Die Autoren bedanken sich bei Andres Salazar (Oncovir, Inc.) für das Geschenk des Poly-ICLC danken.

1. Isolation und Kryokonservierung von PBMCs 4 <ol><li> Aseptisch Spike einer der Access-Ports in der Leukapherese Tasche mit einem Plasma-Transfer-Set. Mit einem 60-ml-Spritze, übertragen Sie die Leukapherese von Patienten in eine sterile 500 ml-Flasche erhältlich. </li><li> Stellen Sie die Lautstärke des Leukapherese auf sein ursprüngliches Volumen mit Raumtemperatur RPMI 2x. Gründlich mischen. </li><li> Vorsichtig mischen die Flasche Ficoll-Paque PLUS. In 12 ml Ficoll-Paque PLUS in sterile 50 ml konischen Röhrchen. </li><li> Vorsichtig Schicht 30 ml des verdünnten Leukapherese Produkt zu jedem sterile 50 ml konischen Röhrchen mit Ficoll. Achten Sie darauf, die Schnittstelle zwischen dem Ficoll und Zellsuspension stören. </li><li> Schritte 1.3 und 1.4 bis alle leukapheresis überlagert worden ist. </li><li> Zentrifugieren Sie die 50 ml konischen Röhrchen bei 1.000 × g für 20 min bei Raumtemperatur ohne Bremse. </li><li> Sorgfältig ernten die trübe Schicht von PBMCs aus jedem Röhrchen und transfer neue sterile 50 ml Röhrchen. </li><li> In RPMI in jedes Röhrchen auf ein Endvolumen von 50 ml. Vorsichtig mischen durch Umdrehen. </li><li> Zentrifugieren Sie die Zellen bei 500 × g für 10 min bei 4 ° C mit Vollbremsung. </li><li> Entfernen Sie die Überstände aus jeder Röhre. Resuspendieren der Zellpellets aus jedem Röhrchen und fügen RPMI auf ein Gesamtvolumen von 50 ml. Vorsichtig mischen durch Umdrehen. </li><li> Zentrifugieren Sie die Zellen bei 500 × g für 6 min bei 4 ° C. </li><li> Entfernen Sie die Überstände aus jeder Röhre. Resuspendieren und bündeln die Zellsuspensionen in einem 50 ml konischen Röhrchen. </li><li> Bringen Sie die Lautstärke der gepoolten PBMCs bis zu 50 ml mit RPMI. Vorsichtig mischen durch Umdrehen. </li><li> Zentrifugieren Sie die Zellen bei 300 × g für 6 min bei 4 ° C. </li><li> Entfernen Sie den Überstand. Zellpellet in 50 ml RPMI. Vorsichtig mischen, um eine gleichmäßige Zellsuspension gewährleisten. </li><li> Zählen Sie die Anzahl von Zellen und die Lebensfähigkeit der Zellen zu bestimmen. Berechnen der Gesamtzahl lebensfähiger Zellen. </li><li> Zentrifugieren Sie die Zellen bei 300× g für 6 min bei 4 ° C. Bereiten Sie die gefrierenden Medien. Freezing Medien besteht aus 10% Dimethylsulfoxid (DMSO; Miltenyi Biotec) in humanem AB-Serum (Tal Biomedical). </li><li> Entfernen Sie den Überstand. Resuspendieren der Zellen in einer Endkonzentration von 2 × 10 8 Zellen / ml in kaltem Einfriermedien. </li><li> Als 1 ml Aliquots in 1,8 ml Kryoröhrchen. </li><li> Übertragen Sie die Kryoröhrchen in den kontrollierten Rate Gefrierfach und beginnen das Einfrieren run, Programm 1. Am Ende des Laufs, übertragen Sie die eingefrorenen Kryoröhrchen sofort in die Gasphase des flüssigen Stickstoffs Gefrierschrank. </li></ol> 2. Tag 0: Differenzierung von dendritischen Zellen aus Monozyten <ol><li> 30 ml RPMI / 1% autologem Plasma in ein steriles 50 ml konischen Röhrchen. </li><li> Tau-Aliquots von gefrorenen PBMCs durch vorsichtiges Schütteln in 37 ° C warmes Wasserbad. Wenn vollständig aufgetaut und der Inhalt der Fläschchen in den sterilen 50 ml konischen Röhrchen. Gründlich mischen. </li><li> Zentrifugieren Sie die Zellen für 6 minbei 500 × g bei Raumtemperatur. Entfernen des Überstandes und Resuspendieren der pelletierten Zellen in einem kleinen Volumen RPMI / 1% autologes Plasma. Dann fügen Sie mehr RPMI / 1% autologem Plasma auf ein Gesamtvolumen von 50 ml. Gründlich mischen. </li><li> Zentrifugieren Sie die Zellen wieder für 6 min bei 500 × g bei Raumtemperatur. Saugen Sie den Überstand und das Pellet in 50 ml RPMI / 1% autologem Plasma. Gründlich mischen. </li><li> Zählen Sie die Anzahl von Zellen und die Lebensfähigkeit der Zellen zu bestimmen. Berechnen der Gesamtzahl lebensfähiger Zellen. </li><li> Platte 1,40 x 10 8 Zellen in 40 ml RPMI / 1% autologem Plasma auf 225 cm 2 EasYFlask. Wiederholen, bis alle Zellen überzogen worden. </li><li> Inkubieren der EasyFlasks flach auf die Seite für 1 - 2 Stunden bei 37 ° C in der Gewebekultur-Inkubator mit 5% CO 2, um die Monozyten zu dem Kolben zu halten. </li><li> Wenn die Inkubation abgeschlossen ist, entfernen Sie die EasYFlask aus dem Inkubator und waschen zweimal zur Entfernung des nicht-haftenden Zellen using 30 ml vorgewärmtem RPMI. </li><li> Nach dem zweiten Waschen, 40 ml RPMI / 1% autologem Plasma mit 400-1.000 IU / ml IL-4 und 100-1000 IU / ml GM-CSF 5-8. Aus diesem Protokoll werden wir mit 400 IU / ml IL-4 und 100 IU / ml GM-CSF. </li><li> Inkubieren der EasyFlasks für 2 Tage bei 37 ° C in der Gewebekultur-Inkubator mit 5% CO 2. </li></ol> 3. Tag 2: Fütterung von dendritischen Zellen mit IL-4 und GM-CSF <ol><li> 4 ml RPMI / 1% autologem Plasma mit 400-1.000 IU / ml IL-4 und 100-1000 IU / ml GM-CSF zu jedem EasYFlask. Mix durch Verwirbelung und rockt auf seiner Seite. </li><li> Inkubieren Sie die EasyFlasks für 3 weitere Tage (bis Tag 5) bei 37 ° C in der Gewebekultur-Inkubator mit 5% CO 2. </li></ol> 4. Tag 5: Ernten von unreifen dendritischen Zellen <ol><li> Ernten Sie die Kulturen durch kräftiges Schwenken der Kolben und durch Auf-und Abpipettieren nicht-haftend und lose anhaftenden Zellen resuspendieren. Übertragen Sie diegeernteten Zellen auf neue 50 ml konischen Röhrchen. </li><li> Zentrifugieren bei 500 × g für 6 min bei Raumtemperatur. Entfernen Sie die Überstände und Zellpellet in 50 ml RPMI / 1% autologem Plasma. Gründlich mischen. </li><li> Zählen Sie die Anzahl von Zellen und die Lebensfähigkeit der Zellen zu bestimmen. Berechnen der Gesamtzahl lebensfähiger Zellen. </li><li> Zentrifugieren bei 500 × g für 6 min bei Raumtemperatur. Platte 1-2 x 10 6 Zellen pro Vertiefung in 3 ml RPMI / 1% autologem Plasma mit 400 IU / ml IL-4 und 100 IU / ml GM-CSF in 6-Well-Gewebekulturplatten. </li><li> Die Inkubation bei 37 ° C in der Gewebekultur-Inkubator mit 5% CO 2 über Nacht. </li></ol> 5. Tag 6: Reifung und Antigen Laden von dendritischen Zellen <ol><li> In 10 ug / ml KLH bis 1/3 der Vertiefungen in den 6-well Kulturplatten. Swirl die Platten zu mischen. KLH wird nur 1/3 in die Vertiefungen gegeben, weil KLH ist nur für den ersten DC-Impfstoff verwendet. Nachfolgende DC Impfstoffe conTain nur die Tumor-Antigen-Peptide. </li><li> DCs stimuliert, um zu reifen mit verschiedenen Stimuli werden. Die häufigsten Reifung Reize, die in klinischen Studien verwendet wurde, ist ein Cocktail von Zytokinen (IL-1 β, TNF und IL-6) und Prostaglandin E2 (PGE 2) 9,10. In jüngerer Zeit haben die Verwendung von Toll-like Rezeptor (TLR)-Agonisten Beliebtheit 11,12 gewonnen. In diesem Protokoll, fügen 2 mg / ml Polyinosin-Polycytidylsäure-Poly-L-Lysin Carboxymethylcellulose (Poly-ICLC) in die Vertiefungen und gut mischen. Poly-ICLC ist klinischem Formulierung von Poly-IC, die mit Poly-L-Lysin *-und Carboxymethylcellulose (hergestellt von Oncovir, Inc.) stabilisiert wird. </li><li> In 100 ug / ml lange Peptid-Antigene (NY-ESO-1 und / oder Melan-A/MART-1) bezeichnet, um ihre Brunnen, jede Vertiefung, die nur eine einzelne Peptid an Cross-Wettbewerb 13 zu vermeiden. </li><li> Die Inkubation erfolgt bei 37 ° C in der Gewebekultur-Inkubator mit 5% CO &lt;sub&gt; 2 O / N. </li></ol> 6. Tag 7: Kryokonservierung von dendritischen Zellen <ol><li> Bereiten DC Freezing Lösung mit autologen Plasma und 10% DMSO. Zentrifugieren Sie die DC Freezing Lösung bei 2.000 × g für 20 min bei 4 ° C, um Partikel zu entfernen. Überstand in ein neues 15 ml beschriftet konischen Rohr. Lagerung bei 4 ° C bis zur Verwendung. </li><li> Wenn die Inkubation über Nacht beendet ist, Ernte und Pool alle Brunnen, die DCs mit den Peptiden in 50 ml konische Röhrchen gepulst. </li><li> Zentrifugieren bei 500 × g für 6 min. Entfernen Sie die Überstände. Zellpellet in 5 ml RPMI / 1% autologem Plasma. Bringen Sie das Gesamtvolumen auf 50 ml mit RPMI / 1% autologem Plasma. Gründlich mischen. </li><li> Zählen Sie die Anzahl von Zellen und die Lebensfähigkeit der Zellen zu bestimmen. Berechnen Gesamtzahl der lebensfähigen Zellen. </li><li> Zentrifugieren bei 500 × g für 6 min bei Raumtemp. Entfernen Sie die Überstände und resuspendieren jedes Pellet in 5 ml Sterile 0,9% NaCl, USP und Transfer zum neuen 15 ml konischen Röhrchen. Bringen Sie jede Zellsuspension auf 14 ml mit steriler 0,9% NaCl, USP. Cap und durch Umdrehen mischen. </li><li> Wiederholen Sie den vorhergehenden Schritt zweimal. Nach der letzten Zentrifugation entfernen Überstände, so nahe wie möglich an den Zellpellets, ohne die Pellets. </li><li> Resuspendieren der Zellpellets in DC Freezing-Lösung bei 4 - 20 x 10 6 Zellen / ml und 1 ml Aliquot jeder 1,8 ml CryoTube Fläschchen. </li><li> Verwenden Sie den kontrollierten Rate Gefrierschrank, Programm 1, zu frieren unten die Portionen von DC Impfstoffe und übertragen sofort in der Gasphase von flüssigem Stickstoff einen Gefrierschrank für die langfristige Lagerung. </li></ol> 7. Lot Veröffentlichung Testing <ol><li> Jede Charge von DC Impfstoff muss ausgewertet werden und erfüllen spezifische Menge Release Kriterien (Tabelle 1), bevor es für die Injektion in Patienten in der Studie, in der Regel 5 bis 6 Wochen nach der Herstellung von Impfstoffen veröffentlicht. </li><li> Lebensfähigkeit:Bewerten Lebensfähigkeit des DC-Impfstoff unter Verwendung einer automatisierten Zelle Zähler. Die minimale akzeptable Rentabilität Spezifikation ist&gt; 70%. </li><li> Identität: Bewerten Sie die Identität des reifen DCs (CD11c + CD14-CD83 +) mittels Durchflusszytometrie. Der Anteil der CD11c + Zellen sollte&gt; 50% sein. Der Anteil der CD11c + und CD14 +-Zellen sollte &lt;30% und der Anteil der CD11c +, CD14-, und CD83 +-Zellen, sollte&gt; 50%. </li><li> Sterilität: Testen der DC-Impfstoff für das Vorhandensein von aeroben und anaeroben Bakterien und Pilze durch direkte Kultur und Gramfärbungen. Bewerten auf die Anwesenheit von Mycoplasma über eine direkte Kultur und DNA Fluorochrom Färbungsanalyse mit Indikator-Zelllinie. Für diese Untersuchungen ist die minimale Spezifikation festgelegt, dass kein Wachstum von allen Kulturen beobachtet. </li><li> Endotoxin: Beurteilen Sie die Endotoxingehalts mit dem kinetischen QCL-kinetischen chromogenen LAL-Test und es muss &lt;50 EU / ml sein, um die minimalen Kriterien erfüllen. </li><li&gt; Funktion: Bewerten DC-Funktion in einer gemischten Lymphozyten-Reaktion durch Inkubation mit allogenen T-Zellen. Die Anwesenheit der Proliferation im Vergleich zu unstimulierten DCs wird berichtet (Abbildung 3). </li></ol>

<ol>
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Die Autoren haben die finanzielle Unterstützung aus der folgenden erhalten: NIH (AI061684, AI071078, AI044628 und K08 AI084578 (Miller)), die Bill and Melinda Gates Foundation, Doris Duke Charitable Foundation, Cancer Research Institute, Alliance for Lupus Research und der Smaragd Foundation. Nina Bhardwaj ist ein Miterfinder von Patenten im Zusammenhang mit der Herstellung und Verwendung von dendritischen Zellen, die Immunität zu manipulieren. Die Autoren haben keine anderen relevanten Zugehörigkeiten oder finanzielle Beteiligung mit keiner Organisation oder Einrichtung mit einer finanziellen Beteiligung an oder finanzielle Konflikt mit dem Gegenstand oder Materialien im Manuskript abgesehen von denen offenbart diskutiert.

Phase-I-und-II-Studien von Monozyten-abgeleiteten DCs haben gezeigt, dass sie Immunreaktionen induzieren in Patienten jedoch klinische Erfolg wurde 1 beschränkt. Dies kann zum Teil auf den Mangel an Konsens darüber, wie die optimalen DCs für die Tumor-Immuntherapie Verwendung erzeugen. Obwohl es zahlreiche Möglichkeiten, um klinische Studien zu DCs zu erzeugen, unterscheiden sich diese Verfahren im Hinblick auf die Verwendung von Zytokinen, die Monozyten, Stimuli, um die Reifung zu induzieren, und die Met...

<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

Obwohl klinische Studien festgestellt haben, dass Antigen-beladenen DC Impfstoffe sicher und viel versprechende Therapie für Tumore 1 sind, bleibt ihre klinische Wirksamkeit festgelegt werden. Das nachfolgend beschriebene Verfahren, hergestellt gemäß Good Manufacturing-Verfahren (GMP)-Richtlinien, ist eine Optimierung der häufigste ex vivo Herstellungsverfahren zur Erzeugung einer großen Anzahl von DCs für klinische Studien 2. Unsere Methode nutzt die synthetischen TLR-Agonisten Polyinosin 3-Polycytidylsäure-Poly-L-Lysin Carboxymethylcellulose (Poly-ICLC), um die DCs zu stimulieren. Unsere früheren Studie festgestellt, dass Poly-ICLC die stärkste einzelne Reifung Stimulus für den menschlichen DCs ist, wie durch eine Hochregulation von CD83 und CD86, Induktion von Interleukin-12 (IL-12), Tumor-Nekrose-Faktor (TNF), Interferon-gamma-induzierten beurteilt Protein 10 (IP-10), interleukmin 1 (IL-1) und Typ-I-Interferonen (IFN) und minimal Interleukin 10 (IL-10) Produktion. </p&gt; DCs sind aus gefrorenem peripheren mononukleären Blutzellen (PBMCs) von Leukapherese erhalten differenziert. PBMCs durch Ficollgradientenzentrifugation isoliert und in Aliquots eingefroren. Am Tag 1 werden PBMCs aufgetaut und überzogen auf Gewebekulturflaschen für Monozyten, die auf der Kunststoffoberfläche nach 1-2 h Inkubation bei 37 ° C in der Gewebekulturinkubator haften wählen. Nach der Inkubation werden die Lymphozyten durch Waschen entfernt und die anhaftenden Monozyten werden für 5 Tage in Gegenwart von Interleukin-4 (IL-4) und Granulozyten-Makrophagen-Kolonie-stimulierender Faktor (GM-CSF) zu unreifen DC differenzieren kultiviert. Am Tag 6 werden unreife DCs mit Keyhole-Limpet-Hämocyanin (KLH)-Protein, das als eine Kontrolle für die Qualität der Impfstoff dient und die Immunogenität der Impfstoff 3 steigern gepulst. Die DCs stimuliert werden, um zu reifen, beladen mit Peptid-Antigene, und über Nacht inkubiert. Am 7. Tag wurden die Zellen gewaschen und in 1 ml eingefroren Aliquots, welche4 - 20 x 10 6 Zellen mit einer kontrollierten Rate Gefrierschrank. Lot Release-Tests für die Chargen von DCs durchgeführt und müssen Mindestanforderungen erfüllen, bevor sie in die Patienten injiziert werden.

Zwischen 10 - 20% Ausgangsmaterial PBMCs differenzieren in DCs am Ende der Kulturperiode. Reife DCs sind CD11c +, CD14-, CD83 +, CD40 + und CCR7 + (Abbildung 1). Sie exprimieren hohe Mengen von MHC-Klasse-I und-II-Molekülen und den kostimulatorischen Moleküle CD80 und CD86. Poly-ICLC auch unteren Ebenen der PDL-1 induziert zu anderen TLR-Agonisten 14 verglichen. Darüber hinaus führen diese Poly-IC-gereiften DCs sezernieren große Mengen an IL-12 (Abbildung 2 und 15,1...

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

Das am häufigsten verwendete Verfahren zum Erzeugen einer großen Anzahl von autologen dendritischen Zellen (DCs) zur Verwendung bei Tumor-Immuntherapie beschrieben. Das Verfahren nutzt IL-4 und GM-CSF zu DCs aus Monozyten differenzieren. Die unreifen DCs sind, um zu reifen und dann mit Antigenen beladen stimuliert, bevor sie zurück in den Patienten injiziert werden.

Herstellung von Tumor-Antigen-beladenen reifen dendritischen Zellen für die Immuntherapie

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 Aufrufe.  NYU Langone Medical Center. Das am häufigsten verwendete Verfahren zum Erzeugen einer großen Anzahl von autologen dendritischen Zellen (DCs) zur Verwendung bei Tumor-Immuntherapie beschrieben. Das Verfahren nutzt IL-4 und GM-CSF zu DCs aus Monozyten differenzieren. Die unreifen DCs sind, um zu reifen und dann mit Antigenen beladen stimuliert, bevor sie zurück in den Patienten injiziert werden.

Serie: 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.

Mausmodell der CD40-Aktivierung von B-Zellen

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

Forschung

Immunologie und Infektion

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.

Optimiertes Protokoll für die effiziente Transfektion von dendritischen Zellen ohne Zell-Reifung

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.

Isolierung von Maus Lung Dendritischen Zellen

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.

Isolierung von myeloider dendritischer Zellen und Epithelzellen Menschliche 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 ...

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.

Generierung von CAR T-Zellen für die adoptive Therapie im Kontext von Glioblastoma Standardtherapie

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.

Die Erzeugung von Unreife, Ältere und Tolerogene Dendritischen Zellen mit unterschiedlichen Metabolic Phänotypen

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.

Ein CFSE-basierten Assay die Migration von murinen Haut Dendritischen Zellen in Lymphknoten während der Infektion zu studieren mit<em&gt; Mycobacterium bovis</em&gt; Bazillus 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 Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Generation von diskriminierendem menschlichen monoklonalen Antikörpern aus seltenen Antigen-spezifische B-Zellen im Blut zirkulierenden

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

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.

Entwicklung und Validierung eines empfindsamen Einzelmolekül Array digitale Enzym-linked Immunosorbentprobe Assay für menschliche Interferon-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.

Neuartiges Protokoll zur Generierung physiologischer immunogener Dendritischer Zellen

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

Methods Article

Herstellung von Tumor-Antigen-beladenen reifen dendritischen Zellen für die Immuntherapie