Rôle de la réorganisation du cytosquelette des cellules T CD4+ à la synapse immunologique dans les fonctions T

L activation d un lymphocyte T (LT) CD4+ par une cellule présentatrice d antigène (CPA) estune étape cruciale pour la mise en place d une réponse immune adaptatrice efficace contre unpathogène ou une cellule tumorale. Elle nécessite un contact prolongé entre les deux types cellulaires,initié par la reconnaissance, par le récepteur des LT (TCR), d un complexe CMH-peptide spécifiqueprésenté à la surface de la CPA. Cette interaction LT-CPA induit la formation d une zone de contactorganisée dans le temps et l espace, appelée la synapse immunologique. La mise en place de cettestructure entraîne le remodelage des cytosquelettes d actine et de microtubules dans les LT. Quelquesminutes après la reconnaissance de l antigène par le TCR, l actine se polymérise à la zone synaptiqueet le centre organisateur des microtubules (MTOC) se polarise en face de la CPA.Le but du travail de thèse présenté ici a été d étudier le rôle de la réorganisation ducytosquelette des LT lors de la mise en place de la synapse, dans la sécrétion des cytokines et lemaintien de l interaction permettant l activation des LT. Nous nous sommes particulièrementintéressés au rôle de deux protéines qui régulent le remodelage du cytosquelette : la petite RhoGTPaseCdc42 et un de ses partenaires IQGAP1. Cette étude a été réalisée essentiellement dans des LT CD4+primaires humains, grâce au développement d une approche permettant d inhiber l expression de cesdeux protéines par introduction de shRNAs à l aide de vecteurs lentiviraux.Nous avons ainsi mis en évidence que le remaniement du cytosquelette d actine à la synapseétait dépendant de Cdc42, et contrôlait : 1/ la formation d un anneau d actine polymérisée enpériphérie de la synapse, 2/ le recrutement et la concentration des vésicules contenant l IFN-g aucentre de la synapse, 3/ la sécrétion de l IFN-g dans la zone synaptique. Nous avons également montréque la protéine IQGAP1 contrôlait le remaniement de l actine des LT à la synapse, la signalisation enaval du TCR, et la dynamique de contact entre LT et CPA.Cette étude participe à une meilleure compréhension du rôle du remodelage du cytosqueletted actine et de sa régulation dans la mise en place de l interaction entre LT et CPA, l activation des LTet une de leur fonction clé : la sécrétion de lymphokines.CD4+ T lymphocyte activation by an antigen presenting cell (APC) is a crucial step in theestablishment of an adaptive immune response against pathogens or tumor cells. It requires contactbetween the two cell types, initiated by the recognition, by the T cell receptor (TCR), of a specificpeptide-MHC complex presented by the APC. This T cell-APC interaction induces the formation of aparticular zone organized in time and space, called the immunological synapse. The establishment ofthis structure induces the remodeling of the actin and microtubule cytoskeleton in T cells. Few minutesafter antigen recognition by the TCR, actin polymerizes at the synaptic zone, and the microtubuleorganizing center (MTOC) polarizes toward the APC.The goal of the work presented here was to study the role of T cell cytoskeleton remodelingduring the establishment of the synapse, in cytokine secretion and in the maintenance of the interactionallowing T cell activation. We mainly studied the role of two proteins regulating the cytoskeleton: thesmall RhoGTPase Cdc42 and one of its partners IQGAP1. This study was performed mostly in humanprimary CD4+ T cells, thanks to the development of an approach allowing the inhibition of theexpression of these two proteins by introducing shRNAs through lentiviral vectors.Altogether, our results show that remodeling of the actin cytoskeleton at the synapse isdependent on Cdc42 and controls : 1/ the formation of a polymerized actin ring at the periphery of thesynapse, 2/ the recruitment and concentration of vesicles containing IFN-g at the center of the synapse,3/ the secretion of IFN-g in the synaptic cleft. They also show that IQGAP1 controls T cell actinremodeling at the synapse, signaling downstream the TCR, and the dynamic of interactions between Tcells and APC.This study allows a better understanding of the role of cytoskeleton remodeling and of itsregulation in the establishment of Tcell-APC interaction, the activation of T cells and one of their keyfunctions: lymphokine secretion.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

Dynamic palmitoylation events following T-cell receptor signaling

Palmitoylation is the reversible addition of palmitate to cysteine via a thioester linkage. The reversible nature of this modification makes it a prime candidate as a mechanism for regulating signal transduction in T-cell receptor signaling. Following stimulation of the T-cell receptor we find a number of proteins are newly palmitoylated, including those involved in vesicle-mediated transport and Ras signal transduction. Among these stimulation-dependent palmitoylation targets are the v-SNARE VAMP7, important for docking of vesicular LAT during TCR signaling, and the largely undescribed palmitoyl acyltransferase DHHC18 that is expressed in two isoforms in T cells. Using our newly developed On-Plate Palmitoylation Assay (OPPA), we show DHHC18 is capable of palmitoylating VAMP7 at Cys183. Cellular imaging shows that the palmitoylation-deficient protein fails to be retained at the Golgi and to localize to the immune synapse upon T cell activation

Characterization of endogenous Kv1.3 channel isoforms in T cells

Producción CientíficaVoltage-dependent potassium channel Kv1.3 plays a key role on T-cell activation; however, lack of reliable antibodies has prevented its accurate detection under endogenous circumstances. To overcome this limitation, we created a Jurkat T-cell line with endogenous Kv1.3 channel tagged, to determine the expression, location, and changes upon activation of the native Kv1.3 channels. CRISPR-Cas9 technique was used to insert a Flag-Myc peptide at the C terminus of the KCNA3 gene. Basal or activated channel expression was studied using western blot analysis and imaging techniques. We identified two isoforms of Kv1.3 other than the canonical channel (54 KDa) differing on their N terminus: a longer isoform (70 KDa) and a truncated isoform (43 KDa). All three isoforms were upregulated after T-cell activation. We focused on the functional characterization of the truncated isoform (short form, SF), because it has not been previously described and could be present in the available Kv1.3−/− mice models. Overexpression of SF in HEK cells elicited small amplitude Kv1.3-like currents, which, contrary to canonical Kv1.3, did not induce HEK proliferation. To explore the role of endogenous SF isoform in a native system, we generated both a knockout Jurkat clone and a clone expressing only the SF isoform. Although the canonical isoform (long form) localizes mainly at the plasma membrane, SF remains intracellular, accumulating perinuclearly. Accordingly, SF Jurkat cells did not show Kv1.3 currents and exhibited depolarized resting membrane potential (VM), decreased Ca2+ influx, and a reduction in the [Ca2+]i increase upon stimulation. Functional characterization of these Kv1.3 channel isoforms showed their differential contribution to signaling pathways involved in formation of the immunological synapse. We conclude that alternative translation initiation generates at least three endogenous Kv1.3 channel isoforms in T cells that exhibit different functional roles. For some of these functions, Kv1.3 proteins do not need to form functional plasma membrane channels.Ministerio de Economía y Competitividad (grant PID 2020‐118517RB‐I00)Junta de Castilla y León (grants VA172P20) and (CLU-2019-02)Funds from Institut Curie, INSERM,Agence Nationale de la Recherche RetroTact (ANR‐20CE15‐0009‐01,ANR‐10‐IDEX‐0001‐02 PSL*, and ANR‐11‐LABX‐0043)Fondation pour la Recherche Médicale FRM (EQU202003010280

T-Cell Artificial Focal Triggering Tools: Linking Surface Interactions with Cell Response

T-cell activation is a key event in the immune system, involving the interaction of several receptor ligand pairs in a complex intercellular contact that forms between T-cell and antigen-presenting cells. Molecular components implicated in contact formation have been identified, but the mechanism of activation and the link between molecular interactions and cell response remain poorly understood due to the complexity and dynamics exhibited by whole cell-cell conjugates. Here we demonstrate that simplified model colloids grafted so as to target appropriate cell receptors can be efficiently used to explore the relationship of receptor engagement to the T-cell response. Using immortalized Jurkat T cells, we monitored both binding and activation events, as seen by changes in the intracellular calcium concentration. Our experimental strategy used flow cytometry analysis to follow the short time scale cell response in populations of thousands of cells. We targeted both T-cell receptor CD3 (TCR/CD3) and leukocyte-function-associated antigen (LFA-1) alone or in combination. We showed that specific engagement of TCR/CD3 with a single particle induced a transient calcium signal, confirming previous results and validating our approach. By decreasing anti-CD3 particle density, we showed that contact nucleation was the most crucial and determining step in the cell-particle interaction under dynamic conditions, due to shear stress produced by hydrodynamic flow. Introduction of LFA-1 adhesion molecule ligands at the surface of the particle overcame this limitation and elucidated the low TCR/CD3 ligand density regime. Despite their simplicity, model colloids induced relevant biological responses which consistently echoed whole cell behavior. We thus concluded that this biophysical approach provides useful tools for investigating initial events in T-cell activation, and should enable the design of intelligent artificial systems for adoptive immunotherapy

Force Generation upon T Cell Receptor Engagement

T cells are major players of adaptive immune response in mammals. Recognition of an antigenic peptide in association with the major histocompatibility complex at the surface of an antigen presenting cell (APC) is a specific and sensitive process whose mechanism is not fully understood. The potential contribution of mechanical forces in the T cell activation process is increasingly debated, although these forces are scarcely defined and hold only limited experimental evidence. In this work, we have implemented a biomembrane force probe (BFP) setup and a model APC to explore the nature and the characteristics of the mechanical forces potentially generated upon engagement of the T cell receptor (TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon contact with a model APC coated with antibodies towards TCR-CD3, after a short latency, the T cell developed a timed sequence of pushing and pulling forces against its target. These processes were defined by their initial constant growth velocity and loading rate (force increase per unit of time). LFA-1 engagement together with TCR-CD3 reduced the growing speed during the pushing phase without triggering the same mechanical behavior when engaged alone. Intracellular Ca2+ concentration ([Ca2+]i) was monitored simultaneously to verify the cell commitment in the activation process. [Ca2+]i increased a few tens of seconds after the beginning of the pushing phase although no strong correlation appeared between the two events. The pushing phase was driven by actin polymerization. Tuning the BFP mechanical properties, we could show that the loading rate during the pulling phase increased with the target stiffness. This indicated that a mechanosensing mechanism is implemented in the early steps of the activation process. We provide here the first quantified description of force generation sequence upon local bidimensional engagement of TCR-CD3 and discuss its potential role in a T cell mechanically-regulated activation process

Tout ce que vous avez toujours voulu savoir sur la protéine ZAP-70

La tyrosine kinase ZAP-70 (zeta-chain-associated protein of 70 kDa) a connu son heure de gloire au début des années 1990, lorsque des mutations du gène codant pour cette protéine ont été mises en évidence chez de très jeunes patients présentant des déficits immunitaires combinés sévères. La caractérisation, chez ces patients, de déficits fonctionnels des lymphocytes T signait le rôle crucial de cette protéine dans l’activation de ces cellules. Très récemment, un regain d’intérêt pour ZAP-70 est apparu lorsque son expression, dans un premier temps considérée comme spécifique des lymphocytes T et des cellules NK (natural killer), a été mise en évidence dans certaines leucémies lymphoïdes chroniques B et associée à un facteur pronostique. Enfin, la découverte d’une mutation ponctuelle spontanée de ZAP-70 dans une souche de souris présentant une arthrite rhumatoïde révélait également le rôle potentiel de ZAP-70 dans des maladies auto-immunes. Les diverses maladies associées à des modifications d’expression ou d’activité de ZAP-70 démontrent le rôle clé de cette kinase dans la mise en place et la régulation d’une réponse immunitaire.The ZAP-70 tyrosine kinase has been described more than ten years ago. Its key role in thymocytes development and mature T lymphocytes activation has been illustrated by the characterization of several human immunodeficiencies presenting with mutations in the zap-70 gene resulting in the absence of ZAP-70 expression. More recently, it has been shown that deregulation of ZAP-70 activity can induce autoimmune diseases. Finally, ZAP-70 expression has been shown in some B chronic lymphocytic leukaemia and correlated with bad prognosis of the disease. The diversity of pathologies associated with deregulation of ZAP-70 demonstrates its key role in immune responses. Research aiming at deciphering the different signalling pathways regulated by ZAP-70 will not only shed some lights on these pathologies, but will also help finding new pharmacological tools, targeting ZAP-70, designed to induce immunosuppression or tolerance

Contribution a l'etude de l'activation et de la proliferation des cellules B de leucemie lymphoiede chronique humaine

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc