Ce travail a été soutenu par des subventions du NIH (5U01CA137070) et l&#39;American Cancer Society (RSG-09-070-01-LIB), un prix Cancer Research Institute chercheur (MK), une association américaine de coeur de pré-doctorat (KM) et un ministère espagnol de l&#39;Education et des Sciences Fellowship (APG). Nous aimerions remercier le Dr Nicholas Restifo et le Dr Yu Zhiya (NIH / NCI) pour des suggestions utiles pour le protocole.

1. Préparer Construire rétroviraux <ol><li> Sous-clonage du récepteur des cellules T (TCR) du gène d&#39;intérêt dans un vecteur rétroviral (figure 1, par exemple des vecteurs pMSG 7, pMIGII 5,8, pMXs partir Cellbiolabs). L&#39;α TCR et gène de la chaîne β devrait être sur le même vecteur, sous le contrôle du même promoteur pour garantir l&#39;expression égale. Si le TCR d&#39;intérêt est humain, les domaines constante humaine doivent être remplacés par des domaines de souris constant 9. Notre observation est que TCR pleine longueur humains ne expriment de façon stable sur les lymphocytes T de souris. </li><li> Préparer l&#39;ADN plasmidique de haute qualité en utilisant Qiagen Maxi Prep Kit ou un produit similaire. La concentration du plasmide devrait être d&#39;environ 1 ug / uL. </li></ol> 2. La transfection et la T-cellule d&#39;isolement Jour -1 (cellules emballage Plate) <ol><li> Déloger Platinum-E (Plat-E, Cellbiolabs) les cellules d&#39;encapsidation rétrovirale à la trypsine-EDTA et neutraliser avec du DMEM médias. </li><li> Centrifuger les cellules à 1000 xg pendant 5 min. Aspirer le surnageant et remettre en suspension les cellules dans du milieu DMEM à granulés. </li><li> Déterminer le nombre de cellules et de diluer les cellules à 0.6x10 6 / mL. Planche 10 ml de suspension cellulaire dans un 10mm poly-lysine plaque de culture de tissus enduits et pousser la nuit dans un incubateur de cellules à 37 ° C, 5% de CO 2. </li></ol> Jour 0 (transfection) <ol start="4"><li> Le lendemain matin, examiner les cellules sous un microscope optique. Les cellules doivent être d&#39;environ 80% de confluence. </li><li> Retirer délicatement le support de la plaque. Laver les cellules une fois avec du PBS 1x et ajouter 10 ml pré-chauffé, frais DMEM médias sans pénicilline-streptomycine (Pen / Strep). (Remarque: Pen / Strep peut interférer avec la transfection) </li><li> Préparer complexes de transfection. Préparer le mélange A et B comme suit: <table border="0"><tbody><tr><td> Mix A: L&#39;ADN plasmidique </td><td> 9 pg </td></tr><tr><td> PCL-Eco plasmide helper </td><td> 6,3 mg </td></tr><tr><td> OptiMEM </td><td> 1,5 ml </td></tr><tr><td colspan="2"></td></tr><tr><td> Mix B: Lipopfectime 2000 </td><td> 60 uL </td></tr><tr><td> OptiMEM </td><td> 1,5 ml </td></tr></tbody></table> Incuber A et B séparément pendant 5 minutes à température ambiante Mix A et B ensemble doucement et incuber pendant 20 minutes à température ambiante pour former un complexe de transfection. </li><li> Lentement goutte à goutte le mélange (total 3 ml) dans les cellules Plat-E. Secouez doucement la plaque avant en arrière pour répartir le mélange de transfection uniformément. </li><li> Incuber les cellules à 37 ° C CO / 5% 2 dans un incubateur à cellules. </li><li> Après 6-8 heures, remplacez moyenne avec 10 ml de milieu DMEM frais (avec Pen / Strep) pour la production virale. </li></ol> Plaque Manteau avec des anticorps <ol start="10"><li> Souris T-cellules ont besoin d&#39;être activé avant de transduction virale. Il ya plusieurs façons différentes pour activer les lymphocytes T. Ici, nous utilisons la plaque liée anticorps anti-CD3e et anti-CD28. Préparer un mélange d&#39;anticorps de 1 pg / mL d&#39;anti-CD3e et 2 pg / ml d&#39;anti-CD28 dans du PBS stérile. </li><li> Distribuer 250 uL du mélange d&#39;anticorps à chaque puits d&#39;une plaque de 24 puits de culture de tissu. La plaque peut être enduit nuit à 4 heures C ou 2 ° à 37 ° C. </li></ol> Préparation de unicellulaire suspension de rate de souris <ol start="12"><li> Récolte de rate de souris et de transfert en milieu RPMI. </li><li> Placez la rate dans une passoire cellule. Mash la rate avec un piston de la seringue dans une plaque de 5 cm de culture de tissu. Rincer la crépine cellules hors des cellules avec du PBS stérile. </li><li> Centrifugeuse 1000 xg, 5 min. </li><li> Rejeter le surnageant. Resuspendre le culot cellulaire dans un tampon de lyse ACK (2 ml par rate). Incuber 2 à 3 min à température ambiante. Ajouter milieu RPMI jusqu&#39;à 20 ml et centrifuger 1000 xg pendant 5 min. </li><li> Rejeter le surnageant. Resuspendre le culot cellulaire dans du PBS stérile. Déterminer le nombre de cellules et centrifugation 1000 g pendant 5 min à 4 ° C. Procéder à l&#39;isolement de cellules T. </li></ol> Isolement et activation des lymphocytes T de souris <ol start="17"><li> Magnétiquement marquer les cellules selon le manuel du produit (CD8 + kit d&#39;isolation à cellules T de Miltenyi Biotec). </li><li> Placer une colonne LS (Miltenyi Biotec) dans la mchamp agnetic. Appliquer suspension cellulaire étiquetés sur la colonne. Recueillir accréditives (enrichie de souris T CD8 + cellules). Laver la colonne trois fois avec 3 ml de tampon selon le manuel du produit et de combiner avec les précédents accréditives. </li><li> Colorer les cellules avec des anticorps anti-CD3e et anti-CD8a et analyser par cytométrie en flux (Figure 2a). Pour une séparation typique, ~ 8-10% des cellules totales peuvent être isolés. ~ 90% des cellules isolées sont CD8 + T-cellules. </li><li> Centrifuger les cellules à 1000 xg, 5 min. Jeter le surnageant. Resuspendre la cellule dans le milieu RPMI culot avec recombinante humaine IL-2 (rhIL2, 20 ng / ml) à 1x10 6 / ml. </li><li> Juste avant d&#39;ajouter les cellules, enlever la solution d&#39;anticorps à partir de la plaque (préalablement préparé en 2.11). Rincez chaque puits avec du PBS stérile et retirer du PBS. </li><li> Ajouter 1 mL de la suspension cellulaire dans chaque puits. Mettez la plaque à 37 ° C / 5% de CO 2 dans un incubateur à cellules. </li></ol> 3. (Jour 2 et Jour 3) L&#39;infection de cellules T activées <ol><li> Après 2 jours de production de virus, le milieu de la plaque de cellules Plat-E devrait devenir jaune. Transférer le surnageant viral pour un tube de 15 ml conique. Ajouter 10 ml de milieu DMEM frais à la plaque pour la production virale supplémentaire (en option). </li><li> Centrifuger le surnageant de virus à 1000 xg pendant 5 min pour enlever les débris des cellules. Soigneusement surnageant transfert de virus à un nouveau tube. Laissez un peu de liquide au fond du tube et ne pas déranger les débris des cellules. </li><li> Recueillir des cellules T activées de la plaque dans un tube conique de 50 ml. Déterminer le nombre de cellules. Enregistrer certaines cellules dans un tube séparé pour être utilisé comme négatif (non transduites) de contrôle. Centrifuger à 1000 xg pendant 5 min et éliminer le surnageant. </li><li> Reprendre le surnageant cellulaire par le virus de bouletage à 10 6 cellules par mL avec 1 à 20 ng / ml rhIL2 et 10 pg / ml de sulfate de protamine. Ajouter 1 ml de suspension cellulaire dans chaque puits d&#39;une plaque de 24 puits. Resuspendre les cellules dans le tube de contrôle en milieu RPMI à la même densité cellulaire avec la même quantité de sulfate de protamine et rhIL2. Ajouter aux cellules de la même plaque. </li><li> Enveloppez la plaque avec du film plastique. Centrifuger 90 minutes à 2000 xg, à 32 ° C sans frein. </li><li> Après la centrifugation, retirer le film plastique. Ajouter 1 mL de milieu RPMI frais avec 20 ng / mL rhIL2 à chaque puits. Mettez la plaque arrière à l&#39;incubateur. </li><li> (Facultatif) niveau supérieur d&#39;expression du TCR peut être atteint par la collecte du surnageant de virus et répéter la procédure d&#39;infection au jour 3. </li></ol> 4. L&#39;expansion des cellules et l&#39;évaluation de l&#39;efficacité Transduction <ol><li> Examinez la transduction des lymphocytes T quotidienne. Fractionner les cellules à des taux de 01h03 lorsque cela est nécessaire dans un milieu RPMI rhIL2. Ne laissez pas l&#39;envahir les cellules (milieu devient jaune). </li><li> Au jour 6 ou 7 jours, les taches les cellules avec des anticorps et tétramère CMH spécifiques pour le TCR pour évaluer l&#39;expression du TCR sur les cellules T de surface. Utilisez le non transduites cellules T comme témoin (figure 2c). </li><li> La transduction des lymphocytes T sont prêts pour d&#39;autres applications en aval. </li></ol> 5. Les résultats représentatifs Il est souvent avantageux de purifier les cellules T des sous-ensembles, avant de transduction bien splénocytes peuvent être transduites sans purification. Haute pureté des lymphocytes T des sous-ensembles peuvent être obtenus en utilisant des billes magnétiques commerciales (figure 2a). Pour évaluer le niveau d&#39;expression de TCR sur les cellules T, des anticorps spécifiques pour TCR alpha ou beta des chaînes peuvent être utilisés. Typiquement 30% à 80% des cellules transduites peuvent exprimer TCR (figure 2b) selon les gènes TCR et les titres de virus. CMH tétramère spécifiques pour le TCR peuvent aussi être utilisés pour vérifier davantage l&#39;expression fonctionnelle du TCR (figure 2c). <img src="/files/ftp_upload/2307/2307fig1.jpg" alt="Figure 1" /> Figure 1. Exemple de gène du récepteur des cellules T de construire des sous-cloné dans un vecteur rétroviral. Alpha pleine longueur et les gènes de la chaîne bêta sont reliés par un linker auto-clivable 2A peptide 8. <img src="/files/ftp_upload/2307/2307fig2.jpg" alt="Figure 2" /> Figure 2. Exemple d&#39;isolation réussie et la transduction des lymphocytes T de souris. (A) cellules T CD8 ont été isolés à partir de splénocytes utilisant CD8a + T isolement des cellules Kit (Miltenyi Biotec) et colorées avec des anticorps anti-CD3e et anti-CD8a. Isolée cellules T CD8 ont été transduites avec R6C12 TCR spécifiques pour l&#39;homme gp100: 209 -217 4 peptide et colorées avec anti-humain Vbeta 8 anticorps (b) et HLA-A2kb gp100 tétramère (c).

<ol>
	<li>Krogsgaard, M., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+T+cells+'see'+antigen.">How T cells 'see' antigen.</a> Nat. Immunol. 6, 239-245 (2005).</li><li>Berg, 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=Antigen/MHC-specific+T+cells+are+preferentially+exported+from+the+thymus+in+the+presence+of+their+MHC+ligand.">Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand.</a> Cell. 58, 1035-1046 (1989).</li><li>Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li><li>Yu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+immunogenicity+of+a+self/tumor+antigen+derives+from+peptide-MHC-I+instability+and+is+independent+of+tolerance.">Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.</a> J. Clin. Invest. 114, 551-559 (2004).</li><li>Holst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T-cell+receptor+retrogenic+mice.">Generation of T-cell receptor retrogenic mice.</a> Nat Protoc. 1, 406-417 (2006).</li><li>Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+regression+in+patients+after+transfer+of+genetically+engineered+lymphocytes.">Cancer regression in patients after transfer of genetically engineered lymphocytes.</a> Science. 314, 126-129 (2006).</li><li>Hawley, R. G., Lieu, F. H., Fong, A. Z., Hawley, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+retroviral+vectors+for+potential+use+in+gene+therapy.">Versatile retroviral vectors for potential use in gene therapy.</a> Gene Ther. 1, 136-138 (1994).</li><li>Szymczak, 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=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat. Biotechnol. 22, 589-594 (2004).</li><li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66, 8878-8886 (2006).</li><li>Letourneur, F., Malissen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+a+T+cell+hybridoma+variant+deprived+of+functional+T+cell+receptor+alpha+and+beta+chain+transcripts+reveals+a+nonfunctional+alpha-mRNA+of+BW5147+origin.">Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin.</a> Eur. J. Immunol. 19, 2269-2274 (1989).</li><li>Bendle, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+graft-versus-host+disease+in+mouse+models+of+T+cell+receptor+gene+therapy.">Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.</a> Nat. Med. 16, 565-570 (2010).</li></ol>

Plusieurs étapes sont essentielles pour obtenir des résultats optimaux. Plat-E lignée cellulaire d&#39;emballage doivent être maintenus en bon état sain. Si nécessaire, les cellules peuvent être passées à quelques reprises dans du milieu DMEM avec 1 ug / ml de puromycine, 10 pg / ml blasticidine pour prévenir la perte d&#39;expression structure d&#39;une protéine rétrovirale. Le jour de la transfection, les cellules doivent être confluentes ~ 80%. Trop peu de cellules ou d&#39;un trop grand nombre peut diminuer le titre viral. La qualité du virus peut être vérifiée par des essais sur les lignées cellulaires qui peuvent être facilement transduites. Depuis TCR nécessitent CD3 complexes pour être exprimée à la surface cellulaire, nous avons régulièrement recours 58α-/β- hybridomes 10 (une lignée cellulaire qui n&#39;exprime pas endogènes du TCR α ou de la chaîne β, mais exprime CD3 complexe). Nous obtenons près de 100% d&#39;efficacité de transduction des cellules d&#39;hybridomes lors de transduction des cellules avec 1 ml de surnageant de virus. Enfin, les cellules T doivent être activés et prolifèrent en raison des rétrovirus ne peut infecter que les cellules en division active. La méthode présentée ici pour la production spécifique de l&#39;antigène des cellules T a plusieurs limites. Premièrement, il est difficile d&#39;atteindre une efficacité de transduction de 100% pour principal de la souris T-cellules. Une partie des cellules n&#39;expriment pas le TCR d&#39;intérêt. Deuxièmement, le TCR α transduites et des chaînes β peut mispair avec TCR endogènes 9, ce qui peut réduire le niveau d&#39;expression du TCR d&#39;intérêt. En outre, le TCR mispaired peut causer l&#39;auto-immunité in vivo 11. Enfin, il ya seulement un laps de temps limité de la transduction des lymphocytes T peuvent être utilisés. Après environ une semaine, les cellules en apoptose à moins d&#39;être re-stimulé. Cependant, les cellules peuvent s&#39;épuiser et perdre avec les fonctions répétitives re-stimulations.

Médias: Milieu DMEM (chaleur DMEM 10% FBS inactivé + Pen / Strep + L-Glu + Sodium Pyruvate + non-acides aminés essentiels). Milieu DMEM sans Pen / Strep (Idem milieu DMEM, mais sans ajouter Pen / Strep). Milieu RPMI (chaleur RPMI 10% de FBS inactivé + L-Glu + non-acides aminés essentiels + β-Mercaphoethanol + Pen / Strep pyruvate de sodium +).

retroviral transduction of t-cell receptors in mouse t-cells

Récepteurs des cellules T (TCR) jouent un rôle central dans le système immunitaire. TCR sur les cellules T surfaces peuvent reconnaître spécifiquement des antigènes peptidiques présentés par les cellules présentatrices d&#39;antigène (CPA) 1. Cette reconnaissance conduit à l&#39;activation des cellules T et une série de résultats fonctionnels (production de cytokines, par exemple, tuer des cellules cibles). Comprendre le rôle fonctionnel des TCR est essentielle pour exploiter la puissance du système immunitaire pour traiter une variété de maladies liées immunologie (par exemple le cancer ou d&#39;auto-immunité). Il est commode d&#39;étudier TCR dans des modèles murins, ce qui peut être accompli de plusieurs façons. Faire TCR transgénique modèles de souris est coûteux et prend du temps et actuellement il ya seulement un nombre limité d&#39;entre eux disponibles 2-4. Alternativement, des souris avec des antigènes spécifiques des cellules T peuvent être générés par la moelle osseuse chimère. Cette méthode prend également plusieurs semaines et nécessite une expertise 5. Transduction rétrovirale des TCR en activées in vitro de lymphocytes T de souris est une méthode rapide et relativement facile d&#39;obtenir des cellules T peptide-CMH désirée spécificité. Antigène spécifique de cellules T peuvent être générés en une semaine et utilisé dans toutes les applications en aval. Etudier transduites cellules T a aussi une application directe à l&#39;immunothérapie humaine, comme un transfert adoptif de lymphocytes T humains transduites avec l&#39;antigène spécifique TCR est une stratégie émergente pour le traitement du cancer 6. Nous présentons ici un protocole à un retrovirus transduire TCR en activées in vitro de lymphocytes T de souris. Les deux gènes humains et de souris TCR peuvent être utilisés. Les rétrovirus transportant des gènes spécifiques du TCR sont générés et utilisés pour infecter la souris de cellules T activées par des anticorps anti-CD3 et anti-CD28. Après l&#39;expansion in vitro, les lymphocytes T transduits sont analysés par cytométrie en flux.

Watch this Scientific Journal Video about Retroviral Transduction of T-cell Receptors in Mouse T-cells at JoVE.com

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Nous présentons un protocole pour produire l&#39;antigène spécifique de la souris de cellules T à l&#39;aide transduction rétrovirale

Transduction rétrovirale des récepteurs de cellules T dans les lymphocytes T de souris

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

retroviral-transduction-of-t-cell-receptors-in-mouse-t-cells

Research

JoVE Journal

Immunology and Infection

34.8K vues.  New York University School of Medicine. Nous présentons un protocole pour produire l'antigène spécifique de la souris de cellules T à l'aide transduction rétrovirale

Vidéo: Retroviral Transduction of T-cell Receptors in Mouse T-cells

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&#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 ...

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Immunologie et infection

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

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 ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Transduction Adenoviral de CD4 naïfs cellules T pour étudier la différenciation Treg

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Dérivation des cellules T<em&gt; In Vitro</em&gt; De la souris les cellules souches embryonnaires

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Souris Naïf CD4<sup&gt; +</sup&gt; Isolement des cellules T et<em&gt; In vitro</em&gt; Différenciation en sous-ensembles de cellules T

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Rétrovirale transduction des cellules progénitrices de la moelle osseuse pour générer des cellules T Receptor Retrogenic Souris

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Générateur<em&gt; De Novo</em&gt; Spécifiques de l&#39;antigène T Human cellulaires Receptors par rétrovirale transduction de Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Rétrovirale transduction des cellules T auxiliaires comme une approche génétique pour étudier les mécanismes contrôlant leur différenciation et la fonction

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Isolement des cellules CD8+ T résidentes du rein de souris pour l’analyse de cytométrie de flux

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...