Gli autori desiderano ringraziare Andres Salazar (Oncovir, Inc.) per il dono della Poli-ICLC.

1. Isolamento e Crioconservazione di PBMC 4 <ol><li> Asetticamente Spike una delle porte di accesso nella borsa leucoaferesi utilizzando un set di trasferimento del plasma. Usando una siringa da 60 ml, trasferire il leucaferesi ottenute da pazienti in una bottiglia sterile da 500 ml. </li><li> Regolare il volume della leucaferesi a 2x il suo volume originale con temperatura ambiente RPMI. Mescolare accuratamente. </li><li> Mescolare delicatamente il flacone di Ficoll-Paque PLUS. Aggiungere 12 ml PLUS Ficoll-Paque in sterile da 50 ml tubo conico. </li><li> Strato delicatamente 30 ml di prodotto diluito leucaferesi per ogni tubo da 50 ml conica sterile contenente Ficoll. Fare attenzione a non disturbare l&#39;interfaccia tra il Ficoll e la sospensione cellulare. </li><li> Ripetere i passi 1.3 e 1.4 fino a quando tutta la leucaferesi è stato stratificato. </li><li> Centrifugare le provette coniche da 50 ml a 1.000 xg per 20 min a temperatura ambiente senza freno. </li><li> Raccolta accuratamente lo strato nuvoloso di PBMC da ciascuna provetta e transfer per nuovi ml provette sterili 50. </li><li> Aggiungere a ciascun tubo RPMI ad un volume finale di 50 ml. Mescolare delicatamente per inversione. </li><li> Centrifugare le cellule a 500 xg per 10 min a 4 ° C con freno pieno. </li><li> Rimuovere il surnatante da ogni provetta. Risospendere il pellet di cellule provenienti da ogni provetta e aggiungere RPMI ad un volume finale di 50 ml. Mescolare delicatamente per inversione. </li><li> Centrifugare le cellule a 500 g per 6 minuti a 4 ° C. </li><li> Rimuovere il surnatante da ogni provetta. Risospendere e piscina insieme le sospensioni di cellule in una provetta da 50 ml. </li><li> Portare il volume delle PBMC aggregati fino a 50 ml con più RPMI. Mescolare delicatamente per inversione. </li><li> Centrifugare le cellule a 300 g per 6 minuti a 4 ° C. </li><li> Rimuovere il surnatante. Risospendere il pellet di cellule in 50 ml di RPMI. Mescolare delicatamente per garantire la sospensione cellulare uniforme. </li><li> Contare il numero di cellule e determinare la vitalità cellulare. Calcolare il numero totale di cellule vitali. </li><li> Centrifugare le cellule a 300G per 6 minuti a 4 ° C. Preparare il supporto di congelamento. Congelamento dei media è costituito da 10% di dimetilsolfossido (DMSO; Miltenyi Biotec) in AB umana sierica (Valle Biomedica). </li><li> Rimuovere il surnatante. Risospendere le cellule ad una concentrazione finale di 2 x 10 8 cellule / ml in mezzi congelamento fredde. </li><li> Fai aliquote da 1 ml a 1,8 ml cryovials. </li><li> Trasferire i cryovials nel congelatore a velocità controllata e iniziare la corsa di congelamento, Programma 1. Al termine della corsa, trasferire le cryovials congelati immediatamente nella fase vapore del congelatore azoto liquido. </li></ol> 2. Giorno 0: la differenziazione delle cellule dendritiche da monociti <ol><li> Aggiungere 30 ml di RPMI / 1% di plasma autologo di una sterile 50 ml tubo conico. </li><li> Aliquote disgelo di PBMC congelati da parte di agitazione a 37 ° C bagnomaria. Quando completamente scongelati, trasferire il contenuto dei flaconi per la sterile da 50 ml tubo conico. Mescolare accuratamente. </li><li> Centrifugare le cellule per 6 mina 500 xg a temperatura ambiente. Rimuovere il surnatante e risospendere le cellule pellet in un piccolo volume di RPMI / 1% di plasma autologo. Poi aggiungere più RPMI / 1% di plasma autologo ad un volume finale di 50 ml. Mescolare accuratamente. </li><li> Centrifugare nuovamente le cellule per 6 min a 500 xg a temperatura ambiente. Aspirare il surnatante e risospendere il pellet in 50 ml di RPMI / 1% di plasma autologo. Mescolare accuratamente. </li><li> Contare il numero di cellule e determinare la vitalità cellulare. Calcolare il numero totale di cellule vitali. </li><li> Piastra 1,40 x 10 8 cellule in 40 ml di RPMI / 1% di plasma autologo su 225 centimetri 2 EasyFlask. Ripetere l&#39;operazione fino a che tutte le celle sono stati placcati. </li><li> Incubare le EasyFlasks piatte sul suo lato per 1-2 ore a 37 ° C in coltura tissutale incubatore contenente il 5% di CO 2 per permettere ai monociti di aderire al pallone. </li><li> Quando l&#39;incubazione, rimuovere il EasyFlask dall&#39;incubatore e lavare due volte per rimuovere le cellule non aderenti USIng 30 ml di RPMI preriscaldata. </li><li> Dopo il secondo lavaggio, aggiungere 40 ml di RPMI / 1% di plasma autologo con 400-1,000 IU / ml di IL-4 e 100-1.000 UI / ml di GM-CSF 5-8. Per questo protocollo, stiamo utilizzando 400 UI / ml di IL-4 e 100 UI / ml di GM-CSF. </li><li> Incubare le EasyFlasks per 2 giorni a 37 ° C in incubatore coltura tissutale contenente il 5% di CO 2. </li></ol> 3. Giorno 2: Alimentazione delle cellule dendritiche con IL-4 e GM-CSF <ol><li> Aggiungere 4 ml di RPMI / 1% di plasma autologo con 400-1,000 IU / ml di IL-4 e 100-1.000 UI / ml di GM-CSF per ciascun EasyFlask. Mescolare accuratamente e dondolo su un fianco. </li><li> Incubare le EasyFlasks per ulteriori 3 giorni (fino al giorno 5) a 37 ° C in incubatore coltura tissutale contenente il 5% di CO 2. </li></ol> 4. Giorno 5: Raccolta di cellule dendritiche immature <ol><li> Raccogliere le culture vigorosamente vorticoso i palloni e pipettando su e giù per risospendere le cellule non aderenti e vagamente aderente. Trasferire ilcellule raccolte a nuove provette coniche da 50 ml. </li><li> Centrifugare le provette a 500 g per 6 minuti a temperatura ambiente. Rimuovere il surnatante e risospendere il pellet cellulare in 50 ml RPMI / 1% di plasma autologo. Mescolare accuratamente. </li><li> Contare il numero di cellule e determinare la vitalità cellulare. Calcolare il numero totale di cellule vitali. </li><li> Centrifugare le provette a 500 g per 6 minuti a temperatura ambiente. Piastra di 1-2 x 10 6 cellule per pozzetto in 3 ml RPMI / 1% di plasma autologo con 400 IU / ml di IL-4 e 100 UI / ml di GM-CSF in piastre di coltura tissutale a 6 pozzetti. </li><li> Incubare le piastre a 37 ° C in coltura tissutale incubatore contenente il 5% di CO 2 durante la notte. </li></ol> 5. Giorno 6: Maturazione e Antigen Caricamento delle cellule dendritiche <ol><li> Aggiungere 10 mcg / ml KLH a 1/3 dei pozzetti nelle piastre di coltura a 6 pozzetti. Swirl le piastre per mescolare. KLH viene aggiunto solo a 1/3 dei pozzi perché KLH è usato solo per il primo vaccino DC. Successivamente DC vaccini conTain solo i peptidi antigeni tumorali. </li><li> DC può essere stimolato a maturare con stimoli diversi. Gli stimoli maturazione più comuni che è stato utilizzato in studi clinici è stato un cocktail di citochine (IL-1 β, TNFa e IL-6) e di prostaglandina E2 (PGE2) 9,10. Più di recente, l&#39;uso di agonisti dei recettori Toll-like (TLR) hanno guadagnato popolarità 11,12. In questo protocollo, aggiungere 2 mg / ml Polyinosinic-policitidilico Carbossimetilcellulosa acido poli-L-lisina (Poly-ICLC) ad ogni pozzetto e mescolare bene. Poli-ICLC è la formulazione clinico-grado di Poly-IC che viene stabilizzato con poli-L-lisina e *-carbossimetilcellulosa (fabbricato da Oncovir, Inc.). </li><li> Aggiungere 100 pg / ml antigeni peptidici lunghi (NY-ESO-1 e / o Melan-A/MART-1) nei pozzetti designati, ogni pozzetto riceve solo un singolo peptide per evitare la concorrenza 13. </li><li> Incubare le piastre a 37 ° C in coltura tissutale incubatore contenente il 5% di CO &lt;sub&gt; 2 O / N. </li></ol> 6. Giorno 7: Crioconservazione delle cellule dendritiche <ol><li> Preparare la soluzione di congelamento DC utilizzando plasma autologo e il 10% di DMSO. Centrifugare la soluzione di congelamento DC a 2.000 xg per 20 min a 4 ° C per rimuovere il materiale particolato. Trasferire il surnatante in una nuova etichetta da 15 ml tubo conico. Conservare a 4 ° C fino al momento dell&#39;uso. </li><li> Quando la notte di incubazione è terminata, il raccolto e la piscina tutti i pozzetti contenenti DC pulsate con i peptidi in provette coniche da 50 ml. </li><li> Centrifugare le provette a 500 g per 6 min. Rimuovere il surnatante. Risospendere il pellet in 5 ml di RPMI / 1% di plasma autologo. Portare il volume totale fino a 50 ml con RPMI / 1% di plasma autologo. Mescolare accuratamente. </li><li> Contare il numero di cellule e determinare la vitalità cellulare. Calcolare il numero totale di cellule vitali. </li><li> Centrifugare le provette a 500 × g per 6 min a temperatura ambiente. Rimuovere il surnatante e risospendere ogni pellet in 5 ml steriLe 0,9% NaCl, USP e trasferimento in nuovi tubi da 15 ml. Portare ogni sospensione cellulare a 14 ml con più sterile di NaCl 0,9%, USP. Cap e mescolare per inversione. </li><li> Ripetere il passaggio precedente per due volte. Dopo l&#39;ultima centrifugazione, rimuovere i surnatanti, ottenendo il più vicino possibile al pellet cellulari senza disturbare il pellet. </li><li> Risospendere il pellet di cellule in CC Solution congelamento a 4 - 20 x 10 6 cellule / ml e 1 ml aliquota ad ogni flacone CryoTube 1,8 ml. </li><li> Utilizzare il congelatore a velocità regolata, Programma 1, di congelare le aliquote di vaccini DC e trasferire immediatamente nella fase vapore di un congelatore azoto liquido per la conservazione a lungo termine. </li></ol> 7. Lotto controllo del rilascio <ol><li> Ogni lotto di vaccino DC deve essere valutato e rispettare determinati criteri di lotto specifico (Tabella 1) prima di essere rilasciato per iniezione in pazienti nello studio, in genere 5-6 settimane dopo la preparazione del vaccino. </li><li> Viabilità:Valutare la sostenibilità del vaccino DC utilizzando un contatore automatico di cellule. La specifica minima redditività accettabile è&gt; 70%. </li><li> Identità: Valutare l&#39;identità della DC mature (CD11c + CD14-CD83 +) in citometria a flusso. La percentuale di cellule + CD11c deve essere&gt; 50%. La percentuale di CD11c + e CD14 + cellule dovrebbe essere &lt;30% e la percentuale di CD11c +, CD14-, CD83 + e cellule devono essere&gt; 50%. </li><li> Sterilità: test del vaccino DC per la presenza di batteri anaerobici e aerobici e funghi di cultura diretta e colorazione di Gram. Valutare la presenza di micoplasma utilizzando un sistema di coltura diretta e DNA fluorocromo Assay colorazione con indicatore Cell Line. Per queste prove, il set minimo specifica è alcuna crescita viene osservato da tutte le culture. </li><li> Endotossina: valutare il contenuto di endotossine con il Kinetic-QCL cinetico cromogenico LAL test e deve essere &lt;50 EU / ml per soddisfare i criteri minimi. </li><li&gt; Funzione: Valutare la funzione DC in una reazione linfocitaria mista mediante incubazione con cellule T allogeniche. La presenza di proliferazione rispetto alle DC non stimolate viene riportato (figura 3). </li></ol>

<ol>
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Gli autori hanno ricevuto un sostegno finanziario dal seguente: NIH (AI061684, AI071078, AI044628 e K08 AI084578 (Miller)), la Bill and Melinda Gates Foundation, Doris Duke Charitable Foundation, Cancer Research Institute, Alliance for Lupus Research, e l&#39;Emerald Fondazione. Nina Bhardwaj è un coinventore su brevetti relativi alla preparazione e utilizzo di cellule dendritiche per manipolare l&#39;immunità. Gli autori non hanno altre affiliazioni rilevanti o coinvolgimento finanziario con qualsiasi organizzazione o ente con un interesse finanziario in conflitto o finanziaria con l&#39;oggetto o materiale discusso nel manoscritto oltre a quelle descritte.

Fase I e II di sviluppo clinico di DC derivate da monociti hanno dimostrato che inducono risposte immunitarie nei pazienti però il successo clinico è stato limitato 1. Ciò può essere dovuto in parte alla mancanza di consenso su come generare il DCS ottimali per uso immunotherapeutic tumore. Sebbene ci siano molti modi per generare DC ad uso clinico, questi metodi variano in termini di uso di citochine utilizzati per differenziare i monociti, stimoli usati per indurre la maturazione, e metodi di antigene c...

<table border="1">
<tbody>
 <tr>
 <td fo:width="2in">Reagent/Supplies/Equipment</td>
 <td>Manufacturer</td>
 <td>Catalog No.</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI-1640 medium with L-glutamine</td>
 <td>BioWhittaker</td>
 <td>12-702F</td>
 <td></td>
 </tr>
 <tr>
 <td>1M HEPES buffered saline</td>
 <td>BioWhittaker</td>
 <td>17-737E</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS)</td>
 <td>BioWhittaker</td>
 <td>17-516F</td>
 <td></td>
 </tr>
 <tr>
 <td>Human albumin, 25% solution USP</td>
 <td>Aventis Behring</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ficoll-Hypaque PREMIUM</td>
 <td>GE Healthcare</td>
 <td>17-5442-03</td>
 <td></td>
 </tr>
 <tr>
 <td>Human AB serum</td>
 <td>Valley Biomedical</td>
 <td>HP1022</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile saline USP</td>
 <td>Hospira</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CryoMACS DMSO </td>
 <td>Miltenyi Biotec</td>
 <td>170-076-303</td>
 <td></td>
 </tr>
 <tr>
 <td>Leukine GM-CSF, 0.5 mg/ml</td>
 <td>Berlex</td>
 <td>A02266</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS GMP IL-4</td>
 <td>Miltenyi Biotec</td>
 <td>170-076-101</td>
 <td></td>
 </tr>
 <tr>
 <td>Hiltonol, Poly-ICLC, 2 mg/ml</td>
 <td>Oncovir</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>VACMUNE KLH</td>
 <td>Biosyn</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>225 sq cm EasyFlasks</td>
 <td>Nalgene Nunc</td>
 <td>159934</td>
 <td></td>
 </tr>
 <tr>
 <td>Falcon 6-well tissue culture plates</td>
 <td>Becton Dickinson</td>
 <td>353046</td>
 <td></td>
 </tr>
 <tr>
 <td>1.8 ml CryoTube vials</td>
 <td>Nalgene Nunc</td>
 <td>377267</td>
 <td></td>
 </tr>
 <tr>
 <td>Controlled Rate Freezer</td>
 <td>Thermo</td>
 <td>CryoMed</td>
 <td></td>
 </tr>
 </tbody>
</table>

preparation of tumor antigen-loaded mature dendritic cells for immunotherapy

Mentre gli studi clinici hanno dimostrato che i vaccini DC antigene-caricati sono al sicuro e promettente terapia per i tumori 1, la loro efficacia clinica rimane da stabilire. Il metodo descritto di seguito, redatto in conformità con buon processo di fabbricazione (GMP), è una ottimizzazione del metodo di preparazione ex vivo più comuni per la generazione di un gran numero di cellule dendritiche per gli studi clinici 2. Il nostro metodo utilizza il TLR sintetico 3 agonista Polyinosinic-policitidilico Carbossimetilcellulosa acido poli-L-lisina (Poly-ICLC) per stimolare i PVS. Nostro studio precedente stabilito che Poly-ICLC è il più potente stimolo maturazione individuale per DCs umane valutata da una sovraregolazione di CD83 e CD86, induzione di interleuchina-12 (IL-12), fattore di necrosi tumorale (TNF), interferone gamma-indotta proteina 10 (IP-10), interleukmin 1 (IL-1), e interferoni di tipo I (IFN), e minimale interleuchina 10 (IL-10) di produzione. </p&gt; DC sono differenziati da cellule mononucleate del sangue periferico (PBMC congelati) ottenuti da leucaferesi. PBMC vengono isolati mediante centrifugazione su gradiente Ficoll e congelati in aliquote. Il giorno 1, PBMC vengono scongelati e piastrate su fiasche di coltura tissutale di selezionare per i monociti che aderiscono alla superficie plastica dopo 1-2 ore di incubazione a 37 ° C in incubatore coltura tissutale. Dopo l&#39;incubazione i linfociti vengono lavati via ed i monociti aderenti sono coltivate per 5 giorni in presenza di interleuchina-4 (IL-4) e granulociti macrofagi fattore stimolante le colonie (GM-CSF) per differenziare a DC immature. Il giorno 6, DC immature vengono pulsate con l&#39;emocianina di patella (KLH) keyhole proteina che serve come un controllo per la qualità del vaccino e può aumentare l&#39;immunogenicità del vaccino 3. Le cellule dendritiche sono stimolati a maturare, caricati con antigeni peptidici, e incubate per una notte. Il giorno 7, le cellule vengono lavate, e congelati in aliquote da 1 ml contenente4 - 20 x 10 6 cellule usando un congelatore a velocità controllata. Controllo del rilascio del lotto per i lotti di DCS viene eseguito e deve soddisfare requisiti minimi prima di essere iniettati nel paziente.

Tra il 10 - 20% delle PBMC partire differenziarsi in cellule dendritiche al termine del periodo di coltura. DC mature sono CD11c +, CD14-, CD83 +, CD40 +, e CCR7 + (Figura 1). Essi esprimono alti livelli di MHC di classe I e II e le molecole di molecole co-stimolatorie CD80 e CD86. Poli-ICLC anche indotto livelli inferiori di PDL-1 rispetto ad altri agonisti TLR 14. Inoltre, questi Poly-IC-maturati dendritiche secernono grandi quantità di IL-12 (Figura 2 e 15,16) ...

Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

Il metodo più comunemente utilizzato per generare un elevato numero di cellule dendritiche autologhe (DC) per uso in immunoterapia tumore è descritto. Il metodo utilizza IL-4 e GM-CSF per differenziare DC da monociti. Le DC immature sono stimolati a maturo e poi caricato con antigeni prima di essere iniettati nel paziente.

Preparazione del Tumor Antigen-caricati mature cellule dendritiche per l&#39;immunoterapia

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

preparation-tumor-antigen-loaded-mature-dendritic-cells-for

Research

JoVE Journal

Immunology and Infection

18.1K Views.  NYU Langone Medical Center. Il metodo più comunemente utilizzato per generare un elevato numero di cellule dendritiche autologhe (DC) per uso in immunoterapia tumore è descritto. Il metodo utilizza IL-4 e GM-CSF per differenziare DC da monociti. Le DC immature sono stimolati a maturo e poi caricato con antigeni prima di essere iniettati nel paziente.

Video: Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modello murino di CD40-attivazione delle cellule B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Ricerca

Immunologia e infezioni

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 ...

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 ...

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 CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Generazione di cellule CAR T per adottiva terapia nel contesto di Glioblastoma standard di cura

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

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 CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Un test CFSE-based per studiare la migrazione delle cellule dendritiche murini della pelle in drenanti linfonodi durante l&#39;infezione con<em&gt; Mycobacterium bovis</em&gt; Bacille Calmette-Guérin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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 ...

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university centers worldwide. While ECP&#8217;s clinical efficacy and exemplary safety profile have driven its widespread use, elucidation of the underlying mechanisms has remained a challenge, partly owing to lack of a laboratory ECP model. To overcome this obstacle and create a simple, user-friendly platform for ECP research, we developed a scaled-down version of the clinical ECP leukocyte-processing device, suitable for work with both mouse models, and small human blood samples. This device is termed the Transimmunization (TI) chamber, or plate. In a series of landmark experiments, the miniaturized device was used to produce a cellular vaccine that regularly initiated therapeutic anti-cancer immunity in several syngeneic mouse tumor models. By removing individual factors from the experimental system and ascertaining their contribution to the in vivo anti-tumor response, we then elucidated key mechanistic drivers of ECP immunizing potential. Collectively, our results revealed that anti-tumor effects of ECP are initiated by dendritic cells (DC), physiologically generated through blood monocyte interaction with platelets in the TI plate, and loaded with antigens from tumor cells whose apoptotic cell death is finely titrated by exposure to the photoactivatable DNA cross-linking agent 8-methoxypsoralen and UVA light (8-MOPA). When returned to the mouse, this cellular vaccine leads to specific and transferable anti-tumor T cell immunity. We verified that the TI chamber is also suitable for human blood processing, producing human DCs fully comparable in activation state and profile to those derived from the clinical ECP chamber. The protocols presented here are intended for ECP studies in mouse and man, controlled generation of apoptotic tumor cells with 8-MOPA, and rapid production of physiologic human and mouse monocyte-derived DCs for a variety of applications.

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

Nuovo protocollo per la generazione di cellule dendritiche fisiologiche immunogene

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...