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 Plus em tubo de 50 ml estéril. </li><li> Suavemente camada de 30 ml do produto de leucaferese diluído a cada tubo de 50 ml estéril contendo Ficoll. Ter cuidado para não perturbar a interface entre o Ficoll e suspensão de células. </li><li> Repita os passos 1.3 e 1.4 até que toda a leucaferese foram mergulhados. </li><li> Centrifugar os tubos cónicos de 50 ml a 1000 × g durante 20 minutos à temperatura ambiente sem travagem. </li><li> Colher cuidadosamente a camada turva de PBMCs de cada tubo e transfer para novos tubos estéreis de 50 ml. </li><li> Adicionar RPMI a cada tubo para um volume final de 50 ml. Misture delicadamente por inversão. </li><li> Centrifugar as células a 500 x g durante 10 min a 4 ° C, com travão integral. </li><li> Remove-se os sobrenadantes de cada tubo. Voltar a suspender as pelotas de células de cada tubo e adicionar meio RPMI para um volume final de 50 ml. Misture delicadamente por inversão. </li><li> Centrifugar as células a 500 x g durante 6 min a 4 ° C. </li><li> Remove-se os sobrenadantes de cada tubo. Ressuspender piscina e juntas as suspensões de células em um tubo de 50 ml. </li><li> Levar o volume das CMSPs reunidas até 50 ml com mais meio RPMI. Misture delicadamente por inversão. </li><li> Centrifugar as células a 300 x g durante 6 min a 4 ° C. </li><li> Remover o sobrenadante. Ressuspender o sedimento celular em 50 ml de RPMI. Misture com cuidado para assegurar a suspensão de células uniforme. </li><li> Conte o número de células e determinar a viabilidade celular. Calcular o número total de células viáveis. </li><li> Centrifugar as células em 300× g durante 6 min a 4 ° C. Prepare a mídia de congelamento. Meio de congelação é constituída de 10% de dimetilsulfóxido (DMSO; Miltenyi Biotec) em soro AB humano (Vale Biomedical). </li><li> Remover o sobrenadante. Ressuspender as células a uma concentração final de 2 x 10 8 células / ml em meio de congelamento frio. </li><li> Faça alíquotas de 1 ml em criotubos de 1,8 ml. </li><li> Transfira os criotubos no congelador de taxa controlada e começar a correr congelamento, Programa 1. No final da corrida, a transferência dos criotubos congelados imediatamente para a fase de vapor do congelador de azoto líquido. </li></ol> 2. Dia 0: diferenciação de células dendríticas a partir de monócitos <ol><li> Adicionar 30 ml de RPMI / 1% de plasma autólogo para um tubo de 50 ml estéril. </li><li> Alíquotas descongelamento de PBMCs congelados por agitação suave no banho de água 37 ° C. Quando completamente descongelado, transferir os conteúdos dos frascos com o tubo de 50 ml estéril. Misture bem. </li><li> Centrifugar as células de 6 mina 500 x g à temperatura ambiente. Remover o sobrenadante e ressuspender as células sedimentadas num pequeno volume de RPMI / 1% de plasma autólogo. Em seguida, adicionar mais RPMI / 1% de plasma autólogo até um volume final de 50 ml. Misture bem. </li><li> Centrifugar as células mais uma vez durante 6 minutos a 500 x g à temperatura ambiente. Aspirar o sobrenadante e ressuspender o pellet em 50 ml de RPMI / 1% de plasma autólogo. Misture bem. </li><li> Conte o número de células e determinar a viabilidade celular. Calcular o número total de células viáveis. </li><li> Placa de 1,40 x 10 8 células em 40 ml de RPMI / 1% de plasma autólogo para 225 centímetros 2 EasyFlask. Repetir até que todas as células foram plaqueadas. </li><li> Incubar as EasyFlasks plana no seu lado para 1-2 horas a 37 ° C na incubadora de cultura de tecidos contendo 5% de CO2 para permitir que os monócitos aderir ao balão. </li><li> Quando a incubação estiver completa, remover o EasyFlask da incubadora e lavar duas vezes para remover as células não aderentes using 30 ml de meio RPMI pré-aquecido. </li><li> Após a segunda lavagem, adicionar 40 ml de RPMI / 1% de plasma autólogo, com 400-1.000 UI / ml de IL-4 e 100-1.000 UI / ml de GM-CSF 5-8. Para este protocolo, nós estamos usando 400 UI / ml IL-4 e 100 UI / ml de GM-CSF. </li><li> Incubar as EasyFlasks durante 2 dias a 37 ° C na incubadora de cultura de tecidos contendo 5% de CO 2. </li></ol> 3. Dia 2: A alimentação de células dendríticas com IL-4 e GM-CSF <ol><li> Adicionar 4 ml de RPMI / 1% de plasma autólogo, com 400-1.000 UI / ml de IL-4 e 100-1.000 UI / ml de GM-CSF para cada EasyFlask. Misture agitando e balançando ao seu lado. </li><li> Incubar as EasyFlasks para mais 3 dias (até ao dia 5), a 37 ° C na incubadora de cultura de tecidos contendo 5% de CO 2. </li></ol> 4. Dia 5: Colheita de células dendríticas imaturas <ol><li> Colher as culturas agitando vigorosamente os frascos e pipetando cima e para baixo para voltar a suspender as células não aderentes e pouco aderente. Transferir océlulas colhidas para novos 50 ml tubos cônicos. </li><li> Centrifugar os tubos a 500 x g durante 6 min à temperatura ambiente. Remover o sobrenadante e ressuspender o sedimento celular em 50 ml de RPMI / 1% de plasma autólogo. Misture bem. </li><li> Conte o número de células e determinar a viabilidade celular. Calcular o número total de células viáveis. </li><li> Centrifugar os tubos a 500 x g durante 6 min à temperatura ambiente. Placa de 1-2 x 10 6 culas por po em 3 ml de RPMI / 1% de plasma autólogo, com 400 UI / ml de IL-4 e 100 UI / ml de GM-CSF em placas de cultura de tecidos de 6 poços. </li><li> Incubar as placas a 37 ° C na incubadora de cultura de tecidos contendo 5% de CO 2 durante a noite. </li></ol> 5. Dia 6: Maturação e Antígeno Carregando de células dendríticas <ol><li> Adicionam-se 10 ug / ml de KLH a 1/3 dos poços das placas de cultura de 6 poços. Redemoinho as placas para misturar. KLH só é adicionada a 1/3 dos poços KLH porque é usado apenas para a primeira vacina DC. Subseqüente DC vacinas conTain apenas os péptidos de antigénios tumorais. </li><li> DCs podem ser estimuladas a amadurecer com diferentes estímulos. Os estímulos de maturação mais comum que tem sido usado em estudos clínicos, tem sido um cocktail de citocinas (IL-1 β, TNFa e IL-6) e prostaglandina E2 (PGE2) 9,10. Mais recentemente, a utilização de agonistas do receptor do tipo Toll (TLR) ganharam popularidade 11,12. Neste protocolo, adicionar 2 mg / ml de carboximetilcelulose em ácido poli-L-lisina-Polyinosinic Polycytidylic (Poli-ICLC) aos poços e misturar bem. Poli-ICLC é a formulação clínica grau de poli-CI que é estabilizado com poli-L-*-lisina e carboximetilcelulose (fabricado pela Oncovir, Inc.). </li><li> Adicionam-se 100 ug / ml de antigénios peptídicos longas (NY-ESO-1 e / ou Melan-A/MART-1) designados para os seus poços, cada poço recebe apenas um único péptido a evitar competição cruzada 13. </li><li> Incubar as placas a 37 ° C na incubadora de cultura de tecidos contendo 5% de CO &lt;sub&gt; 2 S / N. </li></ol> 6. Dia 7: Criopreservação de células dendríticas <ol><li> Preparar a solução de congelamento DC usando plasma autólogo e 10% DMSO. Centrifugar a solução de congelação DC a 2000 × g durante 20 min a 4 ° C para remover qualquer matéria em partículas. Transferir o sobrenadante para um novo tubo de 15 ml rotulados cónica. Armazenar a 4 ° C até utilização. </li><li> Quando a incubação durante a noite está terminado, a colheita e piscina todos os poços que contêm as DCs pulsadas com péptidos em tubos cónicos de 50 ml. </li><li> Centrifugar os tubos a 500 × g por 6 min. Remove-se os sobrenadantes. Ressuspender o sedimento de células em 5 ml de RPMI / 1% de plasma autólogo. Levar o volume total a 50 ml com RPMI / 1% de plasma autólogo. Misture bem. </li><li> Conte o número de células e determinar a viabilidade celular. Calcular o número total de células viáveis. </li><li> Centrifugar os tubos a 500 × g por 6 min à temperatura ambiente. Remover o sobrenadante e ressuspender cada pellet em 5 ml sterile 0,9% de NaCl, USP e transferência para novos tubos de 15 mL cónicos. Traga a cada suspensão de células para 14 ml com mais estéril a 0,9% de NaCl, USP. Tampar e misturar por inversão. </li><li> Repetir etapa anterior por duas vezes. Após a última centrifugação, remove-se os sobrenadantes, ficando tão próximo quanto possível para as pelotas de células, sem perturbar os sedimentos. </li><li> Ressuspender as células em peletes DC Solução de congelação em 4 - 20 x 10 6 células / ml e alíquotas de 1 ml a cada frasco 1,8 ml de tubo criogénico. </li><li> Usar o congelador de taxa controlada, um programa, para congelar as alíquotas de vacinas DC e transferir imediatamente para a fase de vapor de um congelador de azoto líquido durante a armazenagem a longo prazo. </li></ol> 7. Lote testes de liberação <ol><li> Cada lote de vacina DC devem ser avaliados e atender aos critérios de liberação de lote específicos (Tabela 1), antes de ser liberado para injeção em pacientes no estudo, tipicamente de 5 a 6 semanas após a preparação da vacina. </li><li> Viabilidade:Avaliar a viabilidade da vacina DC utilizando um contador de células automatizado. A especificação mínima aceitável é de viabilidade&gt; 70%. </li><li> Identidade: Avaliar a identidade do DCs maduras (CD11c + CD14-CD83 +) por citometria de fluxo. A percentagem de células + CD11c deve ser&gt; 50%. A percentagem de células CD14 + e CD11c + deve ser &lt;30%, e a percentagem de CD11c +, CD14-e células CD83 + devem ser&gt; 50%. </li><li> Esterilidade: Testar a vacina DC para a presença de bactérias e fungos anaeróbio e aeróbio por cultura direta e manchas de grama. Avaliar a presença de micoplasma usando um sistema de cultura directa e ADN fluorocromo Ensaio de Coloração com a linha celular indicadora. Para estes testes, o conjunto mínimo de especificação é que não se observa o crescimento de todas as culturas. </li><li> A endotoxina: Avaliar o teor de endotoxina utilizando o ensaio de LAL cromogénico cinético Kinetic QCL-e que deve ser &lt;50 EU / ml para satisfazer os critérios mínimos. </li><li&gt; Função: Avaliar a função DC em uma reação mista de linfócitos através da incubação com as células T alogênicas. A presença de proliferação em comparação com DCs não estimuladas é reportado (Figura 3). </li></ol>

<ol>
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Os autores receberam apoio financeiro a partir do seguinte: NIH (AI061684, AI071078, AI044628 e K08 AI084578 (Miller)), a Fundação Bill e Melinda Gates, Doris Duke Charitable Foundation, Instituto de Pesquisa do Câncer, Alliance for Lupus Research, eo Emerald Foundation. Nina Bhardwaj é um co-inventor em patentes relacionadas com a preparação e utilização de células dendríticas para manipular a imunidade. Os autores não têm outras afiliações relevantes ou envolvimento financeiro com qualquer organização ou entidade com interesse financeiro em ou conflito financeiro com o assunto ou materiais discutidos no manuscrito além daqueles divulgados.

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 ...

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

Research

JoVE Journal

Immunology and Infection

18.1K visualizações.  NYU Langone Medical Center. 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.

Vídeo: 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.

Modelo murino de CD40-ativação de células B

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

Imunologia e Infecção

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.

Protocolo otimizado para transfecção eficiente de células dendríticas, sem maturação das células

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

Isolation of Mouse Lung Dendritic Cells

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

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

Isolamento de células dendríticas pulmonares do rato

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.

O isolamento de células dendríticas mielóides e células epiteliais de timo humano

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.

Geração de células T para CAR adoptivo Therapy no Contexto do Glioblastoma Padrão de Atendimento

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.

Geração de imatura, madura e tolerogênicas células dendríticas com diferentes fenótipos Metabolic

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.

Um ensaio baseado em CFSE para estudar a migração de Murino pele as células dendríticas em drenagem gânglios linfáticos durante a infecção com<em&gt; Mycobacterium bovis</em&gt; Bacilo de 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.

Geração de células dendríticas derivadas de monócitos humanos a partir de sangue inteiro

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.

Desenvolvimento e validação de uma única molécula ultra-sensível de matriz Digital ensaio imunossorvente ligado à enzima para humana 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.

Novo protocolo para a geração de células dendríticas imunogênicas fisiológicas

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