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The EphB4-ephrinB2 signaling axis has been heavily implicated in metastasis across numerous cancer types. Our emerging understanding of the dichotomous roles that EphB4 and ephrinB2 play in head and neck squamous cell carcinoma (HNSCC) poses a significant challenge to rational drug design. We find that EphB4 knockdown in cancer cells enhances metastasis in preclinical HNSCC models by augmenting immunosuppressive cells like T regulatory cells (Tregs) within the tumor microenvironment. EphB4 inhibition in cancer cells also amplifies their ability to metastasize through increased expression of genes associated with epithelial mesenchymal transition and hallmark pathways of metastasis. In contrast, vascular ephrinB2 knockout coupled with radiation therapy (RT) enhances anti-tumor immunity, reduces Treg accumulation into the tumor, and decreases metastasis. Notably, targeting the EphB4-ephrinB2 signaling axis with the engineered EphB4 ligands EFNB2-Fc-His and Fc-TNYL-RAW-GS reduces local tumor growth and distant metastasis in a preclinical model of HNSCC. Our data suggest that targeted inhibition of vascular ephrinB2 while avoiding inhibition of EphB4 in cancer cells could be a promising strategy to mitigate HNSCC metastasis.
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Significance: Stimulated emission depletion (STED) is a powerful super-resolution microscopy technique that can be used for imaging live cells. However, the high STED laser powers can cause significant photobleaching and sample damage in sensitive biological samples. The dynamic intensity minimum (DyMIN) technique turns on the STED laser only in regions of the sample where there is fluorescence signal, thus saving significant sample photobleaching. The reduction in photobleaching allows higher resolution images to be obtained and longer time-lapse imaging of live samples. A stand-alone module to perform DyMIN is not available commercially. Aim: In this work, we developed an open-source design to implement three-step DyMIN on a STED microscope and demonstrated reduced photobleaching for timelapse imaging of beads, cells, and tissue. Approach: The DyMIN system uses a fast multiplexer circuit and inexpensive field-programmable gate array controlled by Labview software that operates as a stand-alone module for a STED microscope. All software and circuit diagrams are freely available. Results: We compared time-lapse images of bead samples using our custom DyMIN system to conventional STED and recorded a â¼ 46 % higher signal when using DyMIN after a 50-image sequence. We further demonstrated the DyMIN system for time-lapse STED imaging of live cells and brain tissue slices. Conclusions: Our open-source DyMIN system is an inexpensive add-on to a conventional STED microscope that can reduce photobleaching. The system can significantly improve signal to noise for dynamic time-lapse STED imaging of live samples.
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Sterile pyogranulomas and heightened cytokine production are hyperinflammatory hallmarks of Chronic Granulomatous Disease (CGD). Using peritoneal cells of zymosan-treated CGD (gp91phox-/-) versus wild-type (WT) mice, an ex vivo system of pyogranuloma formation was developed to determine factors involved in and consequences of recruitment of neutrophils and monocyte-derived macrophages (MoMacs). Whereas WT cells failed to aggregate, CGD cells formed aggregates containing neutrophils initially, and MoMacs recruited secondarily. LTB4 was key, as antagonizing BLT1 blocked neutrophil aggregation, but acted only indirectly on MoMac recruitment. LTB4 upregulated CD11b expression on CGD neutrophils, and the absence/blockade of CD11b inhibited LTB4 production and cell aggregation. Neutrophil-dependent MoMac recruitment was independent of MoMac Nox2 status, BLT1, CCR1, CCR2, CCR5, CXCR2, and CXCR6. As proof of concept, CD11b-deficient CGD mice developed disrupted pyogranulomas with poorly organized neutrophils and diminished recruitment of MoMacs. Importantly, the disruption of cell aggregation and pyogranuloma formation markedly reduced proinflammatory cytokine production.
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Naïve T cell activation in secondary lymphoid organs such as lymph nodes (LNs) occurs upon recognition of cognate antigen presented by antigen presenting cells (APCs). T cell activation requires cytoskeleton rearrangement and sustained interactions with APCs. Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are a family of cytoskeletal effector proteins responsible for actin polymerization and are frequently found at the leading edge of motile cells. Ena/VASP proteins have been implicated in motility and adhesion in various cell types, but their role in primary T cell interstitial motility and activation has not been explored. Our goal was to determine the contribution of Ena/VASP proteins to T cell-APC interactions, T cell activation, and T cell expansion in vivo. Our results showed that naïve T cells from Ena/VASP-deficient mice have a significant reduction in antigen-specific T cell accumulation following Listeria monocytogenes infection. The kinetics of T cell expansion impairment were further confirmed in Ena/VASP-deficient T cells stimulated via dendritic cell immunization. To investigate the cause of this T cell expansion defect, we analyzed T cell-APC interactions in vivo by two-photon microscopy and observed fewer Ena/VASP-deficient naïve T cells interacting with APCs in LNs during priming. We also determined that Ena/VASP-deficient T cells formed conjugates with significantly less actin polymerization at the T cell-APC synapse, and that these conjugates were less stable than their WT counterparts. Finally, we found that Ena/VASP-deficient T cells have less LFA-1 polarized to the T cell-APC synapse. Thus, we conclude that Ena/VASP proteins contribute to T cell actin remodeling during T cell-APC interactions, which promotes the initiation of stable T cell conjugates during APC scanning. Therefore, Ena/VASP proteins are required for efficient activation and expansion of T cells in vivo.
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Actinas , Linfocitos T CD8-positivos , Moléculas de Adhesión Celular , Proteínas de Microfilamentos , Fosfoproteínas , Linfocitos T , Actinas/inmunología , Actinas/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto , Activación de Linfocitos , Ratones , Proteínas de Microfilamentos/inmunología , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Polimerizacion , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Cancer and chronic infections often increase levels of the bioactive lipid, lysophosphatidic acid (LPA), that we have demonstrated acts as an inhibitory ligand upon binding LPAR5 on CD8 T cells, suppressing cytotoxic activity and tumor control. This study, using human and mouse primary T lymphocytes, reveals how LPA disrupts antigen-specific CD8 T cell:target cell immune synapse (IS) formation and T cell function via competing for cytoskeletal regulation. Specifically, we find upon antigen-specific T cell:target cell formation, IP3R1 localizes to the IS by a process dependent on mDia1 and actin and microtubule polymerization. LPA not only inhibited IP3R1 from reaching the IS but also altered T cell receptor (TCR)induced localization of RhoA and mDia1 impairing F-actin accumulation and altering the tubulin code. Consequently, LPA impeded calcium store release and IS-directed cytokine secretion. Thus, targeting LPA signaling in chronic inflammatory conditions may rescue T cell function and promote antiviral and antitumor immunity.
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Linfocitos T CD8-positivos , Sinapsis Inmunológicas , Infecciones , Lisofosfolípidos , Neoplasias , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/inmunología , Humanos , Sinapsis Inmunológicas/efectos de los fármacos , Sinapsis Inmunológicas/inmunología , Infecciones/inmunología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Ratones , Neoplasias/inmunología , Receptores del Ácido Lisofosfatídico/metabolismoRESUMEN
Autoimmunity arises when mechanisms of immune tolerance fail. Here we discuss mechanisms of T cell activation and tolerance and the dynamics of the autoimmune response at the site of disease. Live imaging of autoimmunity provides the ability to analyze immune cell dynamics at the single-cell level within the complex intact environment where disease occurs. These analyses have revealed mechanisms of T cell activation and tolerance in the lymph nodes, mechanisms of T cell entry into sites of autoimmune disease, and mechanisms leading to pathogenesis or protection in the autoimmune lesions. The overarching conclusions point to stable versus transient T cell antigen presenting cell interactions dictating the balance between T cell activation and tolerance, and T cell restimulation as a driver of pathogenesis at the site of autoimmunity. Findings from models of multiple sclerosis and type 1 diabetes are highlighted, however, the results have implications for basic mechanisms of T cell regulation during immune responses, tumor immunity, and autoimmunity.
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Autoinmunidad , Diabetes Mellitus Tipo 1 , Humanos , Tolerancia Inmunológica , Activación de Linfocitos , Linfocitos TRESUMEN
During their life span, T cells are tasked with patrolling the body for potential pathogens. To do so, T cells migrate through numerous distinct anatomical sites and tissue environments with different biophysical characteristics. To migrate through these different environments, T cells use various motility strategies that rely on actin network remodeling to generate shape changes and mechanical forces. In this review, we initially discuss the migratory journey of T cells and then cover the actin polymerization effectors at play in T cells, and finally, we focus on the function of these effectors of actin cytoskeleton remodeling in mediating T-cell migration through diverse tissue environments. Specifically, we will discuss the current state of the field pertaining to our understanding of the roles in T-cell migration played by members of the three main families of actin polymerization machinery: the Arp2/3 complex; formin proteins; and Ena/VASP proteins.
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Complejo 2-3 Proteico Relacionado con la Actina , Actinas , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Movimiento Celular , Forminas , Polimerizacion , Linfocitos T/metabolismoRESUMEN
Understanding mechanisms of immune regulation is key to developing immunotherapies for autoimmunity and cancer. We examined the role of mononuclear phagocytes during peripheral T cell regulation in type 1 diabetes and melanoma. MERTK expression and activity in mononuclear phagocytes in the pancreatic islets promoted islet T cell regulation, resulting in reduced sensitivity of T cell scanning for cognate antigen in prediabetic islets. MERTK-dependent regulation led to reduced T cell activation and effector function at the disease site in islets and prevented rapid progression of type 1 diabetes. In human islets, MERTK-expressing cells were increased in remaining insulin-containing islets of type 1 diabetic patients, suggesting that MERTK protects islets from autoimmune destruction. MERTK also regulated T cell arrest in melanoma tumors. These data indicate that MERTK signaling in mononuclear phagocytes drives T cell regulation at inflammatory disease sites in peripheral tissues through a mechanism that reduces the sensitivity of scanning for antigen leading to reduced responsiveness to antigen.
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Autoinmunidad/fisiología , Islotes Pancreáticos/enzimología , Fagocitos/fisiología , Linfocitos T/inmunología , Tirosina Quinasa c-Mer/inmunología , Adenina/análogos & derivados , Adenina/farmacología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos/inmunología , Antígenos/metabolismo , Antígenos CD11/metabolismo , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 1/patología , Femenino , Humanos , Islotes Pancreáticos/inmunología , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Neoplasias Experimentales/enzimología , Neoplasias Experimentales/inmunología , Fagocitos/inmunología , Piperazinas/farmacología , Tirosina Quinasa c-Mer/genética , Tirosina Quinasa c-Mer/metabolismoRESUMEN
Pneumococcal infections are common and serious complications of HIV-1 disease. Prevention has been compromised by the limited magnitude and quality of Ab responses to T cell-independent type 2 pneumococcal capsular polysaccharides (PPS). The pneumococcal polysaccharide-protein conjugate vaccine-13 (PCV-13) contains PPS conjugated to the T cell-dependent protein (diphtheria toxoid [DT] [CRM197]). We investigated the differential response to PPS and DT by human Ab-secreting B cells (ASC) after immunization with PCV-13 in newly diagnosed healthy HIV+ and control adults. The numbers of PPS-specific IgG ASC increased significantly and similarly in HIV+ and controls. However, DT-specific IgG ASC increased in controls but not HIV+ subjects. To determine the cellular basis of these disparate responses to DT and PPS, we characterized the frequency and activation of T follicular helper (Tfh) cells, the predominant T cell subset providing B cell help. Expression of inducible T cell costimulator (ICOS), which sustains Tfh function and phenotype, increased significantly among controls, when compared with the HIV+ group. Increases in ICOS+ Tfh correlated with changes in T-dependent, DT-specific IgG ASC in controls but not in HIV+ In contrast, ICOS expression did not correlate with T cell-independent type 2 PPS-specific ASC in either group. Of note, upon optimized ex vivo stimulation, CD4 T cells from HIV+ subjects differentiated into Tfh cells and formed synapses with Raji B cells at frequencies similar to that of controls. In summary, PCV-13-induced increase in ICOS expression on Tfh was associated with responses to DT, which was compromised in recently diagnosed healthy HIV+ adults and can be restored ex vivo by providing effective Tfh-differentiating signals.
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Infecciones Oportunistas Relacionadas con el SIDA/prevención & control , Inmunidad Adaptativa , VIH-1/inmunología , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/inmunología , Streptococcus pneumoniae/inmunología , Células T Auxiliares Foliculares/inmunología , Vacunación/métodos , Vacunas Conjugadas/inmunología , Infecciones Oportunistas Relacionadas con el SIDA/sangre , Infecciones Oportunistas Relacionadas con el SIDA/inmunología , Infecciones Oportunistas Relacionadas con el SIDA/virología , Adolescente , Adulto , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Linfocitos B/inmunología , Estudios de Casos y Controles , Femenino , Humanos , Inmunogenicidad Vacunal , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Infecciones Neumocócicas/sangre , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/microbiología , Resultado del Tratamiento , Adulto JovenRESUMEN
Lymphocyte migration is essential for the function of the adaptive immune system, and regulation of T cell entry into tissues is an effective therapy in autoimmune diseases. Little is known about the specific role of cytoskeletal effectors that mediate mechanical forces and morphological changes essential for migration in complex environments. We developed a new Formin-like-1 (FMNL1) knock-out mouse model and determined that the cytoskeletal effector FMNL1 is selectively required for effector T cell trafficking to inflamed tissues, without affecting naïve T cell entry into secondary lymphoid organs. Here, we identify a FMNL1-dependent mechanism of actin polymerization at the back of the cell that enables migration of the rigid lymphocyte nucleus through restrictive barriers. Furthermore, FMNL1-deficiency impairs the ability of self-reactive effector T cells to induce autoimmune disease. Overall, our data suggest that FMNL1 may be a potential therapeutic target to specifically modulate T cell trafficking to inflammatory sites.
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Autoinmunidad , Movimiento Celular , Forminas/metabolismo , Inflamación/metabolismo , Linfocitos T/fisiología , Animales , Línea Celular , Células Endoteliales , Forminas/genética , Sistema Linfático/citología , Ratones , Ratones NoqueadosRESUMEN
Type 1 diabetes (T1D) is a T cell mediated autoimmune disease that affects more than 19 million people with incidence increasing rapidly worldwide. For T cells to effectively drive T1D, they must first traffic to the islets and extravasate through the islet vasculature. Understanding the cues that lead to T cell entry into inflamed islets is important because diagnosed T1D patients already have established immune infiltration of their islets. Here we show that CD11c+ cells are a key mediator of T cell trafficking to infiltrated islets in non-obese diabetic (NOD) mice. Using intravital 2-photon islet imaging we show that T cell extravasation into the islets is an extended process, with T cells arresting in the islet vasculature in close proximity to perivascular CD11c+ cells. Antigen is not required for T cell trafficking to infiltrated islets, but T cell chemokine receptor signaling is necessary. Using RNAseq, we show that islet CD11c+ cells express over 20 different chemokines that bind chemokine receptors expressed on islet T cells. One highly expressed chemokine-receptor pair is CXCL16-CXCR6. However, NOD. CXCR6-/- mice progressed normally to T1D and CXCR6 deficient T cells trafficked normally to the islets. Even with CXCR3 and CXCR6 dual deficiency, T cells trafficked to infiltrated islets. These data reinforce that chemokine receptor signaling is highly redundant for T cell trafficking to inflamed islets. Importantly, depletion of CD11c+ cells strongly inhibited T cell trafficking to infiltrated islets of NOD mice. We suggest that targeted depletion of CD11c+ cells associated with the islet vasculature may yield a therapeutic target to inhibit T cell trafficking to inflamed islets to prevent progression of T1D.
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Antígeno CD11c/inmunología , Diabetes Mellitus Tipo 1/inmunología , Islotes Pancreáticos/inmunología , Linfocitos T/inmunología , Animales , Femenino , Ratones Endogámicos NOD , Ratones NoqueadosRESUMEN
Leukemias typically arise in the bone marrow and then spread to the blood and into other tissues. To disseminate into tissues, leukemia cells migrate into the blood stream and then exit the circulation by migrating across vascular endothelial barriers. Formin proteins regulate cytoskeletal remodeling and cell migration of normal and malignant cells. The Formin mDia1 is highly expressed in transformed lymphocytes and regulates lymphocyte migration. However, the role of mDia1 in regulating leukemia progression in vivo is unknown. Here, we investigated how mDia1 mediates the ability of leukemia cells to migrate and disseminate in vivo. For these studies, we used a mouse model of Bcr-Abl pre-B cell acute lymphoblastic leukemia. Our data showed that mDia1-deficient leukemia cells have reduced chemotaxis and ability to complete transendothelial migration in vitro. In vivo, mDia1 deficiency reduced the ability of leukemia cells to engraft in recipient mice. Furthermore, leukemia dissemination to various tissues and leukemia progression were inhibited by mDia1 depletion. Finally, mDia1 depletion in leukemia cells resulted in prolonged survival of recipient mice in a leukemia transfer model. Overall, our data show that the Formin mDia1 mediates leukemia cell migration, and drives leukemia engraftment and progression in vivo, suggesting that targeting mDia1 could provide a new method for treatment of leukemia.
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The generation of class-switched, high-affinity, antibody-producing B cells plays a critical role in the establishment of type 2 immunity to intestinal helminths as well as in the pathogenesis of allergy and asthma. The generation of these high-affinity, antibody-producing B cells occurs in germinal centers (GC) and relies on interactions with follicular dendritic cells (FDCs) and T follicular helper (Tfh) cells. One critical mediator produced by Tfh cells in GCs is interleukin-4 (IL-4). Tfh-derived IL-4 drives class switching to type 2 antibody isotypes IgE and IgG1 and is required for high-affinity IgG1 production. In vivo detection of IL-4-expressing Tfh cells is required to better understand the role of these cells during the GC response. Detection of IL-4-expressing cells has been greatly improved by the generation of the IL-44get reporter mice, which read out IL-4 expression as green fluorescent protein (GFP). Much has been learned from these mice with regard to type 2 immunity using flow cytometry and immunohistochemistry. However, these methods do not allow the study of cellular behavior and interactions in real time. In contrast, multi-photon microscopy allows for deep tissue imaging and tracking of multiple cell types in intact tissues over time. Here, we describe a protocol for in vivo detection of IL-4-expressing Tfh cells in an explanted popliteal lymph node by multi-photon microscopy. The dynamics of Tfh cell motility and their interactions with FDC networks in the GCs were analyzed.
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Expresión Génica , Interleucina-4/genética , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Imagen Molecular , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Genes Reporteros , Centro Germinal/inmunología , Centro Germinal/metabolismo , Procesamiento de Imagen Asistido por Computador , Interleucina-4/metabolismo , Ratones , Ratones Transgénicos , Microscopía , Programas Informáticos , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner.
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Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Linfocitos T/fisiología , Migración Transendotelial y Transepitelial/fisiología , Actinas/metabolismo , Animales , Linfocitos T CD4-Positivos/fisiología , Adhesión Celular , Moléculas de Adhesión Celular/genética , Quimiotaxis/fisiología , Inflamación/patología , Integrina alfa4/metabolismo , Activación de Linfocitos , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Fosfoproteínas/genéticaRESUMEN
Cell migration has two opposite faces: although necessary for physiological processes such as immune responses, it can also have detrimental effects by enabling metastatic cells to invade new organs. In vivo, migration occurs in complex environments and often requires a high cellular deformability, a property limited by the cell nucleus. Here we show that dendritic cells, the sentinels of the immune system, possess a mechanism to pass through micrometric constrictions. This mechanism is based on a rapid Arp2/3-dependent actin nucleation around the nucleus that disrupts the nuclear lamina, the main structure limiting nuclear deformability. The cells' requirement for Arp2/3 to pass through constrictions can be relieved when nuclear stiffness is decreased by suppressing lamin A/C expression. We propose a new role for Arp2/3 in three-dimensional cell migration, allowing fast-moving cells such as leukocytes to rapidly and efficiently migrate through narrow gaps, a process probably important for their function.
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Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Movimiento Celular , Núcleo Celular/metabolismo , Células Dendríticas , Neutrófilos , Lámina Nuclear/metabolismo , Animales , Immunoblotting , Lamina Tipo A/metabolismo , Ratones , PolimerizacionRESUMEN
Leukemia dissemination (the spread of leukemia cells from the bone marrow) and relapse are associated with poor prognosis. Often, relapse occurs in peripheral organs, such as the CNS, which acts as a sanctuary site for leukemia cells to escape anti-cancer treatments. Similar to normal leukocyte migration, leukemia dissemination entails migration of cells from the blood circulation into tissues by extravasation. To extravasate, leukemia cells cross through vascular endothelial walls via a process called transendothelial migration, which requires cytoskeletal remodeling. However, the specific molecular players in leukemia extravasation are not fully known. We examined the role of myosin-IIA a cytoskeletal class II myosin motor protein, in leukemia progression and dissemination into the CNS by use of a mouse model of Bcr-Abl-driven B cell acute lymphoblastic leukemia. Small hairpin RNA-mediated depletion of myosin-IIA did not affect apoptosis or the growth rate of B cell acute lymphoblastic leukemia cells. However, in an in vivo leukemia transfer model, myosin-IIA depletion slowed leukemia progression and prolonged survival, in part, by reducing the ability of B cell acute lymphoblastic leukemia cells to engraft efficiently. Finally, myosin-IIA inhibition, either by small hairpin RNA depletion or chemical inhibition by blebbistatin, drastically reduced CNS infiltration of leukemia cells. The effects on leukemia cell entry into tissues were mostly a result of the requirement for myosin-IIA to enable leukemia cells to complete the transendothelial migration process during extravasation. Overall, our data implicate myosin-IIA as a key mediator of leukemia cell migration, making it a promising target to inhibit leukemia dissemination in vivo and potentially reduce leukemia relapses.
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Encéfalo/inmunología , Movimiento Celular , Modelos Animales de Enfermedad , Leucemia Experimental/inmunología , Miosina Tipo IIA no Muscular/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Animales , Apoptosis , Encéfalo/metabolismo , Encéfalo/patología , Adhesión Celular , Células Cultivadas , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Leucemia Experimental/metabolismo , Leucemia Experimental/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologíaRESUMEN
RATIONALE: The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors. OBJECTIVES: Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs. METHODS: We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. MEASUREMENTS AND MAIN RESULTS: We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood. CONCLUSIONS: Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics.
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Citometría de Flujo , Pulmón/inmunología , Ganglios Linfáticos/inmunología , Sistema Mononuclear Fagocítico/inmunología , Fagocitos/inmunología , Adulto , Cadáver , Femenino , Humanos , MasculinoRESUMEN
In addition to the secretion of Ag-specific Abs, B cells may play an important role in the generation of immune responses by efficiently presenting Ag to T cells. We and other investigators recently described a subpopulation of CD11c(+) B cells (Age/autoimmune-associated B cells [ABCs]) that appear with age, during virus infections, and at the onset of some autoimmune diseases and participate in autoimmune responses by secreting autoantibodies. In this study, we assessed the ability of these cells to present Ag and activate Ag-specific T cells. We demonstrated that ABCs present Ag to T cells, in vitro and in vivo, better than do follicular B cells (FO cells). Our data indicate that ABCs express higher levels of the chemokine receptor CCR7, have higher responsiveness to CCL21 and CCL19 than do FO cells, and are localized at the T/B cell border in spleen. Using multiphoton microscopy, we show that, in vivo, CD11c(+) B cells form significantly more stable interactions with T cells than do FO cells. Together, these data identify a previously undescribed role for ABCs as potent APCs and suggest another potential mechanism by which these cells can influence immune responses and/or the development of autoimmunity.
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Envejecimiento/inmunología , Células Presentadoras de Antígenos/inmunología , Autoinmunidad , Linfocitos B/inmunología , Antígeno CD11c/inmunología , Bazo/inmunología , Envejecimiento/genética , Animales , Células Presentadoras de Antígenos/citología , Autoanticuerpos/biosíntesis , Linfocitos B/citología , Antígeno CD11c/genética , Quimiocina CCL19/genética , Quimiocina CCL19/inmunología , Quimiocina CCL21/genética , Quimiocina CCL21/inmunología , Femenino , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Receptores CCR7/genética , Receptores CCR7/inmunología , Transducción de Señal , Bazo/citología , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space.