The author would like to thank the NIH Tetramer Core Facility for tetramer production. The author would also like to thank E.D. O&#8217;Connor and K.E. Marquez at the UCSD Human Embryonic Stem Cell Core Facility flow cytometry laboratory for assistance in cell sorting. This work was funded by National Institutes of Health grant K08AI085039 (G.P.M.).

NOTE: Ce protocole nécessite l&#39;utilisation d&#39;échantillons de sang périphérique chez des volontaires humains. Toutes les recherches sur des sujets humains doit être examiné et approuvé par un comité d&#39;examen institutionnel études humaines pour assurer la conformité avec la Déclaration d&#39;Helsinki (2013) et la Health Insurance Portability and Accountability Act de 1996. 1. Isolement de cellules de sang total T <ol><li> Avant de commencer, chauffer le milieu à gradient de densité à la température ambiante. Aliquote de 4 ml de milieu de gradient de densité dans 2-4 stériles tubes de 15 ml à centrifuger coniques (1 tube sera utilisé pour chaque 10 ml de volume total de sang dilué). </li><li> Obtenir 10-20 ml de sang dans l&#39;héparine 1-2 sodium enrobés par pulvérisation (vert-haut) tubes de prélèvement de sang veineux. Recueillir des échantillons humains sous la supervision de personnes qualifiées et selon un Institutional Review Board protocole approuvé. </li><li> Essuyez l&#39;extérieur des tubes avec 70% d&#39;éthanol. Retirez délicatement les sommets de la firempli les tubes de prélèvement et décanter le sang dans un tube de 50 ml à centrifuger stérile. </li><li> Ajouter 10 ml de solution saline tamponnée au phosphate stérile (PBS) dans chaque tube de collecte de sang. Recueillir le PBS, ajouter le sang tout décanté, et mélanger doucement. </li><li> Ajouter 25 ul T Cellule humaine enrichissement cocktail / 2 ml volume total. Incuber à température ambiante pendant 20 min. </li><li> L&#39;utilisation d&#39;une pipette de 10 ml, la couche de 10 ml de 1: 1 de sang dilué légèrement au-dessus du milieu à gradient de densité. Attention à ne pas perturber la surface du milieu de gradient de densité. </li><li> Centrifugeuse sang en couches et le milieu de gradient de densité à 1200 g pendant 20 minutes à 20 o C. </li><li> Retirer les tubes de la centrifugeuse, en prenant soin de ne pas perturber l&#39;interface entre le milieu à gradient de densité et la couche de globules blancs à l&#39;interface entre le milieu à gradient de densité et le plasma dilué. L&#39;utilisation d&#39;un pipette de 5 ml, de recueillir soigneusement la couche de leucocytes et de les transférer à un nouveau 50 ml baignoire conique stérilee. </li><li> Ajouter PBS pour porter le volume de la PBL recueilli 50 ml et mélanger doucement. </li><li> Centrifuger à 600 g pendant 5 min à 20 ° C. Décanter. </li><li> Remettre en suspension les cellules en culot dans 10 ml flux stérile cytométrie tampon (PBS stérile filtrée contenant 1% de BSA) de tri. Échantillon de 10 pi de suspension cellulaire, diluer 01h10 (ajouter 90 pi) au bleu trypan à compter les cellules en utilisant un hématimètre (rendement attendu: 1 - 4 x 10 6 cellules / tubes de sang total). <ol><li> Retirer aliquote de 1 ml, transférer à 5 ml tube, et garder sur la glace pour l&#39;analyse des cellules T non étiquetés par tétramère. Gardez la suspension cellulaire sur la glace. </li></ol></li></ol> 2. Enrichissement magnétique de cellules T allo-antigène-spécifiques <ol><li> Diluer allo pMHC tétramère à 1: 100 dans le tampon de tri stérile. </li><li> Centrifuger la suspension de cellules (9 ml de l&#39;étape 1.11) à 600 g pendant 5 min à 20 ° C. Décanter. </li><li> Ajouter 50 ul de dilution allo pMHC tétramère à granulésles cellules (à 10 7 cellules). Mélanger par pipetage doux. Transférer dans un tube de 5 ml stérile. Incuber pendant 30 min à température ambiante. </li><li> Ajouter 2 ml sorte tampon. Centrifuger à 600 g pendant 5 min à 20 ° C. Décanter. </li><li> cellules de remettre en suspension dans 100 ul de tampon de tri. Ajouter 10 ul biotine sélection Cocktail et incuber pendant 15 min à température ambiante. </li><li> Ajouter 5 ul magnétique Nanoparticules et incuber pendant 10 min à température ambiante. </li><li> Ajouter 2,5 ml sorte de tampon et mélanger doucement à la pipette. Retirer 100 pi aliquote, transférer à 5 ml tube, et garder sur la glace pour l&#39;analyse des cellules pré-enrichissement. </li><li> Placer le tube de 5 ml contenant la suspension de cellules dans l&#39;aimant de séparation de cellules. Le plafond devrait être vaguement au sommet du tube. Incuber pendant 5 min à température ambiante. </li><li> Retirez délicatement le bouchon du tube. En tenant le tube et aimant ensemble, décanter le contenu du tube dans un nouveau tube de 5 ml. Ne touchez pas ou ne le secouez le contenu du tube àéliminer les dernières gouttes de liquide. </li><li> Enlevez le tube de l&#39;aimant. Ajouter 2 ml de tampon de tri froid et mélanger doucement à la pipette. Échantillon de 10 pi de suspension cellulaire, diluer 1: 2 (ajouter 10 pi) au bleu trypan à compter les cellules en utilisant un hématimètre. </li></ol> 3. Préparation des cellules T pour une seule cellule cytométrie en flux Tri cellulaire <ol><li> Ajouter tampon de tri froid à tous les tubes de cellules (tétramère non marquée, tétramère marqué non enrichi, et tétramère marqué enrichi) pour porter le volume à 3 ml. </li><li> Centrifugeuse à 600 g pendant 5 min à 4 ° C et tampon décanter pour récupérer les cellules. </li><li> Reprendre culot de cellules dans 25 tampon de tri ul froid. Ajouter 5 ul de bloc Fc humaine. Incuber sur de la glace pendant 20 min. </li><li> Préparer des anticorps pour la cytométrie en flux tri. Mélanger 15 pi de tampon de tri, 15 pl anti-CD5, 15 pl anti-CD14, et 15 pi anti-CD19. </li><li> Ajouter 20 ul de mélange d&#39;anticorps à des cellules de chaque tube. Incuber sur de la glace pendant 20 min. </li><li> Ajouter 2 ml de tampon de tri froid dans chaque tube. </li><li> Centrifugeuse à 600 g pendant 5 min à 4 ° C et tampon décanter pour récupérer les cellules. </li><li> Remettre en suspension les cellules à une concentration de 1 à 2 x 10 6 cellules / ml dans du tampon de tri. </li><li> Aliquote de 100 ul de milieu de culture de cellules humaines T (DMEM d&#39;Iscove complété avec 2 mM de Glutamax, HEPES 10 mM, 50 ug / ml de gentamycine, 50 uM de 2-mercaptoéthanol, 10% de sérum humain AB inactivé par la chaleur, et de 2,5 ng ml humaine recombinante / IL-2) à chaque puits d&#39;une plaque ronde à 96 puits de culture de cellules de base. Garder sur la glace. </li></ol> 4. Isolement de cellules T tétramère marqué par l&#39;unité de cytométrie en flux des cellules Tri <ol><li> Établir cytométrie en flux paramètres de déclenchement pour identifier les cellules allo-pMHC tétramères marqués T (Figure 2.A). Utilisez la fraction de tétramère de pré-enrichissement marqué à établir gating des stratégies pour éliminer les doublets, porte sur les lymphocytes, les cellules exprimant CD19 exclure B et CD14exprimant les monocytes, et identifier les cellules T par l&#39;expression CD5. Utilisez l&#39;échantillon non étiqueté avec tétramère comme une fluorescence moins un contrôle d&#39;établir le déclenchement pour l&#39;identification de cellules tétramère contraignant T (avec 0% de CD5 + CD14 - CD19 - les cellules de l&#39;échantillon ne sont pas étiquetés par tétramère doivent se situer dans cette porte) . REMARQUE: un noise gate stratégies qui ne sont pas suffisamment strictes seront conduire à l&#39;isolement de cellules non-T ou cellules T qui ne sont pas l&#39;antigène spécifique, tandis que les stratégies de déclenchement trop restrictives permettront de réduire le nombre de cellules T isolées. </li><li> Programmer les paramètres de la plaque de la sorte d&#39;une seule cellule. Dirigez la trieuse de placer 1 CD5 + CD14 - CD19 - tétramère + cellule dans chaque puits. Dirigez la plaque mis en place pour contenir 1 puits du contrôle négatif (pas de cellules triées dans le puits) et 1 puits du contrôle positif (100 CD5 + CD14 - CD19 - tétramère - cellules) dans chaque rangée (Figure 2.B </strong&gt;). </li><li> Utilisation de l&#39;écoulement établi cytométrie stratégie de déclenchement et de la configuration de la plaque, isoler des cellules individuelles de tétramère contraignant T directement dans la plaque de 96 puits en utilisant un trieur de cellules par cytométrie en flux. </li></ol> 5. La culture et l&#39;expansion de clones allo-spécifiques des cellules T <ol><li> Après l&#39;achèvement de tri cellulaire, la plaque centrifuge à 600 g pendant 5 min à 20 ° C. Placer la plaque de culture à 37 ° C dans un incubateur à CO2 de 6%. </li><li> Vortex stimulation microbilles anti-CD3 / anti-CD28 pour 30 secondes pour remettre en suspension les perles. </li><li> Calculer le volume de billes d&#39;activateur nécessaires pour la stimulation des cellules T recueillies (0,5 ul de microbilles activateur / puits). Transférer le volume calculé de billes stimulatrices dans un tube de 5 ml stérile. Ajouter 1 ml de milieu de culture humaine de cellules T et de vortex. </li><li> Placez le tube avec microbilles suspension dans l&#39;aimant. Le plafond devrait être vaguement au sommet du tube. Incuber pendant 2 min à la température ambiante. </li><li> Retirez délicatementle bouchon du tube. En tenant le tube et aimant ensemble, le contenu du tube de décantation dans un nouveau tube de 5 ml. Ne touchez pas ou ne le secouez le contenu du tube. </li><li> Enlevez le tube de l&#39;aimant. Ajouter milieu de culture de cellules humaines T (100 ul de milieu / 0,5 ul de microbilles ajoutées initialement). </li><li> Aliquote de 100 pi d&#39;activateur suspension de billes dans chaque puits de la plaque de 96 puits. Incuber la plaque de culture à 37 ° C dans un incubateur à CO 2 à 6%. </li><li> Surveiller la croissance des cellules quotidiennement par l&#39;examen microscopique. Petits amas de cellules proliférantes peuvent être observées au microscope après 5 - 7 jours de culture (Figure 3.A). REMARQUE: Visualisation des cultures de cellules T à ce point peut être difficile, et l&#39;absence d&#39;amas cellulaires observables à ce stade ne peut être le signe d&#39;un manque de cellules T croissance. </li><li> 14 jours après l&#39;isolement cellulaire, les cultures d&#39;alimentation en retirant soigneusement 100 pi de milieu off de la partie supérieure de la culture et de la remplacer par 100 μ; L de milieu de culture de cellules T humaines fraîches. Continuer l&#39;incubation de la plaque de culture à 37 ° C dans un incubateur à CO 2 à 6%. </li><li> Surveiller la croissance cellulaire tous les jours par examen et l&#39;évaluation de la couleur du milieu de culture microscopique. Les grands groupes de cellules doivent être visibles au microscope 2 - 3 jours après le changement de milieu. Macroscopiquement, culots cellulaires devraient devenir visible pendant les 14 jours suivants le changement moyen. </li><li> A 28 jours suivants l&#39;isolement cellulaire, identifier les clones de plus en plus par l&#39;examen microscopique. Transférer le volume de 200 ul de la plaque à 96 puits de cultures de croissance positive à des puits individuels d&#39;une plaque de culture tissulaire à 48 puits contenant 200 milieu de culture de cellules T humaines ul. </li><li> Ajouter 100 ul contenant 12,5 ul de microbilles anti-CD3 / anti-CD28 de stimulation (préparé comme décrit dans les sections 05.02 à 05.06). Incuber la plaque de culture à 37 ° C dans un incubateur à CO 2 à 6%. </li><li> Surveiller la croissance de la culture quotidienne par ex microscopiqueamination et l&#39;évaluation de la couleur du milieu de culture. Quand la culture de cellules T atteint&gt; 2x10 6 / ml (généralement 3-5 jours après re-stimulation, peut être estimée par la journée que les médias commence à se tourner jaune paille), la culture de transfert à un puits d&#39;une plaque de culture de 24 puits tissu et ajouter 500 milieu de cellule T humaine ul. </li><li> À 10-14 jours suivre re-stimulation, recueillir 200 pi de culture de cellules T à évaluer allo spécificité par cytométrie en flux de pMHC tétramère contraignant (en utilisant la stratégie de l&#39;étiquetage et de déclenchement décrites dans les sections 3 et 4.1). </li></ol> 6. long terme Re-stimulation et la culture des clones de cellules T <ol><li> Continuellement re-stimuler et de développer les cultures de cellules T clonales avec la spécificité désirée peut être tous les 14 jours. Recueillir les cultures de cellules T dans un tube de 5 ml stérile à 14 jours suivant la dernière stimulation de microbilles anti-CD3 / CD28. Combinaison de plusieurs puits d&#39;un même clone de cellules T dans un seul tube, jusqu&#39;à un volume de 2,5 ml. </li&gt; <li> Ajouter moyen pour amener le volume final de 2,5 ml et mélanger doucement à la pipette. </li><li> Placer le tube 5 ml de la culture contenant des cellules T dans l&#39;aimant de séparation de cellules pour éliminer les vieilles microbilles de la culture. Le plafond devrait être vaguement au sommet du tube. Incuber pendant 5 min à température ambiante. </li><li> Retirez délicatement le bouchon du tube. En tenant le tube et aimant ensemble, décanter la suspension de cellules dans un nouveau tube de 5 ml. Ne touchez pas ou ne le secouez le contenu du tube pour éliminer les dernières gouttes de liquide. </li><li> Ajouter moyen pour amener le volume final de 4 ml. Échantillon de 10 pi de compter les cellules en utilisant un hématimètre. </li><li> Récupérer les cellules T par centrifugation à 600 xg pendant 5 min à température ambiante. </li><li> Décanter le surnageant. Remettre en suspension les cellules T à 10 6 cellules / ml de milieu de culture de cellules T humaines. Transfert des aliquotes de 1 ml à chaque puits d&#39;une plaque de culture tissulaire à 24 puits. </li><li> Re-stimuler les cellules T par l&#39;ajout de 100 médias ul contenant 12,5 pi de stimulation anti-CD3 / amicrobilles nti-CD28 (tels que décrits dans les sections 5.2 à 5.6). Incuber à 37 ° C dans un incubateur à CO 2 à 6%. </li></ol>

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The authors declare no competing financial interests.

T cell alloreactivity is a long-studied and clinically-relevant phenomenon. The robust proliferative and effector responses of T cells to allogeneic stimulation has enabled extensive analyses of human T cell responses in vitro through relatively straightforward mixed lymphocyte reactions of peripheral blood T cells against inactivated allogeneic cells. However, these primary alloreactive T cell responses are oligoclonal, comprised of a large number of individual T cells responding to specific alloantigens. This ...

Les lymphocytes T sont des composantes essentielles du système immunitaire adaptatif. Les cellules T sont responsables de la médiation directement non seulement des réponses immunitaires protectrices à des agents pathogènes à travers une variété de mécanismes effecteurs, mais également le maintien activement auto-tolérance immunologique et diriger les réponses des autres cellules dans le système immunitaire. Ces fonctions sont dirigés à travers un certain nombre de signaux intégrés, y compris des récepteurs de cellule T (TCR) ligature, les cytokines et les chimiokines, et les métabolites 1. Parmi ces signaux, le TCR est d&#39;une importance particulière, car il fournit la spécificité caractéristique qui définit le rôle de la cellule T dans l&#39;immunité adaptative. Un TCR interagit avec les antigènes peptidiques linéaires présentés par le CMH (HLA de orthologue humain) molécules (complexes de pMHC) d&#39;une manière hautement spécifique et sensible pour fournir les signaux qui déclenchent la fonction T effectrice des cellules. Les paramètres biochimiques des interactions TCR avec des ligands PMHC fournissent non seulement la spécificité de Tl&#39;activation des cellules, mais aussi avoir un impact qualitatif sur la fonction des cellules T ultérieur 2. Ainsi, l&#39;étude de la fonction des cellules T nécessite souvent l&#39;examen des réponses de lymphocytes T clonaux ayant une spécificité antigénique déterminée. Le compartiment des cellules T humaines, contenant environ 10 12 cellules T αβ, contient environ 7 octobre au 8 octobre αβTCRs distinctes 3-4. Ce répertoire très diversifié offre la possibilité de reconnaissance de la vaste gamme de peptides de pathogènes potentiels qui nécessiterait une réponse des cellules T à une immunité protectrice. On estime que la fréquence de la réponse des cellules T à un antigène étranger présenté donné par auto-MHC est de l&#39;ordre de 10 -4 à 10 -7 en l&#39;absence de réponse immunitaire à cet antigène avant 5. Le répertoire des lymphocytes T naïfs est formée par sélection thymique à assurer la capacité de reconnaître des auto-antigènes MHC de présentation de peptides et de limite réagissentivité contre les antigènes auto-peptide, la maximisation de l&#39;utilité potentielle de la médiation de l&#39;immunité protectrice 2. Cependant, en violation de cette réactivité conçu, une fréquence relativement importante, 10 -3 à 10 -4, de cellules T d&#39;individus immunologiquement naïfs répondre à une stimulation avec des cellules allogéniques, reconnaissant à la fois les molécules du CMH étrangers ainsi que les peptides endogènes qu&#39;ils présentent 6. La reconnaissance de ligands PMHC allogéniques est structurellement similaire à la reconnaissance d&#39;antigènes étrangers présentés par CMH du soi; du TCR permet d&#39;interactions biochimiques essentiels à la fois avec la molécule MHC allogénique aussi bien que le peptide présenté 7. La robustesse de la réponse des cellules T aux résultats de stimulation allogéniques de la diversité de complexes de pMHC présent sur ​​la surface des cellules allogéniques 8. On estime que chaque CMH présente à environ 2 x 10 4 différents antigènes peptidiques endogènes 9. Ce breadth de réponse à une stimulation allogénique est un aspect important de la pathologie cliniquement pertinentes, telles que le rejet d&#39;allogreffe ou la réaction du greffon contre l&#39;hôte (GVHD), résultant de T alloréactivité cellulaire. Étude de cellules T réponses alloréactives humaines a toujours compté sur l&#39;examen des réponses polyclonaux de cellules T naïves après stimulation avec des cellules allogéniques. Stimulation répétée avec la même lignée cellulaire allogénique combiné avec dilution limitante analyse est capable de générer des cellules T clonales de reconnaissance définis allogénique HLA de 10. Cependant, cette approche est problématique pour examiner les réponses aux différents ligands PMHC allogéniques, comme le répertoire vaste et diversifié de endogène pMHC complexes présente un allogénique HLA donné stimule un large répertoire de cellules T. Cette stimulation de la population en vrac et en limitant approche de dilution, il faudrait dépistage d&#39;un grand nombre de clones pour isoler les cellules T avec la Reacti souhaitéevité contre un seul ligand pMHC. En outre, la fréquence des cellules T répondant à un ligand pMHC allogénique individu est relativement faible chez les populations de cellules T naïves, qui présente un obstacle à la génération efficace de clones de lymphocytes T humains en réponse à un antigène donné. L&#39;identification et l&#39;isolement des cellules T spécifiques de l&#39;antigène à partir de populations polyclonales ont été activées par le développement de multimères pMHC 11 marqué par un fluorophore. Cette approche utilise des antigènes peptidiques spécifiques chargés dans des molécules de CMH solubles recombinants biotinylés, qui sont marqués par liaison à un fluorophore de la streptavidine marquée. Multimérisation de pMHC augmente l&#39;avidité, la compensation de l&#39;affinité intrinsèquement faible (M) de TCR pour ligands PMHC solubles. Les cellules marquées peuvent être identifiées et isolées par cytométrie de flux. Cependant, cette approche est encore limitée par la faible fréquence des cellules T spécifiques de l&#39;antigène chez les populations de cellules T naïves, qui sont typiquementordres de grandeur inférieure à la limite de l&#39;identification et quantification précise sur la plupart des cytomètres de flux. Pour remédier à cette limitation, un procédé de marquage pMHC de tétramère et d&#39;enrichissement par billes magnétiques subséquente des cellules tétramère marqué a été mis au point 12. Cette méthode a montré une détection fiable, le dénombrement et l&#39;isolement des cellules T spécifiques de l&#39;antigène à basse fréquence. Ce protocole décrit un protocole efficace pour la génération de clones de cellules T humains qui réagissent spécifiquement aux ligands individuels pMHC allogéniques. Le protocole applique pMHC (HLA) l&#39;étiquetage des multimères et d&#39;enrichissement pour l&#39;isolement de cellules T humaines spécifiques à l&#39;alloantigène de cytométrie de flux de tri cellulaire et un procédé robustes pour la culture in vitro de cellules T humaines pour permettre la production de clones de cellules triées individuelles de cellules T ( Vue d&#39;ensemble de la figure 1).

<table border="0" cellpadding="0" cellspacing="0" width="907">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name of Reagent/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:292px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Sodium heparin venous blood collection tube 16 x 100 mm</td>
			<td style="width:223px;">Becton, Dickenson and Company</td>
			<td style="width:119px;">366480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Lymphoprep</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">7801</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:273px;">Rosette Sep Human T Cell Enrichment Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">15061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Dulbecco&#39;s PBS, 1x without Ca or Mg</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bovine serum albumin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E6635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Fluorophore-labeled pMHC tetramer</td>
			<td style="width:223px;">NIH Tetramer Facility</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Biotin Selection Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Selection magnet</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:273px;">TruStain FcX Human Fc blocking solution</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">422301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD5 PE-Cy7 (clone UCHT2)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">300621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD14 FITC (clone HCD14)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">325603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD19 FITC (clone HIB19)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">302205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Iscove&#39;s DMEM, without b-ME or L-glutamine</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">15-016-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-Mercaptoethanol&#160;</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin sulfate (50 mg/ml)</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">GT-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human AB serum, male donor</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">HS-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2</td>
			<td style="width:223px;">Peprotech</td>
			<td style="width:119px;">AF 200-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Dynabeads Human T-Activator CD3/CD28</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">11131D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Media</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Cell sorting buffer</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">PBS, pH 7.4</td>
			<td style="width:223px;">1 L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BSA</td>
			<td style="width:223px;">10g</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA (0.5 M)</td>
			<td style="width:223px;">2 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human T Cell Culture Medium</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Iscove&#39;s DMEM</td>
			<td style="width:223px;">351.6 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Heat-inactivated human AB serum</td>
			<td style="width:223px;">40 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES (1 M)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax (100 x)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin (50 mg/ml)</td>
			<td style="width:223px;">0.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-mercaptoethanol (14.3 M)</td>
			<td style="width:223px;">1.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2 (1 mg/ml)</td>
			<td style="width:223px;">1 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Ce protocole décrit la génération de cultures de cellules T humaines clonale défini allo spécificité par un enrichissement par billes magnétiques et flux unicellulaire cytométrie tri stratégie. La figure 1 donne un aperçu du processus. <img alt="Figure 1" src="/files/ftp_upload/52257/52257fig1highres.jpg" /> Figure 1: vue d&#39;ensemble Protocole Le protocole décrit ici fournit une méthode fiable pour la...

Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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 ...

generation-of-human-alloantigen-specific-t-cells-from-peripheral-blood

Research

JoVE Journal

Immunology and Infection

12.9K vues.  University of California, San Diego. 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.

Vidéo: Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansion, purification et d&#39;évaluation fonctionnelle des cellules NK humaines périphérique Sang

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Recherche

Immunologie et infection

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Induction de l&#39;alloantigène spécifique anergie dans les cellules mononucléaires du sang périphérique par stimulation alloantigène avec blocage du signal de co-stimulation

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansion du sang périphérique humain cellules T γδ utilisant zolédronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Génération de cellules T régulatrices induites de l&#39;homme primaires cellules T naïves et mémoire

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Application clinique de<em&gt; Belle au Bois Dormant</em&gt; Et artificielle cellules présentatrices d&#39;antigènes pour modifier génétiquement des cellules T du sang de cordon ombilical et périphériques

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

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 ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

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

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

Génération de discriminatif anticorps monoclonaux humains de cellules rares spécifiques de l’antigène B circulant dans le sang

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

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Évaluation de l’Interface synaptique des cellules primaires humaines T du sang périphérique et le tissu lymphoïde

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

Isolement, activation et expansion des lymphocytes T par flottation à partir d’échantillons de cellules mononucléaires du sang périphérique humain à l’aide de microbulles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Différenciation des ostéoclastes fonctionnels des monocytes CD14+ du sang périphérique humain

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...