Ce travail a été soutenu en partie par le NIH (N01 HHSN272201100019C, U19 AI 089992, et le Programme NCRR / GCRC M01-RR00125). Les auteurs déclarent pas d&#39;intérêts financiers concurrents et à remercier le Dr Mark Shlomchik, directeur de l&#39;établissement de cellules de base de Yale trieuse.

1. L&#39;isolement et la stimulation des cellules immunitaires <ol><li> Obtenir 10-50 ml de sang hépariné des volontaires sains après consentement éclairé écrit en vertu des lignes directrices de l&#39;Institutional Review Board locale. Pour les études de vieillissement ou la maladie de la pathogenèse, l&#39;exclusion et les critères d&#39;inclusion pour chaque volontaire ou le patient doit être explicitement déterminée. </li><li> Isoler les cellules mononucléées du sang périphérique (CMSP) en utilisant des tubes ou CPT Ficoll-Paque Plus conformément aux instructions du fabricant (GE Healthcare, NJ) et mener des expériences sur le jour de l&#39;isolement 7 (réactifs, tableau 1). </li><li> Incuber PBMC (3 x 10 6 / tube) dans des tubes de 1,5 ml à 0,6 ml de milieu seul ou stimulés par les ligands de TLR ou des agents d&#39;activation d&#39;autres, par exemple le ligand TLR7 / 8 R848 (0,6 ml de milieu plus 0,6 ul de 10 mM TLR7 / 8 ligand R848, la concentration finale de 10 pM) à 37 ° C pendant 10-60 min (InvivoGen, CA) 9. </li></ol> 2. LaboratoireEling des cellules <ol><li> Lignée cellulaire d&#39;étiquettes et d&#39;autres marqueurs de surface sur les suspensions de cellules vivantes en utilisant des anticorps par fluorescence-conjugués. Label 3 x 10 6 PBMC pendant 20 min à 4 ° C dans 100 ul de PBS / 2% SVF dans un tube Eppendorf de 1,5 ml avec un cocktail d&#39;anticorps spécifiques de surface pour les lignées de monocytes ou DC (tableau 2). Dilutions d&#39;anticorps optimales peuvent être déterminées dans des expériences préliminaires et les échantillons doivent être traités par lots pour réduire lot à lot de variation. Pour le stock d&#39;anticorps vendeur de 0,1 mg / ml utiliser 5 pi d&#39;anticorps (dilution 1:20). Utilisez un tube séparé de cellules marquées avec un seul conjugué seule couleur pour mettre en place les canaux de fluorescence sur l&#39;instrument. </li><li> Après marquage de surface, fixer les cellules dans 4% PFA / PBS pendant 10 min à température ambiante. Pour le stockage jusqu&#39;à ce moment de l&#39;analyse, les cellules centrifuger à 500 xg et remettre les cellules dans un tampon de gel (90% de FBS contenant 10% de DMSO) à -80 ° C jusqu&#39;à ce que le jour de test. </li><li> Pour l&#39;évaluation, le processus de lots of cellules non traitées et stimulé à partir d&#39;un groupe de bailleurs de fonds ainsi que pour minimiser la variabilité. Le jour de l&#39;analyse, les cellules dégel rapidement à 37 ° C bain-marie, centrifugeuse à g 500x à éliminer le tampon de congélation. </li><li> Pour perméabiliser les cellules pour la détection des cytokines intracellulaires ou d&#39;autres marqueurs, les suspensions cellulaires remettre en suspension dans 100 BD Perm / Wash pi de tampon (BD Biosciences, NJ). Label pour intracellulaires de signalisation avec des composants par exemple, dilution 1:20 de lapin anti-NF-kB (p65) antibody (concentration finale de 10 pg / ml, Santacruz Biotechnology, CA) pendant 20 min à température ambiante, centrifuger à 500 xg, Aspirer le surnageant et remettre les cellules dans 100 ul Perm BD / tampon de lavage contenant de dilution 1:250 de chèvre anti-IgG de lapin-Alexa647 (Invitrogen, CA) et incuber pendant 20 min à température ambiante. </li><li> Immédiatement avant l&#39;imagerie, recolorer noyaux au DAPI (0,2 pg / ml, Invitrogen, CA) ou l&#39;iodure de propidium (PI, 20 ng / ml, Invitrogen, CA). </li></ol> 3. Analyse ImageStream <ol><li>Mener l&#39;imagerie par ImageStream sur des échantillons dosés à réduire la variabilité entre les échantillons humains. Réglages de l&#39;instrument pour pouvoir les lasers étaient comme suit: 200 mW pour 488 nm, 10 mW pour 658 nm et 250 MW pour 405 nm. </li><li> Pour analyser les fichiers de données (figure 1), d&#39;abord, porte sur les événements avec une intensité normale et élevée PI ratio d&#39;aspect PI (largeur: hauteur) pour distinguer les cellules simples (R1) à partir de débris (de faible intensité PI) ou multi-cellulaires des événements ( haute intensité PI et un ratio faible). Les monocytes sont dans une grille pour CD14 des cellules positives. PID sont dans une grille de cellules qui sont élevés pour CD11c et faible pour CD4 (R2) et également faible pour Lin-1 marqueurs (R3). </li><li> Utilisez le logiciel IDEAS (Amnis, WA) pour fournir une mesure efficace quantitative du degré d&#39;activation de chaque cellule. Déterminer similitude (ou co-localisation) du facteur de transcription NF-kB cytoplasmique avec des armes nucléaires colorant PI pour indiquer la translocation dans le noyau (R4). Une forte corrélation entre NF-κB/PI localisation se traduit par un score de similarité élevé et indique le degré d&#39;activation 10. </li><li> Comparer les ratios de similitude entre les différents groupes de traitement ou de populations de patients d&#39;indiquer par rapport efficacité fonctionnelle des cellules. Quantifier des données entre les échantillons grâce à la comparaison statistique des valeurs absolues ou des différences de pliage. Utilisez un logiciel IDEAS pour afficher des images de cellules à partir de segments clés de la population histogrammes (Fig. 2). </li></ol> 4. Les résultats représentatifs Nous avons quantifié les voies de signalisation en réponse à un ligand modèle viral en stimulant les PBMC avec le ligand TLR7 / 8, R848 (Fig. 1). Nous avons recueilli les deux images de localisation cellulaire et statistiques sur la population dans nos groupes de l&#39;échantillon (Fig. 2). ImageStream nous permet de sous-ensembles de cellules de grille directement à partir de PBMC et les réponses de cellules d&#39;image à partir de populations de cellules gated, mais non triés. Ici, nous avons quantifié l&#39;effet de TLR signaling sur la localisation nucléaire de NF-kB (p65) dans Lin1-, CD4 faible, CD11c + myéloïde PED (MDC). Au départ, nous avons observé un pourcentage élevé de cellules non stimulées avec le facteur de transcription NF-kB (p65) dans le cytoplasme. Après la stimulation, les cellules traitées ont un score significativement plus élevé de similitude NF-B (p65) avec PI colorant nucléaire, indiquant que le NF-kB (p65) transloqué dans le noyau après stimulation (scores de similarité médianes sont 1,52 et 3,01, respectivement, Fig. 2). Des résultats similaires sont notés dans TLR5 des monocytes stimulés 9. <img alt="Figure 1" src="/files/ftp_upload/3741/3741fig1.jpg" /> Figure 1. Coloration et de la stratégie d&#39;ouverture de porte pour quantifier les effets de la stimulation sur MDC de l&#39;homme. Le facteur de transcription NF-kB régule l&#39;expression de nombreux gènes du système immunitaire. Cette étude mesure la localisation nucléaire de NF-kB (p65) dans MDC d&#39;un sujet représentant après TLR7 / 8 liga<html> la stimulation ème. De mise au point des cellules individuelles ont été identifiés par le déclenchement PI événements positifs avec des ratios élevés de l&#39;aspect nucléaire (R1). MDC (Lin1-, CD4dim, CD11c +) étaient fermée dans R3. La localisation nucléaire de NF-kB (p65) est tracée sur la R4. <a href="http://www.jove.com/files/ftp_upload/3741/3741fig1large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . <img alt="Figure 2" src="/files/ftp_upload/3741/3741fig2.jpg" /> Figure 2. . La translocation de NF-kB dans les pays plus développés après la stimulation de la translocation de NF-kB (p65) dans le noyau après stimulation (R4) est représenté dans les populations de MDC non traités et stimulé; le score de similarité médiane est de 1,52 dans l&#39;échantillon non traité et 3,01 en l&#39;échantillon stimulée (A). Les images numériques collectées simultanément sur les populations de cellules non traitées ou R848-stimulé montrent des cellules représentatives et l&#39;intensité de la norme NF-B transloqué dans le noyau de la cellule (B)._upload/3741/3741fig2large.jpg &quot;target =&quot; _blank &quot;&gt; Cliquez ici pour agrandir la figure.

<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>

Pas de conflits d&#39;intérêt déclarés.

Les étapes essentielles de l&#39;utilisation de ImageStream pour les études translationnelles sont la sélection des groupes de comparaison appropriés et l&#39;optimisation et la validation de la spécificité des anticorps. Les différences entre les groupes de sujets dans la cible marqué sera évidente par le biais des histogrammes et des images de cellules. En combinaison avec une analyse approfondie de l&#39;évolution des récepteurs concernés et les voies de signalisation, ces données fourniront des renseign...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </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> Paraformaldéhyde (16%) </td><td> Sciences Electron Microscopy </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> 1 cocktail de Lineage (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 lapin anti-NF-kB (p65) </td><td> Santacruz </td><td> SC-372 </td></tr><tr><td> Alexa647 F (ab &#39;) 2 de chèvre anti-IgG de lapin </td><td> Invitrogen </td><td> A21246 </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D21490 </td></tr><tr><td> X ImageStream </td><td> Amnis </td><td></td></tr></tbody></table> Tableau 1. Réactifs et équipements spécifiques. <table border="1"><tbody><tr><td> Type de cellule </td><td> FITC </td><td> PE </td><td> Alexa 647 </td><td> PacBlue </td></tr><tr><td> Monocyte </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> Panel d&#39;anticorps Tableau 2. Pour la coloration lignée cellulaire des voies de signalisation Monocytes: CD14 + MDC: Lin1-, CD4dim, CD11c + PDC: Lin1-, CD4dim, CD123 + Lin1 (lignage) marqueur comprend CD3, CD14, CD19, CD20, CD56 marqueurs pour les monocytes, cellules NK, lymphocytes T et B.

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

Les variations individuelles dans l&#39;état immunitaire de déterminer les réponses à l&#39;infection et de contribuer à la gravité des maladies et des résultats. Le vieillissement est associé à une susceptibilité accrue aux infections virales et bactériennes et une diminution de la réactivité aux vaccins avec un déclin bien documenté dans humorale ainsi que 1,2 à médiation cellulaire réponses immunitaires. Nous avons récemment évalué les effets du vieillissement sur ​​récepteurs Toll-like (TLR), les composants clés du système immunitaire inné qui permettent de détecter l&#39;infection microbienne et de déclenchement des réponses antimicrobiens défense de l&#39;hôte 3. Dans une large cohorte de donneurs humains sains, nous avons montré que les monocytes du sang périphérique de personnes âgées ont diminué expression et la fonction des TLR 4 et certains niveaux similaires TLR réduits et les réponses de signalisation dans les cellules dendritiques (CD), cellules présentatrices d&#39;antigène qui sont déterminant dans le lien entre l&#39;immunité innée et adaptative 5. Nous avons montré une dérégulation du TLR3 dans les macrophages uned baisse de la production d&#39;IFN par les DC provenant de donneurs âgés en réponse à l&#39;infection par le virus West Nile 6,7. Paramount à notre compréhension de immunosénescence et à l&#39;intervention thérapeutique est une compréhension détaillée des types de cellules spécifiques répondant et le mécanisme (s) de la transduction du signal. Les études traditionnelles de réponses immunitaires à travers l&#39;imagerie de cellules primaires et des marqueurs de cellules d&#39;arpentage par FACS ou immunoblot ont fait avancer notre compréhension de manière significative, cependant, ces études sont généralement limitées techniquement par le faible volume d&#39;échantillon disponible pour les patients et l&#39;incapacité de procéder à des techniques de laboratoire complexes sur de multiples échantillons humains. ImageStream combine la cytométrie en flux quantitative simultanée imagerie à haute résolution numérique et facilite ainsi l&#39;enquête dans des populations cellulaires multiples simultanément pour une capture efficace de la susceptibilité du patient. Ici, nous montrent l&#39;utilisation de ImageStream dans les PED afin d&#39;évaluer TLR7 / 8activation à médiation augmentation de la phosphorylation et la translocation nucléaire d&#39;un facteur de transcription clé, NF-kB, qui déclenche la transcription de nombreux gènes qui sont essentiels pour les réponses immunitaires 8. Grâce à cette technologie, nous avons aussi récemment démontré une altération précédemment non reconnu de TLR5 signalisation et de la voie NF-kB dans les monocytes de donneurs âgés qui peuvent contribuer à la réponse immunitaire altérée au cours du vieillissement 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

Nous décrivons l&#39;utilisation de la technologie ImageStream (<a href="http://www.amnis.com/" target="_blank"&gt; Www.amnis.com</a&gt;), Qui combine la cytométrie en flux quantitative simultanée à haute résolution d&#39;imagerie numérique, de quantifier les mécanismes cellulaires de cellules immunitaires primaires de cohortes de patients bien définis. Nos études fournissent un modèle pour les recherches translationnelles de quantifier la lignée spécifiques des réponses cellulaires dans de petits échantillons à partir de cohortes soumises.

Imagerie quantitative de Lineage spécifiques Toll-like Receptor la signalisation médiée par les monocytes et les cellules dendritiques à partir de petits échantillons de sang humain

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

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JoVE Journal

Immunology and Infection

35.0K vues.  Yale University School of Medicine. Nous décrivons l'utilisation de la technologie ImageStream ( Www.amnis.com), Qui combine la cytométrie en flux quantitative simultanée à haute résolution d'imagerie numérique, de quantifier les mécanismes cellulaires de cellules immunitaires primaires de cohortes de patients bien définis. Nos études fournissent un modèle pour les recherches translationnelles de quantifier la lignée spécifiques d...

Vidéo: 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.

Détection de Neu1 activité sialidase dans la régulation activation du récepteur Toll-like

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

Recherche

Immunologie et infection

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

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

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of 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.

Génération de cellules T humaines allo-spécifiques du sang périphérique

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.

Isolement d&#39;anticorps murins péritonéale macrophages pour effectuer Analyse expression génique Sur Toll-like récepteurs Stimulation

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.

Génération de cellules humaines dérivées de monocytes dendritiques du sang total

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

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.

Utilisation d&#39;un monocytes monocouche Assay pour évaluer phagocytose Fcy Receptor médiée

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.

Dextrane augmente l’efficacité de la Transduction des gènes des cellules NK primaires murines et humaines

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.

Imagerie en direct de l&#39;activité par Human Antifongique primaire neutrophiles et des monocytes en réponse 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.

Isolement de cellules dendritiques de l’appareil reproducteur féminin humain pour des études phénotypiques et fonctionnelles

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.

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

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...