RESUMEN
BACKGROUND: Circulating tumor DNA (ctDNA) positivity at diagnosis, which is associated with worse outcomes in multiple solid tumors including stage I-III non-small cell lung cancer (NSCLC), may have utility to guide (neo)adjuvant therapy. METHODS: In this retrospective study, 260 patients with clinical stage I NSCLC (180 adenocarcinoma, 80 squamous cell carcinoma) were allocated (2:1) to high- and low-risk groups based on relapse versus disease-free status ≤5 years post-surgery. We evaluated the association of preoperative ctDNA detection by a plasma-only targeted methylation-based multi-cancer early detection (MCED) test with NSCLC relapse ≤5 years post-surgery in the overall population, followed by histology-specific subgroup analyses. RESULTS: Across clinical stage I patients, preoperative ctDNA detection did not associate with relapse within 5 years post-surgery. Sub-analyses confined to lung adenocarcinoma suggested a histology-specific association between ctDNA detection and outcome. In this group, ctDNA positivity tended to associate with relapse within 2 years, suggesting prognostic implications of MCED test positivity may be histology- and time-dependent in stage I NSCLC. Preoperative ctDNA detection was associated with upstaging of clinical stage I to pathological stage II-III NSCLC. CONCLUSIONS: Our findings suggest preoperative ctDNA detection in patients with resectable clinical stage I NSCLC using MCED, a pan-cancer screening test developed for use in an asymptomatic population, has no detectable prognostic value for relapse ≤5 years post-surgery. MCED detection may be associated with early adenocarcinoma relapse and increased pathological upstaging rates in stage I NSCLC. However, given the exploratory nature of these findings, independent validation is required.
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Carcinoma de Pulmón de Células no Pequeñas , ADN Tumoral Circulante , Metilación de ADN , Neoplasias Pulmonares , Estadificación de Neoplasias , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/patología , ADN Tumoral Circulante/sangre , ADN Tumoral Circulante/genética , Neoplasias Pulmonares/cirugía , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/patología , Masculino , Femenino , Anciano , Persona de Mediana Edad , Estudios Retrospectivos , Pronóstico , Recurrencia Local de Neoplasia/sangre , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genéticaRESUMEN
In man, two CC chemokine receptor isoforms, CCR2a and CCR2b, are present that belong to the rhodopsin-like G protein-coupled receptor family, and couple to Gi and Gq family members. The CCR2 receptors are known to regulate canonical functions of chemokines such as directed migration of leukocytes, and to potentially control non-canonical functions such as differentiation, proliferation, and gene transcription of immune and non-immune cells. We recently reported on the activation of phospholipase C isoenzymes and RhoA GTPases by coupling of the two CCR2 receptors to members of the Gq family, in particular Gαq and Gα14. So far little is known about the structural requirements for the CCR2/Gq/14 interaction. Interestingly, the CCR2 receptor isoforms are identical up to arginine 313 (R313) that is part of the putative 8th helix in CCR2 receptors, and the 8th helix has been implicated in the interaction of rhodopsin-like G protein-coupled receptors with Gαq. In the present work we describe that the 8th helix of both CCR2a and CCR2b is critically involved in selectively activating Gαq/14-regulated signaling. Refined analysis using various CCR2a and CCR2b mutants and analyzing their cellular signaling, e.g. ligand-dependent (i) activation of phospholipase C isoenzymes, (ii) stimulation of serum response factor-mediated gene transcription, (iii) activation of mitogen-activated protein kinases, (iv) internalization, and (v) changes in intracellular calcium concentrations, identified arginine 313 within the amino terminal portion of helix 8 to play a role for the agonist-mediated conformational changes and the formation of a Gαq/14 binding surface. We show that R313 determines Gαq/14 protein-dependent but not Gi protein-dependent cellular signaling, and plays no role in Gq/Gi-independent receptor internalization, indicating a role of R313 in biased signaling of CCR2 receptors.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Receptores CCR2/metabolismo , Animales , Arginina/análisis , Arginina/metabolismo , Células COS , Chlorocebus aethiops , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Células HEK293 , Humanos , Conformación Proteica en Hélice alfa , Mapas de Interacción de Proteínas , Receptores CCR2/química , Transducción de SeñalRESUMEN
Employing live cells as therapeutics is a direction of future drug discovery. An easy and robust method to modify the surfaces of cells directly to incorporate novel functionalities is highly desirable. However, genetic methods for cell-surface engineering are laborious and limited by low efficiency for primary cell modification. Here we report a chemoenzymatic approach that exploits a fucosyltransferase to transfer bio-macromolecules, such as an IgG antibody (MWâ¼ 150 KD), to the glycocalyx on the surfaces of live cells when the antibody is conjugated to the enzyme's natural donor substrate GDP-Fucose. Requiring no genetic modification, this method is fast and biocompatible with little interference to cells' endogenous functions. We applied this method to construct two antibody-cell conjugates (ACCs) using both cell lines and primary cells, and the modified cells exhibited specific tumor targeting and resistance to inhibitory signals produced by tumor cells, respectively. Remarkably, Herceptin-NK-92MI conjugates, a natural killer cell line modified with Herceptin, exhibit enhanced activities to induce the lysis of HER2+ cancer cells both ex vivo and in a human tumor xenograft model. Given the unprecedented substrate tolerance of the fucosyltransferase, this chemoenzymatic method offers a general approach to engineer cells as research tools and for therapeutic applications.
RESUMEN
The adaptive immune response involves T cell differentiation and migration to sites of inflammation. T cell trafficking is initiated by rolling on inflamed endothelium. Tethers and slings, discovered in neutrophils, facilitate cell rolling at high shear stress. Here, we demonstrate that the ability to form tethers and slings during rolling is highly inducible in T helper 1 (Th1), Th17, and regulatory T (Treg) cells but less in Th2 cells. In vivo, endogenous Treg cells rolled stably in cremaster venules at physiological shear stress. Quantitative dynamic footprinting nanoscopy of Th1, Th17, and Treg cells uncovered the formation of multiple tethers per cell. Human Th1 cells also showed tethers and slings. RNA sequencing (RNA-seq) revealed the induction of cell migration and cytoskeletal genes in sling-forming cells. We conclude that differentiated CD4 T cells stabilize rolling by inducible tether and sling formation. These phenotypic changes approximate the adhesion phenotype of neutrophils and support CD4 T cell access to sites of inflammation.
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Estrés Mecánico , Linfocitos T Reguladores/patología , Animales , Diferenciación Celular , Movimiento Celular/genética , Citoesqueleto/metabolismo , Selectina E/metabolismo , Humanos , Recuento de Linfocitos , Ratones Endogámicos C57BL , Microvellosidades/metabolismo , Neutrófilos/metabolismo , Selectina-P/metabolismo , Linfocitos T Reguladores/metabolismo , Células TH1/metabolismo , Transcriptoma/genéticaRESUMEN
Activated leukocyte cell adhesion molecule (ALCAM) has been proposed to mediate leukocyte migration across the blood-brain barrier (BBB) in multiple sclerosis or experimental autoimmune encephalomyelitis (EAE). Here, we confirmed vascular ALCAM expression in human brain tissue samples in situ and on two different human in vitro BBB models. Antibody-mediated inhibition of ALCAM reduced diapedesis of human CD4+ Th1 but not of Th17 cells across the human BBB in vitro. In accordance to human Th1 cells, mouse Th1 cells showed reduced diapedesis across an ALCAM-/- in vitro BBB model under static but no longer under flow conditions. In contrast to the limited role of ALCAM in T cell extravasation across the BBB, we found a contribution of ALCAM to rolling, adhesion, and diapedesis of human CD14+ monocytes across the human BBB under flow and static conditions. Taken together, our study highlights the potential differences in the CNS expression of ALCAM in mouse and human and supports a prominent role for ALCAM in the multi-step extravasation of monocytes across the BBB.
Asunto(s)
Antígenos CD/metabolismo , Barrera Hematoencefálica/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas Fetales/metabolismo , Monocitos/inmunología , Linfocitos T/inmunología , Migración Transendotelial y Transepitelial/inmunología , Animales , Antígenos CD/genética , Barrera Hematoencefálica/inmunología , Moléculas de Adhesión Celular Neuronal/genética , Células Cultivadas , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Proteínas Fetales/genética , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Linfocitos T/metabolismo , Migración Transendotelial y Transepitelial/fisiologíaRESUMEN
PURPOSE OF REVIEW: We review P-selectin glycoprotein ligand-1 (PSGL-1) as a selectin and chemokine-binding adhesion molecule. PSGL-1 is widely studied in neutrophils. Here, we focus on T cells, because PSGL-1 was recently described as a major immunomodulatory molecule during viral infection. PSGL-1 also plays a crucial role in T-cell homeostasis by binding to lymphoid chemokines, and can induce tolerance by enhancing the functions of regulatory T cells. RECENT FINDINGS: PSGL-1 was originally described as a leukocyte ligand for P-selectin, but it is actually a ligand for all selectins (P-, L- and E-selectin), binds chemokines, activates integrins and profoundly affects T-cell biology. It has been shown recently that PSGL-1 can modulate T cells during viral infection by acting as a negative regulator for T-cell functions. Absence of PSGL-1 promotes effector CD4 and CD8 T-cell differentiation and prevents T-cell exhaustion. Consistent with this, tumor growth was significantly reduced in PSGL-1-deficient mice because of an enhanced number of effector T cells together with reduced levels of inhibitory receptors that induce T-cell exhaustion. SUMMARY: PSGL-1 is the best-studied selectin ligand and has become a posterchild of versatility in leukocyte adhesion, inflammation and immunology. The direct involvement of PSGL-1 in T-cell biology suggests that it might be a drug target. Indeed, PSGL-1 has been tested in some clinical trials and recently, PSGL-1 blockers were proposed as a potential cotherapy in cancer immunotherapy.
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Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Quimiocinas/metabolismo , Citoesqueleto/metabolismo , Glicosilación , Humanos , Tolerancia Inmunológica , Inmunomodulación , Integrinas/metabolismo , Glicoproteínas de Membrana/química , Microdominios de Membrana/metabolismo , Unión Proteica , Transporte de Proteínas , Selectinas/metabolismo , Transducción de SeñalRESUMEN
Activated leukocyte cell adhesion molecule (ALCAM) is a cell adhesion molecule found on blood-brain barrier endothelial cells (BBB-ECs) that was previously shown to be involved in leukocyte transmigration across the endothelium. In the present study, we found that ALCAM knockout (KO) mice developed a more severe myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis (EAE). The exacerbated disease was associated with a significant increase in the number of CNS-infiltrating proinflammatory leukocytes compared with WT controls. Passive EAE transfer experiments suggested that the pathophysiology observed in active EAE was linked to the absence of ALCAM on BBB-ECs. In addition, phenotypic characterization of unimmunized ALCAM KO mice revealed a reduced expression of BBB junctional proteins. Further in vivo, in vitro, and molecular analysis confirmed that ALCAM is associated with tight junction molecule assembly at the BBB, explaining the increased permeability of CNS blood vessels in ALCAM KO animals. Collectively, our data point to a biologically important function of ALCAM in maintaining BBB integrity.
Asunto(s)
Molécula de Adhesión Celular del Leucocito Activado/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Células Endoteliales/metabolismo , Molécula de Adhesión Celular del Leucocito Activado/genética , Animales , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Encefalomielitis Autoinmune Experimental/patología , Femenino , Homeostasis , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos , Índice de Severidad de la Enfermedad , Médula Espinal/metabolismo , Proteínas de Uniones Estrechas/metabolismoRESUMEN
The extravasation of CD4(+) effector/memory T cells (TEM cells) across the blood-brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) or multiple sclerosis (MS). Endothelial ICAM-1 and ICAM-2 are essential for CD4(+) TEM cell crawling on the BBB prior to diapedesis. Here, we investigated the influence of cell surface levels of endothelial ICAM-1 in determining the cellular route of CD4(+) TEM -cell diapedesis across cytokine treated primary mouse BBB endothelial cells under physiological flow. Inflammatory conditions, inducing high levels of endothelial ICAM-1, promoted rapid initiation of transcellular diapedesis of CD4(+) T cells across the BBB, while intermediate levels of endothelial ICAM-1 favored paracellular CD4(+) T-cell diapedesis. Importantly, the route of T-cell diapedesis across the BBB was independent of loss of BBB barrier properties. Unexpectedly, a low number of CD4(+) TEM cells was found to cross the inflamed BBB in the absence of endothelial ICAM-1 and ICAM-2 via an obviously alternatively regulated transcellular pathway. In vivo, this translated to the development of ameliorated EAE in ICAM-1(null) //ICAM-2(-/-) C57BL/6J mice. Taken together, our study demonstrates that cell surface levels of endothelial ICAM-1 rather than the inflammatory stimulus or BBB integrity influence the pathway of T-cell diapedesis across the BBB.
Asunto(s)
Barrera Hematoencefálica/inmunología , Linfocitos T CD4-Positivos/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Migración Transendotelial y Transepitelial/inmunología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/inmunología , Adhesión Celular/inmunología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Células Endoteliales/inmunología , Femenino , Memoria Inmunológica/inmunología , Inflamación/inmunología , Molécula 1 de Adhesión Intercelular/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/inmunologíaRESUMEN
Activated T cells use very late antigen-4/α4ß1 integrin for capture, rolling on, and firm adhesion to endothelial cells, and use leukocyte function-associated antigen-1/αLß2 integrin for subsequent crawling and extravasation. Inhibition of α4ß1 is sufficient to prevent extravasation of activated T cells and is successfully used to combat autoimmune diseases, such as multiple sclerosis. Here we show that effector T cells lacking the integrin activator Kindlin-3 extravasate and induce experimental autoimmune encephalomyelitis in mice immunized with autoantigen. In sharp contrast, adoptively transferred autoreactive T cells from Kindlin-3-deficient mice fail to extravasate into the naïve CNS. Mechanistically, autoreactive Kindlin-3-null T cells extravasate when the CNS is inflamed and the brain microvasculature expresses high levels of integrin ligands. Flow chamber assays under physiological shear conditions confirmed that Kindlin-3-null effector T cells adhere to high concentrations of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, albeit less efficiently than WT T cells. Although these arrested T cells polarize and start crawling, only few remain firmly adherent over time. Our data demonstrate that the requirement of Kindlin-3 for effector T cells to induce α4ß1 and αLß2 integrin ligand binding and stabilization of integrin-ligand bonds is critical when integrin ligand levels are low, but of less importance when integrin ligand levels are high.
Asunto(s)
Proteínas del Citoesqueleto/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Integrina alfa4beta1/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Linfocitos T/inmunología , Animales , Adhesión Celular/genética , Adhesión Celular/inmunología , Proteínas del Citoesqueleto/genética , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Integrina alfa4beta1/genética , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Antígeno-1 Asociado a Función de Linfocito/genética , Ratones , Linfocitos T/patología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.