Questo lavoro è stato sostenuto da The Will e Jeanne Caldwell Endowed Research Fund dell'University of Michigan Comprehensive Cancer Center e in parte dalla sovvenzione NIH CA82529 e dalla Gillson Longenbaugh Foundation.

1. Colorazione ALDEFLUOR
<ol><li>Preparazione del campione cellulare: Preparare la sospensione a singola cellula delle cellule tumorali, sia da cellule tumorali coltivate o da campioni di tumore appena raccolti. Contare le cellule e regolare la sospensione cellulare a una concentrazione di 1 x 106 celle/ml nel tampone di dosaggio ALDEFLUOR.</li>
	<li>Etichettare quattro provette in polistirolo da 12 mm x 75 mm come provette di controllo come segue: #1: Non macchiato, #2: ALDEFLUOR, #3: ALDEFLUOR più DEAB e #4: 7AAD.</li>
	<li>Per questi tubi di controllo, posizionare 1 ml di sospensione cellulare #1 e #4, ma 2 ml nel tubo #2. Aggiungere 5 μl DEAB (inibitore ALDH) nel tubo #3 e tenere sul ghiaccio. Quindi aggiungere 10 μl di substrato ALDEFLUOR attivato al tubo #2, mescolare e trasferire immediatamente 1 ml della miscela al tubo #3.</li>
	<li>Allo stesso tempo, aggiungere 2 μl di substrato ALDEFLUOR attivato per milione di cellule al resto delle cellule (tubi campione) anche a 1 x 106 celle/ml nel tampone di dosaggio ALDEFLUOR.</li>
	<li>Incubare sia i 4 tubi di controllo che i tubi campione per 30 minuti in bagno d'acqua a 37 °C.</li>
	<li>Dopo l'incubazione, aggiungere 5 μl 7AAD nel tubo di controllo #4 e 1 μl 7AAD 1 x 106 celle al tubo campione. Incubare 10 min a 4 °C.</li>
	<li>Centrifugare tutti i tubi per 5 minuti a 250 x g e rimuovere il supernatante.</li>
	<li>Cellule resuspend nel tampone di dosaggio ALDEFLUOR.</li>
	<li>Eseguire l'ordinamento basato sulla citometria a flusso.</li>
	<li>Impostare i cancelli di smistamento utilizzando celle macchiate di ALDEFLUOR trattate con DEAB come controllo negativo e le celle macchiate di propididio (PI) 7AAD per il controllo della vitalità. Sulla base di questi controlli, è stato istituito un cancello per distinguere le cellule ALDEFLUOR+/ALDHhigh,D5 e SCC7. Questi cancelli verranno quindi utilizzati per ordinare tutte le celle campione preparate sopra per le celle ALDEFLUOR+/ALDHhigh D5 e SCC7.</li>
</ol>2. Preparazione del vaccino contro le cellule staminali tumorali a impulsi dendritici a impulsi (CSC-TPDC)
<ol><li>Eutanasia dei topi (sigeneico B6 e C3H, rispettivamente) usando CO2 e isolare femore e ossa di tibia.</li>
	<li>Mettere le ossa in etanolo al 75% per 1 minuto a temperatura ambiente. Quindi lavare le ossa usando HBSS.</li>
	<li>Tagliare la testa dalle ossa e utilizzare un ago da 21 G e una siringa da 10 ml per spingere le cellule in un piatto.</li>
	<li>Aspirare e soffiare le cellule usando la siringa per effettuare la sospensione a singola cella.</li>
	<li>Dopo la centrifugazione in 1.500 giri/min per 5 minuti, scartare il supernatante. Quindi aggiungere 5 ml di tampone di lisi RBC per lisiizzare l'RBC per 1 min in bagno d'acqua a 37 °C.</li>
	<li>Contare il numero di cellule e regolare la sospensione cellulare ad una concentrazione di 1 milione di cellule/ml in mezzo completo (CM) contenente 10 ng/ml DI GM-CSF e 10 ng/ml IL-4.</li>
	<li>Coltura delle cellule e compensazione CM più IL-4 e GM-CSF 3 giorni dopo.</li>
	<li>Al5 ° giorno, raccogliere le cellule dendritiche immature (DC) e preparare il mezzo di isolamento CC contenente 4,2 ml di soluzione C più 1 ml di mezzo gradiente di densità OptiPrep.</li>
	<li>Sospendere lentamente il pellet di cella in CM da 5 ml. Centrifuga a 2.000 giri/min a temperatura ambiente.</li>
	<li>Raccogliere i TC tra il CM e il supporto di isolamento. Contare il numero di cellule e regolare la sospensione cellulare ad una concentrazione di 1 milione di cellule/ml in un mezzo di coltura contenente 10 ng/ml di GM-CSF e 10 ng/ml IL-4.</li>
	<li>Preparare i lisati tumorali sospendendo le celluleALDH D5 o SCC7 alte o nonsorte nel terreno di coltura e soggette a esposizioni rapide di congelamento-disgelo quattro volte seguite da spin a ∼100 x g per 5 minuti per raccogliere la porzione di membrana dei lisati.</li>
	<li>Per preparare CSC-TPDC, pulsare i CD con il lysate delle cellule alteALDH autologhe. Per preparare h-TPDC, i PC a impulsi con lisato di cellule tumorali eterogenee nonsortite. Il rapporto tra DC e il llysato delle cellule tumorali è lo stesso.</li>
</ol>3. Vaccinazione e valutazione dell'efficacia
<ol><li>Vaccinare i normali topi B6 rispettivamente con 2.500 cellule tumorali D5 CSC-TPDC o D5 H-TPDC per via sottocutanea (s.c.). Vaccinare i normali animali C3H s.c. con 5.000 cellule tumorali SCC7 CSC-TPDC o SCC7 H-TPDC, rispettivamente.</li>
	<li>Dopo la vaccinazione, sfida i topi B6 con le cellule tumorali D5 eterogenee, cioè eutanasia i topi usando CO2 20 giorni dopo. Raccogliere i polmoni ed enumerare le metastasi polmonari.</li>
	<li>Nel modello SCC7, sfida i topi C3H con cellule tumorali SCC7 non .c sul lato opposto del vaccino DC. Monitorare le dimensioni del tumore.</li>
	<li>Al termine degli esperimenti eutanasiare i topi B6 e C3H utilizzando CO2. Al giorno 34 dopo il primo vaccino, eutanasiare i topi con CO2e allo stesso tempo raccogliere le milza di ogni gruppo con una procedura aseptica.</li>
	<li>Attivare celle T e/o B milza con ascite anti-CD3 immobilizzate più anti-CD28 mAb in CM contenenti hrIL-2 o LPS più ascite anti-CD40 (FGK45) mAb.</li>
	<li>Dopo l'attivazione, raccogliere i supernatanti e utilizzarli per il test ELISA.</li>
	<li>Per il test ELISA, rivestire piastre con anticorpi per IFNγ, GM-CSF e IgG a 4 °C O/N.</li>
	<li>Dopo l'applicazione del tampone di blocco, aggiungere campioni e standard e incubare a temperatura ambiente.</li>
	<li>Lavare la piastra e aggiungere anticorpi di rilevamento HRP e substrato TMB per l'incubazione.</li>
	<li>Misurare l'assorbanza su un lettore di piastre ELISA a 450 nm entro 30 minuti dopo aver interrotto la reazione.</li>
</ol>

<ol>
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STEMCELL Technologies ha sostenuto le tariffe di pubblicazione open access.

Gli ospiti immunocomprosibili, come i topi SCID, impediscono valutazioni immunologiche dei CSC a causa della mancanza di immunità adattiva all'interno degli ospiti. In questo studio, abbiamo valutato l'immunogenicità dei CSC negli ospiti immunocompetenti, che potrebbe imitare più da vicino le impostazioni del paziente. I CSC arricchiti sono immunogenici e potrebbero indurre un'immunità protettiva tumorale più efficace quando i loro lisati vengono caricati nei DC come vaccino rispetto ai DC pulsati a cellule tumorali non selezionati.  Meccanicamente, la protezione è stata conferita dall'induzione selettiva di anticorpi reattivi CSC e cellule T8, nonché dalla produzione di citochine di tipo 1, ad esempio IFNγ e GM-CSF.
La maggior parte delle attuali immunoterapie, compresi i vaccini a base di cellule dendritiche e il trasferimento adottivo delle cellule T, sono progettate per colpire gli antigeni differenziati per il tumore. I CSC, che non possono esprimere questi antigeni differenziati, possono quindi sfuggire a questi obiettivi immunologici. Al contrario, il vaccino CSC progettato per colpire specificamente le cellule staminali tumorali può distruggere questa popolazione speciale delle cellule tumorali, e quindi migliorare l'efficacia terapeutica del vaccino prevenendo la ricaduta e la metastasi tumorale.
Sia nelle cellule tumorali coltivate che nei tumori appena raccolti, abbiamo identificato la popolazione arricchita di CSC dalla citometria a flusso basata sull'alta attività dell'aldeide deidrogenasi. Tali cellule alteALDH potrebbero essere isolate dallo smistamento del flusso da utilizzare come fonte di antigene per pulsare DC per generare CSC-TDC. Il confronto con cellule alteALDH isolate da cellule tumorali coltivate rispetto ai tumori appena raccolti non ha dimostrato alcuna differenza significativa in termini di induzione dell'immunità anti-CSC8. Questi risultati hanno rivelato il potenziale di utilizzare CSC isolati da cellule tumorali coltivate o da tumori appena raccolti per l'applicazione clinica.
Per essere clinicamente rilevante, un vaccino deve essere esaminato in un contesto terapeutico. Questi esperimenti vengono ora eseguiti nel nostro laboratorio.

Le cellule staminali tumorali sono relativamente resistenti alla chemioterapia convenzionale e alla radioterapia1,2. D'altra parte, questa popolazione di cellule potrebbe essere le cellule responsabili della ricaduta e della progressione dei tumori dopo le terapie oncologichetradizionali 1-4. A causa della mancanza di espressione di antigeni tumorali differenziati sulle cellule staminali tumorali, le cellule staminali tumorali possono sfuggire agli attuali interventi immunologici della terapia per il cancro, che sono per lo più progettati per colpire gli antigeni sulle cellule tumorali differenziate. Pertanto, lo sviluppo di nuove strategie specificamente mirate e distruggono le cellule staminali tumorali può mantenere promesse di aumentare l'efficacia terapeutica dell'attuale trattamento del cancro. A tal fine, abbiamo isolato le popolazioni arricchite di cellule staminali tumorali (CSC) da due tumori animali (melanoma D5 e cancro delle cellule squamose SCC7), e le abbiamo utilizzate come fonte di antigene per pulsare le cellule che presentano l'antigene (cellule dendritiche, DC) per preparare il vaccino CSC-TPDC. Abbiamo quindi valutato l'immunità antitumorale indotta dal vaccino CSC-TPDC negli ospiti immunocompetenti sigeneici, nei topi B6 e nei topi C3H rispettivamente. L'efficacia antitumore indotta dal CSC-TPDC è stata confrontata con il tradizionale vaccino CC pulsato con lisato da cellule tumorali eterogenee nonsortite (H-TPDC), che è stato precedentemente utilizzato dalnostro gruppo 5,6, nonché da altriricercatori 7 sia in studi preclinici che in studi clinici.

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		<col />
		<col />
		<col />
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	<tbody>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">ALDHEFLOUR KIT</td>
			<td style="width:399px;">Stemcell Technologies</td>
			<td style="width:219px;">1700</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine IL-4</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">214-14</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Murine GM-CSF</td>
			<td style="width:399px;">Pepro Tech</td>
			<td style="width:219px;">315-03</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse IFN-&#611; ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555138</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Mouse GM-CSF ELISA KIT</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555167</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">OptiPrep Density Gradient Medium</td>
			<td style="width:399px;">Sigma Aldrich</td>
			<td style="width:219px;">D1556</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">BD OptEIA TMB Substrated Reagent Set</td>
			<td style="width:399px;">BD Biosciences</td>
			<td style="width:219px;">555214</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;"></td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Equipment</td>
			<td style="width:399px;"></td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">BD FACS Aria Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>336834</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:405px;">Kcjunior</td>
			<td style="width:399px;">Bio-Tek Instruments</td>
			<td style="width:219px;">176058</td>
		</tr>
	</tbody>
</table>

generation of a novel dendritic-cell vaccine using melanoma and squamous cancer stem cells

Abbiamo identificato le popolazioni arricchite di cellule staminali tumorali (CSC) dai topi murini melanoma D5 sigeneici a C57BL/6 e il cancro squamoso da SCC7 sigeneico a topi C3H usando ALDEFLUOR/ALDH come marcatore e ne abbiamo testato l'immunogenicità usando il lisato cellulare come fonte di antigeni per pulsare le cellule dendritiche (DC). I DC pulsati con glisatiALDH ad alto CSC hanno indotto un'immunità antitumorale protettiva significativamente più elevata rispetto ai DCs pulsati con i liti di liti di cellule tumorali intere nonsortite in entrambi i modelli e in un ambiente di metastasi polmonare e un s.c. crescita tumorale, rispettivamente. Questo fenomeno era dovuto alle risposte umoriali indotte dal vaccino CSC e alle risposte anti-CSC cellulari. In particolare, gli splenociti isolati dall'ospite sottoposti a vaccino CSC-DC hanno prodotto una quantità significativamente maggiore di IFNγ e GM-CSF rispetto agli splenociti isolati dall'ospite sottoposti a vaccino a impulsi-DC a cellule tumorali nonsorte. Questi risultati supportano gli sforzi per sviluppare un vaccino terapeutico autologo a base di CSC per uso clinico in un ambiente adiuvante.

ALDEFLUOR/ALDH è stato utilizzato come singolo marcatore per isolare le cellule staminali in molteplici neoplasiemaligne 8-11. Abbiamo identificato popolazioni arricchite di cellule staminali tumorali in due modelli tumorali D5 e SCC7 usando ALDEFLUOR come marcatore. Abbiamo rilevato cellule ALDEFLUOR+ nel melanoma murino B16-D5 e nel cancro delle cellule squamose SCC7. La Corte ha riscontrato che le cellule ALDEFLUOR+ contribuiscono rispettivamente a circa lo 0,5% e il 5,2% nelle linee cellulari tumorali D5 e SCC7 coltivate (Figura 1). Le cellule tumorali appena raccolte da tumori affermati sono state analizzate per confermare l'esistenza delle cellule ALDEFLUOR+. Come mostrato anche nella figura 1, c'erano rispettivamente il 2,5% e il 4,2% delle cellule ALDEFLUOR+ provenienti da tumori D5 e SCC7 stabiliti in vivo. L'genicità dei tumori e la capacità di autorilu rinnovamento di queste popolazioni elevate ordinate D5 e SCC7 ALDEFLUOR+/ALDH sono state valutate nell'ospite immunocompetente sigeneico, i topi C57BL/6 e C3H,rispettivamente 8.
Abbiamo usato lisato di cellule tumorali eterogenee non disomogenee per pulsare i DC (H-TPDC) sia negli studi sugli animali che negli studi clinici5,6. Per esaminare l'immunogenicità dei CSC, abbiamo isolato l'ALTOCSC di ALDH e il CC pulsato con il lisato dei CSC al CSC-TPDC generato (Figura 2) e abbiamo utilizzato H-TPDC come controllo convenzionale del vaccino contro il cancro per verificare se CSC-TPDC ha qualche effetto benefico nel prevenire la crescita del tumore.
Abbiamo valutato l'immunogenicità dei CSC esaminando l'immunità protettiva antitumorale indotta dal CSC-TPDC. Nel modello D5, i topi immunocompetenti ingenui sono stati vaccinati s.c con CSC-TPDC o H-TPDC (allo stesso rapporto lysate: DC). I gruppi di controllo hanno ricevuto saline (PBS). Una settimana dopo l'ultimo vaccino, i topi sono stati sfidati con cellule tumorali D5 nonsortite pervia endovenosa ( v.v.), e ipolmoni sono stati raccolti 3 settimane dopo per enumerare le metastasi polmonari. Come mostrato nella tabella 1, rispetto al gruppo PBS, i topi trattati con l'H-TPDC hanno sviluppato meno metastasi polmonari. È importante sottolineare che i topi trattati con CSC-TPDC avevano significativamente meno metastasi polmonari rispetto al gruppo vaccinale H-TPDC in entrambi gli esperimenti eseguiti. Nel modello SCC7, i normali animali C3H sono stati vaccinati s.c con SCC7 CSC-TPDC o H-TPDC rispettivamente sul fianco destro, seguiti da sfide con cellule tumorali SCC7 nonsortite s.c sul fianco sinistro. Rispetto al gruppo PBS, H-TPDC ha indotto una modesta immunità antitumorale contro la crescita tumorale. Tuttavia, c'è stata una significativa inibizione della crescita tumorale nei topi che sono stati trattati con CSC-TPDC rispetto sia al gruppo di controllo che al gruppo H-TPDC (p<0.05, Figura 3). Questi risultati indicano che i CSC potrebbero essere utilizzati come fonte di antigene più efficace per caricare i PC rispetto alle tradizionali cellule tumorali nonsortite nell'indurre l'immunità protettiva contro la sfida delle cellule tumorali.
Per comprendere ulteriormente i meccanismi alla base dell'immunità protettiva antitumorale indotta dal CSC osservata, abbiamo raccolto le milza dagli animali sottoposti a vaccinazioni CC alla fine degli esperimenti. Le cellule di milza sono state poi attivate da anti-CD3/CD28/IL-2 o anti-CD3/CD28/IL-2 + LPS/anti-CD40. Quindi i supernatici di coltura sono stati raccolti per rilevare l'espressione di citochine e anticorpi.  Le produzioni di IFNγ e GM-CSF da parte delle cellule di milza provenienti dagli animali vaccinati con D5 CSC-TPDC o SCC7 CSC-TPDC(Figura 4)sono state significativamente più elevate. Inoltre, la produzione di IgG da parte delle cellule di milza attivate LPS/anti-CD40 è stata significativamente (p<0,05) aumentata rispetto a D5 H-TPDC o SCC7 H-TPDC rispetto a D5 H-TPDC o SCC7 H-TPDC. Questi anticorpi si sono dati legare rispettivamente ai CSC D5 e SCC7 e tale legame potrebbe comportare la lisi CSC in presenza delcomplemento 8.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig1highres.jpg" src="/files/ftp_upload/50561/50561fig1.jpg"> Figura 1. Alte popolazioni di ALDHFLUOR+/ALDH sono state rilevate in tumori delle cellule squamose coltivati e di nuova raccolta di melanoma murino D5 e SCC7. Le cellule tumorali trattate con 50 mmol/L DEAB, uno specifico inibitore dell'ALDH, sono state utilizzate come controllo negativo.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig1highres.jpg" target="_blank">Clicca qui per visualizzare l'immagine più grande.</a> 
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig2highres.jpg" src="/files/ftp_upload/50561/50561fig2.jpg"> Figura 2. Generazione di vaccini contro le cellule staminali tumorali a base di cellule dendritiche. Per la preparazione di CSC-TPDC e H-TPDC, le cellule dendritiche derivate dal midollo osseo sono state pulsate rispettivamente con liti delle cellule tumorali alte o nonsortiteALDH.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig2highres.jpg" target="_blank">Clicca qui per visualizzare l'immagine più grande.</a> 
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig3highres.jpg" src="/files/ftp_upload/50561/50561fig3.jpg"> Figura 3. Il vaccino csc lysate cc pulsato (CSC-TPDC) potrebbe indurre un'immunità protettiva antitumorale più efficace nel s.c. Modello tumorale SCC7. <a href="https://www.jove.com/files/ftp_upload/50561/50561fig3highres.jpg" target="_blank">Clicca qui per visualizzare l'immagine più grande.</a> 

<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50561/50561fig4highres.jpg" src="/files/ftp_upload/50561/50561fig4.jpg"> Figura 4. Risposte cellulari sistemiche più potenti nell'ospite immunocompetente vaccinato con CSC-TPDC.  Gli splenociti sono stati raccolti dagli animali sottoposti a vaccinazione H-TPDC o CSC-TPDC e sono stati attivati come indicato. I supernatici di coltura sono stati quindi raccolti per il rilevamento delle citochine utilizzando ELISA.
<a href="https://www.jove.com/files/ftp_upload/50561/50561fig4highres.jpg" target="_blank">Clicca qui per visualizzare l'immagine più grande.</a>

Watch this Scientific Journal Video about Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells at JoVE.com

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

Valutato in ospiti immunocompetenti sigeneici, il vaccino a cellule dendritiche (DC) a base di cellule staminali tumorali (CSC) ha dimostrato un'immunità antitumorale significativamente più elevata rispetto ai tradizionali vaccini CC pulsati con cellule tumorali alla rinfusa eterogenee.

Generazione di un nuovo vaccino a cellule dendritiche con melanoma e cellule staminali tumorali squamose

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

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Research

JoVE Journal

Immunology and Infection

11.2K Views.  University of Michigan. Valutato in ospiti immunocompetenti sigeneici, il vaccino a cellule dendritiche (DC) a base di cellule staminali tumorali (CSC) ha dimostrato un'immunità antitumorale significativamente più elevata rispetto ai tradizionali vaccini CC pulsati con cellule tumorali alla rinfusa eterogenee.

Video: Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

Analisi della maturazione polmonare delle cellule dendritiche e migrazione durante l&#39;infiammazione allergica delle vie aeree

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

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Immunologia e infezioni

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Spettrometria di massa, di antigeni glicosfingolipide

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Enumerazione delle popolazioni dei leucociti del sangue periferico per le principali studi clinici multicentrici utilizzando un intero saggio Fenotipizzazione Sangue

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Monitoraggio migrazione delle cellule dendritiche usando<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Magnetic Resonance Imaging

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

Generazione di Arenavirus ricombinante per lo sviluppo del vaccino nel approvati dalla FDA cellule Vero

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

L&#39;identificazione del DNA Mutazioni in depurata ematopoietiche cellule staminali / progenitrici

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Utilizzando Clicca Chimica per misurare l&#39;effetto dell&#39;infezione virale sulla cellula ospite RNA Sintesi

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Arricchimento cellulare automatizzata di Citomegalovirus specifici linfociti T per le applicazioni cliniche che utilizzano il sistema di citochine cattura

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

In Vivo Test per la determinazione della funzione citolitica di cellule T antigene-specifiche utilizzando un modello di vaccinazione

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Generazione di un gran numero di progenitori mieloidi e precursori delle cellule dendritiche dal midollo osseo murino utilizzando una romanzo cella ordinamento strategia

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...