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>
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The authors have nothing to disclose.

Insgesamt ist dieses Protokoll beschreibt Methoden zur Untersuchung Endothelzellen als i) under physiologischen APCs und ii) als eine neue Art von &quot;planare Zell APC-Modell&quot;. In Bezug auf die frühere, wurde es zunehmend klar, daß nicht-hämatopoetischen peripheren (oder &quot;Stromazellen&quot;) APCs spielen kritische, nicht redundanten Rollen (dh gegenüber hämatopoetischen APCs) bei der Gestaltung der adaptiven Immunantwort 16-18. Unter solchen &#39;semi-professionellen &quot;APCs, vask...

T-Lymphozyten sind ein Zweig des adaptiven Immunsystems gekennzeichnet durch die Fähigkeit, Peptidantigen (Ag) effizient zu erkennen Haupthistokompatibilitätskomplex (MHC) gebundenen Moleküle durch ihre T-Zell-Rezeptoren (TCRs) 1. Naive Lymphozyten konstitutiv migrieren und scannen Berufs Ag-präsentierende Zellen &quot;(APC; zB dendritische Zellen) innerhalb von Lymphknoten, während Memory / Effektor-T-Zellen benötigen, um eine extrem breite Palette von APCs und potentiellen Zielzellen im peripheren Gewebe effektiv zu überwachen. Im min nach dem erstmaligen Ansatz des verwandten Ag auf einer APC, Lymphozyten zu verhaften ihre Migration und beginnen, eine spezialisierte intime Zell-Zell-Schnittstelle genannt &#39;immunologischen Synapse&#39; zu bilden (IS). Anhaltende (dh 30-60 min) Kontakte sind erforderlich, um zu verstärken und zu erhalten Signalisierungs 2-7. Schwellen Studien identifizieren, die innerhalb der IS, ist es die kontinuierliche und schnelle Bildung remodeling diskreter subzellulären Signalisierungsmikrocluster (dh enthaltend MHC / Ag-TCR, F-Actin, Adhäsion und Signalmolekülen), die die Stärke und Qualität der resultierenden Immunantworten 2-7 zu bestimmen. Allerdings sind dynamische Details und regulatorischen Mechanismus dieses Prozesses unvollständig verstanden 8,9. Dies ergibt sich im Wesentlichen aus technischen Herausforderungen mit unregelmäßigen Topologien von APC Oberflächen und schlecht kontrollierte Ausrichtung der Zell-Zell-Interaktion Flugzeuge, Fragen, die zutiefst begrenzen die erforderliche Raumzeit-Bildgebung assoziiert nähert 8-10 (Abbildung 1a). <img alt="Abbildung 1" src="/files/ftp_upload/53288/53288fig1.jpg" /> Abbildung 1. Ein Physiologische Planar Handy APC-Modell für Imaging immunologischen Synapse Dynamics. Die Abbildung zeigt, traditionellen Bildgebung von immunologischen Synapse zwischen einer T-Zelle und einer professio<html> nal APC (A) und T-Zell-und ein traditionelles planaren Lipid-Doppelschicht APC-Modell (B) im Vergleich zu diesem Roman Endothelzellen planar APC-Modell (C). Professionelle APCs bereitzustellen physiologischen immunologischen Synapsen bieten aber schlecht orientierte Zell-Zell-Schnittstelle (dh in Bezug auf die optimale xy Abbildungsebene; Auflösung ~ 0,2 &amp; mgr; m), die dramatisch beeinträchtigt räumliche (z Abbildungsebene Auflösung ~ 1 &amp; mgr; m) und zeitliche (dh aufgrund der Notwendigkeit, immer wieder zu scannen durch alle z Abbildungsebenen) Auflösung der Bildgebung. Doppelschicht-Modelle haben eine planare Topologie, die eine optimale Raumzeit-Bildauflösung bietet, werden aber auch in stark vereinfachter, nicht-physiologischen und starr. Diese Endothelzellen-Modell vereint die planare Topologie der Lipid-Doppelschichten mit dem physiologischen Substrat eines klassischen APC, um eine optimale räumliche und zeitliche Bildauflösung in einer physiologischen Umgebung zu liefern.m / files / ftp_upload / 53288 / 53288fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen. Frühere Arbeiten haben zum Teil diese Hindernisse durch Entwicklung planaren Substrat Modellen (dh Lipiddoppelschichten und Antikörper-beschichteten Oberflächen), welche optimale Raum-Zeit-Auflösung (dh bereitzustellen umgangen über Fixierung des T-Zell-Aktivierungsfläche in einem einzigen Plan, die parallel zu der optimalen xy Bildgebung Ebene) 11-15 (1B). Diese Modelle haben wichtige Einblicke in die subzelluläre / ​​Molekulardynamik, die Antigen-Signalisierung in T-Zellen, einschließlich der Entdeckung der dynamischen Aktin / TCR Signalisierung Mikro-Clustern 7,11-14 Kontrolle erleichtert. Allerdings sind solche Modelle von Natur aus stark vereinfacht, sowie starre (Ausschluss der Entwicklung / Untersuchung der 3-dimensionale topologische Merkmale) (1B). Daher bleibt es unsicher, wie man solche Feststellungen phy beziehensiologic Zell-Zell-Immunüberwachung. Obwohl noch under werden vaskulären und lymphatischen Endothelzellen als großen Schwellen (dh mehr in den Zahlen als alle professionellen APCs, die von ~ 1.000-fach) Peripheriefach &#39;semi-professionellen &quot;APCs 16-18. Diese Zellen exprimieren MHC-I- MHC-II- und einer Vielzahl von Co-Stimulator Moleküle (zB CD40, LFA3, ICOSL, 4-1BB, OX40L, TL1A, PD-L1, nicht aber CD80 und CD86) und sind strategisch an der Blut-Gewebe-Grenzfläche, wo sie dienen spezialisierte Sentinel-Funktionen 16-18 positioniert. Frühere Studien zeigten, dass Endothelzellen wirksam neu zu stimulieren Effektor / Memory, aber nicht naiv, T-Zellen 19-25. Somit sind Endothelzellen wahrscheinlich einzigartig APC Rollen in Effektorphase adaptive Immunantworten in den peripheren Geweben, wie etwa lokale Einfluss auf T-Zellaktivierung, Differenzierung, Speicher und Toleranz 16,17,26 spielen. Crimatisch, wenn sie in vitro gezüchtet, Endothelzellen bilden praktisch planaren Zelloberflächen und leicht transfektierbare (beispielsweise mit fluoreszierenden Reporterproteine). Diese Eigenschaften sind ideal für hohe Raum-Zeit-auflösende Bildgebung der topologischen Dynamik während der Zell-Zell-Wechselwirkungen 19,27. So Endothelzellen als physiologische &#39;planaren Zell APC-Modell ausgesprochen geeignet für die Untersuchung der subzellulären / molekulare Umbaumechanismen, die Antigen-Erkennung zu fahren und zu regulieren Antworten (1C) 19,20 dienen könnte. Vorher festgelegten komplementären Bildgebungstechniken (einschließlich Transfektion der Endothelzellen mit fluoreszierenden Protein Trägern der Plasmamembran und Cytosol) zur Untersuchung der Details der Leukozyten-Endothel-Interaktion während der Adhäsion und transendotheliale Migration 27, zeigte, daß Leukozyten die Oberfläche des Endothels Sonde aktiv durch dynamische Insertion eind Einfahren der Submikron-Maßstab, Actin-reiche zylindrischen Vorsprünge (~ 200-1000 nm Durchmesser und Tiefe) bezeichnet invadosome artigen Vorsprüngen (dh &quot;ILPs &#39;) 27,28. Diese bildgebenden Ansätze wurden weitere mit der Erstellung von Protokollen erweitert, um die Vorteile der endothelialen APC-Funktion, um die ersten Methoden zur hohen Raum-Zeit-auflösende Bildgebung des T-Zell-endothelialen immunologischen Synapse wie berichtet 19,20 entwickeln und weiter zu beschreiben hier. Eine zentrale Erkenntnis aus diesem neuartigen planaren Zell APC-Modell abgeleitet ist, dass T-Zell-ILPs funktionieren sowohl bei der Förderung der anfänglichen Ag Nachweis und bei der Aufrechterhaltung nachfolgende Signalisierung. Tatsächlich Arrays von mehreren ILPs (die stabilisiert und als Reaktion auf aufgelaufene Calciumfluss initial wurden) zeigen die Anreicherung in TCR und Molekülen andeutend aktives Melde wie PKC-Q, ZAP-70, Phosphotyrosin und HS1. Daher ILPs scheinen eine dreidimensionale physiologischen äquivalent der TCR-Signalgebung Mikro darstellenCluster in planare Doppelschicht-Modellen gesehen. Dieser Ansatz, so zeigt sensibel / Berichte molekularen und architektonischen (und stillschweigende biomechanischen) Dynamik sonst nicht nachweisbar. Das hier beschriebene Verfahren ist nützlich, um die Lücke zwischen professionellen APC und künstliche APC-Substrat-Modelle, um unsere Fähigkeit, grundlegende Mechanismen der adaptiven Immunantwort zu verhören zu verbessern. Während hier der Schwerpunkt auf der Aktivierung von CD4 + Th1-Typ-Effektor / Speicherzelle kann dieser Ansatz leicht modifiziert werden, um eine breite Palette von T-Zell-Typen und Ags zu studieren, wie unten diskutiert.

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		<tr height="21">
			<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

Adaptiven Immunität wird von dynamischen Wechselwirkungen zwischen T-Zellen und Antigen-präsentierenden Zellen (&quot;APCs&quot;) reguliert die so genannte &quot;immunologischen Synapsen. Innerhalb dieser intimen Zell-Zell-Schnittstellen diskreten subzellulärer Cluster von MHC / Ag-TCR, F-Actin, Adhäsion und Signalmoleküle bilden und umgestalten schnell. Diese Dynamik werden gedacht, um kritische Determinanten der sowohl die Effizienz und die Qualität der Immunantwort, die sich entwickeln und damit der Schutz gegen pathologische Immunität. Aktuelle Verständnis der immunologischen Synapsen mit physiologischer APCs wird durch die Unzulänglichkeit der erhältliche Bildauflösung beschränkt. Obwohl künstlichen Substrat-Modelle (zB planaren Lipid-Doppelschichten) bieten hervorragende Auflösung und waren äußerst wertvolle Instrumente, sie sind von Natur aus nicht-physiologischen und vereinfacht. Vaskulären und lymphatischen Endothelzellen haben sich als wichtige peripheren Gewebe (oder Stroma) Abteil &quot;semi-Beruf entstandenal APCs &quot;. Diese APCs (die meisten der molekularen Maschinerie der professionellen APCs Express) haben die einzigartige Eigenschaft von nahezu planaren Zelloberfläche bilden und sind (mit fluoreszierenden Proteins Reportern zB) leicht transfizierbar. Hier ein grundlegendes Konzept für Endothelzellen als neuartige und physiologische &#39;planaren Zell APC-Modell &quot;für eine verbesserte Bildverarbeitung und Abfrage der Grundantigenen Signalprozesse zu implementieren beschrieben.

Eine neuartige Abbildungsansatz mit Endothelzellen und Kombinieren der Auflösung Vorteile der planaren Lipiddoppelschicht-Modell mit der physiologischen Komplexität und Verformbarkeit professionelle APCs wurde (Figur 1) entwickelt. 2 stellt Beispiele von typischen Migration, Calciumflusses und topologische Dynamik bei dieser beobachtete Ansatz. In Abwesenheit der SAG auf Endothel SAG-spezifische CD4 + Th1-Lymphozyten schnell verbreiten, zu p...

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'.

Ein Endothelial Planar Zellmodell für Imaging immunologischen Synapse Dynamics

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 Aufrufe.  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'.

Serie: 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.

Imaging von HIV-1 Envelope-induzierte virologische Synapse und Signalisierung auf Synthetic Lipiddoppelschichten

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

Forschung

Immunologie und Infektion

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.

Visualisierung der immunologischen Synapse von Dual Color Zeitgesteuerte Stimulated Emission Depletion (STED) Nanoskopie

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.

Echtzeit-Bildgebung von Zell-Zell-Endothelial Junctions Während Neutrophile Transmigration unter physiologischen Fluss

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.

Ein<em&gt; Ex-vivo-</em&gt; Modell eines Oligodendrozyten-gerichtete T-Cell Attack in akuten Hirnschnitten

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-Resolution Imaging der natürlichen Killerzell Immunologische Synapse auf einem Glas-gestützten Planar Lipiddoppelschicht

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.

Ein<em&gt; In Vitro</em&gt; Modell der Blut-Hirn-Schranke Mit Impedanzspektroskopie: Ein Fokus auf T-Zell-Endothelzellinteraktion

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.

Studium der Organelle-Dynamik in B-Zellen während der Immunsynapse-Bildung

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.

Implantation und Überwachung eines orthotopischen Modells des menschlichen Pleuralmesothelioms bei athymischen Mäusen durch PET/CT

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).

Bildgebung der humanen immunologischen 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) ...

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.

Ein immunologisches Modell für heterotopische Herz- und Herzmuskelzelltransplantation bei Ratten

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