Ce travail est soutenu par une aide à la production pour les thérapies cellulaires (contrat NIH-NHLBI (HB-10-03) HHSN26820100000C) (CMR), un des centres spécialisés pour thérapie cellulaire subvention du NIH-NHLBI 1 U54 HL081007 (CMR), une ASBMT Young Investigator Award (UG et JV), Fellow Société de leucémie et de lymphome spécial de bourses de recherche clinique (UG), et un prix Amy Strelzer Scholar Manasevit (LAM).

1. Nucléofection DC <ol><li> Récolte dérivées de monocytes PED, qui ont été enrichies avec l&#39;adhésion de plastique, cultivées pendant 5 jours à l&#39;aide de cellules-Genix-sur des milieux supplémentés avec IL4 (1000U/ml), GMCSF (800IU/ml) et encore mûri pendant 24h en utilisant les cytokines IL4 maturation des DC (1000U / ml), GMCSF (800IU/ml), IL6100ng/ml, le TNF-α 10ng/ml, IL1-β 10ng/ml et la PGE2 (1μg/ml) 1, par resuspension doux avec une pipette 3 ml. </li><li> Comptez PED viables en utilisant le bleu trypan, le transfert dans des tubes 15ml 3x avec pas moins de 6 0.5x10 et pas plus de 2x10 6 cellules / tube. </li><li> Centrifuger les PED pour les 10mins @ 200g. Pendant ce temps préchauffer cellulaire Genix des milieux supplémentés avec les cytokines maturation des DC (médias maturation des DC) - 2ml/well dans trois puits d&#39;une culture de tissus de 12 puits dans une plaque de traiter 37 ° C / 5% de CO 2 incubateur. </li><li> Une fois que les cellules ont fini de filage, aspirer le surnageant et ajouter des plasmides d&#39;ADN correspondant à chacun des tubes dans une concentration finale de 5 pg d&#39;ADN / tube. Dans ce cas, ajoutez le plasmide codant pour IE1-pp65 au tube n ° 1, Hexon-Penton au tube n ° 2, et EBNA1-LMP2-BZLF1 au tube n ° 3. </li><li> Resuspendre PED et l&#39;ADN avec 100 pi de solution de nucloefection Amaxa, bien mélanger et transférer les cuvettes nucléofection. </li><li> Cuvettes place dans le Nucleofector 4D, choisissez le programme CB150 (Amaxa / Lonza), et appuyez sur Start. </li><li> Immédiatement après nucléofection ajouter 500μl de 2ml préchauffé médias Genix cellulaire maturation des DC dans la cuvette, mélanger doucement par aspiration et refoulement 2-3 fois, et le transfert PED nucleofected le prêt de 12 puits plaque contenant le reste de 1,5 ml de préchauffée les médias maturation des DC. Transfert à la 37 ° C / 5% de CO 2 incubateur pour un autre 12-18hrs. </li></ol> 2. Stimulation des cellules T <ol><li> Récolte et de compter les PED nucleofected, et irradier au 30 Gy. Laver une fois avec 10ml de milieu CTL (45% du RPMI, 45% Clics EHAA, 10% de FBS, 2mM Glutamax) et resuspendre @ 3 x 10 5 PED par ml de médias CTL. </li><li> Piscine un minimum de 5 7.5x10 (2.5ml) et un maximum de 15x10 5 (5 ml) contenant des PED chacun des plasmides et de transférer les PED en commun à l&#39;appareil G-Rex qui seront ensuite placés dans l&#39;incubateur. </li><li> Pour la préparation des cellules répondeuses utiliser soit CMSP préalablement congelé ou non adhérente des cellules mononucléées qui restent après la sélection DC (adhésion ou CD14 sélection). Décongeler les cellules, le transfert d&#39;un milieu de culture préchauffée, se laver une fois avec les médias CTL. Resuspendre les cellules dans les médias CTL, compter les cellules et les amener à une concentration de 2x10 6 cellules par ml. Prenez 15x10 6 cellules ou 7,5 ml et compléter avec 30000U IL4 (1000U/ml -. Concentration finale) et 300ng IL7 (10ng/ml - conc finale.). </li><li> Transfert 7,5 ml de PBMC (15x10 6 cellules) pour le G-Rex et compléter le bioréacteur avec les médias de CTL à un volume total de 30ml. </li><li> Culture du G-Rex pendant 6-7 jours dans un 37 ° C / 5% de CO 2 incubateur humidifié. </li></ol> 3. L&#39;expansion des cellules T <ol><li> Le jour de 6-7, 10ml aspiration des médias, puis mélanger les cellules dans le reste de 20ml médias avec une pipette de 10ml et de compter les cellules viables en utilisant du bleu trypan. S&#39;il ya &lt;50x10 6 reconstituer avec les médias frais + cytokines. S&#39;il ya&gt; 50x10 6 cellules retirer 10ml de suspension cellulaire, le transfert à un nouveau G-Rex, puis nourrir les deux G-Rexs avec Fresh Media CTRL + cytokines. </li><li> Culture pour une 4-6 jours supplémentaires. Une fois que suffisamment de cellules ont été élargis, effectuer la caractérisation phénotypique et fonctionnelle de l&#39;excès de CTL et cryoconserver pour une utilisation future. </li></ol> 4. Les résultats représentatifs: Un schéma de notre approuvé par la FDA multivirus spécifique processus de génération de CTL est montré dans la figure 1. Contrairement à la convention multivirus protocoles CTL qui utilisent adénovecteurs et EBV-LCL pour stimuler le virus réactive les cellules T 2, nous avons remplacé matériel viral infectieux avec des plasmides d&#39;ADN qui codent des antigènes multiples issus de chacun des trois virus. Pour stimuler CTL trivirus nous avons conçu trois multicistroniques plasmides codant Hexon et Penton de l&#39;adénovirus, IE1 et pp65 du CMV, et EBNA1, LMP2, et BZLF1 de l&#39;EBV. Ces antigènes ont été choisis sur la base des résultats cliniques encourageants de notre propre et d&#39;autres groupes qui montrent que les cellules T dirigées contre ADV-hexon et penton 2,4-6, et à CMV-1E1 et à CMV pp65 sont protecteurs in vivo 7. Pour l&#39;EBV, EBNA1 est un CD4 + antigène T immunodominant cellule cible exprimée dans toutes les tumeurs malignes associées à l&#39;EBV et dans les cellules normales B EBV-infectés 8,9, LMP2 est immunogène pour tous les types HLA multiples et exprimée dans la plupart des tumeurs malignes EBV, 10,11 tandis BZLF1 encode une immunodominants, immédiate antigène précoce du cycle lytique qui stimule à la fois CD4 + et CD8 + T de la plupart des individus et est probablement important pour la coopérationntrol de cellules répliquant le virus 12. Pour optimiser encore davantage nos méthodes de fabrication, nous avons collaboré avec la technologie de la nature qui a généré minimisée, sans antibiotique (conforme FDA) pour la stimulation de CTL plasmides 13,14. En utilisant cette stratégie nous avons constamment réaliser des économies d&#39;nucléofection&gt; 35% tout en maintenant la viabilité cellulaire élevée (données non présentées) 3. La figure 2 montre que la fréquence des cellules T spécifiques du virus en réponse à des plasmides d&#39;ADN optimisée telle que mesurée par IFN ELISPOT, était supérieure qu&#39;en réponse à des plasmides d&#39;expression conventionnels pShuttle basé exprimant les mêmes antigènes (n = 8 adénovirus, CMV n = 4 et n = 2 EBV). Le ratio optimal de DC: PBMC était important pour la stimulation des cellules T puissants comme le montre la figure 3, où un ratio de 01:50 produite sous-optimale par rapport à une activation 01:20 S: ratio R (n = 2 donneurs). Production de nombres suffisants CTL avec une spécificité d&#39;antigène large est un pré-requis pour l&#39;efficacité clinique contre les trois virus. Ce résultat est obtenu par la culture CTL dans le G-Rex, qui soutient l&#39;expansion des cellules T supérieure par rapport aux traditionnels des plaques 24 puits (figure 4A) 15, alors que l&#39;addition d&#39;IL4 et IL7 à des augmentations des cultures et la spécificité du répertoire comme le montre la figure 4B où la fréquence des cellules T réactives contre le CMV pp65-dérivés de HLA-A2 resticted NLV peptide a été évaluée dans des cultures produites dans la présence ou l&#39;absence d&#39;IL4 et / ou IL7 16,17. Pour évaluer la capacité phénotype et fonctionnelle des cellules élargi que nous effectuons une analyse de cytométrie en flux, cytokines intracellulaires coloration / IFN ELISPOT, et des essais Cr communiqué 51 sur le produit final pour la cryoconservation / perfusion. Généralement, les cellules générées sont polyclonaux avec une population mixte de cellules CD4 + et CD8 + T avec l&#39;antigène de spécificité détectable dans les deux compartiments cellulaires T. Les CTL sont capables de tuer exprimant l&#39;antigène viral des cellules cibles du virus, mais pas négative partiellement HLA cibles appariés, ce qui indique qu&#39;ils ne doivent pas induire une réaction du greffon contre l&#39;hôte (GvHD) in vivo (figure 5). <img src="/files/ftp_upload/2736/2736fig1.jpg" alt="Figure 1"/> Figure 1. Protocole de génération de rctl. Tout d&#39;abord, les PED sont nucleofected avec les antigènes viraux plasmides codant et ensuite mélangés avec des PBMC autologues à un R: S de 10 ou 20h01. Les cellules sont développées dans le G-Rex pendant 10-14 jours, en présence d&#39;IL4 et IL7, puis récoltées, comptées, testé pour la fonction, l&#39;identité et la stérilité, et ensuite cryoconservés pour une utilisation clinique. <img src="/files/ftp_upload/2736/2736fig2.jpg" alt="Figure 2"/> Figure 2. Plasmides d&#39;ADN optimisée induisent supérieure activation des cellules T in vitro. PED ont été optimisés avec nucleofected, conformes à la FDA Hexon plasmides codant et Penton (ADV), IE1 et pp65 (CMV), et EBNA1, LMP2, et BZLF1 (EBV) ou les plasmides pShuttle conventionnels codant pour les antigènes mêmes. Ils ont été utilisés pour stimuler les cellules T et la spécificité a été analysée par ELISPOT IFN 10 jours post-stimulation. <img src="/files/ftp_upload/2736/2736fig3.jpg" alt="Figure 3"/> Figure 3 DC optimale:. Ratios des lymphocytes T pour l&#39;activation de CTL. PED à partir de 2 donneurs ont été nucleofected avec les trois plasmides optimisée et ensuite utilisé pour stimuler les PBMC autologues à 1:20 ou 1:50 DC: ratio de PBMC. La fréquence des cellules T réactivé a été évaluée sur 10 jours par IFN ELISPOT. <img src="/files/ftp_upload/2736/2736fig4.jpg" alt="Figure 4"/> Figure 4. Expansion des cellules T dans le G-Rex en utilisant des cytokines amélioration. Le panneau A montre l&#39;appareil G-Rex ainsi que l&#39;apparence de CTL sur la membrane perméable au gaz, évaluée par microscopie. Une comparaison entre la production de cellules obtenus en culture tissulaire Convention traitées plaques vs G-Rex est également représentée. Groupe B montre la fréquence du CMV pentamère CTL positifs obtenus dans les cultures élargi, en présence de cytokines pas, IL4 seul, seul et IL7 IL4 + IL7. <img src="/files/ftp_upload/2736/2736fig5.jpg" alt="Figure 5"/> Figure 5. Phénotype et la fonction des CTL élargi. Le panneau A montre un exemple représentatif du phénotype de l&#39;CTL élargi multivirus, qui sont polyclonaux avec un mélange de cellules CD4 + (45% - helper) et CD8 + (42% - cytotoxiques) des cellules T, dont la majorité (95%) ont exprimé le la mémoire de marqueur CD45RO + / CD62L +. panneau B montre que ces cellules sont spécifiques pour tous les antigènes stimulant et sont polyfonctionnels évaluée par coloration des cytokines intracellulaires pour détecter la production d&#39;IFNy et TNF après stimulation antigénique. panneau C montre que les CTL élargi sont fonctionnels tels que mesurés par Cr 51 dosage. LCL autologues, soit seule ou transduites avec un vecteur nul ou un vecteur adénoviral exprimant CMV pp65 ont été utilisés comme du goudronobtient. Alloréactivité été évaluée en utilisant allogéniques explosions PHA comme une cible.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N. Engl. J. Med. 333, 1038-1044 (1995).</li></ol>

Juan F. Vera est un consultant pour Wilson Loup fabrication. Les autres auteurs n&#39;ont aucune divulgation pertinente.

Les infections virales représentent une morbidité et une mortalité importantes chez les patients qui sont immunodéprimés par leur maladie ou son traitement. Après HSCT, par exemple, les infections causées par les herpèsvirus persistants tels que l&#39;EBV et le CMV, ainsi que par des virus respiratoires comme le virus respiratoire syncytial (VRS), sont bien connus, tandis que l&#39;importance des infections causées par Adv, virus BK, et humain herpesvirus (HHV) -6 ont plus récemment été appréciée. Alors que les agents pharmacologiques sont le traitement standard pour certaines infections, ils ont des toxicités importantes, de générer des variants résistants, et sont souvent inefficaces. En revanche, les cellules T spécifiques du virus provenant de donneurs de cellules souches se sont avérés sûrs et efficaces pour la prévention et le traitement de l&#39;infection virale ou une maladie de la greffe de cellules souches hématopoïétiques (HSCT) la mise en 2,5,6,18-21. Cependant, la mise en œuvre plus large de l&#39;immunothérapie cellulaire T est finalement limitée par le coût, la complexité et le temps requis pour la production de CTL. Notre approche nouvelle et rapide de générer des CTL multivirus, décrite dans le manuscrit en cours, devrait accroître considérablement la faisabilité de la thérapie cellulaire T cytotoxique pour les maladies virales, permettant à la stratégie visant à devenir une norme de soins pour l&#39;hôte immunodéprimé. L&#39;utilisation de plasmide PED nucleofected comme APC permet la présentation des antigènes sur les deux CMH de classe I et II, sans la concurrence des vecteurs viraux ou bien à partir de multiples antigènes viraux exprimés dans une cellule unique, depuis les différentes populations DC sont utilisés pour chacune des 3 plasmide. L&#39;utilisation de l&#39;IL-4 / 7 augmente la survie des cellules T et la prolifération, qui permet une augmentation correspondante de la fréquence et le répertoire de réponse spécifique de l&#39;antigène des cellules T 16,17. Enfin, la culture des pays du G-Rex réduit considérablement l&#39;apoptose des cellules T au cours de la culture. Les échanges gazeux (O 2 et de CO 2 dans la sortie) se produit à travers une membrane de silicium perméables au gaz, à la base du flacon, ce qui empêche l&#39;hypoxie, tout en permettant une plus grande profondeur de milieu au-dessus des cellules, fournissant plus de nutriments et de diluer les déchets. Cette plate-forme peut aussi être étendu à d&#39;autres virus comme lorsque des antigènes protecteurs ont été identifiés.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> CellGenix </td><td> CellGenix </td><td> 2005 </td></tr><tr><td> IL4 </td><td> R + D Systems </td><td> 204-IL/CF </td></tr><tr><td> IL7 </td><td> Peprotech </td><td> 200-15 </td></tr><tr><td> Hyclone RPMI 1640 </td><td> Thermo Scientific </td><td> SH30096.01 </td></tr><tr><td> Sérum humain AB </td><td> Vallée Biomedical Inc </td><td> HP1022 </td></tr><tr><td> Nucleofector </td><td> Amaxa / Lonza </td><td> AAF-1001B &amp; AAF-1001X </td></tr><tr><td> Kit nucléofection </td><td> Amaxa / Lonza </td><td> V4XP-3012 </td></tr><tr><td> Les plasmides </td><td> NTC </td><td> n / a </td></tr><tr><td> GM-CSF </td><td> R &amp; D </td><td> 215-GM/CF </td></tr><tr><td> IL1 </td><td> R &amp; D </td><td> 201-LB-025 </td></tr><tr><td> IL6 </td><td> R &amp; D </td><td> 206-IL-FC </td></tr><tr><td> TNF </td><td> R &amp; D </td><td> 210-TA-010 </td></tr><tr><td> PGE2 </td><td> Sigma </td><td> P6532-1MG </td></tr><tr><td> G-Rex </td><td> Wilson Loup fabrication </td><td> AY11-00027 </td></tr></tbody></table>

generation of multivirus-specific t cells to prevent/treat viral infections after allogeneic hematopoietic stem cell transplant

Les infections virales cause de morbidité et de mortalité dans allogreffe de cellules souches hématopoïétiques (HSCT). Nous et les autres ont réussi à produire et infusé cellules T spécifiques de virus d&#39;Epstein Barr (EBV), le cytomégalovirus (CMV) et l&#39;adénovirus (Adv) en utilisant les monocytes et transformés par l&#39;EBV de cellules lymphoblastoïdes (LCL EBV) modifiées génétiquement avec un vecteur adénovirus comme cellules présentatrices d&#39;antigènes (CPA). Aussi peu que 5 2x10 / kg trivirus-lymphocytes T cytotoxiques spécifiques (CTL) ont proliféré par plusieurs journaux après la perfusion et semblait prévenir et traiter les maladies virales graves, même résistants aux autres traitements disponibles. La plus large mise en œuvre de cette approche est encourageant limitée par les coûts de production élevés, la complexité de fabrication et le temps prolongé (4-6 semaines pour l&#39;EBV LCL génération, et 4-8 semaines pour la fabrication de CTL - total 10-14 semaines) pour la préparation. Pour surmonter ces limitations, nous avons développé un nouveau GMP protocole de production de CTL. Tout d&#39;abord, à la place de adénovecteurs pour stimuler les cellules T, nous utilisons des cellules dendritiques (CD) nucleofected avec l&#39;ADN des plasmides codant LMP2, EBNA1 et BZLF1 (EBV), Hexon et Penton (ADV), et de pp65 et IE1 (CMV) en tant que présentatrice d&#39;antigène cellules. Ces APC de réactiver les cellules T spécifiques pour tous les antigènes stimulant. Deuxièmement, la culture des lymphocytes T activés en présence d&#39;IL-4 (1000 U / ml) et de l&#39;IL-7 (10ng/ml) augmente et soutient le répertoire et la fréquence des cellules T spécifiques dans nos lignes. Troisièmement, nous avons utilisé un nouveau dispositif de gaz à la culture perméable (G-Rex) qui favorise l&#39;expansion et la survie d&#39;un nombre à grandes cellules après une stimulation unique, éliminant ainsi l&#39;exigence de l&#39;EBV et LCL en réduisant l&#39;intervention de technicien. En mettant en œuvre ces changements, nous pouvons maintenant produire CTL multispécifiques ciblant EBV, CMV, et Adv à un coût par 10 6 cellules qui est réduit par&gt; 90%, et en seulement 10 jours plutôt que 10 semaines en utilisant une approche qui peut être étendue à d&#39;autres protection des antigènes viraux. Notre approuvé par la FDA approche doit être de valeur pour les applications prophylactiques et le traitement pour les bénéficiaires de risque élevé GCSH allogénique.

Watch this Scientific Journal Video about Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant at JoVE.com

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Un rapide, protocole simple et rentable pour la génération de donateurs issus multivirus des CTL spécifiques (rctl) pour perfusion à allogéniques greffe de cellules souches hématopoïétiques (HSCT) à risque de développer des infections à CMV, Adv ou EBV. Ce processus de fabrication est conforme aux BPF et devrait assurer la plus large mise en œuvre des cellules T immunothérapie delà des centres spécialisés.

Génération de cellules T spécifiques Multivirus pour prévenir / traiter les infections virales, après allogreffe de cellules souches hématopoïétiques

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

generation-multivirus-specific-t-cells-to-preventtreat-viral

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JoVE Journal

Immunology and Infection

17.1K vues.  Baylor College of Medicine. Un rapide, protocole simple et rentable pour la génération de donateurs issus multivirus des CTL spécifiques (rctl) pour perfusion à allogéniques greffe de cellules souches hématopoïétiques (HSCT) à risque de développer des infections à CMV, Adv ou EBV. Ce processus de fabrication est conforme aux BPF et devrait assurer la plus large mise en œuvre des cellules T immunothérapie delà des centres spécialisés.

Vidéo: Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Après Cell-sort<em&gt; E. coli</em&gt; Après infection par le phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

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Immunologie et infection

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 ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

Identifier ADN mutations dans purifié hématopoïétiques cellules souches / progénitrices

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

Transplantation de queue peau d&#39;étudier allogéniques CD4 réponses des cellules T chez la souris

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Cellules T d&#39;enrichissement de cellules automatisé de cytomégalovirus spécifique pour des applications cliniques utilisant le système de capture de cytokine

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Génération et multi-phénotypique criblage à haut contenu de<em&gt; Coxiella burnetii</em&gt; Mutants de transposon

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Développement de cellules souches dérivées de cellules T régulatrices spécifiques de l&#39;antigène contre Autoimmunité

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

Mesure de cellules T alloréactivité basés sur l&#39;imagerie cytométrie en flux

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Évaluation des réponses de cellules T spécifiques du Virus Zika dans les organes Immunoprivileged des Ifnar1 infecté les souris- / -

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...