Gli autori desiderano ringraziare Michael Chou, Bingbing Dai, e Liang Xiao per contribuire dati per questo manoscritto. Riconosciamo anche il Dr. John Gray per i doni generosi di reagenti utilizzati in questo studio. PB è sostenuto da una borsa di studio post-dottorato dal National Cancer Center. Questa ricerca è stata sostenuta da sovvenzioni dal National Institute of Health (R01AI68978, P01CA132681 e RCA170820A), una borsa di studio dalla Fondazione Bill e Melinda Gates Foundation, una borsa di accelerazione traslazionale dal Centro comune di Medicina Traslazionale e di una sovvenzione da parte del California HIV / AIDS programma di ricerca.

DC-LV cellule produttori stabili sono costruite sulla base della confezione cella linea 11 GPR che contiene la necessaria componenti lentivirale gagpol, rev e il sistema tet-off. Prima, trasduzione retrovirale viene utilizzato per generare una linea cellulare di packaging GPRS che codifica un tet-dipendente SVGmu glicoproteina. Poi, concatemer matrice trasfezione è usato per trasfettare la linea cellulare GPRS con un vettore lentivirale come transgene GFP. Questa linea cellulare stabile produttore, designato come LV-MGFP, può essere testato in vitro e in vivo per la sua capacità di produrre un vaccino DC-LV contro GFP. 1. Generazione di un Tet-dipendente SVGmu Cell Line Plasmide PRX-SVGmu è un costrutto in cui DC-specifico SVGmu glicoproteina è clonato a valle del promotore tTA-avanzata di plasmide retrovirale pRetroX-Tet-off 1 (Figura 1C). Cultura 293T cellule in D10 (medium di Eagle modificato da Dulbecco con10% di siero fetale bovino, 2 mM L-glutammina). Coltura GPR linea cellulare di packaging in D10 con doxiciclina (1 ng / ml) e puromicina (2 mg / ml). Tutte le cellule sono coltivate in un umidificata 37 ° C incubatore con il 5% di CO 2. <ol><li> 16-18 hr prima della transfezione transiente, piastra 2 x 10 6 cellule 293T in 4 ml di D10 in un piatto di coltura tissutale a 6 cm in modo che le cellule si avvicinano 90% di confluenza al momento della transfezione. </li><li> Trasfezione di plasmidi retrovirali: mescolare 100 ml 1,25 M CaCl 2 soluzione, acqua Milli-Q sterile e seguenti plasmidi: 5 mcg PRX-SVGmu, 2,5 mcg pGag-Pol, 2,5 mcg pVSV-G. Il volume finale è di 500 microlitri. Per un ml polistirolo tubo a fondo rotondo 5, aggiungere 500 microlitri 2X HBS (HEPES 50 mm, 10 mm, 12 mm KCl Destrosio, 280 mM NaCl, 1,5 mM Na 2 HPO 4 * 7H 2 O, pH 7,05). Aggiungere goccia a goccia la miscela plasmide nel buffer HBS 2X mentre ribolle il buffer energicamente con una pipetta Pasteur di vetro. Dopo aver aggiunto il mixture, continuare spumeggiante per altri 30 sec. Quindi, aggiungere l&#39;intera miscela sulle cellule 293T in colture di 6 cm, e incubare a 37 ° C. </li><li> 4 ore dopo la transfezione, sostituire attenzione il mezzo nel piatto cultura con 4 ml D10 preriscaldato. </li><li> Spin infezione: 48 ore dopo la trasfezione, raccolto SVGmu-codifica particelle retrovirali nel surnatante facendo passare il surnatante attraverso un filtro di 0,45 micron. Piastra le cellule di packaging GPR in un piatto da 24 pozzetti con 2 x 10 4 cellule / pozzetto. Aggiungere surnatante filtrato per cellule di packaging GPR nel piatto (2 ml / pozzetto) e cellule centrifugare per 90 min a 1050 xg e 25 ° C. Cambiare mezzo in D10 fresco con doxiciclina (1 ng / ml) dopo spin-infezione (2 ml / pozzetto). </li><li> 72 ore post-trasfezione, espandere la cultura di cellule trasdotte confezionamento in D10 con doxiciclina (1 ng / ml) e puromicina (2 ng / ml). </li><li> Per confermare l&#39;espressione di SVGmu, coltivare le cellule senza doxiciclina per 48 ore. Misurare ex superficiepressione di SVGmu con citometria a flusso utilizzando siero anti-Sindbis. Queste cellule sono designati come GPRS linea cellulare di packaging. </li></ol> 2. Costruire DC-LV Produttore Cells da Concatemer Array Transfezione Lentiviral trasferimento plasmide TL20-GFP è una auto-inattivanti lentivirali trasferimento plasmide vettore sulla base di pCL20c-MSCV-GFP con un sistema di Dox-regulatable virale RNA del genoma espressione e la sostituzione del citomegalovirus (CMV) enhancer con 7 tet operatori (Figura 1C) 11 , 12,15. Plasmide PGK-bile è un resistente (BLE) cassette bleomicina guidato da un debole promotore PGK 2. <ol><li> Digest 20 mcg TL20-GFP plasmide con l&#39;enzima di restrizione SfiI a 50 ° C. Digest 20 mcg plasmide PGK-bile con PflMI a 37 ° C. </li><li> Purificare i frammenti di DNA (la cassetta PGK-bile è 1011 bp e il vettore TL20-GFP è 6861 bp) mediante elettroforesi su gel di agarosio. </li><li> Legare il vettore TL20-GFP e PGK-cassetta bile ad un rapporto molare di 25:1 (NEB DNA ligasi T4). Incubare per una notte a temperatura ambiente. </li><li> Dopo una notte legatura, purificare il DNA dalla miscela di ligazione (Qiagen DNeasy Blood and Tissue Kit). Garantire la quantità di DNA purificato è di circa 5 mg. </li><li> 16-18 hr prima della transfezione, le cellule piastra GPRS confezionamento in un piatto di coltura tissutale a 6 cm in modo che la confluenza saranno circa il 90% al momento della transfezione. </li><li> Utilizzare il metodo di trasfezione fosfato di calcio descritto nel passo 1.2 trasfezione 5 pg di DNA purificato concatemeric nelle cellule confezionamento GPRS nel piatto di coltura tissutale di 6 cm. 4 ore dopo la trasfezione, rimuovere con attenzione il mezzo e sostituirlo con 4 ml D10 pre-riscaldata con doxiciclina (1 ng / ml). </li><li> 48 ore dopo la trasfezione, cambiare il mezzo in D10 con doxiciclina (1 ng / ml) e puromicina (2 mg / ml). Seleziona per cloni trasfettati aggiungendo Zeocin (50 mcg / ml) nel mezzo di coltura. Cultura le cellule per circa 2 settimane fino cella Colonies può essere visto sul fondo dei piatti. </li><li> Etichettare le colonie di cellule sul fondo dei piatti. Prendere 24 pozzetti piatti di coltura di tessuto, il numero dei pozzetti e aggiungere a ciascun pozzetto contenente 2 ml D10 Zeocin (50 mcg / ml), doxiciclina (1 ng / ml), e puromicina (2 mg / ml). Aspirare il terreno delle cellule trasdotte, quindi aggiungere una goccia di tripsina su ciascuna delle colonie per meno di un minuto. Poi, aggiungere una o più gocce di D10 sulle stesse colonie. Prelevare colonie uno ad uno con una pipetta e trasferirli in pozzetti separati della piastra di coltura tissutale a 24 pozzetti. </li><li> Cultura e espandere tutti cloni cellulari in D10 contenente Zeocin (50 mcg / ml), doxiciclina (1 ng / ml) e puromicina (2 mg / ml) per la valutazione della capacità di produzione virale. </li></ol> 3. Valutare virale produzione di ogni clone cellulare <ol><li> Trypsinize le cellule di produttori e piastra in tutto 4x10 6 celle a 6 cm di tessuto cultura piatto in D10 senza doxiciclina tale che la confluenzasupera il 90%. Sostituire il supporto con fresca D10 preriscaldato quotidianamente e raccogliere il supporto per il titolo test 72 ore dopo la rimozione dox. </li><li> Raccogliere il surnatante virale filtrando il mezzo con un filtro di 0,45 micron. </li><li> Cellule bersaglio targa che esprime il recettore DC-SIGN (es. 293T.hDC-SIGN o il mouse derivate dal midollo osseo cellule dendritiche 16) e 293T come controllo negativo in piatti di coltura da 96 pozzetti, 1 x 10 4 cellule / pozzetto. Aggiungere diluizioni seriali di 2 volte del surnatante virale ai pozzetti (100 pl / pozzetto). Solitamente, 6-8 diluizioni sono sufficienti per raggiungere l&#39;intervallo in cui trasdotte cellule GFP-positive sono in un rapporto lineare alla quantità di aggiunta di virus. </li><li> Centrifugare le cellule e sostituire il mezzo come descritto al punto 1.4. BMDCs dovrebbero essere coltivate in terreno RPMI con 10% di FBS e GM-CSF (01:20 J558L terreno condizionato). </li><li> Misurare l&#39;espressione di GFP in cellule trasdotte mediante citometria di flusso 4-6 giorni post-trasduzione. Il vecto lentiviralir titolo è calcolata nell&#39;intervallo di diluizione vettore quando la percentuale di cellule GFP-positive e quantità vettoriali sono in una relazione lineare. Scegliere il clone cellulare con la produzione virale più alta per le applicazioni successive. </li></ol> 4. Produrre e Concentrato vettori lentivirali <ol><li> Cultura della linea cellulare produttore (LV-MGFP) in 15 cm di tessuto piatti della cultura. </li><li> Trypsinize le cellule produttrici e le cellule in piastre da 15 cm piatti coltura di tessuti a più di 90% di confluenza in D10 fresca senza doxiciclina. Cambiare media con fresco D10, preriscaldato quotidiana. </li><li> Al momento della produzione virale picco (come determinato dall&#39;utente), raccogliere il surnatante virale e filtrare utilizzando un filtro di 0,45 micron. Caricare il surnatante filtrato in spessore parete 32,5 tubi ultracentrifuga ml, sigillare con parafilm e centrifugare a 50.000 xg, a 4 ° C per 90 min. Accuratamente risospendere il pellet in 50 microlitri o un volume appropriato di PBS o HBSS seconda dell&#39;applicazione. &lt;/ Li&gt; </ol> 5. Immunizzare topi in vivo e analisi antigene-specifica risposta immunitaria <ol><li> Produrre virus ad alto titolo come descritto nel capitolo 4. </li><li> Iniettare topi BALB / c (6-8 settimane di età, femmina) attraverso un percorso footpad con il vettore di sospensione (25 microlitri per footpad). </li><li> 2 settimane dopo l&#39;immunizzazione, isolare milza e splenociti raccolto. Splenociti coltura con GFP peptidi epitopi e analizzare la presenza di GFP-specifiche cellule T CD8 + mediante colorazione intracellulare delle citochine, come precedentemente descritto 17 (Figura 3B). </li></ol>

<ol>
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<p class="jove_content"&gt; Gli autori dichiarano di non avere interessi finanziari in competizione.</p

Qui, abbiamo delineato un metodo per la produzione di grandi quantità di vettori lentivirali utilizzando 293T cellule trasdotte stabilmente con tutti i componenti lentivirali sotto il tet off sistema di regolamentazione. Ad oggi, la maggior parte dei protocolli per produrre vettori lentivirali basano su standard di trasfezione transiente di calcio fosfato (cfr. 18, per esempio). Questo approccio ha avuto successo su scala clinico, ma può soffrire di alcune limitazioni che possono non essere presenti in scal...

Molti sistemi vettore sono stati sviluppati per il trasferimento genico. Vettori basati su lentivirus sono stati tra il sistema virale più comunemente studiate. Questi vettori sono vantaggiose perché possono trasdurre efficientemente sia divisione e non divisione delle cellule 1, ottenere l&#39;espressione a lungo termine a causa di integrazione nel genoma dell&#39;ospite, mostrare bassa immunità anti-vettore naturale nella maggior parte delle popolazioni umane 2, e hanno un basso potenziale di genotossicità di mutagenesi inserzionale 3,4. Produzione di lentivirus è sempre stata colorata da preoccupazioni di sicurezza. Vettori lentivirali derivano generalmente da HIV-1, l&#39;agente eziologico dell&#39;AIDS. Trasfezione transiente dei singoli componenti del lentivector (trasferimento, busta e plasmidi di imballaggio) è un mezzo comune e flessibile di fornire materiale genetico in laboratorio. Tuttavia, scaling-up transfezioni transitori per le applicazioni cliniche è ingombrante e may portare allo sviluppo di replicazione-competenti lentivirus 5,6. Per superare questi ostacoli, vari imballaggio stabile e linee cellulari di produttori sono state sviluppate 6-11. Una di queste linee, la linea di confezionamento GPR 11, ha il vantaggio di essere attraente regolato dalla tetraciclina. In questo lavoro, dimostriamo come adattare questo sistema per la produzione di auto-inattivanti vettori lentivirali che sono specificamente indirizzate verso le cellule dendritiche (DC-VL) 12. Le cellule dendritiche (DC) sono le più robuste cellule presentanti l&#39;antigene del sistema immunitario. Essi sono stati oggetto di grande interesse per lo sviluppo di vaccini cancro perché essi avviano direttamente, programmi, e regolano le risposte immunitarie tumore-specifici 13. Inserimento di un protocollo di vaccinazione per includere PVS ha il potenziale di provocare una risposta immunitaria antitumorale forte di peptide o DNA vaccini. Recentemente, abbiamo sviluppato un vettori lentivirali che specifically obiettivi di cellule dendritiche attraverso un Sindbis modificato virus glicoproteina, SVGmu 14. Questi vettori sono unici nel senso che mostrano elevata specificità per le cellule dendritiche e di generare risposte immunitarie più forti che non specifici, vettori VSVG-pseudotyped. Qui riportiamo un metodo per produrre grandi quantità di questi vettori lentivirali DC-mirati. Dimostriamo che questi DC-LV può infettare cellule dendritiche e generare forti CD8 + risposta immunitaria delle cellule T. Tutte le procedure che coinvolgono animali sono stati eseguiti con umanità, con l&#39;approvazione della USC istituzionale dell&#39;Ateneo cura degli animali e del Comitato Istituzionale Usa. Per svolgere esperimenti in vivo e clinici, è fondamentale per creare una linea cellulare che può produrre virus ad un alto titolo. Di eseguire la procedura di trasduzione e trasfezione esattamente come descritto consentirà di massimizzare le probabilità di generare una tale clone.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutamine</td>
			<td>Mediatech Inc.</td>
			<td>25-005-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxycycline</td>
			<td>Clontech Laboratories, Inc.</td>
			<td>631311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>R250-01</td>
			<td>Toxic</td>
		 </tr>
		 <tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		 </tr>
		 <tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DNeasy Blood &amp; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

Vettori lentivirali (LV) sono un mezzo efficace per la fornitura di materiale genetico di molti tipi di cellule. A causa di problemi di sicurezza associati a questi HIV-1 vettori derivati, la produzione di grandi quantità di volumi logici è impegnativo. In questo lavoro, riportiamo un metodo per produrre alti titoli di LV auto-inattivanti. Abbiamo retrovirally trasdurre il tet-off stabile produttore cella linea GPR per generare una linea cellulare, GPRS, che può esprimere tutti i componenti virali, compresa una cellula dendritica glicoproteina-specifico, SVGmu. Poi usiamo trasfezione del DNA concatemeric trasfezione trasferimento plasmide codificante GFP LV un gene reporter in combinazione con un marcatore selezionabile. Molti dei cloni risultanti possono produrre LV ad un titolo 10 volte più grande di quello che otteniamo con trasfezione transiente. In più, questi virus efficientemente trasdurre cellule dendritiche in vitro e generano una cellula T forte risposta immunitaria all&#39;antigene nostra giornalista. Questo metodo può essere una buona opzione for la produzione di vaccini forti LV-based per gli studi clinici di cancro o di malattie infettive.

La linea cellulare stabile descritto nel presente metodo può produrre grandi quantità di vettori lentivirali mirati specificatamente alle cellule dendritiche. Come mostrato nella Figura 1B, isolamento di cloni individuali ha prodotto linee cellulari stabili di qualità variabile 12. Tra 26 cloni saggiati, 8 prodotte particelle lentivirali ad un titolo maggiore di 10 6 unità di trasduzione per ml (TU / ml), che è un punto di riferimento tipico per vettori lentivirali SVG-pseudot...

Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Qui, usiamo trasduzione retrovirale e trasfezione concatemeric per creare una linea cellulare che può esprimere le componenti di un vettore lentivirale (LV) in assenza di tetraciclina. Questo LV codifica GFP ed è pseudotyped con una glicoproteina, SVGmu, che è specifico per un recettore sulle cellule dendritiche.

Una linea cellulare tetraciclina regolata produce vettori lentivirali ad alto titolo che si rivolgono specificamente cellule dendritiche

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

a-tetracycline-regulated-cell-line-produces-high-titer-lentiviral

Research

JoVE Journal

Immunology and Infection

14.4K Views.  University of Southern California, Los Angeles. Qui, usiamo trasduzione retrovirale e trasfezione concatemeric per creare una linea cellulare che può esprimere le componenti di un vettore lentivirale (LV) in assenza di tetraciclina. Questo LV codifica GFP ed è pseudotyped con una glicoproteina, SVGmu, che è specifico per un recettore sulle cellule dendritiche.

Video: A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Protocollo ottimizzato per Transfection efficiente delle cellule dendritiche, senza la maturazione delle cellule

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Ricerca

Immunologia e infezioni

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

L&#39;introduzione di shear stress nello studio di adesione batterica

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Isolamento di cellule polmonari mouse dendritiche

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Espansione linfociti T citotossici da sangue del cordone ombelicale che Target Citomegalovirus, virus di Epstein-Barr virus, e Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

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

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 mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

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 ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Isolamento di cellule dendritiche mieloidi e cellule epiteliali di timo umano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Generazione di Immaturo, matura e tollerogenico dendritiche cellule con differenti fenotipi metabolica

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Un protocollo Facile generare proteine site-specifically acetilati in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Sviluppo e validazione di una singola molecola ultrasensibile di matrice digitale analisi enzima-collegata dell'immunosorbente per umana dell'interferone-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...