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.

Nel complesso, questo protocollo descrive i metodi per indagare le cellule endoteliali come i) understudied APC fisiologici e ii) come un nuovo tipo di &#39;planare cellulare modello APC&#39;. Per quanto riguarda il primo, è diventato sempre più apprezzato che APC non ematopoietiche periferiche (o &#39;stromali&#39;) svolgono, un ruolo non ridondanti critici (ad esempio, rispetto a ematopoietiche APC) nel modellare le risposte immunitarie adattive 16-18. Tra tali &#39;semi-professionale&#39; APC, v...

Linfociti T sono un ramo del sistema immunitario adattativo caratterizzato dalla capacità di riconoscere efficientemente antigene peptide (Ag) vincolato al complesso maggiore di istocompatibilità (MHC) molecole attraverso i recettori delle cellule T (TCR) 1. Linfociti naïve costitutivamente migrano e Scan &#39;Ag cellule presentanti professionali »(APC, ad esempio, cellule dendritiche) all&#39;interno dei linfonodi, mentre le cellule di memoria / T effettrici devono sorvegliare efficacemente una gamma estremamente ampia di APC e potenziali cellule bersaglio all&#39;interno dei tessuti periferici. Nel min seguente iscrizione iniziale delle cognate Ag su un APC, linfociti arrestare la loro migrazione e cominciano a formare un professionista intimo un&#39;interfaccia cellula-cellula chiamato &#39;sinapsi immunologica&#39; (IS). Sostenuta (cioè, 30-60 min) È contatti sono tenuti per amplificare e sostenere segnalazione 2-7. Studi emergenti identificano che all&#39;interno della IS, è la formazione continua e rapida remodeling di discrete segnalazione sub-cellulare micro-cluster (ad esempio, contengono MHC / Ag-TCR, F-actina, adesione e molecole di segnalazione), che determinano la forza e la qualità della conseguente risposta immunitaria 2-7. Tuttavia, i dettagli dinamici e meccanismo di regolazione di questo processo non sono completamente compresi 8,9. Ciò deriva in gran parte da problemi tecnici connessi con topologie irregolari di superfici APC e l&#39;orientamento mal controllata dei piani di interazione cellula-cellula, i problemi che limitano profondamente imaging spazio-temporale richiesto avvicina 8-10 (Figure1A). <img alt="Figura 1" src="/files/ftp_upload/53288/53288fig1.jpg" /> Figura 1. Una fisiologica Planar APC cellulare Modello for Imaging immunologiche Synapse Dynamics. Lo schema mostra l&#39;imaging tradizionale di sinapsi immunologica tra una cellula T e un professio<html> nal APC (A) e cellule T e un modello tradizionale lipidico planare doppio strato APC (B) rispetto a questo romanzo endoteliale modello planare APC (C). APC professionali forniscono sinapsi immunologiche fisiologiche ma offrono interfaccia mal orientata cellula-cellula (cioè, rispetto al piano ottimale di imaging xy; risoluzione ~ 0,2 micron), che compromette notevolmente spaziale (piano di imaging z risoluzione ~ 1 micron) e temporale (cioè, a causa della necessità di eseguire la scansione ripetutamente attraverso tutti i piani di imaging z) Risoluzione di imaging. Modelli a doppio strato hanno una topologia planare che fornisce la risoluzione ottimale delle immagini spazio-temporale, ma sono anche molto semplificate, non fisiologica e rigida. Questo modello delle cellule endoteliali combina la topologia planare di doppi strati lipidici con il substrato fisiologico di un classico APC per fornire la risoluzione ottimale di imaging spaziale e temporale in un ambiente fisiologico.m / files / ftp_upload / 53288 / 53288fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. Il lavoro precedente ha parzialmente aggirato questi ostacoli attraverso lo sviluppo di modelli di substrato planare (ad esempio, doppi strati lipidici e superfici rivestiti con anticorpo) che forniscono la risoluzione spazio-temporale ottimale (cioè, mediante il fissaggio alla superficie di attivazione delle cellule T in un unico piano che è parallelo al di imaging ottimale xy piano) 11-15 (Figura 1B). Questi modelli hanno facilitato importanti conoscenze sulle dinamiche subcellulari / molecolari che controllano la segnalazione antigenico nelle cellule T, tra cui la scoperta di actina dinamico / TCR segnalazione micro-cluster 7,11-14. Tuttavia, tali modelli sono intrinsecamente avevano semplificato, così come rigida (precludendo sviluppo / studio delle caratteristiche topologiche 3-dimensionali) (Figura 1B). Pertanto, rimane incerto su come relazionarsi tali risultati al PHYsiologic cellula-cellula sorveglianza immunitaria. Sebbene ancora poco studiato, vascolari e cellule endoteliali linfatiche stanno emergendo come un grande (vale a dire, maggiore in numero rispetto a tutti APC professionali, da ~ 1.000 volte) compartimento periferico di &#39;semi-professionale&#39; APC 16-18. Queste cellule esprimono MHC-I-, MHC-II e una moltitudine di molecole co-stimolatore (ad esempio, CD40, LFA3, ICOSL, 4-1BB, OX40L, TL1A, PD-L1, ma non CD80 e CD86) e sono strategicamente posizionato all&#39;interfaccia sangue tessuto in cui servono le funzioni di sentinella specializzate 16-18. Studi precedenti hanno dimostrato che le cellule endoteliali possono effettivamente ri-stimolare effettori / memoria, ma non ingenua, le cellule T 19-25. Così, le cellule endoteliali sono suscettibili di svolgere ruoli unici APC in fase effettrice delle risposte immunitarie adattive all&#39;interno dei tessuti periferici, come ad esempio l&#39;influenza locale all&#39;attivazione delle cellule T, la differenziazione, la memoria e la tolleranza 16,17,26. Criticamente, quando coltivate in vitro, le cellule endoteliali formano superfici cellulari praticamente piane e sono immediatamente transfectable (ad esempio, con i reporter proteina fluorescente). Queste caratteristiche sono ideali per imaging ad alta risoluzione spazio-temporale delle dinamiche topologiche durante le interazioni cellula-cellula 19,27. Così le cellule endoteliali potrebbe servire come &#39;cellulare planare APC&#39; modello fisiologico distintamente adatto per lo studio dei meccanismi di rimodellamento sottocellulari / molecolare che guidano riconoscimento dell&#39;antigene e regolano le risposte (Figura 1C) 19,20. Precedentemente stabilito tecniche di imaging complementari (comprese trasfezione di cellule endoteli con accessori proteina fluorescente della membrana plasmatica e citosol) per studiare i dettagli di interazione leucociti-endotelio durante adesione e migrazione transendoteliale 27, hanno mostrato che i leucociti sondare attivamente superficie dell&#39;endotelio dalla dinamica l&#39;inserimento di und retrazione del sub-micron-scala, sporgenze cilindriche ricco di actina (~ 200-1.000 nm di diametro e profondità) definito invadosome-come sporgenze (vale a dire, &#39;ILPS&#39;) 27,28. Questi approcci di imaging sono state ulteriormente espansa insieme alla creazione di protocolli di sfruttare funzione endoteliale APC per sviluppare i primi metodi per un&#39;alta risoluzione spazio-temporale della cellula endoteliale sinapsi immunologica T come riportato 19,20 e ulteriormente descrivere qui. Un risultato centrale derivato da questo romanzo planare cellulare modello APC è che ILPS cellule T funzionano sia nel promuovere il riconoscimento iniziale Ag e nel sostenere la successiva segnalazione. In effetti, gli array di più ILPS (che sono stati stabilizzati e maturati in risposta alla prima flusso di calcio) spettacolo di arricchimento in TCR e molecole suggestivi della segnalazione attiva tale PKC-Q, ZAP-70, phosphotyrosine e HS1. Pertanto, ILPS sembrano rappresentare un equivalente fisiologica tridimensionale al micro TCR segnalazionecluster visto in modelli doppio strato planari. Questo approccio, quindi, sensibilmente rivela / report dinamica molecolare e architettonici (e implicite biomeccanica) non altrimenti rilevabile. Il metodo qui descritto dovrebbe essere utile per colmare il divario tra APC professionali e modelli substrato artificiale APC, al fine di migliorare la nostra capacità di interrogare meccanismi di base della risposta immunitaria adattativa. Mentre qui l&#39;attenzione è sull&#39;attivazione di CD4 + di tipo Th1 effettore / cella di memoria, questo approccio di base può essere facilmente modificato per studiare una vasta gamma di tipi di cellule T e Ags, come discusso di seguito.

<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
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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

Immunità adattativa è regolata da interazioni dinamiche tra cellule T e cellule presentanti l&#39;antigene (&#39;APC&#39;) denominati «sinapsi immunologica. All&#39;interno di queste interfacce cellula-cellula intime discrete sub-cellulari cluster di MHC / Ag-TCR, F-actina, adesione e molecole di segnalazione si formano e rimodellano rapidamente. Queste dinamiche si pensa siano fattori determinanti sia per l&#39;efficienza e la qualità delle risposte immunitarie che sviluppano e quindi di protezione contro l&#39;immunità patologica. Attuale comprensione delle sinapsi immunologiche con fisiologica APC è limitata dalla inadeguatezza della risoluzione delle immagini ottenibili. Anche se i modelli substrato artificiale (ad esempio, doppi strati lipidici planari) offrono un&#39;eccellente risoluzione e sono stati strumenti estremamente utili, essi sono intrinsecamente non-fisiologica e semplicistica. Cellule endoteliali vascolari e linfatiche sono emersi come un importante tessuto periferico (o stromale) Vano di &#39;semi-professionaleAl APC &#39;. Questi APC (che esprimono la maggior parte del macchinario molecolare di professionisti APC) hanno la caratteristica unica di formare superficie cellulare praticamente piana e sono immediatamente transfectable (ad esempio, con i giornalisti proteina fluorescente). Qui un approccio di base per l&#39;attuazione cellule endoteliali come un romanzo e fisiologica &#39;planare modello APC cellulare&#39; per migliorare l&#39;imaging e l&#39;interrogazione dei processi di segnalazione antigeniche fondamentali sarà descritto.

Un nuovo approccio di imaging utilizzando cellule endoteliali e combinando i vantaggi risoluzione della planare bistrati lipidici modello con la complessità fisiologica e deformabilità professionale APC stato sviluppato (Figura 1). La Figura 2 fornisce esempi di migrazione tipica, flusso di calcio e dinamiche topologici osservato con questo approccio. In assenza di SAG in endotelio, SAG-specifici linfociti CD4 + Th1 rapidame...

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 modello cellule endoteliali Planar for Imaging immunologiche 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 Views.  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.

Imaging di HIV-1 Envelope indotta Synapse virologica e segnalazione sui doppi strati lipidici sintetici

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

Ricerca

Immunologia e infezioni

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.

Visualizzazione della immunologica Synapse da Dual Color Time-gated emissione stimolata Depletion (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.

In tempo reale Imaging del endoteliali giunzioni cellula-cellula Durante neutrofili Trasmigrazione Sotto fisiologico flusso

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.

Un<em&gt; Ex vivo</em&gt; Modello di un attacco T-Cell Oligodendrocyte-diretto nel acute cervello fette

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 di cellule Natural Killer immunologica Sinapsi su un vetro supportato Planar doppio strato lipidico

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; Modello della barriera emato-encefalica Utilizzando spettroscopia di impedenza: un focus sui T Cell-endoteliale cellulare Interazione

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.

Studiare le dinamiche di Organelle nelle cellule B durante la formazione immunitaria di sinapsi

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.

Impianto e monitoraggio da PET/CT di un modello ortotopico di mesotelioma pliologico umano in topi atimici

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).

Immaginare la sinapsi immunologica umana

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 modello immunologico per il trapianto di cellule cardiache e cardiache cardiache cardiache cardiache cardiache cardiache

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