE, 2008

How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major His... more How the T cell antigen receptor (TCR) discriminates between molecularly related peptide/Major Histocompatibility Complex (pMHC) ligands and converts this information into different possible signaling outcomes is still not understood. One current model proposes that strong pMHC ligands, but not weak ones, induce a conformational change in the TCR. Evidence supporting this comes from a pull-down assay that detects ligand-induced binding of the TCR to the N-terminal SH3 domain of the adapter protein Nck, and also from studies with a neoepitope-specific antibody. Both methods rely on the exposure of a polyproline sequence in the CD3e subunit of the TCR, and neither indicates whether the conformational change is transmitted to other CD3 subunits. Using a protease-sensitivity assay, we now show that the cytoplasmic tails of CD3e and CD3f subunits become fully protected from degradation upon TCR triggering. These results suggest that the TCR conformational change is transmitted to the tails of CD3e and CD3f, and perhaps all CD3 subunits. Furthermore, the resistance to protease digestion suggests that CD3 cytoplasmic tails adopt a compact structure in the triggered TCR. These results are consistent with a model in which transduction of the conformational change induced upon TCR triggering promotes condensation and shielding of the CD3 cytoplasmic tails.

The Journal of Immunology, 2014

On TCR ligation, the adaptor Nck is recruited through its src homology 3.1 domain to a proline-ri... more On TCR ligation, the adaptor Nck is recruited through its src homology 3.1 domain to a proline-rich sequence (PRS) in CD3ε. We have studied the relevance of this interaction for T cell activation in vitro and in vivo by targeting the interaction sites in both partners. The first approach consisted of studying a knockin (KI) mouse line (KI-PRS) bearing a conservative mutation in the PRS that makes the TCR incompetent to recruit Nck. This deficiency prevents T cell activation by Ag in vitro and inhibited very early TCR signaling events including the tyrosine phosphorylation of CD3ζ. Most important, KI-PRS mice are partly protected against the development of neurological symptoms in an experimental autoimmune encephalitis model, and show a deficient antitumoral response after vaccination. The second approach consisted of using a high-affinity peptide that specifically binds the src homology 3.1 domain and prevents the interaction of Nck with CD3ε. This peptide inhibits T cell prolifera...

The Journal of Immunology, 2006

The translocation of the microtubule-organizing center (MTOC), its associated signaling complex, ... more The translocation of the microtubule-organizing center (MTOC), its associated signaling complex, and the secretory apparatus is the most characteristic early event that involves the tubulin cytoskeleton of T or NK cells after their interaction with APC or target cells. Our results show that Fyn kinase activity is essential for MTOC reorientation in an Ag-dependent system. Moreover, T cells from Fyn-deficient mice are unable to rearrange their tubulin cytoskeleton in response to anti-CD3-coated beads. Analysis of conjugates of T cells from transgenic OT-I mice with dendritic cells revealed that an antagonist peptide induces translocation of the MTOC, and that this process is impaired in T cells from Fyn−/− OT-I mice. In addition, Fyn deficiency significantly affects the MTOC relocation mediated by agonist peptide stimulation. These results reveal Fyn to be a key regulator of tubulin cytoskeleton reorganization in T cells.

The Journal of Immunology, 2008

TCR gene therapy is adversely affected by newly formed TCRαβ heterodimers comprising exogenous an... more TCR gene therapy is adversely affected by newly formed TCRαβ heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCRαβ dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3ζ with specificities for three different Ags. Transfer of either TCRα:CD3ζ or β:CD3ζ genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCRαβ:ζ does not compromise surface expression and functions of an endogenous TCRαβ. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCRαβ:CD3ζ is the first strategy that results in highly preferred pairing between CD3ζ-modified TCRα and β chains as well as absence of TCR mispairing between TCR:CD3ζ and nonmodified TCR chains. Intrace...

Journal of Immunological Methods, 2007

Characterization of multiprotein complexes (MPCs) is an important step toward an integrative view... more Characterization of multiprotein complexes (MPCs) is an important step toward an integrative view of protein interaction networks and prerequisite for a molecular understanding of how a certain MPC functions. Here, we present a technique utilizing monoclonal subunit-specific antibodies for an electrophoretic immunoshift assay in Blue Native-gels (NAMOS-assay), which allows the determination of the stoichiometry of MPCs. First, we use the B cell antigen receptor as a model MPC whose stoichiometry is known, confirming the HC 2 LC 2 Igα/β 1 stoichiometry. Second, we demonstrate that the digitonin-extracted T cell antigen receptor (TCR) extracted from T cells has a stoichiometry of αβε 2 γδζ 2. We then show that the NAMOS-assay does not require purified MPCs, since it can determine the stoichiometry of an MPC in cell lysates. The NAMOS-assay is also compatible with use of epitope tags appended to the protein of interest, as e.g. the widely used HA-tag, and anti-epitope antibodies for the assay. Given its general applicability, this method has a wide potential for MPC research.