Questo lavoro è supportato dal NIH PBS (R21 AI095484). Ringraziamo core facility tetramer NHI per CD1d tetramero.

1. Preparazione degli strumenti, Cultura Media e reagenti <ol><li> Strumenti: Preparare i reagenti in condizioni sterili e utilizzare sotto una cappa a flusso laminare. </li><li> Preparare 1 L di terreno di coltura RPMI, aggiungere 5% di siero fetale bovino, 2,1 g di bicarbonato, 10 ml glutammato 100x, 10 mg di HEPES, 1 mg di piruvato di sodio e 10 ml di 100x penicillina / streptomicina. Nota: il terreno di coltura tissutale raccomandata, RPMI, è intercambiabile con DMEM. </li><li> Preparare soluzione al 5% di collagenasi &quot;D&quot; (5 ml di collagenasi &quot;D&quot; a 9 ml di terreno di coltura tissutale). </li><li> Preparare la soluzione di Percoll a tre diverse concentrazioni: 100%, 80% e 40%. I seguenti concentrazioni sono calcolati per 1 campione. Mantenere le soluzioni a temperatura ambiente. <ol><li> Soluzione Percoll: aggiungere 9 ml di diluito Percoll (da soluzione stock); aggiungere 1 ml di PBS 10x. </li><li> Soluzione Percoll: aggiungere 8 ml di Percoll 100%; aggiungere 2 ml di PBS 1x. </li><li> Soluzione Percoll:aggiungere 4 ml di Percoll 80%; aggiungere 4 ml di PBS 1x. </li></ol></li><li> Fare 1 L di cella fluorescenza-attivato (FACS) tampone; aggiungere 5 ml di 10% di BSA (0,1% BSA), sodio azide 1 ml 10% (0,1%), 2 agente ml EDTA, portare a 1000 ml con PBS 1x. </li></ol> 2. Preparazione del rene Digestione <ol><li> Avendo acquisito alcuna approvazione istituzionale necessario, sacrificare e exsanguinate i topi con metodi standard e seguendo le normative vigenti e le linee guida per la cura degli animali. <ol><li> Anestetizzare il mouse con pentobarbital (0,07 mg / kg). </li><li> Procedere al dissanguamento. </li><li> Posizionare il mouse in posizione supina su un tavolo operatorio. </li><li> Spruzzare la zona addominale con etanolo al 70%. </li></ol></li><li> Aggiungere 10 ml di soluzione D collagenasi al piatto di Petri. </li><li> Rimuovere e decapsulate entrambi i reni da ogni mouse. <ol><li> Aprire la cavità abdomimal facendo un&#39;incisione mediana attraverso la pelle addominale e del peritoneo dallo sternoil pube. </li><li> Esporre il rene sinistro spostando l&#39;intestino lateralmente al lato. </li><li> Separare con cautela la capsula sinistra con le pinze. Nota: La capsula appare come uno strato trasparente che circonda il rene e parzialmente coperta da tessuto adiposo perirenale. </li><li> Rimuovere il rene sinistro tagliando le navi peduncolo con una forbice chirurgica. </li><li> Ripetere la stessa procedura per il rene destro. Nota: Il rene destro è più craniale e più vicino alla linea mediana (pedicelli sono più corti). </li></ol></li><li> Posizionare il rene nel piatto di Petri. </li><li> Tagliare il tessuto renale in piccoli pezzi (1-2 mm di dimensione) utilizzando una normale lama formatura dei metalli e disporre i pezzi nella soluzione di collagenasi per 30-45 min in incubatore a 37 ° C (Figura 1). </li></ol> 3. Preparazione di cellule mononucleate del rene (KMNC) dalla digestione rene <ol><li> Ottenere una sospensione di cellule di renedigestione interrompendo meccanicamente il tessuto utilizzando un filtro (70 micron) e risospendere in 25 ml di terreno di coltura tissutale e mescolare. </li><li> Centrifugare a 400 xg per 10 min a 4 ° C per sedimentare la sospensione cellulare. </li><li> Risospendere il pellet in 4 ml di 40% soluzione di Percoll e delicatamente sovrapporre su 4 ml di 80% soluzione di Percoll con una pipetta di plastica trasferimento, molto lentamente e con attenzione. Questo comporta due fasi nettamente separate da uno strato traslucido. Sulla parte superiore sarà uno strato giallo corrispondente ai lipidi. </li><li> Centrifugare a 1500 xg per 30 min a temperatura ambiente con la centrifuga in modalità off freno. </li><li> Rimuovere con aspirazione lo strato superiore di colore giallo e denso (circa 1-2 ml). </li><li> Raccogliere solo il leggero strato biancastro traslucido dall&#39;interfaccia delle due fasi con una pipetta di trasferimento. Trasferire molta attenzione questa sospensione in una provetta conica da 15 ml contenente 2 ml di terreno di coltura tissutale e quindi aggiungere 13 ml di terreno per fare un volume totale di15 ml. </li><li> Centrifugare a 400 xg per 10 min a 4 ° C. </li><li> Risospendere i campioni in 1 ml del mezzo. </li><li> Contare il numero di KMNC utilizzando trypan blu su un emocitometro ed esprimere i risultati come numero di KMNC per ml al rene. </li><li> Risciacquare una volta come sopra. </li></ol> 4. Isolamento del emopoietiche (CD45 +) Celle dal KMNC (Fase I) <ol><li> Risospendere le cellule fino a 10 7 in 90 ml di tampone di corsa. </li><li> Aggiungere 10 ml di microsfere CD45 e incubare per 15 min a 4 ° C. </li><li> Aggiungere 1 ml di tampone di corsa per arrestare la reazione. Centrifugare a 400 xg per 10 min a 4 ° C e risospendere in 500 ml di tampone di corsa. </li><li> Preparare la colonna magnetico mettendo a magnete e risciacquare con 3 ml di tampone di corsa. </li><li> Passare la sospensione cellulare attraverso la colonna magnetica e raccogliere le cellule non marcati che passano attraverso la colonna. </li><li> Lavare le cellule tre volte con 3 ml di Running buffer. Poi raccogliere l&#39;effluente. </li><li> Rimuovere la colonna dal separatore e pipetta 5 ml di tampone di corsa sulle colonne e scovare la frazione etichettato immediatamente. Questa frazione contiene le cellule CD45 +. Contare le cellule. </li><li> Per calcolare il numero assoluto di linfociti T diversi sottoinsiemi in reni moltiplicare il numero totale di KMNC per la percentuale di cellule positive determinata mediante citometria di flusso (Figura 2). </li></ol> 5. Isolamento di DN cellule T CD45 + da Preparazione per selezione negativa (Fase II) <ol><li> Aggiungere 2,5 ml (0,5 mg / ml, 1,25 microgrammi) di ciascuno dei seguenti anticorpi biotinilati: anti-CD4 e anti-CD8, anti-recettore Fc (CD16/32), anti-MHC di classe II (IA b), anti- CD1d PBS-57 tetramer ogni 10 cellule ematopoietiche 7. </li><li> Incubare le cellule per 30 minuti nella camera fredda. </li><li> Aggiungere 1 ml microsfere anti-biotina. </li><li> Incubare per 30 min in una camera fredda. </li><li> Porre il tubo in tegli magnete e procedere a esaurimento. </li><li> Contare le cellule vitali per determinare il numero totale di cellule T DN nella frazione negativa utilizzando un emocitometro. </li><li> Controllare la purezza delle cellule DN T con citometria a flusso seguendo questa strategia di gating: primo cancello CD45 +, secondo cancello TCRαβ +. Escludi + cellule CD1d. Plot CD4 + CD8 + vs identificare cellule DN T (Figura 3). </li><li> Moltiplicare il numero totale per la percentuale determinata in purezza. Nota: utilizzando questo protocollo i risultati in una popolazione di cellule pure oltre il 90% DN T. </li></ol>

<ol>
	<li>Zhang, Z. X., Yang, L., Young, K. J., DuTemple, B., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+previously+unknown+antigen-specific+regulatory+T+cell+and+its+mechanism+of+suppression.">Identification of a previously unknown antigen-specific regulatory T cell and its mechanism of suppression.</a> Nat Med. 6, 782-789 (2000).</li><li>Ford, M. S., Young, K. J., Zhang, Z., Ohashi, P. S., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+regulatory+function+of+lymphoproliferative+double+negative+T+cells+in+vitro+and+in+vivo.">The immune regulatory function of lymphoproliferative double negative T cells in vitro and in vivo.</a> J Exp Med. 196, 261-267 (2002).</li><li>Ford McIntyre, M. S., Young, K. J., Gao, J., Joe, B., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+in+vivo+trogocytosis+as+a+mechanism+of+double+negative+regulatory+T+cell-mediated+antigen-specific+suppression.">Cutting edge: in vivo trogocytosis as a mechanism of double negative regulatory T cell-mediated antigen-specific suppression.</a> J Immunol. 181, 2271-2275 (2008).</li><li>Young, K. J., Yang, L., Phillips, M. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-lymphocyte+infusion+induces+transplantation+tolerance+by+activating+systemic+and+graft-infiltrating+double-negative+regulatory+T+cells.">Donor-lymphocyte infusion induces transplantation tolerance by activating systemic and graft-infiltrating double-negative regulatory T cells.</a> Blood. 100, 3408-3414 (2002).</li><li>Chen, W., Ford, M. S., Young, K. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+in+vitro-generated+DN+T+regulatory+cells+induces+permanent+cardiac+allograft+survival+in+mice.">Infusion of in vitro-generated DN T regulatory cells induces permanent cardiac allograft survival in mice.</a> Transplant Proc. 35, 2479-2480 (2003).</li><li>Young, K. J., DuTemple, B., Phillips, M. J., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+graft-versus-host+disease+by+double-negative+regulatory+T+cells.">Inhibition of graft-versus-host disease by double-negative regulatory T cells.</a> J Immunol. 171, 134-141 (2003).</li><li>Mohamood, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gld+mutation+of+Fas+ligand+increases+the+frequency+and+up-regulates+cell+survival+genes+in+CD25+CD4++TR+cells.">Gld mutation of Fas ligand increases the frequency and up-regulates cell survival genes in CD25+CD4+ TR cells.</a> Int Immunol. 18, 1265-1277 (2006).</li><li>Ascon, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+mouse+kidneys+contain+activated+and+CD3+CD4-+CD8-+double-negative+T+lymphocytes+with+a+distinct+TCR+repertoire.">Normal mouse kidneys contain activated and CD3+CD4- CD8- double-negative T lymphocytes with a distinct TCR repertoire.</a> J Leukoc Biol. 84, 1400-1409 (2008).</li><li>Hamad, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B220++double-negative+T+cells+suppress+polyclonal+T+cell+activation+by+a+Fas-independent+mechanism+that+involves+inhibition+of+IL-2+production.">B220+ double-negative T cells suppress polyclonal T cell activation by a Fas-independent mechanism that involves inhibition of IL-2 production.</a> J Immunol. 171, 2421-2426 (2003).</li><li>Rabb, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiological+role+of+T+lymphocytes+in+renal+ischemia-reperfusion+injury+in+mice.">Pathophysiological role of T lymphocytes in renal ischemia-reperfusion injury in mice.</a> Am J Physiol-Renal. 279, 525-531 (2000).</li><li>Burne, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+CD4(+)+T+cell+as+a+major+pathogenic+factor+in+ischemic+acute+renal+failure.">Identification of the CD4(+) T cell as a major pathogenic factor in ischemic acute renal failure.</a> J Clin Invest. 108, 1283-1290 (2001).</li><li>Yokota, N., Daniels, F., Crosson, J., Rabb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+T+cell+depletion+in+murine+renal+ischemia-reperfusion+injury.">Protective effect of T cell depletion in murine renal ischemia-reperfusion injury.</a> Transplantation. 74, 759-763 (2002).</li><li>Crispin, J. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+FAS+mutations+are+common+in+patients+with+genetically+undefined+autoimmune+lymphoproliferative+syndrome.">Somatic FAS mutations are common in patients with genetically undefined autoimmune lymphoproliferative syndrome.</a> Blood. 115, 5164-5169 (2010).</li><li>Juvet, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FcRgamma+promotes+T+cell+apoptosis+in+Fas-deficient+mice.">FcRgamma promotes T cell apoptosis in Fas-deficient mice.</a> J Autoimmun. 42, 80-93 (2013).</li><li>Thomson, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FcR+gamma+presence+in+TCR+complex+of+double-negative+T+cells+is+critical+for+their+regulatory+function.">FcR gamma presence in TCR complex of double-negative T cells is critical for their regulatory function.</a> J Immunol. 177, 2250-2257 (2006).</li></ol>

Gli autori non hanno nulla da rivelare.

Vi è un crescente interesse in cellule T DN poiché sono stati implicati in diverse condizioni patologiche, come disturbi autoimmuni, il cancro, la tolleranza del trapianto e malattie primarie di rene compresa insufficienza renale acuta (AKI), glomerulonefrite 8, 13. Pertanto, non vi è necessità di comprendere meglio e caratterizzare le funzioni fisiopatologiche delle cellule T DN. Tuttavia, attualmente non vi è una mancanza di comprensione della funzione di queste cellule rispetto ai CD4 e le cellule T C...

A correction was made to: <a href="http://www.jove.com/video/51192/isolation-of-double-negative-t-cells-from-the-kidney">Isolation of Double Negative &#945;&#946; T Cells from the Kidney</a>.
There was an error with an author's name. The author's given name had a typo, this was corrected to:
Samatha Bandapelle
instead of:
Samantha Bandapelle

A correction was made to: Isolation of Double Negative &#945;&#946; T Cells from the Kidney.

Erratum: Isolation of Double Negative &#945;&#946; T Cells from the Kidney

Periferico αβTCR + CD3 + CD4 - CD8 - doppio negativo cellule (DN) T sono suddivisi in vari sottogruppi che possiedono fenotipi e funzioni 1-4 distinte. Cellule DN T sono poco conosciuti, ma sempre più spesso coinvolti nelle risposte immunitarie fisiopatologiche in differenti modelli di malattia 4-6. Cellule DN T sono un tipo di cellula immunitaria unico che è sempre più coinvolto nella regolazione delle varie risposte immunitarie e autoimmuni e la modulazione della tolleranza allotrapianto. 4-6, 9 sono rari nel sangue periferico e negli organi linfoidi secondari (milza e dei linfonodi ). Tuttavia, essi sono i principali abitanti della normale rene e intestino epitelio 10-12. Molto poco si sa circa la loro funzione 7 del rene in stato stazionario e in condizioni patologiche come danno renale acuto (AKI) associata a Transpl reneant. A causa dei ruoli intricati di diverse cellule immunitarie nel regolare le risposte immunitarie, tra cui alloresponses, la definizione del ruolo di ogni giocatore è fondamentale per comprendere alloresponses e la progettazione di nuove terapie. Dato numero significativo di cellule DN T presenti nel rene in condizioni fisiologiche e diverse malattie, le cellule T DN sono suscettibili di svolgere un ruolo critico nel regolare le risposte immunitarie e autoimmuni nei topi e nell&#39;uomo, e alloresponses nei pazienti trapiantati. Accumulare se prova ancora sparse implica cellule DN T in entrambe le funzioni patogeni e immunosoppressori, ma è poco conosciuta perché e come mostrano una funzione dannoso o soppressiva specifico e come l&#39;ambiente li influenza. A causa della loro bassa abbondanza nel rene, sono necessari metodi migliorati di isolamento. Attualmente non esiste un protocollo standard per l&#39;isolamento di cellule T DN da tegli tessuti non linfoidi. Il nostro protocollo descrive un nuovo metodo per l&#39;isolamento di cellule T DN dal rene; Tuttavia, il metodo può essere utilizzata anche per vari tessuti non linfoidi.

<table border="0" cellpadding="0" cellspacing="0" width="1172">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Laminar flow hood&#160;</td>
			<td style="width:257px;">Baker Company, Inc</td>
			<td style="width:276px;"></td>
			<td style="width:200px;">Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Hemocytometer</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:276px;">63514-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Atmosphere-controlled incubator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td style="width:200px;">&#160;(37&#176;C with 5% CO2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Microscope&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Analytical flow cytometer</td>
			<td style="width:257px;">LSR II&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:439px;">Name of the reagent</td>
			<td style="width:257px;">Company&#160;</td>
			<td style="width:276px;">Catalog&#160; Number</td>
			<td style="width:200px;">Comments&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">RPMI 1640</td>
			<td style="width:257px;">Media Tech</td>
			<td style="width:276px;">10-040-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Collagenase D</td>
			<td style="width:257px;">Roche</td>
			<td style="width:276px;">11088858001</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Percoll&#160;</td>
			<td style="width:257px;">GE Healthcare</td>
			<td style="width:276px;">&#160;17-0891-01</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Fetal bovine serum&#160;</td>
			<td>Corning Cellgro</td>
			<td>35-011-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">0.1% sodium azide (NaN3)</td>
			<td style="width:257px;">Sigma-Aldrich&#160;</td>
			<td style="width:276px;">S2002</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Buffer phosphate buffered saline (PBS)</td>
			<td style="width:257px;">Corning Cellgro</td>
			<td>21-040-CV</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">PBS 10X</td>
			<td>Mediatech</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">EDTA buffer&#160;</td>
			<td style="width:257px;">Sigma-Aldrich&#160;</td>
			<td style="width:276px;">E1161</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">LS columns&#160;</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">CD45+ microbeads</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">6.78E-51</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">Biotin microbeads&#160;</td>
			<td style="width:257px;">MiltenyiBiotec</td>
			<td style="width:276px;">130-090-485</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; PerCP&#160;</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td style="width:276px;">G1397</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD45 (clone: 30-F11)&#160; APC-Cy7</td>
			<td style="width:257px;">Biolegend&#160;&#160;</td>
			<td style="width:276px;">103116</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-TCR Pacific Blue&#160; (ab-chain, clone: H57-597)</td>
			<td style="width:257px;">Invitrogen</td>
			<td style="width:276px;">HM3628</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 PE</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>12-0043</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 FITC</td>
			<td style="width:257px;">eBioscience&#160;</td>
			<td>8011-0087</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD4 biotinylated (GK1.5)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553728</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD8 biotinylated&#160; (clone 53-6.7)</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553029</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Fc receptor (CD16/32) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553143</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-MHC class II (I-A d) biotinylated</td>
			<td style="width:257px;">BD</td>
			<td style="width:276px;">553609</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:439px;">anti-CD1d PBS-57 tetramer</td>
			<td style="width:257px;">NHI tetramer&#160;</td>
			<td style="width:276px;">lote # 15621-PBS57</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:439px;">AutoMACS Running Buffer-&#160; MACS Separation Buffer&#160;</td>
			<td style="width:257px;">MIlteny I Biotec</td>
			<td style="width:276px;">130-091-221</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">C57BL/6J mice</td>
			<td>Jackson Labs</td>
			<td style="width:276px;">000664</td>
			<td>Kidneys - Lymph Nodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri dish&#160;</td>
			<td>BD Biosciences</td>
			<td style="width:276px;">356517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">70mc filter&#160;</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plunger&#160;</td>
			<td style="width:257px;"></td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GeneMate Tubes 50 ml</td>
			<td>Bioexpress</td>
			<td>C-3394-3</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">GeneMate Tubes 15 ml</td>
			<td>Bioexpress</td>
			<td style="width:276px;">C-3394-1</td>
			<td>Or equivalent equipment&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Petri Dish&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:276px;">S33580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:439px;">Plate 24 wells&#160;</td>
			<td style="width:257px;">BD Biosciences</td>
			<td style="width:276px;">354723</td>
			<td style="width:200px;"></td>
		</tr>
	</tbody>
</table>

isolation of double negative &#945;&#946; t cells from the kidney

Attualmente non esiste un protocollo standard per l&#39;isolamento delle cellule T DN dai tessuti non linfoidi nonostante il loro coinvolgimento sempre segnalato in varie risposte immunitarie. Cellule DN T sono un tipo di cellula immunitaria unico che è stato implicato nella regolazione delle risposte immunitarie e la tolleranza e autoimmuni di allotrapianto 1-6. Cellule DN T sono, tuttavia, rare nel sangue periferico e negli organi linfoidi secondari (milza e linfonodi), ma sono i principali abitanti del rene normale. Molto poco si sa circa la loro funzione fisiopatologico 7 a causa della loro scarsità in periferia. Abbiamo recentemente descritto una vasta analisi fenotipica e funzionale di questa popolazione nel rene 8 in stato stazionario e durante ischemia riperfusione. Analisi della funzione delle cellule T DN sarà notevolmente migliorata con lo sviluppo di un protocollo per il loro isolamento dal rene. Qui, descriviamo un nuovo protocollo che consente isolation di CD8 + cellule altamente puri ab CD4 + T e le cellule T DN dal rene murino. In breve, abbiamo digerire tessuto renale con collagenasi e isolare le cellule mononucleate del rene (KMNC) dal gradiente di densità. Questo è seguito da due passaggi per arricchire cellule T ematopoietiche dal 3% al 70% dal KMNC. La prima fase consiste in una selezione positiva di cellule ematopoietiche CD45 + utilizzando un kit di isolamento. Nella seconda fase, le cellule T sono DN negativamente isolati mediante rimozione delle cellule non desiderati utilizzando CD4, CD8, e MHC di classe II anticorpi monoclonali e CD1d α-GalCer tetramero. Questa strategia porta ad una popolazione di cellule pure oltre il 90% DN T. Colorazione della superficie con gli anticorpi suddetti seguita mediante analisi FACS è utilizzato per confermare la purezza.

Wild type C57BL / 6 (B6) rene contiene circa 1,5-2,1 x 10 6 cellule mononucleate al rene. Circa meno del 10% sono CD45 + cellule ematopoietiche. Per la preparazione di cellule mononucleate rene (KMNC), i reni sono tagliati in piccoli pezzi, come mostrato in Figura 1 seguito da digestione con collagenasi 5%. Questo è seguito eseguendo una centrifugazione in gradiente di densità per raccogliere KMNC strato (figura 2, pannello di sinistra).</stro...

Watch this Scientific Journal Video about Isolation of Double Negative Î±Î² T Cells from the Kidney at JoVE.com

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

Cellule DNabT sono rari tra cellule T periferiche; tuttavia, sono abbondanti in alcuni tessuti non linfoidi. Difficoltà di isolare le cellule T DN dal tessuto linfoide non ostacola la loro analisi funzionale, nonostante l&#39;aumento riconosciuto significato fisiopatologico. Descriviamo un nuovo protocollo per l&#39;isolamento di cellule altamente purificate DN T di rene murino.

Isolamento di Double Negative Cellule T αβ dal rene

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

isolation-of-double-negative-t-cells-from-the-kidney

Research

JoVE Journal

Immunology and Infection

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

12.1K Views.  Johns Hopkins University School of Medicine.  Cellule DNabT sono rari tra cellule T periferiche; tuttavia, sono abbondanti in alcuni tessuti non linfoidi. Difficoltà di isolare le cellule T DN dal tessuto linfoide non ostacola la loro analisi funzionale, nonostante l'aumento riconosciuto significato fisiopatologico. Descriviamo un nuovo protocollo per l'isolamento di cellule altamente purificate DN T di rene murino. 

Video: Isolation of Double Negative &#945;&#946; T Cells from the Kidney

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generazione di cellule T regolatrici indotte da cellule primarie umane T naive e memoria

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Ricerca

Immunologia e infezioni

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Isolamento e differenziazione di Th17 Naïve linfociti T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Derivazione di cellule T<em&gt; In Vitro</em&gt; Da cellule staminali embrionali di topo

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generazione di Human alloantigene-specifiche cellule T da sangue periferico

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Mouse Naïve CD4<sup&gt; +</sup&gt; Isolamento T cellulare e<em&gt; In vitro</em&gt; Differenziazione in T sottopopolazioni cellulari

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Cellule T Capture batteri da Transinfection da cellule dendritiche

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Efficiente protocollo Isolamento da B e T linfociti umani da Palatine Tonsille

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

L&#39;isolamento e la citometria a flusso Analisi di Human endocervicale Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Valutazione dell'interfaccia sinaptica primaria umana delle cellule di T dal sangue periferico e tessuto linfoide

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Formazione immagine confocal di RNA Double-Stranded e Pattern Recognition Receptors in infezione Virus RNA negativo

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...