Name: preparation of tumor antigen-loaded mature dendritic cells for immunotherapy
Uploaded: 2013-08-01T12:00:00
Description: Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

Os autores gostariam de agradecer Andres Salazar (Oncovir, Inc.) para o dom do Poly-ICLC.

1. O isolamento e a criopreservação de PBMCs 4 <ol><li> Assepticamente espiga de uma das portas de acesso na bolsa leucaferese utilizando um conjunto de transferência de plasma. Usando uma seringa de 60 ml, a transferência da leucaferese obtidos de pacientes em um frasco de 500 ml estéril. </li><li> Ajustar o volume da leucaferese para 2x o seu volume original usando RPMI temperatura ambiente. Misture bem. </li><li> Misture delicadamente a garrafa de Ficoll-Paque PLUS. Adicionar 12 ml de Ficoll-Paque P...

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B., Bhardwaj, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+agonists:+are+they+good+adjuvants?.">Toll-like receptor agonists: are they good adjuvants?.</a> Cancer J. 16, 382-391 (2010).</li><li>Kedl, R. M., Kappler, J. W., Marrack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+dominance,+competition+and+T+cell+affinity+maturation.">Epitope dominance, competition and T cell affinity maturation.</a> Curr. Opin. Immunol. 15, 120-127 (2003).</li><li>Bogunovic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR4+engagement+during+TLR3-induced+proinflammatory+signaling+in+dendritic+cells+promotes+IL-10-mediated+suppression+of+antitumor+immunity.">TLR4 engagement during TLR3-induced proinflammatory signaling in dendritic cells promotes IL-10-mediated suppression of antitumor immunity.</a> Cancer Research. 71, 5467-5476 (2011).</li><li>Verdijk, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyriboinosinic+polyribocytidylic+acid+(poly(I:C))+induces+stable+maturation+of+functionally+active+human+dendritic+cells.">Polyriboinosinic polyribocytidylic acid (poly(I:C)) induces stable maturation of functionally active human dendritic cells.</a> J. Immunol. 163, 57-61 (1999).</li><li>Rouas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(I:C)+used+for+human+dendritic+cell+maturation+preserves+their+ability+to+secondarily+secrete+bioactive+IL-12.">Poly(I:C) used for human dendritic cell maturation preserves their ability to secondarily secrete bioactive IL-12.</a> Int. Immunol. 16, 767-773 (2004).</li><li>Jongmans, W., Tiemessen, D. M., van Vlodrop, I. J., Mulders, P. F., Oosterwijk, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th1-polarizing+capacity+of+clinical-grade+dendritic+cells+is+triggered+by+Ribomunyl+but+is+compromised+by+PGE2:+the+importance+of+maturation+cocktails.">Th1-polarizing capacity of clinical-grade dendritic cells is triggered by Ribomunyl but is compromised by PGE2: the importance of maturation cocktails.</a> J. Immunother. 28, 480-487 (2005).</li><li>Krause, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E2+is+a+key+factor+for+monocyte-derived+dendritic+cell+maturation:+enhanced+T+cell+stimulatory+capacity+despite+IDO.">Prostaglandin E2 is a key factor for monocyte-derived dendritic cell maturation: enhanced T cell stimulatory capacity despite IDO.</a> J. Leukoc. Biol. 82, 1106-1114 (2007).</li><li>Morelli, A. E., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+under+the+spell+of+prostaglandins.">Dendritic cells under the spell of prostaglandins.</a> Trends Immunol. 24, 108-111 (2003).</li><li>Adams, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+(DC)+based+therapy+for+cervical+cancer:+use+of+DC+pulsed+with+tumour+lysate+and+matured+with+a+novel+synthetic+clinically+non-toxic+double+stranded+RNA+analogue+poly+[I]:poly+[C(12)U]+(Ampligen+R).">Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)U] (Ampligen R).</a> Vaccine. 21 (12), 787-790 (2003).</li><li>Colombo, M. P., Trinchieri, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-12+in+anti-tumor+immunity+and+immunotherapy.">Interleukin-12 in anti-tumor immunity and immunotherapy.</a> Cytokine Growth Factor Rev. 13, 155-168 (2002).</li><li>Mayordomo, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+dendritic+cells+pulsed+with+synthetic+tumour+peptides+elicit+protective+and+therapeutic+antitumour+immunity.">Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.</a> Nat Med. 1, 1297-1302 (1995).</li><li>Dhodapkar, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+generation+of+broad+T-cell+immunity+in+humans+after+a+single+injection+of+mature+dendritic+cells.">Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.</a> J. Clin. Invest. 104, 173-180 (1999).</li><li>Schuler-Thurner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+induction+of+tumor-specific+type+1+T+helper+cells+in+metastatic+melanoma+patients+by+vaccination+with+mature,+cryopreserved,+peptide-loaded+monocyte-derived+dendritic+cells.">Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells.</a> J. Exp. Med. 195, 1279-1288 (2002).</li></ol>

Fase I e II de ensaios clínicos de DCs derivadas de monócitos têm mostrado que induzem respostas imunitárias em pacientes, no entanto o sucesso clínico tem sido limitada 1. Isto pode ser em parte devido à falta de consenso sobre a forma de gerar os DCs óptimas para a utilização de imunoterapia tumoral. Embora haja várias maneiras de gerar DCs clínico grau, estes métodos variam em termos do uso de citocinas utilizadas para diferenciar os monócitos, os estímulos utilizados para induzir a maturaç?...

<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

Embora estudos clínicos têm demonstrado que as vacinas DC antígeno-carregados são seguros e promissora terapia para tumores 1, sua eficácia clínica ainda não foi estabelecida. O método descrito abaixo, preparadas de acordo com as Boas Processo de Fabricação (GMP), é uma otimização do método de preparação ex vivo mais comum para a geração de um grande número de DCs para estudos clínicos 2. Nosso método utiliza o TLR sintético 3 agonista Carboxymethylcellulose ácido poli-L-lisina Polyinosinic-Polycytidylic (Poly-ICLC) para estimular os DCs. O nosso estudo anterior estabeleceu que a poli-ICLC é o estímulo da maturação individual mais potente para DCs humanas, tal como avaliado por uma sobre-regulação de CD83 e CD86, a indução da interleucina-12 (IL-12), factor de necrose tumoral (TNF), o interferão-gama induzido proteína 10 (IP-10), interleukmin 1 (IL-1), e os interferões tipo I (IFN), e interleucina mínima 10 (IL-10) de produção. </p&gt; DCs são diferenciados a partir de células mononucleares do sangue periférico (PBMC congelados), obtidos por leucaferese. As PBMC são isoladas por centrifugação em gradiente de Ficoll e congelado em alíquotas. No dia 1, as CMSPs são descongelados e semeados em frascos de cultura de tecido para seleccionar os monócitos que aderiram à superfície plástica após 1-2 horas de incubação a 37 ° C na incubadora de cultura de tecido. Após a incubação, os linfócitos são lavados e os monócitos aderentes foram cultivadas durante 5 dias na presença de interleucina-4 (IL-4) e de granulócitos factor de estimulação de colónias de macrófagos (GM-CSF) para se diferenciarem para DCs imaturas. No dia 6, DCs imaturas são pulsadas com a proteína hemocianina de lapa buraco de fechadura (KLH), que serve como um controlo para a qualidade da vacina e pode aumentar-se a imunogenicidade da vacina contra a 3. As DCs são estimuladas a amadurecer, carregado com antígenos peptídeos, e incubadas durante a noite. No dia 7, as células são lavadas, e congelado em alíquotas de 1 ml contendo4-20 x 10 6 células, utilizando um congelador de taxa controlada. Testes de liberação de lote para os lotes de DCs é realizada e deve atender a especificações mínimas, antes de serem injetadas em pacientes.

Entre 10 - 20% a partir de PBMCs diferenciarem em DCs no final do período de cultura. DCs maduras são CD11c +, CD14-, CD83 +, CD40 + e CCR7 + (Figura 1). Eles expressam altos níveis de moléculas MHC classe II, as moléculas co-estimulatórias CD80 e CD86 e eu e. Poli-ICLC também induziu níveis baixos de PDL-1, em comparação com outros agonistas de TLR 14. Além disso, estas DCs segregam grandes quantidades de poli-IC-maturados de IL-12 (Figura 2 e 15,16) e ...

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

O método mais vulgarmente usado para a geração de grandes números de células dendríticas autólogas (DCs), para uso na imunoterapia de tumores é descrita. O método utiliza a IL-4 e GM-CSF a partir de monócitos diferenciam DCs. As DC imaturas são estimuladas a maduro e, em seguida, carregado com os antigénios, antes de serem injectadas de novo no paciente.

Preparação do antigénio tumoral-carregados células dendríticas maduras para a imunoterapia

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

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