SS est soutenu en partie par le HHMI Med en Grad Subvention de formation par l&#39;intermédiaire du Programme TBMM. MHW est soutenu en partie par une bourse de développement de carrière du ministère des Anciens combattants et le soutien généreux de M. et Mme Harold M. Selzman. Ce travail a également été soutenu en partie par le NIH lymphome SPORE P50CA126752 subvention et le NIH R01 DK093660.

Jour 0 1. Isolation des PBMC à partir de sang humain <ol><li> Recueillir 20 ml de sang humain frais en utilisant ponction veineuse dans des tubes Vacutainer Na-héparine. </li><li> Mélanger le sang et avancée RPMI 1640 en 1:1 (v / v). </li><li> Ajouter 20 ml Lymphoprep moyen d&#39;un tube à centrifuger de 50 ml (25 ° C). Lentement couche de 25-30 ml de sang RPMI 1640 mélange sur le dessus du Lymphoprep. </li><li> Centrifuger à 400 xg pendant 40 min sans freins. </li><li> Recueillir les deux couches distinctes et floue à l&#39;aide d&#39;une pipette jetable dans 10 ml de PBS 1x (25 ° C) et porter le volume à 50 ml avec du PBS 1x. </li><li> Centrifuger à 450 xg pendant 10 min. </li><li> Aspirer le surnageant et ajouter 20 ml de RPMI 1640 avancée. </li><li> Centrifuger à 400 xg pendant 5 min. </li><li> Aspirer le surnageant. Ajouter 10 ml de milieu complet de lymphocytes T additionné de 5 ng / ml rhIL-15 préchauffé à 37 ° C. </li><li> Compter le nombre de cellules et la plaque en 24 manteau de culture de tissus ainsied plaque à 2 x 10 6 cellules / puits dans les médias complets de lymphocytes T supplémenté avec 5 ng / ml rhIL-15 Ajouter de l&#39;eau stérile pour puits environnants. Incuber une nuit dans un incubateur humidifié à 37 ° C, 5% de CO 2. </li></ol> Jour 1 2. Les plaques de revêtement avec un anticorps anti-CD28 et anti-CD3 pour stimuler les lymphocytes T <ol><li> Diluer anticorps anti-CD28 et anti-CD3 dans de l&#39;eau stérile à une concentration de 1 pg / ml. </li><li> Ajouter 500 ul de solution d&#39;anticorps à chaque 5 puits marqués d&#39;une culture de tissu non enduit plaque à 24 puits. </li><li> Ajouter de l&#39;eau stérile pour se reposer des puits. Enveloppez la plaque dans une pellicule rétractable et placer au réfrigérateur 4 ° C. </li></ol> 3. Nucléofection de cellules T non stimulés <ol><li> Préchauffer médias lymphocytes T à 37 ° C et un supplément de 5 ng / ml de rhIL-15. Préparer une solution complète Nucleofector en ajoutant 500 ul de supplément Nucleofector 1 à 2,25 ml de solution Nucleofector. </li><li> Aliquot 5 ug chacun des transposon (Zeo-pT-CMV-eGFP) et la transposase (pCMV-piggyBac) dans un tube de 1,5 ml. Remarque: il peut être nécessaire d&#39;optimiser la transposase et la quantité d&#39;ADN du transposon pour la livraison génique optimale et de minimiser toxicité cellulaire. Les plasmides peuvent être obtenus auprès des auteurs sur demande. </li><li> PBMC récolte de la plaque 24 tissus et la culture dans un tube de 50 ml. Compter le nombre de cellules. Enregistrer 2 x 10 6 cellules pour une utilisation en tant que commande au cours de la cytométrie en flux. </li><li> Ajouter 7-10 x 10 6 cellules dans un tube de 15 ml et centrifuger à 400 xg, 5 min, aspirer le surnageant et le doigt du coup de la pastille. </li><li> Ajouter 100 ul de cellules T nucléofection solution complète à la cellule desserré granulés. </li><li> Ajouter le mélange de solution de cellules dans le tube contenant les plasmides. </li><li> Ajouter le mélange de solution de cellule de plasmide au fond de la cuvette nucléofection. Veillez à ne pas introduire de bulles. </li><li> Nucleofect les cellules en utilisant programme U-014 (cellules T non stimulés, l&#39;homme, <a href="http://bio.lonza.com/resources/product-instructions/protocols/" target="_blank">http://bio.lonza.com/resources/product-instructions/protocols/</a> ). </li><li> Immédiatement ajouter 500 ul de médias préchauffé avec rhIL-15 à la cuvette. Transférer les cellules à un puits d&#39;une plaque à 24 puits avec 1,5 ml de milieu préchauffé à rhIL15. </li><li> Incuber une nuit dans un incubateur humidifié à 37 ° C, 5% de CO 2. </li></ol> Jour 2 4. La stimulation non spécifique des cellules T <ol><li> Récolte des cellules et de déterminer le nombre de cellules. Annuler 0,5 x 10 6 cellules par cytométrie en flux pour déterminer la fréquence de cellules GFP-positives. Utilisez les PBMC non transfectées comme témoins. </li><li> Aspirer la solution CD3/28 anticorps de la plaque de culture de tissu non enduit et rincer chaque puits avec les médias de lymphocytes T. </li><li> Remettre en suspension les cellules nucleofected à 0,5 x 10 6 cellules par ml dans les médias complets CTLadditionné de 5 ng / ml de rhIL-15. Ajouter 2,0 ml de chacune des cellules nucleofected à 4 puits de la plaque de culture de tissus non. Ajouter 0,5 x 10 6 cellules non nucleofected au 5 bien. </li><li> Incuber pendant 3 jours dans un incubateur humidifié à 37 ° C, 5% de CO 2. </li></ol> Jour 5 <ol><li> Récolter les cellules T stimulées à partir de la plaque non revêtue de la culture tissulaire. </li><li> Compter et Réensemencement les cellules dans un 24 plaque de culture tissulaire pour bien les enrober à 0,7 x 10 6 cellules / ml dans un milieu de lymphocytes T avec 5 ng / ml d&#39;IL-15. </li></ol> Jour 7 7. L&#39;analyse de l&#39;expression des gènes <ol><li> Cellules de récolte et se colorent avec anti-humain CD8 (peut également utiliser l&#39;anti-CD3 et anti-CD4) et d&#39;analyser avec la cytométrie en flux pour l&#39;expression de la GFP%. </li></ol> Jour 8 (Facultatif) 8. L&#39;expansion des cellules T <ol><li> Le jour 8 après transfection, les cellules T peuvent être réensemencées en TDes cellules support avec l&#39;IL-15 à une densité de 0,7 x 10 6 cellules par puits d&#39;une nouvelle expansion 16. </li></ol>

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	<li>Cary, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon+mutagenesis+of+baculoviruses:+analysis+of+Trichoplusia+ni+transposon+IFP2+insertions+within+the+FP-locus+of+nuclear+polyhedrosis+viruses.">Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses.</a> Virology. 172 (1), 156 (1989).</li><li>Fraser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+for+movement+of+Lepidopteran+transposon+IFP2+in+insect+cells+using+a+baculovirus+genome+as+a+target+DNA.">Assay for movement of Lepidopteran transposon IFP2 in insect cells using a baculovirus genome as a target DNA.</a> Virology. 211 (2), 397 (1995).</li><li>Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transposition+of+the+piggyBac+(PB)+transposon+in+mammalian+cells+and+mice.">Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice.</a> Cell. 122 (3), 473 (2005).</li><li>Saridey, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac+transposon-based+inducible+gene+expression+in+vivo+after+somatic+cell+gene+transfer.">PiggyBac transposon-based inducible gene expression in vivo after somatic cell gene transfer.</a> Mol. Ther. 17 (12), 2115 (2009).</li><li>Nakanishi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac+transposon-mediated+long-term+gene+expression+in+mice.">piggyBac transposon-mediated long-term gene expression in mice.</a> Mol. Ther. 18 (4), 707 (2010).</li><li>Wilson, M. H., Coates, C. J., George, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac+Transposon-mediated+Gene+Transfer+in+Human+Cells.">PiggyBac Transposon-mediated Gene Transfer in Human Cells.</a> Mol. Ther. 15 (1), 139 (2007).</li><li>Nakazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+PiggyBac+transposon+system+for+the+sustained+genetic+modification+of+human+T+lymphocytes.">Optimization of the PiggyBac transposon system for the sustained genetic modification of human T lymphocytes.</a> J. Immunother. 32 (8), 826 (2009).</li><li>Raja Manuri, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac+transposon/transposase+system+to+generate+CD19-specific+T+cells+for+treatment+of+B-lineage+malignancies.">piggyBac transposon/transposase system to generate CD19-specific T cells for treatment of B-lineage malignancies.</a> Hum. Gene Ther. 21 (4), 427 (2010).</li><li>Doherty, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperactive+piggyBac+gene+transfer+in+human+cells+and+in+vivo.">Hyperactive piggyBac gene transfer in human cells and in vivo.</a> Hum. Gene Ther. , (2011).</li><li>Yusa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hyperactive+piggyBac+transposase+for+mammalian+applications.">A hyperactive piggyBac transposase for mammalian applications.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (4), 1531 (2011).</li><li>Bonini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+modification+of+T+cells.">Genetic modification of T cells.</a> Biol. Blood Marrow Transplant. 17, S15-S20 (2011).</li><li>Hackett, P. B., Largaespada, D. A., Cooper, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transposon+and+transposase+system+for+human+application.">A transposon and transposase system for human application.</a> Mol. Ther. 18 (4), 1531 (2010).</li><li>Galvan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+of+PiggyBac+transposon+integrations+in+primary+human+T+cells.">Genome-wide mapping of PiggyBac transposon integrations in primary human T cells.</a> J. Immunother. 32 (8), 837 (2009).</li><li>Nakazawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PiggyBac-Mediated+Cancer+Immunotherapy+Using+EBV-Specific+Cytotoxic+T-Cells+Expressing+HER2-Specific+Chimeric+Antigen+Receptor.">PiggyBac-Mediated Cancer Immunotherapy Using EBV-Specific Cytotoxic T-Cells Expressing HER2-Specific Chimeric Antigen Receptor.</a> Mol. Ther. 19 (12), 2133 (2011).</li><li>Huye, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+mTor+Inhibitors+With+Rapamycin-resistant+T+Cells:+A+Two-pronged+Approach+to+Tumor+Elimination.">Combining mTor Inhibitors With Rapamycin-resistant T Cells: A Two-pronged Approach to Tumor Elimination.</a> Mol. Ther. 19 (12), 2239 (2011).</li><li>Vera, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+production+of+antigen-specific+T+cells+for+preclinical+and+clinical+applications+using+gas-permeable+rapid+expansion+cultureware+(G-Rex).">Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex).</a> J. Immunother. 33 (3), 305 (2010).</li><li>Kahlig, K. 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Aucun conflit d&#39;intérêt déclaré.

Le procédé décrit ici permet de modifier transgène stable de lymphocytes T humains primaires. Nous avons déjà testé l&#39;utilisation du système piggyBac transposon pour modifier des cellules T pour exprimer un gène rapporteur (pour plus de 4 semaines), un gène suicide non immunogène, un récepteur d&#39;antigène chimérique pour l&#39;immunothérapie adoptive (pour plus de 100 jours), et à concevoir une résistance aux médicaments immunosuppresseurs 7,13-15. Non-virale modification des...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Lympholyte </td><td> Cedarlane </td><td> CL5015 </td><td></td></tr><tr><td> Avancée RPMI 1640 </td><td> LifeTechnologies </td><td> 12633020 </td><td></td></tr><tr><td> Hyclone sérum de veau fœtal </td><td> Fisher Scientific </td><td> SH3008803 </td><td></td></tr><tr><td> Glutamax-je Supplément </td><td> LifeTechnologies </td><td> 35050-061 </td><td></td></tr><tr><td> IL-15 humaine protéine recombinante </td><td> eBioscience </td><td> 14-8159 </td><td></td></tr><tr><td> Kit Endofree Plasmid Maxi </td><td> Qiagen </td><td> 12362 </td><td></td></tr><tr><td> Amaxa Nucleofector </td><td> Lonza </td><td> AAD-1001S </td><td> &amp; Nbsp; </td></tr><tr><td> Cellules T humaines Nucleofector Kit </td><td> Lonza </td><td> APV-1002 </td><td></td></tr><tr><td> CD8-APC </td><td> Southern Biotech </td><td> 9536-11 </td><td></td></tr><tr><td> Anti-Human CD3 </td><td> eBioscience </td><td> 16-0037-81 </td><td></td></tr><tr><td> Anti-Human CD28 </td><td> BD Pharmingen </td><td> 555725 </td><td></td></tr><tr><td> Plate 24 Tissue Culture Eh bien traitée </td><td> BD Falcon </td><td> 353047 </td><td></td></tr><tr><td> 24 Culture tissulaire Eh bien non traitée Plate </td><td> BD Falcon </td><td> 351147 </td><td></td></tr><tr><td></td><td></td><td></td><td> Complète des cellules T médias composition 1x avancée RPMI 1640 5% inactivé par la chaleur du sérum fœtal bovin 2 mM GlutamaxIM-I </td></tr></tbody></table>

piggybac transposon system modification of primary human t cells

Le système piggyBac transposon est active par sa nature, l&#39;origine dérivé de la 1,2 arpenteuse du chou papillon. Ce système non viral est un plasmide à base, le plus souvent en utilisant deux plasmides avec une expression de l&#39;enzyme transposase piggyBac et un transposon plasmide hébergeant le gène (s) d&#39;intérêt entre répétées inversées d&#39;éléments qui sont nécessaires à l&#39;activité de transfert de gène. PiggyBac transfert de gènes par médiation un &quot;couper et coller&quot; mécanisme par lequel la transposase intègre le segment de transposon dans le génome de la cellule cible (s) d&#39;intérêt. PiggyBac a démontré l&#39;efficacité de délivrance de gènes activité dans une large gamme de 1,2 insecte, de mammifère 3-5, et humaine cells6 y compris les cellules T humaines primaires 7,8. Récemment, une transposase hyperactive piggyBac a été générée en améliorant l&#39;efficacité de transfert de gènes 9,10. Lymphocytes T humains sont des intere cliniqueer pour l&#39;immunothérapie adoptive du cancer 11. Fait à noter, le premier essai clinique impliquant une modification transposon des lymphocytes T humains en utilisant le système Sleeping beauty transposon a été approuvée le 12. Nous avons déjà évalué l&#39;utilité de piggyBac comme une méthode non virale pour la modification génétique de cellules T humaines. Nous avons trouvé piggyBac pour être efficace dans la modification génétique de cellules T humaines avec un gène rapporteur et un gène non immunogène inductible suicide 7. L&#39;analyse des sites d&#39;intégration génomique a révélé un manque de préférence pour une intégration dans ou à proximité connues proto-oncogènes 13. Nous avons utilisé piggyBac de gènes modifient les lymphocytes T cytotoxiques pour réaliser un récepteur d&#39;antigène chimérique dirigé contre l&#39;antigène tumoral HER2, et a trouvé que le gène modifiés lymphocytes T médié assassinat ciblé de HER2-positives cellules tumorales in vitro et in vivo dans un modèle de souris orthotopique 14. Nous avons également utilisé piggyBacpour générer des cellules T humaines résistantes à la rapamycine, qui devraient être utiles dans les thérapies du cancer, où la rapamycine est utilisée 15. Nous décrivons ici une méthode pour utiliser piggyBac de modifier génétiquement les cellules T humaines primaires. Ceci inclut l&#39;isolement des cellules mononucléées du sang périphérique (PBMC) à partir de sang humain suivie par la culture, la modification génétique, et l&#39;activation des cellules T. Aux fins du présent rapport, les cellules T ont été modifiées avec un gène rapporteur (eGFP) pour l&#39;analyse et la quantification de l&#39;expression génique par cytométrie en flux. PiggyBac peuvent être utilisées pour modifier des cellules T avec une variété de gènes d&#39;intérêt. Bien que nous ayons utilisé piggyBac de diriger les lymphocytes T aux antigènes tumoraux 14, nous avons également utilisé piggyBac d&#39;ajouter un interrupteur de sécurité inductible afin d&#39;éliminer les cellules gènes modifiés si nécessaire 7. La capacité de chargement importante de piggyBac a également permis le transfert de gènes deune molécule de rapamycine grande résistance mTOR (15 ko) 15. Par conséquent, nous présentons une méthodologie non-virale pour le gène-modification stable de cellules T humaines primaires pour une large variété de fins.

Un schéma montrant les étapes de la modification génétique de lymphocytes T humains avec un gène rapporteur (eGFP) est illustré à la figure 1. Ces plasmides sont disponibles sur demande auprès des auteurs. Un schemtic démontrant les étapes génétiquement modifiés lymphocytes T humains avec un gène rapporteur (eGFP) est showin dans la figure 2. Il est nécessaire d&#39;activer les cellules T dans le but de les amener à se diviser, se développer et se propager dans la cultu...

Watch this Scientific Journal Video about piggyBac Transposon System Modification of Primary Human T Cells at JoVE.com

piggyBac Transposon System Modification of Primary Human T Cells

Nous décrivons une méthode pour modifier génétiquement des cellules T humaines primaires avec un transgène en utilisant la non-virale<em&gt; PiggyBac</em&gt; Transposon système. Lymphocytes T modifiés à l&#39;aide de la<em&gt; PiggyBac</em&gt; Transposon exposition expression du transgène système stable.

Modification piggyBac système transposon des cellules T humaines primaires

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

piggybac-transposon-system-modification-of-primary-human-t-cells

Research

JoVE Journal

Immunology and Infection

piggyBac Transposon System Modification of Primary Human T Cells

17.1K vues.  Baylor College of Medicine. Nous décrivons une méthode pour modifier génétiquement des cellules T humaines primaires avec un transgène en utilisant la non-virale PiggyBac Transposon système. Lymphocytes T modifiés à l'aide de la PiggyBac Transposon exposition expression du transgène système stable.

Vidéo: piggyBac Transposon System Modification of Primary Human T Cells

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 ...

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Immunologie et infection

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Détermination de l&#39;activité cytotoxique CD8 spécifiques optimale des cellules T humaines Her2neu en comparant les Cr51 Essai de libération dans le système de xCELLigence

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

L&#39;évaluation de l&#39;Innate détection du VIH-1 CD4 infectés<sup&gt; +</sup&gt; Cellules T par les cellules dendritiques plasmacytoïdes Utilisation d&#39;un<em&gt; Ex vivo</em&gt; Système de co-culture.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

Identification de nouveaux gènes associés à la production d&#39;alginate dans<em&gt; Pseudomonas aeruginosa</em&gt; Utilisation de Mini-<em&gt; Himar1</em&gt; Mutagenèse Mariner Transposon médiation

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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

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

Génération de cellules T humaines allo-spécifiques du sang périphérique

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

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 ...

Pneumococcus Infection of Primary Human Endothelial Cells in Constant Flow

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von Willebrand Factor (VWF). This glycoprotein changes its molecular conformation in response to shear stress, thereby exposing binding sites for a broad spectrum of host-ligand interactions. In general, culturing of primary endothelial cells under a defined shear flow is known to promote the specific cellular differentiation and the formation of a stable and tightly linked endothelial layer resembling the physiology of the inner lining of a blood vessel. Thus, the functional analysis of interactions between bacterial pathogens and the host vasculature involving mechanosensitive proteins requires the establishment of pump systems that can simulate the physiological flow forces known to affect the surface of vascular cells.
The microfluidic device used in this study enables a continuous and pulseless recirculation of fluids with a defined flow rate. The computer-controlled air-pressure pump system applies a defined shear stress on endothelial cell surfaces by generating a continuous, unidirectional, and controlled medium flow. Morphological changes of the cells and bacterial attachment can be microscopically monitored and quantified in the flow by using special channel slides that are designed for microscopic visualization. In contrast to static cell culture infection, which in general requires a sample fixation prior to immune labeling and microscopic analyses, the microfluidic slides enable both the fluorescence-based detection of proteins, bacteria, and cellular components after sample fixation; serial immunofluorescence staining; and direct fluorescence-based detection in real time. In combination with fluorescent bacteria and specific fluorescence-labeled antibodies, this infection procedure provides an efficient multiple component visualization system for a huge spectrum of scientific applications related to vascular processes.

This study describes the microscopic monitoring of pneumococcus adherence to von Willebrand factor strings produced on the surface of differentiated human primary endothelial cells under shear stress in defined flow conditions. This protocol can be extended to detailed visualization of specific cell structures and quantification of bacteria by applying differential immunostaining procedures.

Infection de Pneumococcus des cellules endothéliales humaines primaires dans le flux constant

Interaction of Streptococcus pneumoniae with the surface of endothelial cells is mediated in blood flow via mechanosensitive proteins such as the Von ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

Production de cellules CAR-T humaines conçues par CRISPR

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.