Loss of ARPC1B impairs cytotoxic T lymphocyte maintenance and cytolytic activity.

CD8 cytotoxic T lymphocytes (CTLs) rely on rapid reorganization of the branched F-actin network to drive the polarized secretion of lytic granules, initiating target cell death during the adaptive immune response. Branched F-actin is generated by the nucleation factor actin-related protein 2/3 (Arp2/3) complex. Patients with mutations in the actin-related protein complex 1B (ARPC1B) subunit of Arp2/3 show combined immunodeficiency, with symptoms of immune dysregulation, including recurrent viral infections and reduced CD8+ T cell count. Here, we show that loss of ARPC1B led to loss of CTL cytotoxicity, with the defect arising at 2 different levels. First, ARPC1B is required for lamellipodia formation, cell migration, and actin reorganization across the immune synapse. Second, we found that ARPC1B is indispensable for the maintenance of TCR, CD8, and GLUT1 membrane proteins at the plasma membrane of CTLs, as recycling via the retromer and WASH complexes was impaired in the absence of ARPC1B. Loss of TCR, CD8, and GLUT1 gave rise to defects in T cell signaling and proliferation upon antigen stimulation of ARPC1B-deficient CTLs, leading to a progressive loss of CD8+ T cells. This triggered an activation-induced immunodeficiency of CTL activity in ARPC1B-deficient patients, which could explain the susceptibility to severe and prolonged viral infections

Le rôle d’adénomatous polyposis coli dans les fonctions des lymphocytes T cytotoxiques

Immunological synapses are the result of CD8 T cell polarization toward an antigen presenting cell or tumor cell. This depends on the action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. Actin intensively polymerizes at the synapse, and then is excluded from the center to form a ring at the synapse periphery. Microtubules are repositioned at the synapse, allowing the polarization of the centrosome and its docking to the plasma membrane. Microtubules drive the polarized transport of TCR and several signaling molecules, ensuring CD8 T cell activation, and of lytic granules, ensuring cytotoxic T cell (CTL) functions. We have identified the polarity regulator and tumor suppressor adenomatous polyposis coli (Apc) as a key regulator of actin and microtubule cytoskeleton in CTLs. Apc allows the actin ring formation at the synapse periphery. In addition, Apc regulates microtubule radial organization and centrosome polarization. As a likely consequence, Apc controls synapse shape symmetry and stability. Interestingly, Apc defects reduce early TCR signaling and nuclear translocation of the transcription factor NFAT, with no significant impact in CTL differentiation and cytokine production. Importantly, Apc modulates CTL cytotoxic activity, by allowing efficient lytic granule targeting, dynamics, and fusion at the plasma membrane. Therefore, this work unveils a novel regulatory role of Apc in cytotoxic T cell effector functions, through its action as polarity regulator and cytoskeleton organizer. It provides further insight into the potential impact of Apc mutations in anti-tumor immune response in familial adenomatous polyposis.La synapse immunologique est le résultat de la polarisation du lymphocyte T CD8 vers une cellule présentatrice ou tumorale. Cette polarisation dépend de la réorganisation du cytosquelette d’actine et du réseau de microtubules, mais aussi du trafic vésiculaire intracellulaire. L’actine polymérise à la synapse, puis est exclue de son centre pour former un anneau à sa périphérie. Les microtubules sont repositionnés à la synapse, permettant le transport polarisé de vésicules contenant des TCR et des molécules de signalisation, assurant l’activation des T CD8, ou de granules lytiques, assurant leurs fonctions cytotoxiques. Nous avons identifié le régulateur de polarité cellulaire et suppresseur de tumeur adénomatous polyposis coli (Apc) comme un régulateur clé du cytosquelette d’actine et des microtubules dans les lymphocytes T CD8. Apc régule la formation de l’anneau d’actine à la périphérie de la synapse. De plus, Apc contrôle l’organisation radiale des microtubules à la synapse. En conséquence, Apc module la forme, la symétrie et la stabilité de la synapse. Des défauts de Apc réduisent la signalisation du TCR et empêchent la translocation dans le noyau du facteur de transcription NFAT. Cependant, ces altérations n’induisent pas de défauts de différentiation ou de production de cytokines des T CD8. Néanmoins, Apc contrôle leur activité cytotoxique, facilitant l’adressage, la dynamique, et la fusion des granules lytiques à la membrane synaptique. Ces résultats révèlent donc un nouveau rôle de Apc dans la régulation des fonctions cytotoxiques des lymphocytes T CD8, via son action de régulateur de polarité et d’organisateur du cytosquelette. Ils suggèrent une altération de la réponse immune anti-tumorale des patients atteints de polypose familiale

Chapter 1 - Imaging polarized granule release at the cytotoxic T cell immunological synapse using TIRF microscopy: Control by polarity regulators

International audienceImmunological 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 lead- ing 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 con- trol 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

Coordinating Cytoskeleton and Molecular Traffic in T Cell Migration, Activation, and Effector Functions

International audienceDynamic 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

Chapter 1 - Imaging polarized granule release at the cytotoxic T cell immunological synapse using TIRF microscopy: Control by polarity regulators

International audienceImmunological 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 lead- ing 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 con- trol 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

Adenomatous Polyposis Coli Modulates Actin and Microtubule Cytoskeleton at the Immunological Synapse to Tune CTL Functions

International audienceAdenomatous 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

Loss of ARPC1B impairs cytotoxic T lymphocyte maintenance and cytolytic activity

CD8 cytotoxic T lymphocytes (CTLs) rely on rapid reorganization of the branched F-actin network to drive the polarized secretion of lytic granules, initiating target cell death during the adaptive immune response. Branched F-actin is generated by the nucleation factor actin-related protein 2/3 (Arp2/3) complex. Patients with mutations in the actin-related protein complex 1B (ARPC1B) subunit of Arp2/3 show combined immunodeficiency, with symptoms of immune dysregulation, including recurrent viral infections and reduced CD8+ T cell count. Here, we show that loss of ARPC1B led to loss of CTL cytotoxicity, with the defect arising at 2 different levels. First, ARPC1B is required for lamellipodia formation, cell migration, and actin reorganization across the immune synapse. Second, we found that ARPC1B is indispensable for the maintenance of TCR, CD8, and GLUT1 membrane proteins at the plasma membrane of CTLs, as recycling via the retromer and WASH complexes was impaired in the absence of ARPC1B. Loss of TCR, CD8, and GLUT1 gave rise to defects in T cell signaling and proliferation upon antigen stimulation of ARPC1B-deficient CTLs, leading to a progressive loss of CD8+ T cells. This triggered an activation-induced immunodeficiency of CTL activity in ARPC1B-deficient patients, which could explain the susceptibility to severe and prolonged viral infections

The tumor suppressor adenomatous polyposis coli regulates T lymphocyte migration

International audienceAdenomatous 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

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

DataSheet_1_T cell migration and effector function differences in familial adenomatous polyposis patients with APC gene mutations.pdf

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.</p