RESUMEN
Cytotoxic T lymphocytes (CTLs) kill infected and cancerous cells. We detected transfer of cytotoxic multiprotein complexes, called supramolecular attack particles (SMAPs), from CTLs to target cells. SMAPs were rapidly released from CTLs and were autonomously cytotoxic. Mass spectrometry, immunochemical analysis, and CRISPR editing identified a carboxyl-terminal fragment of thrombospondin-1 as an unexpected SMAP component that contributed to target killing. Direct stochastic optical reconstruction microscopy resolved a cytotoxic core surrounded by a thrombospondin-1 shell of ~120 nanometer diameter. Cryo-soft x-ray tomography analysis revealed that SMAPs had a carbon-dense shell and were stored in multicore granules. We propose that SMAPs are autonomous extracellular killing entities that deliver cytotoxic cargo targeted by the specificity of shell components.
Asunto(s)
Citotoxicidad Inmunológica , Granzimas/metabolismo , Complejos Multiproteicos/metabolismo , Perforina/metabolismo , Linfocitos T Citotóxicos/metabolismo , Trombospondina 1/metabolismo , Sistemas CRISPR-Cas , Exocitosis , Edición Génica , Humanos , Células K562 , Trombospondina 1/genética , Tomografía por Rayos XRESUMEN
It is increasingly recognized that cell signaling, as a chemical process, must be considered at the local, micrometer scale. Micro- and nanofabrication techniques provide access to these dimensions, with the potential to capture and manipulate the spatial complexity of intracellular signaling in experimental models. This review focuses on recent advances in adapting surface engineering for use with biomolecular systems that interface with cell signaling, particularly with respect to surfaces that interact with multiple receptor systems on individual cells. The utility of this conceptual and experimental approach is demonstrated in the context of epithelial cells and T lymphocytes, two systems whose ability to perform their physiological function is dramatically impacted by the convergence and balance of multiple signaling pathways.
Asunto(s)
Transducción de Señal , Ingeniería de Tejidos/métodos , Animales , Materiales Biocompatibles/química , Adhesión Celular , Comunicación Celular , Células Epiteliales/patología , Transición Epitelial-Mesenquimal , Matriz Extracelular/patología , Humanos , Sistema Inmunológico , Huso Acromático , Linfocitos T/citologíaRESUMEN
Dendritic cells (DCs) interface innate and adaptive immunity in nonlymphoid organs; however, the exact distribution and types of DC within the kidney are not known. We utilized CX3CR1GFP/+ mice to characterize the anatomy and phenotype of tissue-resident CX3CR1+ DCs within normal kidney. Laser-scanning confocal microscopy revealed an extensive, contiguous network of stellate-shaped CX3CR1+ DCs throughout the interstitial and mesangial spaces of the entire kidney. Intravital microscopy of the superficial cortex showed stationary interstitial CX3CR1+ DCs that continually probe the surrounding tissue environment through dendrite extensions. Flow cytometry of renal CX3CR1+ DCs showed significant coexpression of CD11c and F4/80, high major histocompatibility complex class II and FcR expression, and immature costimulatory but competent phagocytic ability indicative of tissue-resident, immature DCs ready to respond to environment cues. Thus, within the renal parenchyma, there exists little immunological privilege from the surveillance provided by renal CX3CR1+ DCs, a major constituent of the heterogeneous mononuclear phagocyte system populating normal kidney.
Asunto(s)
Comunicación Celular/inmunología , Células Dendríticas/citología , Riñón/citología , Riñón/inmunología , Receptores de Quimiocina/inmunología , Animales , Receptor 1 de Quimiocinas CX3C , Células Dendríticas/inmunología , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Sistema Inmunológico/citología , Sistema Inmunológico/inmunología , Ratones , Ratones Transgénicos , Fagocitos/citología , Fagocitos/inmunología , Receptores de Quimiocina/genéticaRESUMEN
According to the two-signal model of T cell activation, costimulatory molecules augment T cell receptor (TCR) signaling, whereas adhesion molecules enhance TCR-MHC-peptide recognition. The structure and binding properties of CD28 imply that it may perform both functions, blurring the distinction between adhesion and costimulatory molecules. Our results show that CD28 on naïve T cells does not support adhesion and has little or no capacity for directly enhancing TCR-MHC-peptide interactions. Instead of being dependent on costimulatory signaling, we propose that a key function of the immunological synapse is to generate a cellular microenvironment that favors the interactions of potent secondary signaling molecules, such as CD28.
Asunto(s)
Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Activación de Linfocitos , Linfocitos T/inmunología , Antígeno B7-1/genética , Antígenos CD28/química , Adhesión Celular , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Glicosilfosfatidilinositoles/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Células Jurkat , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Cells in the immune and nervous systems communicate through informational synapses. The two-dimensional chemistry underlying the process of synapse formation is beginning to be explored using fluorescence imaging and mechanical techniques. Early analysis of two-dimensional kinetic rates (k(on) and k(off)) and equilibrium constants (K(d)) provides a number of biological insights. First, there are two regimes for adhesion-one disordered with slow k(on) and the other self-ordered with 10(4)-fold faster k(on). Despite huge variation in two-dimensional k(on), the two-dimensional k(off) is like k(off) in solution, and two-dimensional k(off) is more closely related to intrinsic properties of the interaction than the two-dimensional k(on). Thus difference in k(off) can be used to set signaling thresholds. Early signaling complexes are compartmentalized to generate synergistic signaling domains. Immune antigen receptor components have a role in neural synapse editing. This suggests significant parallels in informational synapse formation based on common two-dimensional chemistry and signaling strategies.
Asunto(s)
Comunicación Celular , Autotolerancia , Sinapsis/química , Sinapsis/inmunología , Antígenos CD4/inmunología , Fluorescencia , Humanos , Cinética , Activación de Linfocitos/inmunología , Transducción de Señal , Transmisión Sináptica , Linfocitos T/inmunologíaRESUMEN
The T cell and antigen-presenting cell communicate to initiate an immune response through formation of an immunological synapse. This specialized cell-cell junction is compartmentalized into adhesion molecule and T cell receptor enriched regions or SMACs. Distinct signals seem to be generated in the T cell receptor and adhesion molecule-dominated regions. This review focuses on how these distinct signaling pathways may be integrated within the T cell to set thresholds for T cell activation, proliferation, and survival.
Asunto(s)
Moléculas de Adhesión Celular/inmunología , Linfocitos T/inmunología , Células Presentadoras de Antígenos/inmunología , Comunicación Celular , Compartimento Celular , División Celular , Supervivencia Celular , Uniones Intercelulares/inmunología , Activación de Linfocitos , Transducción de Señal , Linfocitos T/citologíaRESUMEN
The stable immunological synapse between a T cell and antigen-presenting cell coordinates migration and activation. Three-dimensional collagen gels transform this interaction into a series of transient hit-and-run encounters. Here we integrate these alternative modes of interaction in a model for primary T cell activation and effector function in vivo.
Asunto(s)
Movimiento Celular/inmunología , Matriz Extracelular/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Animales , Humanos , Especificidad de Órganos/inmunologíaRESUMEN
The adaptive immune response is initiated by the interaction of T cell antigen receptors with major histocompatibility complex molecule-peptide complexes in the nanometer scale gap between a T cell and an antigen-presenting cell, referred to as an immunological synapse. In this review we focus on the concept of immunological synapse formation as it relates to membrane structure, T cell polarity, signaling pathways, and the antigen-presenting cell. Membrane domains provide an organizational principle for compartmentalization within the immunological synapse. T cell polarization by chemokines increases T cell sensitivity to antigen. The current model is that signaling and formation of the immunological synapse are tightly interwoven in mature T cells. We also extend this model to natural killer cell activation, where the inhibitory NK synapse provides a striking example in which inhibition of signaling leaves the synapse in its nascent, inverted state. The APC may also play an active role in immunological synapse formation, particularly for activation of naïve T cells.
Asunto(s)
Presentación de Antígeno/inmunología , Membrana Celular/ultraestructura , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/ultraestructura , Animales , Adhesión Celular , Moléculas de Adhesión Celular/fisiología , Comunicación Celular , Polaridad Celular , Quimiocinas/fisiología , Toxina del Cólera/farmacología , Recubrimiento Inmunológico , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/ultraestructura , Microdominios de Membrana/fisiología , Microdominios de Membrana/ultraestructura , Ratones , Modelos Inmunológicos , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/ultraestructura , Receptores de Antígenos de Linfocitos T/ultraestructura , Receptores de Quimiocina/fisiología , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/fisiología , Receptores Inmunológicos/ultraestructura , Transducción de Señal , Subgrupos de Linfocitos T/inmunologíaRESUMEN
The coordination of T-cell migration and antigen recognition is crucial for an effective immune response. We have proposed that this coordination is achieved by formation of an immunological synapse between the T cell and the antigen-presenting cell (APC). Our view contrasts with the serial encounter model also proposed in this issue of Trends in Immunology, which is based on transient T cell-APC interactions when surrounded by collagen. Here, we propose a model that reconciles immunological synapse formation and serial encounters based on environmental control of immunological synapse formation.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Colágeno/inmunología , Matriz Extracelular/inmunología , Activación de Linfocitos/inmunología , Modelos Inmunológicos , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Movimiento Celular/fisiología , Quimiocinas/inmunología , Ganglios Linfáticos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/fisiología , Factores de TiempoRESUMEN
Transmembrane protein tyrosine phosphatases, such as CD45, can act as both positive and negative regulators of cellular signaling. CD45 positively modulates T cell receptor (TCR) signaling by constitutively priming p56lck through the dephosphorylation of the C-terminal negative regulatory phosphotyrosine site. However, CD45 can also exert negative effects on cellular processes, including events triggered by integrin-mediated adhesion. To better understand these opposing actions of tyrosine phosphatases, the subcellular compartmentalization of CD45 was imaged by using laser scanning confocal microscopy during functional TCR signaling of live T lymphocytes. On antigen engagement, CD45 was first excluded from the central region of the interface between the T cell and the antigen-presenting surface where CD45 would inhibit integrin activation. Subsequently, CD45 was recruited back to the center of the contact to an area adjacent to the site of sustained TCR engagement. Thus, CD45 is well positioned within a supramolecular assembly in the vicinity of the engaged TCR, where CD45 would be able to maintain src-kinase activity for the duration of TCR engagement.
Asunto(s)
Antígenos Comunes de Leucocito/inmunología , Activación de Linfocitos , Linfocitos T/inmunología , Animales , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Cinética , Antígenos Comunes de Leucocito/análisis , Ratones , Ratones Transgénicos , Microscopía Confocal , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Bazo/inmunología , Linfocitos T/citología , Linfocitos T/enzimologíaRESUMEN
Chemokines play an important role in establishing the distribution of lymphocyte subpopulations in primary and secondary lymphoid tissues and in the recruitment of leukocytes to sites of inflammation. However, the potential of chemokines to down-regulate immune responses has not been demonstrated. We now show that certain chemokine gradients have the potential to suppress T cell activation by preventing formation of the immunological synapse, the specialized cell-cell junction that forms before a T cell can be fully activated. Our data reveals an immunosuppressive potential of chemokines engaging the CXCR3 and CCR7 receptors, but not the CXCR4, CCR2, CCR4, or CCR5 receptors. These results suggest a novel mechanism for T cell ignorance of agonist MHC-peptide complexes based on dominant chemokine gradients.
Asunto(s)
Movimiento Celular/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Receptores de Quimiocina/fisiología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Inhibición de Migración Celular , Movimiento Celular/genética , Quimiocina CCL21 , Quimiocinas CC/inmunología , Anergia Clonal/genética , Anergia Clonal/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunosupresores/farmacología , Molécula 1 de Adhesión Intercelular/inmunología , Activación de Linfocitos/genética , Ratones , Ratones Transgénicos , Microscopía por Video , Muramidasa/inmunología , Fragmentos de Péptidos/agonistas , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal/genéticaRESUMEN
T cell antigen recognition is accompanied by cytoskeletal polarization towards the APC and large-scale redistribution of cell surface molecules into 'supramolecular activation clusters' (SMACs), forming an organized contact interface termed the 'immunological synapse' (IS). Molecules are arranged in the IS in a micrometer scale bull's eye pattern with a central accumulation of TCR/peptide-MHC (the cSMAC) surrounded by a peripheral ring of adhesion molecules (the pSMAC). We propose that segregation of cell surface molecules on a much smaller scale initiates TCR triggering, which drives the formation of the IS by active transport processes. IS formation may function as a checkpoint for full T cell activation, integrating information on the presence and quality of TCR ligands and the nature and activation state of the APC.
Asunto(s)
Células Presentadoras de Antígenos/fisiología , Antígenos de Superficie/metabolismo , Proteínas del Citoesqueleto/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/fisiología , Animales , Humanos , Transducción de Señal/fisiologíaRESUMEN
The actin cytoskeleton seems to play two critical roles in the activation of T cells. One of these roles is T cell shape development and movement, including formation of the immunological synapse. The other is the formation of a scaffold for signaling components. This review focuses on the recent convergence of cell biology and immunology studies to explain the role of the actin cytoskeleton in creating the molecular basis for immunological synapse formation and T cell signaling.
Asunto(s)
Citoesqueleto/inmunología , Transducción de Señal/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Actinas/inmunología , Animales , Comunicación Celular/inmunología , Citoesqueleto/ultraestructura , HumanosRESUMEN
To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-alpha was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-alpha in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.
Asunto(s)
Movimiento Celular/inmunología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Antígeno de Macrófago-1/fisiología , Neutrófilos/inmunología , Animales , Antígenos CD18/biosíntesis , Adhesión Celular/inmunología , Quimiotaxis de Leucocito , Cámaras de Difusión de Cultivos , Femenino , Inyecciones Subcutáneas , Interfase/inmunología , Activación de Linfocitos/inmunología , Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno de Macrófago-1/biosíntesis , Masculino , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/metabolismo , Neutrófilos/fisiología , Estrés Mecánico , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
CD2-associated protein (CD2AP) is an 80-kilodalton protein that is critical for stabilizing contacts between T cells and antigen-presenting cells. In CD2AP-deficient mice, immune function was compromised, but the mice died at 6 to 7 weeks of age from renal failure. In the kidney, CD2AP was expressed primarily in glomerular epithelial cells. Knockout mice exhibited defects in epithelial cell foot processes, accompanied by mesangial cell hyperplasia and extracellular matrix deposition. Supporting a role for CD2AP in the specialized cell junction known as the slit diaphragm, CD2AP associated with nephrin, the primary component of the slit diaphragm.
Asunto(s)
Glomérulos Renales/metabolismo , Glomérulos Renales/ultraestructura , Síndrome Nefrótico/congénito , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Membrana Basal/ultraestructura , Proteínas del Citoesqueleto , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Proteínas de la Matriz Extracelular/metabolismo , Mesangio Glomerular/metabolismo , Mesangio Glomerular/ultraestructura , Uniones Intercelulares/metabolismo , Uniones Intercelulares/ultraestructura , Glomérulos Renales/irrigación sanguínea , Activación de Linfocitos , Proteínas de la Membrana , Ratones , Ratones Noqueados , Microscopía Electrónica , Síndrome Nefrótico/genética , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patología , Proteínas/genética , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/inmunologíaRESUMEN
The specialized junction between a T lymphocyte and an antigen-presenting cell, the immunological synapse, consists of a central cluster of T cell receptors surrounded by a ring of adhesion molecules. Immunological synapse formation is now shown to be an active and dynamic mechanism that allows T cells to distinguish potential antigenic ligands. Initially, T cell receptor ligands were engaged in an outermost ring of the nascent synapse. Transport of these complexes into the central cluster was dependent on T cell receptor-ligand interaction kinetics. Finally, formation of a stable central cluster at the heart of the synapse was a determinative event for T cell proliferation.