Primary immunodeficiencies as models to decipher the molecular control of actin remodelling in immune cells

Le cytosquelette d'actine est, avec les microtubules et les filaments intermédiaires, un composant essentiel des cellules eucaryotes. Son dynamisme, sa structure et les différents régulateurs qui le contrôlent permettent de soutenir différentes fonctions cellulaires. L'importance de cette structure est d'ailleurs révélée par l'existence d'immunodéficience primaire, maladies génétiques rares, causée par des mutations touchant les régulateurs du cytosquelette d'actine. À ce jour, 20 déficiences en protéines régulatrices ont été découvertes et étudiées, nous permettant ainsi de mieux comprendre les rôles spécifiques de chaque régulateur dans le contrôle des fonctions immunitaire. Durant ma thèse, j'ai eu l'opportunité d'étudier le rôle de plusieurs régulateurs du cytosquelette d'actine dans les fonctions cellulaires immunitaire telle que WIP, WDR1, ou encore ARPC1B. J'ai en effet démontré que les lymphocytes T CD8+ provenant de patient WIP déficient avait un défaut de cytotoxicité et de migration due à une organisation et polarisation de l'actine anormale. J'ai ensuite pu contribuer à une étude collaborative sur 3 patients déficients en la protéine ARPC1B, où nous avons pu décrire un défaut de la formation de la synapse immunologique. Après avoir décrit le rôle des régulateurs de polymérisation de l'actine, j'ai eu l'opportunité d'étudier la protéine WDR1, protéine impliquée dans la dépolymérisation des microfilaments. L'étude du compartiment lymphoïde de 6 patients porteurs de mutations dans le gène WDR1 a permis de démontrer que WDR1 avait un impact dans le développement des lymphocytes B ainsi que leur différenciation mais avait un impact moindre sur le compartiment lymphocytaire T. En conclusion, mes travaux de thèse ont eu pour but de comprendre plus précisément le rôle des régulateurs d'actine dans les fonctions immunitaires. Mieux comprendre ces mécanismes de régulation permettra un meilleur diagnostic des patients immundéficients et de trouver éventuellement de nouvelle cible de traitement pour les maladies auto inflammatoires.Together with microtubules and intermediate filaments, the actin cytoskeleton is an essential structural component of cells. Its dynamism, its structure and the different regulators sustain multiple cellular functions. The crucial role of the actin cytoskelelon in the functioning of the immune system is best pointed out by the severity of primary immunodeficiencies (PID), genetic diseases, caused by mutations in genes encoding actin cytoskeleton regulators. To date, 20 deficiencies in these proteins as well as upstream regulators have recently been discovered and studied, allowing to better understand the roles of individual actin regulators in controlling the immune cell functions.During my PhD, I had the opportunity to study the roles of several actin regulators in immune cell functions such as WIP, WDR1 and ARPC1B. I described that WIP deficient T lymphocytes fail to form a complete immunological synapse and they display a migration capacity defect towards chemokines, due to an abnormal polarization and actin remodeling. I then contributed to a collaborative study on ARPC1B deficiency and observed that T lymphocytes from patients showed an abnormal immunological synapse formation. After studying the role of actin polymerization promoting factors, I investigated the role of WDR1, an actin filament severing protein. I had the opportunity to study 6 patients carrying mutation in the WDR1 gene. In the lymphoid cell compartment, I described that WDR1 deficiency leads to a dysregulation of actin level and has major impact on B cell diffentiation but more limited impact on T cell signaling. In conclusion, my work has been designed in the way to understand more precisely the role of actin regulators and their roles in immune cellular process. Better understand those mechanisms of regulation will help further to the diagnosis of PID patients and eventually find new target for treatment

Mutations affecting the actin regulator WD repeat–containing protein 1 lead to aberrant lymphoid immunity

Background: The actin-interacting protein WD repeat–containing protein 1 (WDR1) promotes cofilin-dependent actin filament turnover. Biallelic WDR1 mutations have been identified recently in an immunodeficiency/autoinflammatory syndrome with aberrant morphology and function of myeloid cells. Objective: Given the pleiotropic expression of WDR1, here we investigated to what extent it might control the lymphoid arm of the immune system in human subjects. Methods: Histologic and detailed immunologic analyses were performed to elucidate the role of WDR1 in the development and function of B and T lymphocytes. Results: Here we identified novel homozygous and compound heterozygous WDR1 missense mutations in 6 patients belonging to 3 kindreds who presented with respiratory tract infections, skin ulceration, and stomatitis. In addition to defective adhesion and motility of neutrophils and monocytes, WDR1 deficiency was associated with aberrant T-cell activation and B-cell development. T lymphocytes appeared to develop normally in the patients, except for the follicular helper T-cell subset. However, peripheral T cells from the patients accumulated atypical actin structures at the immunologic synapse and displayed reduced calcium flux and mildly impaired proliferation on T-cell receptor stimulation. WDR1 deficiency was associated with even more severe abnormalities of the B-cell compartment, including peripheral B-cell lymphopenia, paucity of B-cell progenitors in the bone marrow, lack of switched memory B cells, reduced clonal diversity, abnormal B-cell spreading, and increased apoptosis on B-cell receptor/Toll-like receptor stimulation. Conclusion: Our study identifies a novel role for WDR1 in adaptive immunity, highlighting WDR1 as a central regulator of actin turnover during formation of the B-cell and T-cell immunologic synapses

Actin Dynamics at the T Cell Synapse as Revealed by Immune-Related Actinopathies

International audienceThe actin cytoskeleton is composed of dynamic filament networks that build adaptable local architectures to sustain nearly all cellular activities in response to a myriad of stimuli. Although the function of numerous players that tune actin remodeling is known, the coordinated molecular orchestration of the actin cytoskeleton to guide cellular decisions is still ill defined. T lymphocytes provide a prototypical example of how a complex program of actin cytoskeleton remodeling sustains the spatio-temporal control of key cellular activities, namely antigen scanning and sensing, as well as polarized delivery of effector molecules, via the immunological synapse. We here review the unique knowledge on actin dynamics at the T lymphocyte synapse gained through the study of primary immunodeficiences caused by mutations in genes encoding actin regulatory proteins. Beyond the specific roles of individual actin remodelers, we further develop the view that these operate in a coordinated manner and are an integral part of multiple signaling pathways in T lymphocytes

Morphological profiling of human T and NK lymphocytes by high-content cell imaging

International audienceThe immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ T cells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture

RASGRP1 deficiency causes immunodeficiency with impaired cytoskeletal dynamics

RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes

Nature Communications / Human DEF6 deficiency underlies an immunodeficiency syndrome with systemic autoimmunity and aberrant CTLA-4 homeostasis

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11CTLA-4 vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.(VLID)492061