Les auteurs tiennent à remercier Andres Salazar (Oncovir, Inc.) pour le don de la Poly-ICLC.

1. L&#39;isolement et la cryoconservation des PBMC 4 <ol><li> Aseptiquement pointe l&#39;un des orifices d&#39;accès à la poche de cytaphérèse aide d&#39;un ensemble de transfert de plasma. En utilisant une seringue de 60 ml, transférer la cytaphérèse provenant de patients dans un flacon de 500 ml stérile. </li><li> Réglez le volume de la cytaphérèse 2x son volume initial en utilisant la température ambiante RPMI. Mélanger soigneusement. </li><li> Mélanger délicatement la bouteille de Ficoll-Paque PLUS. Ajouter 12 ml PLUS Ficoll-Paque dans le tube conique stérile de 50 ml. </li><li> Couche doucement 30 ml de produit de leucaphérèse dilué dans chaque tube conique stérile de 50 ml contenant de Ficoll. Veillez à ne pas perturber l&#39;interface entre le Ficoll et la suspension cellulaire. </li><li> Répétez les étapes 1.3 et 1.4 jusqu&#39;à ce que toute la cytaphérèse a été posé. </li><li> Centrifuger les tubes coniques de 50 ml à 1000 g pendant 20 min à température ambiante sans frein. </li><li> Récolter soigneusement la couche nuageuse de CMSP de chaque tube et transfer pour les nouveaux tubes stériles de 50 ml. </li><li> Ajouter dans chaque tube du RPMI pour obtenir un volume final de 50 ml. Mélanger doucement par inversion. </li><li> Centrifuger les cellules à 500 g pendant 10 min à 4 ° C avec plein frein. </li><li> Retirer le surnageant de chaque tube. Remettre en suspension les culots de cellules de chaque tube et ajouter RPMI à un volume final de 50 ml. Mélanger doucement par inversion. </li><li> Centrifuger les cellules à 500 g pendant 6 min à 4 ° C. </li><li> Retirer le surnageant de chaque tube. Remettre en suspension et la piscine ensemble les suspensions de cellules dans un tube conique de 50 ml. </li><li> Porter le volume des CMSP communs jusqu&#39;à 50 ml avec plus RPMI. Mélanger doucement par inversion. </li><li> Centrifuger les cellules à 300 g pendant 6 min à 4 ° C. </li><li> Eliminer le surnageant. Reprendre le culot cellulaire dans 50 ml de RPMI. Mélanger doucement afin d&#39;assurer suspension cellulaire uniforme. </li><li> Compter le nombre de cellules et de déterminer la viabilité cellulaire. Calculer le nombre total de cellules viables. </li><li> Centrifuger les cellules à 300G pendant 6 min à 4 ° C. Préparer les milieux de congélation. Milieux de congélation se compose de 10% de diméthylsulfoxyde (DMSO; Miltenyi Biotec) dans le sérum humain AB (Vallée biomédicale). </li><li> Eliminer le surnageant. Remettre en suspension les cellules à une concentration finale de 2 x 10 8 cellules / ml dans un milieu froid glacial. </li><li> Faire aliquotes de 1 ml dans 1,8 ml cryovials. </li><li> Transférer les tubes cryogéniques dans le congélateur à vitesse contrôlée et commencer la course congélation, programme 1. A la fin de la course, transférer les cryotubes congelés immédiatement dans la phase de vapeur de la congélation à l&#39;azote liquide. </li></ol> 2. Jour 0: La différenciation des cellules dendritiques à partir de monocytes <ol><li> Ajouter 30 ml de RPMI / 1% de plasma autologue dans un tube conique ml stérile 50. </li><li> aliquotes de dégel CMSP gelés par une légère agitation dans le 37 ° C au bain d&#39;eau. Quand il est complètement dégelé, transférer le contenu des flacons au tube conique stérile de 50 ml. Mélanger soigneusement. </li><li> Centrifuger les cellules pendant 6 minà 500 × g à la température ambiante. Retirer le surnageant et remettre le culot cellulaire dans un petit volume de RPMI / 1% de plasma autologue. Ensuite, ajouter de RPMI / 1% de plasma autologue à un volume final de 50 ml. Mélanger soigneusement. </li><li> Centrifuger les cellules pendant 6 min à 500 g à température ambiante. Aspirer le surnageant et remettre le culot dans 50 ml de RPMI / 1% de plasma autologue. Mélanger soigneusement. </li><li> Compter le nombre de cellules et de déterminer la viabilité cellulaire. Calculer le nombre total de cellules viables. </li><li> Plaque 1,40 x 10 8 cellules dans 40 ml de RPMI / 1% de plasma autologue sur 225 cm 2 EasyFlask. Répétez jusqu&#39;à ce que toutes les cellules ont été étalées. </li><li> Incuber les EasyFlasks plat sur ​​le côté pour 1 - 2 heures à 37 ° C dans l&#39;incubateur de culture tissulaire contenant 5% de CO 2 pour permettre les monocytes adhèrent au ballon. </li><li> Lorsque l&#39;incubation est terminée, retirez le EasyFlask de l&#39;incubateur et laver deux fois pour éliminer les cellules non-adhérentes using 30 ml de RPMI préchauffé. </li><li> Après le second lavage, ajouter 40 ml de RPMI / 1% de plasma autologue avec 400-1000 UI / ml d&#39;IL-4 et 100-1000 UI / ml de GM-CSF 5-8. Pour ce protocole, nous utilisons 400 UI / ml d&#39;IL-4 et 100 UI / ml de GM-CSF. </li><li> Incuber les EasyFlasks pendant 2 jours à 37 ° C dans l&#39;incubateur de culture tissulaire contenant 5% de CO 2. </li></ol> 3. Jour 2: Nourrir des cellules dendritiques avec IL-4 et GM-CSF <ol><li> Ajouter 4 ml de RPMI / 1% de plasma autologue avec 400-1000 UI / ml d&#39;IL-4 et 100-1000 UI / ml de GM-CSF à chaque EasyFlask. Mélanger en agitant et en faisant tourner sur le côté. </li><li> Incuber les EasyFlasks pour 3 jours (jusqu&#39;au 5ème jour) à 37 ° C dans l&#39;incubateur de culture tissulaire contenant 5% de CO 2. </li></ol> 4. Jour 5: La récolte des cellules dendritiques immatures <ol><li> Récolter les cultures en agitant vigoureusement les flacons et par pipetage de haut en bas pour remettre en suspension les cellules non adhérentes et peu adhérente. Transférer lecellules récoltées à de nouveaux tubes 50 ml fond conique. </li><li> Centrifuger les tubes à 500 g pendant 6 min à température ambiante. Retirer le surnageant et remettre le culot cellulaire dans 50 ml de RPMI / 1% de plasma autologue. Mélanger soigneusement. </li><li> Compter le nombre de cellules et de déterminer la viabilité cellulaire. Calculer le nombre total de cellules viables. </li><li> Centrifuger les tubes à 500 g pendant 6 min à température ambiante. Plate 1-2 x 10 6 cellules par puits dans 3 ml de RPMI / 1% de plasma autologue avec 400 UI / ml d&#39;IL-4 et 100 UI / ml de GM-CSF dans des plaques de culture tissulaire à 6 puits. </li><li> Incuber les plaques à 37 ° C dans l&#39;incubateur de culture tissulaire contenant 5% de CO 2 pendant la nuit. </li></ol> 5. Jour 6: Maturation et Antigen Chargement des cellules dendritiques <ol><li> Ajouter 10 pg / ml KLH à 1/3 des puits dans des plaques de culture à 6 puits. Swirl les plaques pour bien mélanger. KLH est seulement ajouté à 1/3 du puits, car KLH est utilisé uniquement pour le premier vaccin de courant continu. Après DC vaccins conmaintenir seulement les peptides antigéniques tumoraux. </li><li> PED peut être stimulée à mûrir en utilisant différents stimuli. Les stimuli de maturation plus courants qui ont été utilisés dans les études cliniques a été un cocktail de cytokines (IL-1 β, TNF et IL-6) et de prostaglandine E2 (PGE2) 9,10. Plus récemment, l&#39;utilisation d&#39;agonistes des récepteurs Toll-like (TLR) ont gagné en popularité 11,12. Dans ce protocole, ajouter 2 mg / ml POLYINOSINIQUE-polycytidylique Carboxymethylcellulose acide poly-L-lysine (Poly-ICLC) dans les puits et bien mélanger. Poly-ICLC est la formulation de qualité clinique du Poly-IC qui est stabilisé avec de la poly-L-lysine-* et de la carboxyméthylcellulose (fabriqué par Oncovir, Inc.). </li><li> Ajouter 100 g / antigènes peptidiques longs ml (NY-ESO-1 et / ou Melan-A/MART-1) à leurs puits désignés, chaque puits recevant une seule peptide d&#39;éviter compétition croisée 13. </li><li> Incuber les boîtes à 37 ° C dans l&#39;incubateur de culture tissulaire contenant 5% de CO &lt;sub&gt; 2 O / N. </li></ol> 6. Jour 7: La cryoconservation des cellules dendritiques <ol><li> Préparer la solution de congélation DC en utilisant du plasma autologue et 10% de DMSO. Centrifuger la solution de congélation DC à 2.000 g pendant 20 min à 4 ° C pour éliminer toute matière particulaire. Transférer le surnageant dans un nouveau tube avec étiquette conique de 15 ml. Conserver à 4 ° C jusqu&#39;à utilisation. </li><li> Lorsque la nuit d&#39;incubation est terminée, la récolte et la piscine tous les puits contenant les PED puisées avec les peptides en tubes coniques de 50 ml. </li><li> Centrifuger les tubes à 500 g pendant 6 min. Retirer le surnageant. Reprendre le culot cellulaire dans 5 ml de RPMI / 1% de plasma autologue. Porter le volume total à 50 ml avec du RPMI / 1% de plasma autologue. Mélanger soigneusement. </li><li> Compter le nombre de cellules et de déterminer la viabilité cellulaire. Calculer le nombre total de cellules viables. </li><li> Centrifuger les tubes à 500 g pendant 6 min à température ambiante. Retirer le surnageant et remettre en suspension chaque culot dans 5 ml Sterile NaCl à 0,9%, USP et le transfert de nouvelles 15 ml tubes coniques. Apportez chaque suspension cellulaire à 14 ml de NaCl plus stérile à 0,9%, USP. Cap et mélanger par inversion. </li><li> Répétez l&#39;étape précédente deux fois. Après la dernière centrifugation, éliminer le surnageant, se rapprochant le plus possible de les culots cellulaires sans perturber les pastilles. </li><li> Remettre en suspension les culots cellulaires dans la solution de congélation DC à 4 - 20 x 10 6 cellules / ml et aliquote de 1 ml dans chaque flacon de CryoTube 1,8 ml. </li><li> Utilisez le congélateur à vitesse contrôlée, programme 1, de geler le bas les portions de vaccins DC et transférer immédiatement dans la phase vapeur d&#39;un congélateur à azote liquide pour le stockage à long terme. </li></ol> 7. Lot essais de libération <ol><li> Chaque lot de vaccin DC doit être évalué et répondent à des critères de libération des lots spécifiques (tableau 1) avant qu&#39;il soit libéré pour injection à des patients dans l&#39;étude, typiquement 5 à 6 semaines après la préparation du vaccin. </li><li> Viabilité:Évaluer la viabilité du vaccin DC en utilisant un compteur de cellules automatisé. Les spécifications minimales de viabilité acceptable est&gt; 70%. </li><li> Identité: Évaluer l&#39;identité du CD matures (CD11c + CD14-CD83 +) par cytométrie de flux. Le pourcentage de cellules + CD11c doit être&gt; 50%. Le pourcentage de CD11c + et CD14 + des cellules doit être &lt;30% et le pourcentage de CD11c +, CD14-, CD83 + et les cellules doivent être&gt; 50%. </li><li> La stérilité: Test du vaccin à courant continu pour la présence de bactéries et de champignons anaérobies et aérobies par culture directe et les taches de Gram. Évaluer la présence de mycoplasmes en utilisant un système de culture directe et ADN fluorochrome essai de coloration avec indicateur lignée cellulaire. Pour ces tests, l&#39;ensemble des spécifications minimum est qu&#39;aucune croissance est observée à partir de toutes les cultures. </li><li> Endotoxine: Évaluer la quantité d&#39;endotoxine en utilisant l&#39;énergie cinétique test LAL chromogénique cinétique QCL et il doit être &lt;50 EU / ml pour répondre aux critères minimaux. </li><li&gt; Fonction: évaluer la fonction continu dans une réaction lymphocytaire mixte par incubation avec des cellules T allogéniques. La présence de prolifération par rapport aux PED non stimulées est signalé (Figure 3). </li></ol>

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Les auteurs ont reçu un soutien financier de ce qui suit: NIH (AI061684, AI071078, AI044628 et AI084578 K08 (Miller)), la Fondation Bill et Melinda Gates, Doris Duke Charitable Foundation, Cancer Research Institute, l&#39;Alliance pour Lupus Research, et le Emerald Fondation. Nina Bhardwaj est un co-inventeur de brevets relatives à l&#39;élaboration et à l&#39;utilisation des cellules dendritiques pour manipuler l&#39;immunité. Les auteurs n&#39;ont pas d&#39;autres affiliations pertinentes ou la participation financière de toute organisation ou entité ayant un intérêt financier ou conflit financier avec l&#39;objet ou de matériaux discuté dans le manuscrit en dehors de ceux décrits.

Phase I et II des essais cliniques de DC dérivées de monocytes ont montré qu&#39;ils induisent des réponses immunitaires chez les patients mais le succès clinique a été limitée 1. Cela peut être dû en partie à l&#39;absence de consensus sur la façon de générer les PED optimale pour une utilisation immunothérapie des tumeurs. Bien qu&#39;il existe de nombreuses façons de générer des contrôleurs de qualité clinique, ces méthodes varient en fonction de l&#39;utilisation de cytokines utilisé...

<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

Bien que des études cliniques ont établi que les vaccins DC antigène chargées sont sûrs et thérapie prometteuse pour les tumeurs 1, leur efficacité clinique reste à établir. La méthode décrite ci-dessous, préparé conformément aux bonnes processus de fabrication (BPF) des lignes directrices, est une optimisation de la méthode de préparation ex vivo la plus courante pour générer un grand nombre de pays en développement pour les essais cliniques 2. Notre méthode utilise le TLR synthétique 3 agoniste POLYINOSINIQUE-polycytidylique Carboxymethylcellulose acide poly-L-lysine (Poly-ICLC) afin de stimuler les PED. Notre précédente étude a établi que Poly-ICLC est le stimulus de maturation individuelle la plus puissante pour les PED humains telle qu&#39;elle est évaluée par une régulation positive de CD83 et CD86, l&#39;induction de l&#39;interleukine-12 (IL-12), le facteur de nécrose tumorale (TNF), l&#39;interféron gamma induite Protéines 10 (IP-10), interleukmin 1 (IL-1), et interférons de type I (IFN) et l&#39;interleukine minimal 10 (IL-10) de production. </p&gt; PED sont différenciées à partir de cellules mononucléées du sang périphérique (CMSP surgelés) obtenues par cytaphérèse. PBMC sont isolées par gradient de Ficoll centrifugation et congelées en portions. Le Jour 1, CMSP sont décongelés et étalées sur des flacons de culture de tissus à choisir pour les monocytes qui adhèrent à la surface du plastique après 1-2 heures d&#39;incubation à 37 ° C dans l&#39;incubateur de culture tissulaire. Après incubation, les lymphocytes sont lavés et les monocytes adhérents sont cultivées pendant 5 jours en présence d&#39;interleukine 4 (IL-4) et granulocyte macrophage-colony stimulating factor (GM-CSF) se différencier pour les CD immatures. Au jour 6, DC immatures sont puisées avec la patelle protéines trou de serrure (KLH), qui sert de contrôle pour la qualité du vaccin et peut stimuler l&#39;immunogénicité du vaccin 3. Les pays en développement sont encouragés à mûrir, chargées avec des antigènes peptidiques, et incubées pendant la nuit. Au jour 7, les cellules sont lavées, et congelés dans aliquotes de 1 ml contenant4 - 20 x 10 6 cellules en utilisant un congélateur à vitesse contrôlée. essai de libération du lot pour les lots des PED est effectuée et doit respecter les spécifications minimales avant d&#39;être injectés dans patients.

Entre 10 - 20% des CMSP départ se différencient en DC à la fin de la période de culture. CD matures sont CD11c +, CD14-, CD83 +, CD40 +, et CCR7 + (Figure 1). Ils expriment des niveaux élevés de molécules CMH de classe II et I et les molécules de co-stimulation CD80 et CD86. Poly-ICLC a également induit des niveaux inférieurs de PDL-1 par rapport à d&#39;autres agonistes TLR 14. En outre, ces Poly-IC-mûri PED sécrètent de grandes quantités d&#39;IL-12 (Figure 2...

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

La méthode la plus couramment utilisée pour générer un grand nombre de cellules dendritiques autologues (CD) pour l&#39;utilisation dans l&#39;immunothérapie tumorale est décrite. La méthode utilise IL-4 et GM-CSF pour différencier les PED à partir de monocytes. Les DC immatures sont stimulées à maturité, puis chargé avec des antigènes avant qu&#39;ils ne soient réinjectées au patient.

Préparation de la tumeur cellules dendritiques matures antigène chargées pour l&#39;immunothérapie

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 vues.  NYU Langone Medical Center. La méthode la plus couramment utilisée pour générer un grand nombre de cellules dendritiques autologues (CD) pour l'utilisation dans l'immunothérapie tumorale est décrite. La méthode utilise IL-4 et GM-CSF pour différencier les PED à partir de monocytes. Les DC immatures sont stimulées à maturité, puis chargé avec des antigènes avant qu'ils ne soient réinjectées au patient.

Vidéo: 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.

Modèle murin de CD40-activation des cellules B

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

Recherche

Immunologie et infection

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.

Protocole optimisé pour une transfection efficace des cellules dendritiques, sans maturation cellulaire

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.

L&#39;isolement des cellules de souris poumon dendritiques

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.

Isolement des cellules dendritiques myéloïdes et les cellules épithéliales du thymus humain

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.

Génération de la RCA cellules T pour la thérapie adoptive dans le contexte de glioblastome norme de diligence

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.

Génération de Immature, d&#39;âge mûr et tolérogène cellules dendritiques avec Metabolic Différentes phénotypes

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 basé CFSE pour étudier la migration de la peau murine cellules dendritiques dans les ganglions lymphatiques de drainage lors de l&#39;infection avec<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.

Génération de cellules humaines dérivées de monocytes dendritiques du sang total

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.

Développement et Validation d’une seule molécule ultrasensible de tableau numérique Enzyme-linked Immunosorbent Assay for Human interféron-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.

Nouveau protocole pour la génération de cellules dendritiques immunogéniques physiologiques

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