RESUMEN
Evaluating the ability of cytotoxic T lymphocytes (CTLs) to eliminate tumor cells is crucial, for instance, to predict the efficiency of cell therapy in personalized medicine. However, the destruction of a tumor by CTLs involves CTL migration in the extra-tumoral environment, accumulation on the tumor, antigen recognition, and cooperation in killing the cancer cells. Therefore, identifying the limiting steps in this complex process requires spatio-temporal measurements of different cellular events over long periods. Here, we use a cancer-on-a-chip platform to evaluate the impact of adenomatous polyposis coli (APC) mutation on CTL migration and cytotoxicity against 3D tumor spheroids. The APC mutated CTLs are found to have a reduced ability to destroy tumor spheroids compared with control cells, even though APC mutants migrate in the extra-tumoral space and accumulate on the spheroids as efficiently as control cells. Once in contact with the tumor however, mutated CTLs display reduced engagement with the cancer cells, as measured by a metric that distinguishes different modes of CTL migration. Realigning the CTL trajectories around localized killing cascades reveals that all CTLs transition to high engagement in the 2 h preceding the cascades, which confirms that the low engagement is the cause of reduced cytotoxicity. Beyond the study of APC mutations, this platform offers a robust way to compare cytotoxic cell efficiency of even closely related cell types, by relying on a multiscale cytometry approach to disentangle complex interactions and to identify the steps that limit the tumor destruction.
Asunto(s)
Poliposis Adenomatosa del Colon , Neoplasias , Humanos , Neoplasias/genética , Linfocitos T Citotóxicos , Mutación , Dispositivos Laboratorio en un ChipRESUMEN
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.
Asunto(s)
Poliposis Adenomatosa del Colon , Neoplasias Colorrectales , Linfocitos T , Humanos , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Movimiento Celular/genética , Neoplasias Colorrectales/genética , Genes APC , Mutación , Proyectos Piloto , Linfocitos T/inmunologíaRESUMEN
Immunological 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 leading 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 control 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.
Asunto(s)
Sinapsis Inmunológicas , Linfocitos T Citotóxicos , Microscopía , Gránulos Citoplasmáticos , Citoesqueleto , Polaridad CelularRESUMEN
Adenomatous 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.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon , Poliposis Adenomatosa del Colon , Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/genética , Movimiento Celular , Humanos , Mutación , FenotipoRESUMEN
Adenomatous 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.
Asunto(s)
Actinas/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/genética , Sinapsis Inmunológicas/metabolismo , Microtúbulos/inmunología , Factores de Transcripción NFATC/genética , Poliposis Adenomatosa del Colon/metabolismo , Poliposis Adenomatosa del Colon/patología , Proteína de la Poliposis Adenomatosa del Colon/inmunología , Animales , Diferenciación Celular , Línea Celular Tumoral , Citoesqueleto/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microtúbulos/ultraestructura , Mutación , Factores de Transcripción NFATC/inmunología , Transducción de Señal , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Endosomal traffic of TCR and signaling molecules regulates immunological synapse formation and T cell activation. We recently showed that Rab11 endosomes regulate the subcellular localization of the tyrosine kinase Lck and of the GTPase Rac1 and control their functions in TCR signaling and actin cytoskeleton remodeling. HIV-1 infection of T cells alters their endosomal traffic, activation capacity, and actin cytoskeleton organization. The viral protein Nef is pivotal for these modifications. We hypothesized that HIV-1 Nef could jointly alter Lck and Rac1 endosomal traffic and concomitantly modulate their functions. In this study, we show that HIV-1 infection of human T cells sequesters both Lck and Rac1 in a pericentrosomal compartment in an Nef-dependent manner. Strikingly, the Nef-induced Lck compartment contains signaling-competent forms (phosphorylated on key Tyr residues) of Lck and some of its downstream effectors, TCRζ, ZAP70, SLP76, and Vav1, avoiding the proximal LAT adaptor. Importantly, Nef-induced concentration of signaling molecules was concomitant with the upregulation of several early and late T cell activation genes. Moreover, preventing the concentration of the Nef-induced Lck compartment by depleting the Rab11 effector FIP3 counteracted Nef-induced gene expression upregulation. In addition, Nef extensively sequesters Rac1 and downregulates Rac1-dependent actin cytoskeleton remodeling, thus reducing T cell spreading. Therefore, by modifying their endosomal traffic, Nef hijacks signaling and actin cytoskeleton regulators to dually modulate their functional outputs. Our data shed new light into the molecular mechanisms that modify T cell physiology during HIV-1 infection.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Infecciones por VIH/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Proteína de Unión al GTP rac1/metabolismo , Citoesqueleto de Actina/inmunología , Citoesqueleto de Actina/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Endosomas/inmunología , Endosomas/metabolismo , Endosomas/virología , Infecciones por VIH/metabolismo , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Transporte de Proteínas/inmunología , Transducción de Señal/inmunología , Proteína de Unión al GTP rac1/inmunologíaRESUMEN
Adenomatous polyposis coli (APC) is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer development. Although extensively studied in epithelial transformation, the effect of APC on T lymphocyte activation remains poorly defined. We found that APC ensures T cell receptor-triggered activation through Nuclear Factor of Activated T cells (NFAT), since APC is necessary for NFAT's nuclear localization in a microtubule-dependent fashion and for NFAT-driven transcription leading to cytokine gene expression. Interestingly, NFAT forms clusters juxtaposed with microtubules. Ultimately, mouse Apc deficiency reduces the presence of NFAT in the nucleus of intestinal regulatory T cells (Tregs) and impairs Treg differentiation and the acquisition of a suppressive phenotype, which is characterized by the production of the anti-inflammatory cytokine IL-10. These findings suggest a dual role for APC mutations in colorectal cancer development, where mutations drive the initiation of epithelial neoplasms and also reduce Treg-mediated suppression of the detrimental inflammation that enhances cancer growth.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/genética , Regulación Neoplásica de la Expresión Génica , Microtúbulos/inmunología , Factores de Transcripción NFATC/genética , Linfocitos T Reguladores/inmunología , Poliposis Adenomatosa del Colon/inmunología , Poliposis Adenomatosa del Colon/patología , Proteína de la Poliposis Adenomatosa del Colon/antagonistas & inhibidores , Proteína de la Poliposis Adenomatosa del Colon/inmunología , Animales , Diferenciación Celular , Línea Celular Tumoral , Células HCT116 , Humanos , Interleucina-10/genética , Interleucina-10/inmunología , Células Jurkat , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microtúbulos/ultraestructura , Factores de Transcripción NFATC/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Linfocitos T Reguladores/patologíaRESUMEN
The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production.
Asunto(s)
Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Western Blotting , Endosomas/inmunología , Técnicas de Silenciamiento del Gen , Humanos , Quinasa I-kappa B/inmunología , Sinapsis Inmunológicas/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Microscopía Confocal , Reacción en Cadena de la Polimerasa , Transporte de Proteínas/inmunología , Proteínas de Unión al GTP rab/inmunologíaRESUMEN
The adapter protein SLP76 is a key orchestrator of T cell receptor (TCR) signal transduction. We previously identified a negative feedback loop that modulates T cell activation, involving phosphorylation of Ser376 of SLP76 by the hematopoietic progenitor kinase 1 (HPK1). However, the physiological relevance of this regulatory mechanism was still unknown. To address this question, we generated a SLP76-S376A-expressing knock-in mouse strain and investigated the effects of Ser376 mutation on T cell development and function. We report here that SLP76-S376A-expressing mice exhibit normal thymocyte development and no detectable phenotypic alterations in mature T cell subsets or other lymphoid and myeloid cell lineages. Biochemical analyses revealed that mutant T cells were hypersensitive to TCR stimulation. Indeed, phosphorylation of several signaling proteins, including SLP76 itself, phospholipase Cγ1 and the protein kinases AKT and ERK1/2, was increased. These modifications correlated with increased Th1-type and decreased Th2-type cytokine production by SLP76-S376A T cells, but did not result in significant changes of proliferative capacity nor activation-induced cell death susceptibility. Hence, our results reveal that SLP76-Ser376 phosphorylation does not mediate all HPK1-dependent regulatory effects in T cells but it fine-tunes helper T cell responses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfoproteínas/metabolismo , Serina/metabolismo , Linfocitos T/metabolismo , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación , Transducción de SeñalRESUMEN
The immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation.
Asunto(s)
Actinas/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Quinasa I-kappa B/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Línea Celular , Células Cultivadas , Endosomas/metabolismo , Humanos , Quinasa I-kappa B/genética , Sinapsis Inmunológicas/metabolismo , Interleucina-2/metabolismo , Células Jurkat , ARN Interferente Pequeño/genéticaRESUMEN
Antigen recognition within immunological synapses triggers and sustains T cell activation by nucleating protein microclusters that gather T cell receptors (TCRs), kinases, and adaptors. Dissipation of these microclusters results in signal termination, but how this process is regulated is unclear. In this paper, we reveal that release of the adaptors SLP76 and GADS from signaling microclusters is induced by the serine/threonine protein kinase HPK1 and that phosphorylation of GADS plays a major role in this process. We found that HPK1 was recruited into microclusters and triggered their dissipation by inducing the phosphorylation of a threonine-containing motif of GADS, together with the previously described serine phosphorylation of SLP76. These events induced the cooperative binding of 14-3-3 proteins to SLP76-GADS complexes, leading to their uncoupling from the transmembrane adaptor LAT and consequently reducing microcluster persistence and activation-induced gene transcription. These results demonstrate that serine/threonine phosphorylation of multiple TCR-proximal effectors controls the stability of signaling microclusters, thereby determining the intensity of T cell responses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Activación de Linfocitos , Fosfoproteínas/metabolismo , Linfocitos T/fisiología , Proteínas 14-3-3/metabolismo , Regulación hacia Abajo , Humanos , Sinapsis Inmunológicas , Células Jurkat , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunologíaRESUMEN
T-cell receptor (TCR) signalling is triggered and tuned at immunological synapses by the generation of signalling complexes that associate into dynamic microclusters. Microcluster movement is necessary to tune TCR signalling, but the molecular mechanism involved remains poorly known. We show here that the membrane-microfilament linker ezrin has an important function in microcluster dynamics and in TCR signalling through its ability to set the microtubule network organization at the immunological synapse. Importantly, ezrin and microtubules are important to down-regulate signalling events leading to Erk1/2 activation. In addition, ezrin is required for appropriate NF-AT activation through p38 MAP kinase. Our data strongly support the notion that ezrin regulates immune synapse architecture and T-cell activation through its interaction with the scaffold protein Dlg1. These results uncover a crucial function for ezrin, Dlg1 and microtubules in the organization of the immune synapse and TCR signal down-regulation. Moreover, they underscore the importance of ezrin and Dlg1 in the regulation of NF-AT activation through p38.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/inmunología , Sinapsis Inmunológicas , Activación de Linfocitos/inmunología , Proteínas de la Membrana/metabolismo , Microtúbulos/metabolismo , Linfocitos T , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Homólogo 1 de la Proteína Discs Large , Activación Enzimática , Humanos , Sinapsis Inmunológicas/química , Sinapsis Inmunológicas/metabolismo , Sinapsis Inmunológicas/ultraestructura , Células Jurkat , Proteínas de la Membrana/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Linfocitos T/citología , Linfocitos T/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Dendritic cells (DC) are suspected to be involved in transmissible spongiform encephalopathies, including bovine spongiform encephalopathy (BSE). We detected the disease-specific, protease-resistant prion protein (PrP(bse)) in splenic DC purified by magnetic cell sorting 45 days after intraperitoneal inoculation of BSE prions in immunocompetent mice. We showed that bone marrow-derived DC (BMDC) from wild-type or PrP-null mice acquired both PrP(bse) and prion infectivity within 2 h of in vitro culture with a BSE inoculum. BMDC cleared PrP(bse) within 2 to 3 days of culture, while BMDC infectivity was only 10-fold diminished between days 1 and 6 of culture, suggesting that the infectious unit in BMDC is not removed at the same rate as PrP(bse) is removed from these cells. Bone marrow-derived plasmacytoid DC and bone marrow-derived macrophages (BMM) also acquired and degraded PrP(bse) when incubated with a BSE inoculum, with kinetics very similar to those of BMDC. PrP(bse) capture is probably specific to antigen-presenting cells since no uptake of PrP(bse) was observed when splenic B or T lymphocytes were incubated with a BSE inoculum in vitro. Lipopolysaccharide activation of BMDC or BMM prior to BSE infection resulted in an accelerated breakdown of PrP(bse). Injected by the intraperitoneal route, BMDC were not infectious for alymphoid recombination-activated gene 2(0)/common cytokine gamma chain-deficient mice, suggesting that these cells are not capable of directly propagating BSE infectivity to nerve endings.
Asunto(s)
Células Dendríticas/metabolismo , Encefalopatía Espongiforme Bovina/metabolismo , Priones/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Células de la Médula Ósea/metabolismo , Bovinos , Células Cultivadas , Encefalopatía Espongiforme Bovina/fisiopatología , Subunidad gamma Común de Receptores de Interleucina , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Receptores de Interleucina/deficiencia , Receptores de Interleucina/genética , Bazo/citología , Bazo/metabolismoRESUMEN
To clarify the mechanisms leading to the development of Creutzfeldt-Jakob disease in some recipients of pituitary-derived human growth hormone (hGH), we investigated the effects of repeated injections of low prion doses in mice. The injections were performed, as in hGH-treated children, by a peripheral route at short intervals and for an extended period. Twelve groups of 24 mice were intraperitoneally inoculated one, two, or five times per week for 200 days with 2 x 10(-5) to 2 x 10(-8) dilutions of brain homogenate containing the mouse-adapted C506M3 scrapie strain. Sixteen control mice were injected once a week for 200 days with a 2 x 10(-4) dilution of normal brain homogenate. Of mice injected in a single challenge with a scrapie inoculum of a 2 x 10(-4), 2 x 10(-5), or 2 x 10(-6) dilution, 2/10, 1/10, and 0/10 animals developed scrapie, respectively. Control mice remained healthy. One hundred thirty-five of 135 mice injected with repeated prion doses of a 2 x 10(-5) or 2 x 10(-6) dilution succumbed to scrapie. Of mice injected with repeated scrapie doses of a 2 x 10(-7) or 2 x 10(-8) dilution, 52/59 and 38/67 animals died of scrapie, respectively. A high incidence of scrapie was observed in mice receiving repeated doses at low infectivity, whereas there was no disease in mice that were injected once with the same doses. Repeated injections of low prion doses thus constitute a risk for development of prion disease even if the same total dose inoculated in a single challenge does not induce the disease.