Este trabalho foi financiado em parte pelo NIH (N01 HHSN272201100019C, U19 AI 089992, e do Programa NCRR / GCRC M01-RR00125). Os autores declaram qualquer conflito de interesse financeiros e agradecer ao Dr. Mark Shlomchik, diretor do Núcleo Facilidade Yale celular Sorter.

1. Isolamento e Estimulação de células imunes <ol><li> Obter 10-50 ml de sangue heparinizado de voluntários saudáveis ​​após consentimento informado por escrito sob as diretrizes do Conselho de Revisão Institucional locais. Para estudos de envelhecimento ou a doença de exclusão, patogênese e os critérios de inclusão para cada voluntário ou paciente deve ser explicitamente determinada. </li><li> Isolar células mononucleares do sangue periférico (PBMCs), utilizando tubos de CPT ou Ficoll-Paque Plus acordo com as instruções do fabricante (GE Healthcare, NJ) e as experiências de conduta no dia do isolamento 7 (Reagentes, Tabela 1). </li><li> Incubar PBMCs (3 x 10 6 / tubo) em tubos de 1,5 ml em 0,6 ml de meio por si só ou estimuladas com ligandos TLR ou outros agentes de activação, por exemplo, o ligando TLR7 / 8 R848 (0,6 ml de meio mais 0,6 uL de 10 mM TLR7 / 8 ligando R848 concentração final, 10 uM) a 37 ° C durante 10-60 min (InvivoGen, CA) 9. </li></ol> 2. LabEling de Células <ol><li> Linhagem celular rótulo e outros marcadores de superfície sobre a suspensão de células vivas utilizando anticorpos fluorescente-conjugados. Etiqueta 3 x 10 6 PBMCs de 20 min a 4 ° C em 100 ul de PBS / 2% de FBS em um tubo Eppendorf de 1,5 ml com um cocktail de anticorpos específicos da superfície para linhagens de monócitos ou DC (Tabela 2). Diluições de anticorpos óptimo pode ser determinado em experiências preliminares e as amostras devem ser processado em lotes para minimizar de lote para lote-variação. Para o estoque fornecedor de anticorpo de 0,1 mg / ml utilizar 5 uL de anticorpo (diluição 1:20). Use um tubo separado de células marcadas com apenas um conjugado de cor única para definir os canais fluorescentes no instrumento. </li><li> Após a marcação de superfície, fixar as células em 4% PFA / PBS durante 10 min à TA. Para armazenamento até ao momento da análise, as células de centrifugação a 500 xg e Ressuspender as células em tampão de congelação (90% de FBS contendo DMSO a 10%) a -80 ° C até que o dia do ensaio. </li><li> Para avaliação, processo de lotes of células não tratadas e estimuladas a partir de um grupo de dadores em conjunto para minimizar a variabilidade. No dia da análise, as células descongelamento rapidamente em 37 ° C banho de água, de centrifugação a 500 xg para remover o tampão de congelação. </li><li> Para permeabilizar as células para a detecção de citocinas intracelulares ou outros marcadores, suspensões de células Ressuspenda em 100 uL de tampão BD Perm / Wash (BD Biosciences, NJ). Rótulo para intracelulares componentes de sinalização com, por exemplo, diluição 1:20 de coelho anti-NF-kB anticorpo (p65) (concentração final 10 ug / ml, Santacruz Biotechnology, CA) durante 20 min a RT, de centrifugação a 500 xg, o sobrenadante aspirado e Ressuspender as células em 100 uL Perm BD / Tampão de lavagem contendo 1:250 de diluição de cabra anti-IgG de coelho-Alexa647 (Invitrogen, CA) e incubar durante 20 min à TA. </li><li> Imediatamente antes de imagiologia, contracoloração núcleos com DAPI (0,2 ug / ml, Invitrogen, CA) ou iodeto de propídio (PI; 20 ng / ml, Invitrogen, CA). </li></ol> 3. Análise ImageStream <ol><li>Conduzir imagem por ImageStream em amostras de lote para reduzir a variabilidade entre amostras humanas. As definições do aparelho para alimentação dos lasers eram como se segue: 200 mW para 488 nm, 10 mW para 658 nm e 250 mW para 405 nm. </li><li> Para analisar os arquivos de dados (Fig. 1), em primeiro lugar, portão em eventos com intensidade PI normal e alta relação de aspecto PI (largura: relação da altura) para distinguir células individuais (R1) de detritos (PI intensidade baixa) ou multi-celulares (eventos PI intensidade alta e baixa relação de aspecto). Os monócitos são dentro de um portão de células CD14 positivas. mDCs estão dentro de uma porta para as células que são altos para CD11c e de baixo para CD4 (R2) e também para baixo Lin-1 marcadores (R3). </li><li> Use IDEAS software (Amnis, WA) para fornecer uma medida eficaz quantitativa do grau de activação de cada célula. Determinar semelhança (ou co-localização) do factor de transcrição NF-kB citoplasmática com corante nuclear PI para indicar translocação para o núcleo (R4). A alta correlação de NF-κB/PI localização reflecte-se numa pontuação de semelhança elevado e indica o grau de activação 10. </li><li> Comparar os índices de similaridade entre os grupos de tratamento diferentes ou populações de doentes para indicar a eficiência relativa funcional das células. Quantificar os dados entre as amostras através da comparação estatística dos valores absolutos ou diferenças de dobra. Use software IDEAS para exibir imagens de células a partir de segmentos de chaves de histogramas da população (Fig. 2). </li></ol> 4. Os resultados representativos Temos quantificada vias de sinalização em resposta a um ligando modelo viral, estimulando PBMC com o ligando TLR7 / 8, R848 (Fig. 1). Foram coletadas duas imagens de localização celular e estatísticas populacionais em grupos de nossa amostra (Fig. 2). ImageStream nos permite subpopulações de células porta diretamente de PBMCs e respostas celulares de imagem de populações de células fechadas, mas não classificado. Aqui nós quantificamos o efeito de TLR signaling na localização nuclear do NF-kB (p65) em Lin1, CD4 dim, CD11c + mielóide DCs (mDCs). Na linha de base, observou-se uma percentagem elevada de células não estimuladas com o factor de transcrição NF-kB (p65) no citoplasma. Após a estimulação, as células tratadas têm uma pontuação de semelhança significativamente maior de NF-B (p65) com PI mancha nuclear, indicando que o NF-kB (p65) translocado para o núcleo, após a estimulação (pontuações de similaridade mediana são 1,52 e 3,01, respectivamente, a fig. 2). Resultados semelhantes são observados nas TLR5 monócitos estimulados 9. <img alt="A Figura 1" src="/files/ftp_upload/3741/3741fig1.jpg" /> Figura 1. Coloração e Estratégia Gating para quantificar os efeitos da estimulação em mDCs humanos. O factor de transcrição NF-kB regula a expressão de numerosos genes do sistema imunológico. Este estudo mede a localização nuclear de NF-kB (p65) em mDCs de um sujeito representativo após TLR7 / 8 liga<html> estimulação nd. Em foco células individuais foram identificados pela passagem no PI eventos positivos, com altas proporções nucleares (R1). mDCs (Lin1, CD4dim, CD11c +) foram fechado em R3. Localização nuclear do NF-kB (p65) é representada em R4. <a href="http://www.jove.com/files/ftp_upload/3741/3741fig1large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img alt="A Figura 2" src="/files/ftp_upload/3741/3741fig2.jpg" /> Figura 2. . Translocação de NF-kB em mDCs após a estimulação A translocação de NF-kB (p65) para dentro do núcleo após a estimulação (R4) é descrito em populações de mDCs não tratadas e estimuladas; a pontuação de semelhança mediana é 1,52 na amostra não tratada e 3,01 em a amostra estimulado (A). As imagens digitais colhidas simultaneamente das populações de células não tratadas ou R848-estimulado mostram as células representativas e da intensidade do NF-B translocado para o núcleo da célula (B)._upload/3741/3741fig2large.jpg &quot;target =&quot; _blank &quot;&gt; Clique aqui para ver maior figura.

<ol>
	<li>Linton, P. J., Dorshkind, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+lymphocyte+development+and+function.">Age-related changes in lymphocyte development and function.</a> Nat. Immunol. 5, 133-139 (2004).</li><li>Shaw, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+Human+Toll-like+Receptor+Function+in+Aging.">Dysregulation of Human Toll-like Receptor Function in Aging.</a> Ageing Research Reviews. 10, 346-353 (2011).</li><li>Medzhitov, R. <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+receptors+and+innate+immunity.">Toll-like receptors and innate immunity.</a> Nature Rev. Immunol. 1, 135-145 (2001).</li><li>van Duin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+Defect+in+Human+TLR1+Function+and+Expression.">Age-associated Defect in Human TLR1 Function and Expression.</a> J. Immunol. 178, 970-975 (2007).</li><li>Panda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+decrease+in+TLR+function+in+primary+human+dendritic+cells+predicts+influenza+vaccine+response.">Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response.</a> J. Immunol. 184, 2518-2527 (2010).</li><li>Kong, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+TLR3+impairs+the+innate+immune+response+to+West+Nile+virus+in+the+elderly.">Dysregulation of TLR3 impairs the innate immune response to West Nile virus in the elderly.</a> J. Virol. 82, 7613-7623 (2008).</li><li>Qian, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+interferon+signaling+in+dendritic+cells+from+older+donors+infected+in+vitro+with+West+Nile+virus.">Impaired interferon signaling in dendritic cells from older donors infected in vitro with West Nile virus.</a> J. Infect. Dis. 203, 1415-1424 (2011).</li><li>Hayden, M. S., Ghosh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappaB+in+immunobiology.">NF-kappaB in immunobiology.</a> Cell research. 21, 223-244 (2011).</li><li>Qian, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-associated+elevation+in+TLR5+leads+to+increased+inflammatory+responses+in+the+elderly.">Age-associated elevation in TLR5 leads to increased inflammatory responses in the elderly.</a> Aging Cell. 11, 104-110 (2011).</li><li>George, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+nuclear+translocation+events+using+similarity+analysis+of+multispectral+cellular+images+obtained+in+flow.">Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow.</a> Journal of immunological. 311, 117-129 (2006).</li></ol>

Não há conflitos de interesse declarados.

Os passos críticos no uso de ImageStream para estudos translacionais são a seleção de grupos de comparação relevantes e otimização e validação da especificidade do anticorpo. As diferenças entre os grupos de indivíduos no alvo marcada serão facilmente aparentes através de histogramas e imagens de células. Em combinação com uma análise aprofundada das alterações nos receptores e vias de sinalização relevantes, estes dados fornecerá informações valiosas sobre os mecanismos que estão subjacentes a...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Ficoll-Paque Plus </td><td> GE Healthcare </td><td> 17-1440-03 </td></tr><tr><td> R848 </td><td> Invivogen </td><td> tlrl-r848 </td></tr><tr><td> Paraformaldeído (16%) </td><td> Electron Microscopy Sciences </td><td> 15710 </td></tr><tr><td> BD Perm / Wash </td><td> BD Biosciences </td><td> 554723 </td></tr><tr><td> CD14-PE </td><td> eBioscience </td><td> 12-0149-41 </td></tr><tr><td> CD4-PacBlue </td><td> BD Pharmingen </td><td> 68436 </td></tr><tr><td> Cocktail Lineage 1 (lin 1)-FITC </td><td> BD Biosciences </td><td> 340546 </td></tr><tr><td> CD11c-PE </td><td> BD Biosciences </td><td> 347637 </td></tr><tr><td> CD123-PE </td><td> BD Biosciences </td><td> 340545 </td></tr><tr><td> de coelho anti-NF-kB (p65) </td><td> Santacruz </td><td> SC-372 </td></tr><tr><td> Alexa647 F (ab &#39;) 2 de cabra anti-IgG de coelho </td><td> Invitrogen </td><td> A21246 </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D21490 </td></tr><tr><td> ImageStream X </td><td> Amnis </td><td></td></tr></tbody></table> Tabela 1. Reagentes específicos e equipamentos. <table border="1"><tbody><tr><td> Tipo de célula </td><td> FITC </td><td> PE </td><td> Alexa 647 </td><td> PacBlue </td></tr><tr><td> Monócitos </td><td></td><td> CD14 </td><td> NF-kB (p65) </td><td></td></tr><tr><td> MDC </td><td> Lin1 </td><td> CD11c </td><td> NF-kB (p65) </td><td> CD4 </td></tr><tr><td> pDC </td><td> Lin1</td><td> CD123 </td><td> NF-kB (p65) </td><td> CD4 </td></tr></tbody></table> Tabela 2 painel de anticorpos. Para a coloração de células de linhagem de vias de sinalização Monócitos: CD14 + MDC: Lin1, CD4dim, CD11c + PDC: Lin1, CD4dim, CD123 + Lin1 marcador (linhagem) inclui CD3, CD14, CD19, CD20, CD56 marcadores para monócitos, células NK, células T e B.

quantitative imaging of lineage-specific toll-like receptor-mediated signaling in monocytes and dendritic cells from small samples of human blood

As variações individuais no estado imunitário determinar as respostas à infecção e contribuir para a gravidade da doença eo resultado. O envelhecimento está associado com um aumento da susceptibilidade a infecções virais e bacterianas e diminuiu a capacidade de resposta para vacinas com um declínio bem documentado na humoral assim como mediada por células 1,2 respostas imunes. Recentemente, avaliou os efeitos do envelhecimento sobre receptores Toll-like (TLRs), os principais componentes do sistema imune inato que detectar a infecção microbiana e desencadear respostas de acolhimento de defesa antimicrobianos 3. Em uma grande coorte de doadores humanos saudáveis, nós mostramos que os monócitos do sangue periférico de idosos têm diminuição da expressão e função de determinados TLRs 4 e semelhantes níveis reduzidos TLR e respostas de sinalização em células dendríticas (DCs), células apresentadoras de antígeno que são fundamentais na a ligação entre a imunidade inata e adaptativa 5. Nós mostramos desregulação do TLR3 em macrófagos umad menor produção de IFN por DCs de doadores idosos em resposta à infecção com o vírus do Nilo Ocidental 6,7. Primordial para nossa compreensão da imunossenescência e à intervenção terapêutica é um entendimento detalhado dos tipos de células específicos em resposta e do mecanismo (s) de transdução de sinal. Estudos tradicionais de respostas imunes através de imagens de pilhas e marcadores de células de levantamento por FACS ou immunoblot têm avançado significativamente a nossa compreensão, no entanto, estes estudos são geralmente limitados tecnicamente pelo pequeno volume de amostra disponível a partir de pacientes e da incapacidade de realizar técnicas laboratoriais complexas em vários amostras humanas. ImageStream combina citometria de fluxo quantitativa simultânea com alta resolução de imagem digital e, assim, facilita investigação em populações de células múltiplas contemporaneamente para uma captura eficiente do susceptibilidade do paciente. Aqui temos demonstram o uso de ImageStream em DCs para avaliar TLR7 / 8activação mediada-aumentos na fosforilação e translocação nuclear de um factor de transcrição de chave, NF-kB, a qual inicia a transcrição de numerosos genes que são críticas para as respostas imunes 8. Usando esta tecnologia, temos também demonstrou recentemente uma alteração previamente não reconhecido de TLR5 sinalização e na via de NF-kB em monócitos de doadores mais velhos que possam contribuir para a resposta imune alterada no envelhecimento 9.

Watch this Scientific Journal Video about Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood at JoVE.com

Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Descrevemos uso da tecnologia de ImageStream (<a href="http://www.amnis.com/" target="_blank"&gt; Www.amnis.com</a&gt;), Que combina a citometria de fluxo quantitativa simultânea com alta resolução de imagem digital, para quantificar os mecanismos celulares primárias de células imunes de coortes bem definidas do paciente. Nossos estudos fornecem um modelo para investigações de translação para quantificar linhagem específica respostas celulares em pequenas amostras de coortes de indivíduos.

Imagem quantitativa da linhagem específica Toll-like receptor-mediados sinalização em monócitos e células dendríticas a partir de pequenas amostras de sangue humano

Individual variations in immune status determine responses to infection and contribute to disease severity and outcome.  Aging is associated with an ...

quantitative-imaging-lineage-specific-toll-like-receptor-mediated

Research

JoVE Journal

Immunology and Infection

35.0K visualizações.  Yale University School of Medicine. Descrevemos uso da tecnologia de ImageStream ( Www.amnis.com), Que combina a citometria de fluxo quantitativa simultânea com alta resolução de imagem digital, para quantificar os mecanismos celulares primárias de células imunes de coortes bem definidas do paciente. Nossos estudos fornecem um modelo para investigações de translação para quantificar linhagem específica respostas celulares em pequenas amostr...

Vídeo: Quantitative Imaging of Lineage-specific Toll-like Receptor-mediated Signaling in Monocytes and Dendritic Cells from Small Samples of Human Blood

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

Detecção de Atividade Neu1 sialidase em Regulação Toll-like receptor de ativação

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Imunologia e Infecção

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 Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Geração de Células T humanas alo-específicos a partir de Sangue Periférico

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Isolamento de Murino macrófagos peritoniais de Cumprir a análise de expressão gênica Após Toll-like receptors Estimulação

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

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 ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Uso de um monócito monocamada Ensaio para Avaliar a fagocitose mediada pelo receptor Fcy

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran aumenta a eficiência de Lentivirus transdução das células NK primário murino e humano

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Imagens ao vivo de antifúngicos Atividade por neutrófilos humanos primários e monócitos em resposta à<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Isolamento de células dendríticas do tracto reprodutivo feminino humano para estudos fenotípica e funcionais

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

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 ...