We thank Dr. Peter T. Sage for his assistance in generating some of the representative images. This work was supported by an NIH R01 grant to C.V.C. (HL104006).

<ol>
	<li>von Andrian, U. H., Mackay, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+function+and+migration.+Two+sides+of+the+same+coin.">T-cell function and migration. Two sides of the same coin.</a> N. Engl. J. Med. 343 (14), 1020-1034 (2000).</li><li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+receptors+of+the+immune+system.">Adhesion receptors of the immune system.</a> Nature. 346 (6283), 425-434 (1990).</li><li>Shaw, A. S., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+the+T+cell+receptor+go+the+distance:+a+topological+view+of+T+cell+activation.">Making the T cell receptor go the distance: a topological view of T cell activation.</a> Immunity. 6 (4), 361-369 (1997).</li><li>Monks, C. R., Freiberg, B. A., Kupfer, H., Sciaky, N., Kupfer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+segregation+of+supramolecular+activation+clusters+in+T+cells.">Three-dimensional segregation of supramolecular activation clusters in T cells.</a> Nature. 395 (6697), 82-86 (1998).</li><li>Delon, J., Stoll, S., Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+T-cell+interactions+with+antigen+presenting+cells+in+culture+and+in+intact+lymphoid+tissue.">Imaging of T-cell interactions with antigen presenting cells in culture and in intact lymphoid tissue.</a> Immunol Rev. 189, 51-63 (2002).</li><li>Brossard, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifocal+structure+of+the+T+cell+-+dendritic+cell+synapse.">Multifocal structure of the T cell - dendritic cell synapse.</a> Eur J Immunol. 35 (6), 1741-1753 (2005).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+context+of+T+cell+signaling.">The cellular context of T cell signaling.</a> Immunity. 30 (4), 482-492 (2009).</li><li>Balagopalan, L., Sherman, E., Barr, V. A., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+for+assaying+lymphocyte+activation+in+action.">Imaging techniques for assaying lymphocyte activation in action.</a> Nat Rev Immunol. 11 (1), 21-33 (2011).</li><li>Cebecauer, M., Spitaler, M., Serge, A., Magee, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+complexes+and+clusters:+functional+advantages+and+methodological+hurdles.">Signalling complexes and clusters: functional advantages and methodological hurdles.</a> J Cell Sci. 123, 309-320 (2010).</li><li>Oddos, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-speed+high-resolution+imaging+of+intercellular+immune+synapses+using+optical+tweezers.">High-speed high-resolution imaging of intercellular immune synapses using optical tweezers.</a> Biophys J. 95 (10), L66-L68 (2008).</li><li>Grakoui, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunological+synapse:+a+molecular+machine+controlling+T+cell+activation.">The immunological synapse: a molecular machine controlling T cell activation.</a> Science. 285 (5425), 221-227 (1999).</li><li>Bunnell, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+ligation+induces+the+formation+of+dynamically+regulated+signaling+assemblies.">T cell receptor ligation induces the formation of dynamically regulated signaling assemblies.</a> J Cell Biol. 158 (7), 1263-1275 (2002).</li><li>Yokosuka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+generated+T+cell+receptor+microclusters+initiate+and+sustain+T+cell+activation+by+recruitment+of+Zap70+and+SLP-76.">Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.</a> Nat Immunol. 6 (12), 1253-1262 (2005).</li><li>Seminario, M. C., Bunnell, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+initiation+in+T-cell+receptor+microclusters.">Signal initiation in T-cell receptor microclusters.</a> Immunol Rev. 221, 90-106 (2008).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+bilayers+at+the+vanguard+of+immune+cell+activation+studies.">Supported bilayers at the vanguard of immune cell activation studies.</a> J Struct Biol. 168 (1), 152-160 (2009).</li><li>Martinelli, R., Carman, C. V., Bradshaw, R. A., Stahl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+Endothelilal+Interactions.">Lymphocyte Endothelilal Interactions.</a> Encyclopedia of Cell Biology. , (2015).</li><li>Choi, J., Enis, D. R., Koh, K. P., Shiao, S. L., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+lymphocyte-endothelial+cell+interactions.">T lymphocyte-endothelial cell interactions.</a> Annu Rev Immunol. 22, 683-709 (2004).</li><li>Marelli-Berg, F. M., Jarmin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+presentation+by+the+endothelium:+a+green+light+for+antigen-specific+T+cell+trafficking?.">Antigen presentation by the endothelium: a green light for antigen-specific T cell trafficking?.</a> Immunol Lett. 93 (2-3), 109-113 (2004).</li><li>Sage, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+recognition+is+facilitated+by+invadosome-like+protrusions+formed+by+memory/effector+T+cells.">Antigen recognition is facilitated by invadosome-like protrusions formed by memory/effector T cells.</a> J Immunol. 188 (8), 3686-3699 (2012).</li><li>Kumari, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+foci+facilitate+activation+of+the+phospholipase+C-gama+in+primary+T+lymphocytes+via+the+WASP+pathway+.">Actin foci facilitate activation of the phospholipase C-gama in primary T lymphocytes via the WASP pathway .</a> eLife. , (2015).</li><li>Marelli-Berg, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Major+histocompatibility+complex+class+II-expressing+endothelial+cells+induce+allospecific+nonresponsiveness+in+naive+T+cells.">Major histocompatibility complex class II-expressing endothelial cells induce allospecific nonresponsiveness in naive T cells.</a> J Exp Med. 183 (4), 1603-1612 (1996).</li><li>Ma, W., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+endothelial+cells+effectively+costimulate+cytokine+production+by,+but+not+differentiation+of,+naive+CD4++T+cells.">Human endothelial cells effectively costimulate cytokine production by, but not differentiation of, naive CD4+ T cells.</a> J Immunol. 161 (5), 2158-2167 (1998).</li><li>Perez, V. L., Henault, L., Lichtman, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+antigen+presentation:+stimulation+of+previously+activated+but+not+naive+TCR-transgenic+mouse+T+cells.">Endothelial antigen presentation: stimulation of previously activated but not naive TCR-transgenic mouse T cells.</a> Cell Immunol. 189 (1), 31-40 (1998).</li><li>Epperson, D. E., Pober, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-presenting+function+of+human+endothelial+cells.+Direct+activation+of+resting+CD8+T+cells.">Antigen-presenting function of human endothelial cells. Direct activation of resting CD8 T cells.</a> J Immunol. 153 (12), 5402-5412 (1994).</li><li>Shiao, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+effector+memory+CD4++T+cells+directly+recognize+allogeneic+endothelial+cells+in+vitro+and+in+vivo.">Human effector memory CD4+ T cells directly recognize allogeneic endothelial cells in vitro and in vivo.</a> J Immunol. 179 (7), 4397-4404 (2007).</li><li>Marelli-Berg, F. M., Okkenhaug, K., Mirenda, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-signal+model+for+T+cell+trafficking.">A two-signal model for T cell trafficking.</a> Trends Immunol. 28 (6), 267-273 (2007).</li><li>Carman, C. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcellular+diapedesis+is+initiated+by+invasive+podosomes.">Transcellular diapedesis is initiated by invasive podosomes.</a> Immunity. 26 (6), 784-797 (2007).</li><li>Carman, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+for+transcellular+diapedesis:+probing+and+pathfinding+by+'invadosome-like+protrusions'.">Mechanisms for transcellular diapedesis: probing and pathfinding by 'invadosome-like protrusions'.</a> J Cell Sci. 122 ((Pt 17)), 3025-3035 (2009).</li><li>Dustin, M. L., Tseng, S. Y., Varma, R., Campi, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell-dendritic+cell+immunological+synapses.">T cell-dendritic cell immunological synapses.</a> Curr Opin Immunol. 18 (4), 512-516 (2006).</li><li>Saito, T., Yokosuka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+synapse+and+microclusters:+the+site+for+recognition+and+activation+of+T+cells.">Immunological synapse and microclusters: the site for recognition and activation of T cells.</a> Curr Opin Immunol. 18 (3), 305-313 (2006).</li><li>Gomez, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HS1+functions+as+an+essential+actin-regulatory+adaptor+protein+at+the+immune+synapse.">HS1 functions as an essential actin-regulatory adaptor protein at the immune synapse.</a> Immunity. 24 (6), 741-752 (2006).</li><li>Burbach, B. J., Medeiros, R. B., Mueller, K. L., Shimizu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+receptor+signaling+to+integrins.">T-cell receptor signaling to integrins.</a> Immunol Rev. 218, 65-81 (2007).</li><li>Vicente-Manzanares, M., Sanchez-Madrid, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+cytoskeleton+during+leukocyte+responses.">Role of the cytoskeleton during leukocyte responses.</a> Nat Rev Immunol. 4 (2), 110-122 (2004).</li><li>Ma, Z., Janmey, P. A., Finkel, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+receptor+deformation+model+of+TCR+triggering.">The receptor deformation model of TCR triggering.</a> Faseb J. 22 (4), 1002-1008 (2008).</li><li>Ma, Z., Sharp, K. A., Janmey, P. A., Finkel, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-anchored+monomeric+agonist+pMHCs+alone+trigger+TCR+with+high+sensitivity.">Surface-anchored monomeric agonist pMHCs alone trigger TCR with high sensitivity.</a> PLoS Biol. 6 (2), e43 (2008).</li><li>Groves, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bending+mechanics+and+molecular+organization+in+biological+membranes.">Bending mechanics and molecular organization in biological membranes.</a> Annu Rev Phys Chem. 58, 697-717 (2007).</li><li>Xu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+T+cell+receptor+activation+by+dynamic+membrane+binding+of+the+CD3epsilon+cytoplasmic+tyrosine-based+motif.">Regulation of T cell receptor activation by dynamic membrane binding of the CD3epsilon cytoplasmic tyrosine-based motif.</a> Cell. 135 (4), 702-713 (2008).</li></ol>

The authors have nothing to disclose.

En general, este protocolo describe métodos para la investigación de las células endoteliales como i) APC fisiológicos poco estudiada y ii) como un nuevo tipo de &quot;modelo APC celular plana &#39;. Con respecto a lo primero, se ha vuelto cada vez más apreciada que la APC periférica no hematopoyéticas (o &#39;estroma&#39;) juegan un papel crítico, no redundantes (es decir, en comparación con hematopoyético APC) en la conformación de la respuesta inmune adaptativa 16-18. Entre tales &quot;...

Los linfocitos T son una rama del sistema inmune adaptativo que se caracteriza por la capacidad de reconocer eficientemente péptido antígeno (Ag) unida a complejo mayor de histocompatibilidad (MHC) moléculas a través de sus receptores de células T (TCR) 1. Linfocitos ingenuos constitutivamente migran y escanear &#39;Ag células presentadoras profesionales&#39; (APC; por ejemplo, células dendríticas) en los ganglios linfáticos, mientras que las células de memoria / T efectoras necesitan para estudiar con eficacia una muy amplia gama de vehículos blindados y potenciales células diana dentro de los tejidos periféricos. En el min tras el reconocimiento inicial de cognado Ag en un APC, linfocitos detener su migración y comienzan a formar una interfaz célula-célula íntima especializado denominado &#39;sinapsis inmunológica &quot;(IS). Sostenida (es decir, 30 a 60 min) ES se requieren contactos para ampliar y mantener la señalización 2-7. Estudios emergentes identificar que dentro del IS, que es la formación continua y rápida remodeling de señalización subcelular micro-grupos discretos (es decir, que contienen MHC / Ag-TCR, F-actina, la adhesión y moléculas de señalización) que determinan la fuerza y la calidad de la que resulta la respuesta inmune 2-7. Sin embargo, los detalles dinámicos y mecanismo de regulación de este proceso se conocen por completo 8,9. Esto se debe en gran parte de los desafíos técnicos asociados con topologías irregulares de las superficies de APC y la orientación de un mal control de los planos de interacción célula-célula, las cuestiones que limitan profundamente la imagen espacio-temporal necesaria acerca 10.08 (Figure1A). <img alt="Figura 1" src="/files/ftp_upload/53288/53288fig1.jpg" /> Figura 1. Un fisiológica planar Modelo APC Cell for Imaging inmunológica Synapse Dynamics. El esquema ilustra la imagen tradicional de la sinapsis inmunológica entre una célula T y un professio<html> nal de células APC (A) y T y un modelo tradicional de lípidos planar APC bicapa (B) en comparación con este novedoso modelo de APC planar endotelial (C). APCs profesionales proporcionan sinapsis inmunológicas fisiológicas pero ofrecen interfaz mal orientada célula-célula (es decir, con respecto al plano óptima de imagen xy; resolución ~ 0,2 micras), lo que compromete drásticamente espacial (z plano de la imagen de resolución ~ 1 m) y temporal (es decir, debido a la necesidad de escanear repetidamente a través de todos los planos de formación de imágenes z) resolución de la imagen. Modelos bicapa tienen una topología plana que ofrece óptima de imágenes de resolución espacio-temporal, pero también son muy simplificada, no fisiológico y rígido. Este modelo de células endoteliales combina la topología planar de bicapas lipídicas con el sustrato fisiológico de un APC clásico para entregar óptima de imágenes de resolución espacial y temporal en un entorno fisiológico.m / files / ftp_upload / 53288 / 53288fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Haga clic aquí para ver una versión más grande de esta figura. El trabajo previo ha eludido parcialmente estos obstáculos mediante el desarrollo de modelos sustrato plano (es decir, bicapas lipídicas y las superficies recubiertas de anticuerpo) que proporcionan resolución espacio-temporal óptima (es decir, a través de la fijación de la superficie de activación de las células T en un único plan que es paralela a la proyección de imagen óptima xy avión) 11 a 15 (Figura 1B). Estos modelos han facilitado importantes conocimientos sobre la dinámica subcelulares / moleculares que controlan la señalización antigénico en las células T, incluyendo el descubrimiento de actina dinámica / TCR señalización micro-clusters 7,11-14. Sin embargo, tales modelos están inherentemente simplificaron, así como rígido (que impide el desarrollo / estudio de características topológicas 3 dimensiones) (Figura 1B). Por lo tanto, sigue siendo incierto cómo relacionar estos hallazgos a physiologic célula-célula vigilancia inmune. Aunque todavía poco estudiado, vascular y las células endoteliales linfáticas están emergiendo como un grande (es decir, mayor en número que todos los vehículos blindados profesionales, por ~ 1000 veces) compartimento periférico de &quot;semi-profesional&quot; APC 16-18. Estas células expresan MHC-I, MHC-II- y una multitud de moléculas co-estimuladoras (por ejemplo, CD40, LFA3, ICOSL, el 4-1BB, OX40L, TL1A, PD-L1; pero no CD80 y CD86) y son estratégicamente posicionado en la interfase sangre-tejido donde sirven funciones especializadas centinela 16-18. Estudios previos demostraron que las células endoteliales pueden efectivamente re-estimular efectoras / memoria, pero no ingenuo, las células T 19-25. Así, las células endoteliales es probable que desempeñen un papel único de APC en la fase efectora de la respuesta inmune adaptativa dentro de los tejidos periféricos, tales como la influencia local sobre la activación de células T, la diferenciación, la memoria y la tolerancia 16-17,26. Cricamente, cuando se cultivan in vitro, las células endoteliales forman superficies celulares prácticamente planas y están fácilmente transfectable (por ejemplo, con los reporteros fluorescentes de proteínas). Estas características son ideales para imágenes de alta resolución espacio-temporal de la dinámica topológicos durante las interacciones célula-célula 19,27. Por lo tanto las células endoteliales podrían servir como un &#39;celular plana APC modelo fisiológica claramente adecuado para el estudio de los mecanismos de remodelación subcelulares / molecular que impulsan el reconocimiento de antígenos y regulan las respuestas (Figura 1C) 19,20. Previamente establecidos técnicas de imagen complementarios (incluyendo la transfección de las células endoteliales con los fabricantes de proteínas fluorescentes de la membrana plasmática y citosol) para estudiar los detalles de la interacción de leucocitos endotelial durante la adhesión y migración transendotelial 27, mostraron que los leucocitos sondean activamente la superficie del endotelio por Dynamic una inserciónd retracción de la sub-escala micrométrica, protuberancias cilíndricas de actina-ricos (~ 200-1.000 nm de diámetro y profundidad) denomina-invadosome como salientes (es decir, &#39;ILPS&#39;) 27,28. Estos enfoques de imagen se han ampliado aún más, junto con la creación de protocolos para tomar ventaja de la función endotelial de APC para desarrollar los primeros métodos de alta resolución de imagen espacio-temporal de la célula endotelial sinapsis inmunológica T según lo informado 19,20 y describir con más detalle en el presente documento. Un hallazgo central derivado de esta novela plana celular modelo de APC es que programas de vida independiente de células T funcionan tanto en la promoción de la detección inicial Ag y en el mantenimiento de la señalización posterior. De hecho, las matrices de múltiples programas de vida independiente (que fueron estabilizados y devengados en respuesta a sus iniciales en el flujo de calcio) muestran un enriquecimiento en TCR y moléculas sugestivos de señalización activa tal PKC-Q, ZAP-70, fosfotirosina y HS1. Por lo tanto, programas de vida independiente parecen representar un equivalente fisiológico tridimensional a la micro TCR de señalizaciónracimos visto en los modelos de dos capas planas. Este enfoque, por lo tanto, revela sensibilidad / informes dinámica molecular y arquitectónicos (y biomecánico implícitas) de otro modo no detectable. El método descrito en este documento deberá ser útil para reducir la brecha entre los modelos de sustratos artificiales APC APC profesional y con el fin de mejorar nuestra capacidad de interrogar a los mecanismos básicos de la respuesta inmune adaptativa. Mientras que aquí la atención se centra en la activación de tipo Th1 CD4 + efectoras / célula de memoria, este enfoque básico puede ser modificado fácilmente para estudiar una amplia gama de tipos de células T y Ags, como se discute a continuación.

<table border="0" cellpadding="0" cellspacing="0" width="914">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">BD Vacutainer stretch latex free tourniquet</td>
			<td style="width:149px;">BD Biosciences</td>
			<td style="width:152px;">367203</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD alcohol swabs</td>
			<td style="width:149px;">BD Biosciences</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Vacutainer Safety-Lok</td>
			<td style="width:149px;">BD Biosciences</td>
			<td style="width:152px;">367861</td>
			<td>K2 EDTA</td>
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			<td height="21" style="height:21px;width:392px;">BD Vacutainer Push Button Blood Collection Set</td>
			<td style="width:149px;">BD Biosciences</td>
			<td style="width:152px;">367335</td>
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			<td height="21" style="height:21px;width:392px;">RPMI-1640</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>R8758-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll-Paque&#160;</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">GE17-1440-02</td>
			<td>Bring to RT before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">FCS-Optima</td>
			<td style="width:149px;">Atlanta Biologics</td>
			<td>s12450</td>
			<td>Heat inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>&#160;P4458-100ML&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>T8154-20ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">staphylococcal enterotoxin B&#160;</td>
			<td>Toxin Technology</td>
			<td>BT202RED</td>
			<td>Stock solution 1mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">toxic shock syndrome toxin 1&#160;</td>
			<td>Toxin Technology</td>
			<td>TT606RED</td>
			<td>Stock solution 1mg/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human IL-15</td>
			<td style="width:149px;">R&#38;D Systems</td>
			<td>247-IL-025</td>
			<td>Stock solution 50ug/ml in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">PBS</td>
			<td>Life Technologies</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">Fibronectin</td>
			<td style="width:149px;">Life Technologies</td>
			<td>33016-015</td>
			<td>Stock solution 1mg/ml in H20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HMVEC-d Ad-Dermal MV Endo Cells</td>
			<td style="width:149px;">Lonza</td>
			<td>CC-2543</td>
			<td>Other Human Microvascular ECs can be used, i.e. HLMVECs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGM-2 MV bullet kit</td>
			<td style="width:149px;">Lonza</td>
			<td>CC-3202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Trypsin-EDTA</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>T-4174</td>
			<td>Stock solution 10x, dilute in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">amaxa-HMVEC-L Nucleofector Kit</td>
			<td style="width:149px;">Lonza</td>
			<td>vpb1003</td>
			<td>Required Kit for step 4</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">IFN-g</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>I3265</td>
			<td>Stock solution 1mg/ml in H20</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">TNF-alpha 10ug, human</td>
			<td style="width:149px;">Life Technologies</td>
			<td>PHC3015</td>
			<td>Stock solution 1mg/ml in H20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phenol Red-free HBSS&#160;</td>
			<td style="width:149px;">Life Technologies</td>
			<td>14175-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>BP299-100</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">Calcium Chloride</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">C1016-100G</td>
			<td>Stock solution 1M in H20</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:392px;">Magnesium chloride</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">208337</td>
			<td>Stock solution 1M in H20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Human Serum albumin</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td>A6909-10ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersol 518 F fluorescence free Immersion oil</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>12-624-66A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fura-2 AM 20x50ug</td>
			<td style="width:149px;">Life Technologies</td>
			<td>F1221</td>
			<td>Stock solution 1mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">pEYFP-Mem (Mem-YFP)</td>
			<td style="width:149px;">Clontech</td>
			<td style="width:152px;">6917-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">pDsRed-Monomer (Soluble Cytoplasmic DsRed)</td>
			<td style="width:149px;">Clontech</td>
			<td style="width:152px;">632466</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">pDsRed-Monomer Membrane (Mem-DsRed)</td>
			<td style="width:149px;">Clontech</td>
			<td style="width:152px;">632512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">pEGFP-Actin</td>
			<td style="width:149px;">Clontech</td>
			<td style="width:152px;">6116-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:149px;">Life Technologies</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Formaldehyde solution 37%</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>BP531-500</td>
			<td>Toxic, use fumehood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Triton X-100</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">X100-5ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">&#160;Falcon 15mL Conical Centrifuge Tubes</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>14-959-70C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Falcon 50mL Conical Centrifuge Tubes</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>14-959-49A&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Falcon Tissue Culture Treated Flasks T25</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>10-126-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">&#160;Falcon Tissue Culture Treated Flasks T75</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>&#160; 13-680-65</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">&#160;Corning Cell Culture Treated T175</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:152px;">10-126-61&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Glass coverslips&#160;</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:152px;">12-545-85&#160;</td>
			<td>12 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">&#160;Falcon Tissue Culture Plates 24-well</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>08-772-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Delta-T plates</td>
			<td style="width:149px;">Bioptechs</td>
			<td>04200415B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wheaton Disposable Pasteur Pipets</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:152px;">13-678-8D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 ml Eppendorf tube&#160;</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td>05-402-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">&#160;ICAM1 mouse anti-human</td>
			<td style="width:149px;">BD Biosciences</td>
			<td>555509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HS1 mouse anti-human</td>
			<td>BD Biosciences</td>
			<td>610541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Human CD11a (LFA-1alpha) Purified</td>
			<td>ebioscience</td>
			<td>BMS102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Human CD3 Alexa Fluor&#174; 488</td>
			<td>ebioscience</td>
			<td>53-0037-41</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-MHC Class II antibody&#160;</td>
			<td style="width:149px;">Abcam</td>
			<td>ab55152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Talin 1 antibody</td>
			<td style="width:149px;">Abcam</td>
			<td>ab71333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Anti-PKC theta antibody&#160;</td>
			<td style="width:149px;">Abcam</td>
			<td>ab109481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphotyrosine (4G10 Platinum)</td>
			<td style="width:149px;">Millipore</td>
			<td>50-171-463</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Nucleofector II</td>
			<td style="width:149px;">Amaxa Biosystems</td>
			<td style="width:152px;"></td>
			<td>Required electroporator for step 4</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">Zeiss Axiovert</td>
			<td style="width:149px;">Carl Zeiss MicroImaging</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">Zeiss LSM510&#160;</td>
			<td style="width:149px;">Carl Zeiss MicroImaging</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">Zeiss Axiovison Software</td>
			<td style="width:149px;">Carl Zeiss MicroImaging</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">NU-425 (Series 60) Biological Safety Cabinet</td>
			<td style="width:149px;">NuAIRE</td>
			<td style="width:152px;">Nu-425-600</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">&#160;Forma STRCYCLE 37 &#176;C, 5% CO2 Cell culture Incubator</td>
			<td>Fisher Scientific</td>
			<td>202370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Centrifuge 5810</td>
			<td style="width:149px;">Eppendorf</td>
			<td style="width:152px;">EW-02570-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Hemocytometer</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">&#160;Z359629</td>
			<td>Bright-Line Hemocytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Isotemp Waterbath model 202</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:152px;">15-462-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

an endothelial planar cell model for imaging immunological synapse dynamics

La inmunidad adaptativa está regulado por las interacciones dinámicas entre las células T y las células presentadoras de antígeno (&quot;APC&quot;) refiere como &#39;sinapsis inmunológicas. Dentro de estas interfaces de célula-célula íntimos grupos subcelulares discretos de MHC / Ag-TCR, F-actina, adhesión y moléculas de señalización forman y remodelar rápidamente. Estas dinámicas se cree que son determinantes críticos de la eficiencia y la calidad de las respuestas inmunes que se desarrollan y, por tanto, de protección contra la inmunidad patológica. El conocimiento actual de las sinapsis inmunológicas con fisiológica APC está limitada por la insuficiencia de la resolución de las imágenes se puede obtener. Aunque los modelos de sustratos artificiales (por ejemplo, bicapas lipídicas planas) ofrecen una excelente resolución y han sido herramientas muy valiosas, que son inherentemente no fisiológica y simplificada. Las células endoteliales vasculares y linfáticos se han convertido en un importante tejido periférico (o estroma) Compartimiento de &quot;semi-profesionalal APC &#39;. Estas APCs (que expresan la mayor parte de la maquinaria molecular de APCs profesional) tienen la característica única de la formación de la superficie celular prácticamente planar y están fácilmente transfectable (por ejemplo, con los reporteros fluorescentes de proteínas). Aquí un enfoque básico para implementar las células endoteliales como una novela y fisiológica &#39;planar modelo APC celular&#39; para mejorar la formación de imágenes y el interrogatorio de los procesos de señalización antigénicos fundamentales se describirá.

Un enfoque de formación de imágenes novedoso utilizando células endoteliales y la combinación de las ventajas resolución de la planar bicapas lipídicas modelo con la complejidad fisiológica y la deformabilidad de las APC profesional fue desarrollado (Figura 1). La Figura 2 proporciona ejemplos de migración típica, el flujo de calcio y la dinámica topológicos observado con esta enfoque. En ausencia de la SAG en el endotelio, los linfocitos SAG-...

Watch this Scientific Journal Video about An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics at JoVE.com

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Un modelo de la célula endotelial planar for Imaging inmunológica Synapse Dinámica

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

an-endothelial-planar-cell-model-for-imaging-immunological-synapse

Research

JoVE Journal

Immunology and Infection

8.8K vistas.  Harvard Medical School - BIDMC. Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Video: An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Imagen del VIH-1 sobre inducida sinapsis virológica y señalización en la bicapa lipídica sintéticas

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Investigación

Inmunología e Infección

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

La visualización de la sinapsis inmunológica mediante gated-Tiempo color dual Emisión Estimulada de Agotamiento (STED) Nanoscopia

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

Imágenes en tiempo real de la célula-célula cruces endoteliales Durante neutrófilos Transmigración Bajo Flujo Fisiológica

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Una<em&gt; Ex vivo</em&gt; Modelo de un ataque de células T Oligodendrocyte dirigida en Rebanadas cerebral agudo

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

Super-resolución de imagen de la célula Natural Killer inmunológica Synapse en un apoyada-Glass planar bicapa lipídica

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Un<em&gt; In Vitro</em&gt; Modelo de la barrera sangre-cerebro mediante Espectroscopia de Impedancia: Un enfoque en la interacción de células T de la célula endotelial

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

Estudio de la dinámica de los organelos en células B durante la formación de la sinapsis inmune

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Implantación y monitorización por PET/CT de un modelo ortotópico de mesotelioma pleural humano en ratones atímicos

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

Imagen de la Sinapsis Inmunológica Humana

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

Un modelo inmunológico para el trasplante heterotópico de células musculares cardíacas y cardíacas en ratas

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...