Nous tenons à remercier Aaron Selya de discussion utile. Ce travail a été soutenu par NIH AI29575, CA108835, AI077097 à JS, une subvention pilote de la Johns Hopkins Malaria Research Institute et le ministère de la Défense accorde 040 972 PC au MO

1. Faire, HLA-A2-Ig base AAPC <ol><li> Préparer un tampon de borate stériles <ol><li> Dissoudre l&#39;acide borique dans l&#39;eau pour faire 0,1. Ajuster le pH à 7,0. </li><li> Filtrer à travers un filtre 0.22μm stériles et conserver à 4 ° C. </li></ol></li><li> Préparer le tampon de lavage de billes stériles <ol><li> Prenez 956ml PBS w / o de magnésium ou le calcium. </li><li> Ajouter 30 ml de sérum humain AB. </li><li> Ajouter l&#39;EDTA 2 mM de concentration finale. </li><li> Ajouter 0.1gsodium azoture. </li><li> Filtrer à travers un filtre de 0,22 um stériles et conserver à 4 ° C. </li></ol></li><li> Prenez 1ml de perles Invitrogen époxy M-450 en stock, environ 400 millions de perles (perles peuvent être comptés par hémocytomètre), et mis dans une stérile, flacon en verre à vis. </li><li> Mettez le flacon contre un aimant Dynal MPC-1, tandis que les perles adhèrent à la face du flacon, éliminer le surnageant par aspiration. Laver une fois avec des perles de 1 ml de tampon borate. </li><li> Perles Remettre en suspension dans un mélange de tampon borate de 1 ml et 20 pg de HLA-A2-Ig dimère et 20 ug de CD28 monoclonal anti-humain (clone 9.3). </li><li> Protéines à la conjugaison perle: mettre le flacon en verre sur des rotateurs et tourner à 4 ° C pendant 24 heures. </li><li> Placer le tube dans le MPC-1 aimant et enlever tous tampon borate. </li><li> Laver les billes deux fois avec le tampon de lavage 1ml billes. </li><li> Incuber les perles dans le tampon de lavage 1ml perle, tournez à 4 ° C pendant 24 heures. Parce que le tampon de lavage de billes contient sérum humain AB, il va bloquer tout le site protéine résiduelle contraignant sur les perles. </li><li> Retirer le surnageant de flacon en verre, remplacez-le par 1ml tampon de lavage frais de billes. </li></ol> 2. Le contrôle de qualité des AAPC et le peptide de chargement, le stockage <ol><li> Ajouter ~ 5 × 10 5 perles pour FACS 100 ul du tampon de lavage dans des tubes FACS, et tache avec 1 microlitre d&#39;anti-souris IgG1-PE (reconnaître la partie Fc de la molécule HLA-A2-Ig) et 1 microlitre de l&#39;anti-IgG2a de souris-FITC (reconnaître la partie Fc des anticorps monoclonaux anti-CD28). Après coloration pendant 20 minutes dans un tampon de lavage FACS, laver de nouveau et de lire le résultat de la coloration immédiatement par cytométrie en flux (Figure 4). </li><li> De chargement de peptide sur les billes: Laver les billes deux fois en flacon de verre avec 1ml de PBS stérile. Resuspendre avec 1ml de PBS stérile, puis ajouter 10 ul de peptide CMV (1mg/ml) </li><li> Comptez le nombre de billes par hémocytomètre, et l&#39;étiquette du flacon avec la date et la concentration. </li><li> VAMP sont prêtes à l&#39;emploi après au moins 3 jours d&#39;incubation peptide à 4 ° C, pour laisser le temps suffisant de liaison du peptide sur le dimère HLA-A2-Ig. Les billes peuvent être conservés à 4 ° C et rester fonctionnel pendant au moins 6 mois. </li></ol> 3. Homme d&#39;isolement de CTL <ol><li> Recueillir ~ 100ml de sang périphérique frais de santé des donneurs HLA-A2 positifs dans 10 tubes BD Vacutainer sodium héparine. Utiliser une aiguille de calibre 21 ou plus pour éviter l&#39;hémolyse. </li><li> Centrifuger à 300xg pendant 10 minutes à température ambiante </li><li> Retirez délicatement la couche de plasma dessus par aspiration. Le plasma peut être utilisé comme supplément pour le milieu de culture. </li><li> Remplacer le plasma recueilli avec du PBS stérile et le transfert du sang dans un flacon stérile T75 culture ou 50ml tubes coniques. Mélanger le sang avec du PBS bien par aspiration et refoulement. </li><li> Une fois que tout le sang est collecté, préparer quatre autres tubes coniques 50 ml et ajouter 15 ml de Ficoll-Paque Plus. </li><li> Lentement superposition 30-35ml de cellules sanguines sur le dessus du Ficoll de chaque tube. Gardez à l&#39;interface distincte entre les Ficoll et les cellules sanguines. </li><li> Centrifuger à 500xg pendant 20 minutes à température ambiante. Tourner le frein &quot;off&quot; et l&#39;accélération aussi bas que possible afin de maintenir une interface claire entre les couches. </li><li> Avec une pipette seriological, aspirer délicatement la couche PBMC et recueillir les PBMC dans un nouveau tube 50ml conique. Lorsque tous les PBMC sont récoltées ajoutez 30ml de PBS et centrifuger à 400xg pendant 10 min. Jeter le surnageant et laver encore une fois avec 30ml de PBS pour enlever tous les résidus de Ficoll. </li><li> Procéder à l&#39;isolement CD8 + des lymphocytes T humains en utilisant Miltenyi CD8 + T kit isolement des cellules selon le protocole du fabricant. Ce kit enrichit fortement pour les cellules CD8 + (généralement&gt; 95%) en appauvrissant CD8 - cellules. </li><li> Compter les cellules T CD8 +. La pureté attendue devrait être supérieure à 95%. Pour confirmer cette utilisation 2x10 5 cellules et effectuer une analyse CD4/3/8 FACS. Le CTL restant peut être soit utilisé immédiatement pour antigène spécifique de stimulation AAPC ou ils peuvent être congelés pour un usage futur. </li></ol> 4. In vitro VAMP système de culture basée <ol><li> Préparer un milieu de culture <ol><li> Pour TF (facteur de croissance des lymphocytes T, fait dans le laboratoire 4) 2X milieu de culture: milieu RPMI complet ainsi que des bailleurs de fonds de 5% du plasma autologue et 8% facteur de croissance des lymphocytes T. </li><liPlasma du donneur&gt; autologue peut être remplacée par inactivés par la chaleur de sérum humain AB. </li></ol></li><li> Resuspendre 1000000 CTL dans 8ml de milieu de culture TF 2X plus de 8 ml de milieu RPMI complet, ajoutez 1 × 10 6 VAMP, bien mélanger. </li><li> Utiliser une pipette multi-canaux pour les cellules plaque sur 96 puits U plaques de fond de culture de tissu. (160 pi par puits) </li><li> Culture en cellules 37 ° C, 5% de CO 2 incubater pendant 7 jours. Alimenter les cellules le jour 4 avec 80 ul / moyen et TF 2X. </li><li> Les cellules sont prêts à être récoltés le jour 7. Après la récolte, placer les cellules contre l&#39;aimant et enlever la vieille AAPC. </li><li> Spécificité antigénique peut être déterminée par coloration tétramère selon le protocole du fabricant. Phénotype coloration et la coloration des cytokines intracellulaires sont effectués conformément à notre précédente étude 3. </li><li> Cellules récoltées peuvent être réensemencées avec VAMP nouveau dans les mêmes conditions. Le nombre de cellules et de spécificité antigénique devrait augmenter après stimulation répétée. </li></ol> 5. Les résultats représentatifs: Un exemple de VAMP après conjugaison HLA A2-Ig et anti-CD28 est montré dans la figure 4. Conjugaison réussie protéines est évident par un changement clair de la coloration des anticorps correspondants. Alors que la fréquence de CTL spécifique du CMV dans le sang périphérique est généralement de 0,5 à 1%, après une seule semaine de VAMP médiation de stimulation, la spécificité peut atteindre 55 - 93% (figure 2 et 3). L&#39;expansion de CTL spécifiques de l&#39;antigène peut être très variable entre les différents bailleurs de fonds, mais les résultats sont reproductibles dans le même donneur. Par extrapolation, l&#39;expansion des cellules CMV spécifiques peuvent être des milliers de fois par rapport aux niveaux de précurseur directement ex vivo (données non présentées). Coloration des cytokines intracellulaires (figure 3) montre que ces CTL élargi sont polyfonctionnels, plutôt que épuisée, après culture cellulaire prolongé et la prolifération significative. <img src="/files/ftp_upload/2801/2801fig1.jpg" alt="Figure 1"/> Figure 1. Diagramme représentant du VAMP l&#39;expansion ex vivo de CTL humains basée pour l&#39;immunothérapie adoptive dans allogéniques HSCT <img src="/files/ftp_upload/2801/2801fig2.jpg" alt="Figure 2"/> Figure 2. Résultat représentatif coloration tétramère CMV CTL spécifique générée par VAMP après une semaine de la culture <img src="/files/ftp_upload/2801/2801fig3.jpg" alt="Figure 3"/> Figure 3. Représentant résultat intracellulaire coloration des cytokines du CMV CTL spécifique générée par VAMP (spécificité du CMV était de 61%) <img src="/files/ftp_upload/2801/2801fig4.jpg" alt="Figure 4"/> Figure 4. Résultat de la coloration Représentant du M-450 perles époxy après conjugaison des protéines colorées par anti-souris IgG1-PE et anti-souris IgG2-FITC

<ol>
	<li>Walter, E. A. <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, 1038-1038 (1995).</li><li>Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+cell+transfer:+a+clinical+path+to+effective+cancer+immunotherapy.">Adoptive cell transfer: a clinical path to effective cancer immunotherapy.</a> Nat Rev Cancer. 8, 3669-3669 (2008).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+induction+and+expansion+of+antigen-specific+cytotoxic+T+cells+by+HLA-Ig-coated+artificial+antigen-presenting+cells.">Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.</a> Nat Med. 9, 619-619 (2003).</li><li>Oelke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+antigen-presenting+cells:+artificial+solution+for+real+diseases.">Artificial antigen-presenting cells: artificial solution for real diseases.</a> Trends Mol Med. 11, 412-412 (2005).</li></ol>

Le système VAMP nous décrivons ici est un système efficace pour l&#39;expansion ex vivo de CTL humain contre une variété d&#39;antigènes. Des précautions particulières doivent être prises en ce qui concerne la qualité de la conjugaison des protéines et la distribution même des AAPC et CTL dans la culture plaque de 96 puits. En utilisant cette approche, nous avons pu élargir CTL pour plus de 8 semaines, durant laquelle nous avons élargi l&#39;antigène CTL spécifique jusqu&#39;à un million de fois 4. Il ya eu différents systèmes APC artificielle en utilisant des lignées cellulaires ou autres plates-formes acellulaire 5; toutefois, selon les données publiées chaque système a son profil unique en ce qui concerne l&#39;expansion et la spécificité en charge des applications différentes. Fait important, car la qualité de CTL est aussi importante que la quantité, la polyfonctionnalité du CTL spécifiques du CMV générée par notre système sont attendus à conférer supérieure d&#39;efficacité anti-virale.

<table border="1"><tbody><tr><th> Réactifs </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> Vacutainer tube (contient de l&#39;héparine) </td><td> Becton Dickinson </td><td> 367874 </td></tr><tr><td> Human T CD8 + kit d&#39;isolement cellulaire </td><td> Miltenyi </td><td> 130-094-156 </td></tr><tr><td> Dynabeads M-450 Epoxy </td><td> Invitrogen </td><td> 140,11 </td></tr><tr><td> Dynal MPC-1 Aimant </td><td> Invitrogen </td><td> 120-01D </td></tr><tr><td> Ficoll-Paque plus </td><td> GE Healthcare </td><td> 17-1440-03 </td></tr><tr><td> Milieu RPMI 1640 </td><td> Gibco </td><td> 11875 </td></tr><tr><td> HLA-A2-Ig X dimère </td><td> Becton Dickinson </td><td> 551263 </td></tr><tr><td> iTAgMHC tétramère (HLA-A2-CMV)-PE </td><td> Beckman Coulter </td><td> T20099 </td></tr><tr><td> Falcon claires de 96 puits microplaque </td><td> Becton Dickinson </td><td> 353077 </td></tr><tr><td> Rat anti-souris IgG2a-FITC </td><td> Becton Dickinson </td><td> 553390 </td></tr><tr><td> Chèvre anti-souris IgG1-PE </td><td> Invitrogen </td><td> P21129 </td></tr><tr><td> Sérum humain de type AB </td><td> Atlanta biologiques </td><td> S40110 </td></tr><tr><td> Souris anti-humaine-FITC CD8a </td><td> Sigma-Aldrich </td><td> F0772 </td></tr><tr><td> Souris anti-humain CD8a-APC </td><td> Becton Dickinson </td><td> 340684 </td></tr><tr><td> Souris anti-humain IFN-FITC </td><td> Becton Dickinson </td><td> 340449 </td></tr><tr><td> De souris anti-TNFa humain-PE </td><td> Becton Dickinson </td><td> 340512 </td></tr></tbody></table>

hla-ig based artificial antigen presenting cells for efficient ex vivo expansion of human ctl

CTL avec la fonction d&#39;effecteur optimale jouent un rôle essentiel dans la médiation de la protection contre les différentes infections intracellulaires et le cancer. Toutefois, les particuliers peuvent présenter suppressif microenvironnement immunitaire et, contrairement à l&#39;activation de CTL, leurs cellules autologues présentant l&#39;antigène peuvent avoir tendance à tolerize ou anergize CTL spécifique de l&#39;antigène. En conséquence, bien que toujours en phase expérimentale, basée sur CTL immunothérapie adoptive a évolué pour devenir un traitement prometteur pour diverses maladies comme le cancer et les infections virales. Dans les premières expériences ex vivo élargi CMV (cytomégalovirus) CTL spécifiques ont été utilisés pour le traitement de l&#39;infection à CMV chez les patients immunodéprimés osseuse allogénique greffe de moelle. Alors qu&#39;il est commun d&#39;avoir la vie en danger virémie à CMV chez ces patients, aucun des patients recevant élargi CTL développer des maladies liées au CMV, impliquant l&#39;immunité anti-CMV est établi par le adoptive transférée CTL 1. Des résultats prometteurs ont également été observées pour le mélanome et peut être étendu à d&#39;autres types de cancer 2. Bien qu&#39;il existe de nombreuses façons d&#39;ex vivo de stimuler et de développer CTL humains, les approches actuelles sont restreintes par les limites de coûts et technique. Par exemple, l&#39;étalon-or actuel est basé sur l&#39;utilisation de DC autologues. Cela nécessite à chaque patient de donner un nombre important de leucocytes et est aussi très coûteuse et laborieuse. Par ailleurs, détaillées dans la caractérisation in vitro de DC élargi CTL a révélé que ceux-ci ont seulement suboptimale fonction effectrice 3. Nous présentons ici un très efficace système de base pour la VAMP expansion ex vivo de CMV CTL humains spécifiques pour une immunothérapie adoptive (figure 1). L&#39;AAPC ont été faites par le couplage de cellules de taille humaine avec des billes magnétiques HLA-A2-Ig dimère et anti-CD28mAb 4. Une fois VAMP sont présentées, elles peuvent être chargés avec différents peptides d&#39;intérêt, et rester fonctionnel pendant des mois. Dans ce rapport, AAPC ont été chargées avec un peptide dominant de CMV, pp65 (NLVPMVATV). Après culture humaine purifiée CD8 + CTL à partir d&#39;un donneur sain avec de VAMP pour une semaine, le CMV CTL spécifique peut être augmenté de façon spectaculaire dans la spécificité de 98% (figure 2) et amplifiée plus de 10.000 fois. Si plus spécifiques du CMV CTL sont nécessaires, l&#39;expansion peut être facilement atteint par la stimulation répétitive avec VAMP. La caractérisation phénotypique et fonctionnelle montre que ces cellules ont un phénotype élargi mémoire effecteurs et faire des quantités importantes de deux TNFa et l&#39;IFN (figure 3).

Watch this Scientific Journal Video about HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL at JoVE.com

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Une méthode nouvelle DC indépendante pour l&#39;induction et l&#39;expansion des cellules T spécifiques d&#39;antigènes est décrite. HLA A2-Ig des cellules à base Antigène artificielle Présentation (AAPC) sont chargés avec des peptides HLA-A2 restreint efficacement l&#39;expansion des CTL spécificité antigénique divers. Cette technologie offre un grand potentiel pour CTL à base d&#39;immunothérapie adoptive.

HLA-Ig des cellules à base Antigène artificielle Présentation d&#39;Efficient Ex vivo Extension du CTL humaines

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

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Immunology and Infection

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

15.3K vues.  Johns Hopkins University. Une méthode nouvelle DC indépendante pour l'induction et l'expansion des cellules T spécifiques d'antigènes est décrite. HLA A2-Ig des cellules à base Antigène artificielle Présentation (AAPC) sont chargés avec des peptides HLA-A2 restreint efficacement l'expansion des CTL spécificité antigénique divers. Cette technologie offre un grand potentiel pour CTL à base d'immunothérapie adoptive.

Vidéo: HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Specific Antigen In Vivo</emTuer Assay> en utilisant des cellules CFSE cible marquée

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

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Immunologie et infection

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Expansion ex vivo des cellules tumorales T-réactive par le moyen de 1/Ionomycin Bryostatine et la chaîne gamma commune cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

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

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

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

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

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

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 ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Présentatrice d&#39;antigène artificielle d&#39;activation à médiation cellulaire (AAPC) et l&#39;expansion des cellules T tueuses naturelles

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

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 ...

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 ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Tueur antigène artificielle cellules présentatrices (KaAPC) d&#39;utilisation efficace<em&gt; In Vitro</em&gt; L&#39;épuisement des lymphocytes T humains spécifiques de l&#39;antigène

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Lymphocytes Isolation de la peau humaine pour Analyse phénotypique et<em&gt; Ex Vivo</em&gt; Culture cellulaire

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Développement d&#39;un modèle Colite antigène conduit à étudier la présentation des antigènes par les cellules présentatrices d&#39;antigènes aux cellules T

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...