1. Isolation des PBMC <ol><li> Prélever le sang (7,5 à 8,0 ml) dans un tube BD Vacutainer CPT Préparation des cellules avec de l&#39;héparine de sodium. Le tube contient un anticoagulant héparine sodique et d&#39;un fluide de densité Ficoll-Hypaque, plus une barrière de gel de polyester, qui sépare les deux liquides. Centrifuger le tube / échantillon de sang à température ambiante (18 ° C à 25 ° C) dans un rotor horizontal (swing-out tête) pendant 20 min à 1800 x g. Commutateur centrifugeuse freins hors tension. </li><li> Après centrifugation, la séquence de couches se produit comme suit (vu de haut en bas): un plasma) - b) les cellules mononucléées du sang périphérique (PBMC) et plaquettes - c) la solution de densité - gel de polyester d) - e) les granulocytes - f) globules rouges (fig. 1). </li><li> Recueillir une fraction de la couche de plasma, en laissant 5 à 10 mm de plasma au-dessus de l&#39;interphase sans déranger la couche de cellules. Le plasma peut être utilisé pour la culture (article 2.5). </li><li> Récolte de la fraction enrichie (PBMC) à l&#39;interphase avec une pipette et transférer dans un tube de 15 ml conique. </li><li> Laver les PBMC avec 10 ml de tampon phosphate salin (PBS), en inversant le tube 5 fois, puis centrifuger 5 min à 400 x g. </li><li> Répétez les étapes de lavage à deux reprises, puis remise en suspension le culot cellulaire dans 5 ml de PBS. Déterminer le nombre de cellules. Habituellement 1,3 x10 6 cellules sont récupérées à partir de 1 ml de sang total. </li><li> Déterminer la fréquence et le phénotype des cellules γδ T PBMC par cytométrie en flux (section 3) (Fig. 2). </li></ol> 2. L&#39;expansion des cellules T γδ <ol><li> Centrifugeuse suspensions cellulaires dans 15 ml tubes coniques pendant 5 min à 400 x g à température ambiante, et jeter le surnageant. </li><li> Préparer un milieu de culture (CM) en ajoutant l&#39;IL-2 humaine (IL-2) et le zolédronate (Zometa) à des concentrations finales de 1000 UI / ml et 5 uM, respectivement. ALyS203 (Cell Science &amp; Technology Institute) ou OpTmizer (Invitrogen) des médias de soutenir l&#39;expansion des cellules T bonnes γδ (plus d&#39;informations dans les références 6 et 9). Zometa est fourni sous forme liquide (4 flacon mg/5-ml). Pour préparer une solution à 5 uM, ajouter 50 ul de Zometa à 30 ml de milieu de culture. </li><li> Resuspendre le culot cellulaire dans un milieu de culture et de s&#39;adapter à 1x10 6 cellules / ml. </li><li> Pipeter 1 ml de CM contenant 1x10 6 cellules dans chaque puits d&#39;une plaque de 24 puits. Pour les grandes cultures, les cellules peuvent être ensemencées à 0,5 x 10 6 cellules / cm 2 selon la surface du puits de la plaque, plat, ou le flacon. </li><li> Ajouter plasma autologue (section 1.3), regroupés AB humain sérums ou FCS pour qu&#39;il se trouve à environ 10% du volume de la culture (100 pi pour chaque puits d&#39;une plaque de 24 puits). Placer les plaques dans un 37 humidifié ° C, 5% de CO 2 incubateur de 24-48 heures. </li><li> Maintenir la culture à une densité cellulaire de 0,5-2 x 10 6 cellules / ml. Ajouter un milieu frais contenant l&#39;IL-2 humaine (1000 UI / ml) seule (sans Zometa) tous les 2-3 jours, et le transfert des cellules cultivées dans des puits nouveaux ou flacons que nécessaire, selon le degré de prolifération cellulaire (Fig. 3). Plasmatique d&#39;alimentation ou de sérum dans le milieu de sorte que la concentration sérique peut être maintenu au moins 1%. </li><li> Récolte des cellules le jour 12-14 et déterminer la fréquence, le phénotype et les fonctions des cellules T γδ par cytométrie en flux (voir ci-dessous). </li></ol> 3. L&#39;analyse phénotypique par cytométrie en flux <ol><li> Transfert de 200 échantillons ul contenant 2 x 10 5 cellules pour le tri de cellules activées par fluorescence (FACS) tubes. </li><li> Ajouter 2 ml de PBS froid et centrifuger 5 min à 400 x g. Ensuite, remettre en suspension les boulettes dans 50 ul de tampon FACS (PBS + 1% de FCS azoture de sodium + 0,1%). Ajouter 5 ul de chaque anticorps pour les échantillons (les anticorps monoclonaux sont énumérés dans le tableau 1). </li><li> Incuber sur de la glace dans l&#39;obscurité pendant 20 min. </li><li> Ajouter 2 ml de tampon FACS à chaque échantillon, puis de vortex. Centrifuger les échantillons pendant 5 min à 400 x g à 4 ° C. Décanter avec précaution le surnageant. </li><li> Resuspendre les cellules dans 300 ul de tampon FACS et les vortex. Analyser des échantillons sur un cytomètre en flux (Fig. 2). </li></ol> 4. IFN-γ test de production 10 <ol><li> Le jour avant le dosage, préparer des cellules stimulatrices en cultivant 3-5 cellules Daudi x10 5 / ml dans du milieu RPMI 1640 plus 10% de FCS (RPMI-10) pendant la nuit avec Zometa (5 uM) (ci-après désignée Z-Daudi). </li><li> Recueillir Z-Daudi et remettre en suspension dans du RPMI-10 à 2x10 6 cellules / ml. Ajouter 100 ul de Z-Daudi (2x10 5) à chaque puits d&#39;une plaque à fond rond de 96 puits. </li><li> Préparer les cellules T γδ au 2x10 6 cellules / ml dans du RPMI-10 contenant Bréfeldine A à 20 pg / ml. Transférer 100 ul de suspension cellulaire T γδ (2x10 5) à chaque puits contenant les cellules Daudi-Z ou de contrôler des puits (100 pl de RPMI-10 seulement, ou RPMI-10 avec 20 ng / ml de phorbol 12-myristate 13-acétate[PMA] et 2 pg / ml d&#39;ionomycine). </li><li> Mélanger à la pipette et à plusieurs reprises. Incuber pendant 4 heures dans un 37 ° C, 5% de CO 2 incubateur. </li><li> Centrifuger la plaque pendant 5 min à 400 x g à 4 ° C et remettre les boulettes dans 200 ul de PBS froid. </li><li> Transférer les échantillons dans des tubes FACS. Ajouter 4 ml de PBS froid et centrifuger les tubes pendant 5 min à 400 x g à 4 ° C. </li><li> Resuspendre les culots dans 50 ul de tampon FACS avec FITC conjugué anti-TCRVγ9 (5 pi) et de PE/Cy5-conjugated anti-CD3 mAb (2,5 pi). Incuber abri de la lumière pendant 15 min à température ambiante. </li><li> Ajouter 100 ul de réactif IntraPrep 1 et incuber pendant 15 min à température ambiante. Ajouter 4 ml de PBS dans chaque tube et centrifuger 5 min à 400 x g à température ambiante. </li><li> Retirer le surnageant par aspiration et ajouter 100 l de réactif 2 IntraPrep. Incuber pendant 5 min à température ambiante sans agitation. </li><li> Ajouter 5 ul de PE-conjugué anti-IFN-γ mAb le tube à essai. Incuber abri de la lumière pendant 15 min à température ambiante. </li><li> Ajouter 4 ml de PBS dans chaque tube et centrifuger 5 min à 400 x g à température ambiante. Retirer le surnageant par aspiration et resuspendre le culot cellulaire dans 0,5 ml de tampon FACS. </li><li> Analyser les cellules par cytométrie en flux. Porte sur le CD3 + + TCRVγ9 cellules et d&#39;examiner l&#39;expression d&#39;IFN-γ (Fig. 4). </li></ol> 5. Les résultats représentatifs: Il est important de déterminer le pourcentage de cellules T γδ dans PBMC à l&#39;initiation de la culture. Comme le montre la Fig. 2 A, le pourcentage de cellules CD3 + + TCRVγ9 γδ lymphocytes T dans les PBMC a été de 1,6% au jour 0. Les populations dominantes étaient CD27 + CD45RA + CD27 + naïfs ou CD45RA - phénotypes mémoire centrale. Lorsque les cellules T γδ ont été stimulés efficacement, ils ont formé des grappes sur les jours 3-5 (Fig. 3 A et B). Lorsque la formation de grappes a été retardée, la croissance des autres types cellulaires, comme les cellules CD4 + ou CD8 + αβ des cellules T ou des cellules NK pourrait dominer la croissance des cellules T γδ (Fig. 3 C et D). Après 14 jours de culture, de la fréquence des cellules T γδ augmenté à plus de 93,8% des cellules cultivées dans des cultures de cellules γδ réussie T (fig. 2 E). La culture des cellules T γδ surexprimés NKG2D et expression de CD69 (fig. 2 G et H). Ils s&#39;affichent CD27 - CD45RA - phénotype mémoire effecteurs (Fig. 2 F). Les fonctions des cellules T γδ ont été évalués à l&#39;égard de la production de cytokines et la cytotoxicité. L&#39;IFN-γ intracellulaire coloration démontré que les cellules T γδ IFN-γ produit en réponse au PMA / ionomycine traitement ou Z-Daudi cellules qui IPP ​​accumulés après le traitement zolédronate (fig. 4). Ces résultats indiquent que le zolédronate peut efficacement stimuler et de développer des cellules T γδ fonctionnelle. <table border="1"><tbody><tr><td> Tube </td><td> FITC </td><td> PE </td><td> DPE </td><td> PE/Cy5 </td></tr><tr><td> 1 </td><td> CD3 </td><td> CD19 </td><td> CD45 </td><td> CD14 </td></tr><tr><td> 2 </td><td> CD3 </td><td> TCRap </td><td> CD4 </td><td> CD8 </td></tr><tr><td> 3 </td><td> CD3 </td><td></td><td></td><td> CD56 </td></tr><tr><td> 4 </td><td> TCR Vy9 </td><td> TCRap </td><td> CD45 </td><td> CD3 </td></tr><tr><td> 5 </td><td> TCR Vy9 </td><td> NKG2D </td><td></td><td></td></tr><tr><td> 6 </td><td> TCR Vy9 </td><td> CD69 </td><td></td><td></td></tr><tr><td> 7 </td><td> TCR Vy9 </td><td> IgG1 de souris </td><td></td><td></td></tr><tr><td> 8 </td><td> TCR Vy9 </td><td></td><td> CD45RA </td><td> CD27 </td></tr><tr><td> 9 </td><td> TCR Vy9 </td><td></td><td> IgG1 de souris </td><td> IgG1 de souris </td></tr></tbody></table> Tableau 1. Les anticorps monoclonaux utilisés dans la coloration des cellules T multicolores γδ. Un exemple de l&#39;analyse phénotypique des cellules T γδ effectué dans notre laboratoire est montré dans la Fig. 2. <img src="/files/ftp_upload/3182/3182fig1.jpg" alt="Figure 1"/> Figure 1. Séparation des PBMC. Sang (7,5 à 8,0 ml) est tiré dans un tube BD Vacutainer CPT Préparation des cellules avec de l&#39;héparine de sodium et directement centrifugé pendant 20 min à 1800 x g. Après centrifugation, les couches résultant comme on le voit du haut vers le boTTOM: un écran plasma) - b) PBMC et plaquettes - solution de densité c) - gel de polyester d) - e) Granulocytes - f) Les globules rouges. <img src="/files/ftp_upload/3182/3182fig2.jpg" alt="Figure 2"/> Figure 2. Phénotype de surface typiques des cellules T γδ. PBMC ont été stimulées avec le zolédronate et IL-2 pendant 14 jours. Les cellules ont été colorés avec marqué FITC anti-TCR Vy9 et PE/Cy5-labeled anti-CD3 pour surveiller l&#39;expansion des cellules T γδ (A et E). Les cellules T γδ ont été identifiés par leur expression de TCRVγ9, et leur expression de CD27 et CD45RA (B et F), NKG2D (C et G), ou CD69 (D et H) a été examiné. <img src="/files/ftp_upload/3182/3182fig3.jpg" alt="Figure 3"/> Figure 3. Représentant γδ cultures de cellules T. PBMC ont été stimulées par l&#39;IL-2 (1000 UI / ml) et le zolédronate (5 um). Champs représentatifs sont présentés (IX71 microscope inversé [Olympus] x 200). Les clusters et les agrégats de cellules T γδ peuvent être observés au jour 3 (A) et le jour 5 (B), lorsque les cellules T γδ ont été développé avec succès. En revanche, aucun amas ou d&#39;agrégats ont été observés lorsque la croissance des cellules T γδ n&#39;était pas adéquate (C et D). <img src="/files/ftp_upload/3182/3182fig4.jpg" alt="Figure 4"/> Figure 4. IFN-γ production. Les cellules T γδ ont été incubées avec du RPMI-10 seulement (A) ou PMA / ionomycine (B) ou Z-Daudi (C) pendant 4 heures. Tout d&#39;abord, l&#39;expression de surface de TCRVγ9 était tachée et ensuite de l&#39;IFN-γ de production a été examiné par l&#39;IFN-γ intracellulaire coloration. <img src="/files/ftp_upload/3182/3182fig5.jpg" alt="Figure 5"/> Figure 5. Cinétique de la culture de cellules γδ T. (A) Le nombre absolu de cellules cultivées, (B) le pourcentage de cellules T γδ, et (C) le nombre absolu de lymphocytes T γδ dans les points de temps indiqués.

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Aucun conflit d&#39;intérêt déclaré.

La méthode présentée ici permet l&#39;expansion efficace des cellules T γδ partir des PBMC. γδ cellules T activées et élargies par le zolédronate et IL-2 développent complète des fonctions effectrices, reflétée par la production de cytokines et la cytotoxicité. Il a été rapporté que le pyrophosphate bromhydrine synthétiques phosphoantigènes (BrHPP) et le 2-méthyl-3-butényle-1-pyrophosphate (2M3B1PP) élargira également les cellules T γδ, mais ils ne sont pas disponibles dans le commerce. En reva...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> ZOMETA </td><td> Novartis Pharma K. K </td><td></td><td> zolédronate </td></tr><tr><td> PROLEUKIN </td><td> Novartis Pharmaceuticals </td><td></td><td> IL-2 humaine recombinante </td></tr><tr><td> BD Vacutainer CPT Tube Préparation des cellules avec de l&#39;héparine de sodium </td><td> BD </td><td> 362753 </td><td></td></tr><tr><td> RPMI1640 </td><td> Invitrogen </td><td> 21870-076 </td><td></td></tr><tr><td> ALyS203 moyennes </td><td> Cellule Science &amp; Technology Institute </td><td> 0301-7 </td><td></td></tr><tr><td> OpTmizer </td><td> Invitrogen </td><td> 0080022SA </td><td></td></tr><tr><td> Un bréfeldine </td><td> Sigma </td><td> B5936-200UL </td><td></td></tr><tr><td> phorbol 12-myristate 13-acétate (PMA) </td><td> Sigma </td><td> P1585-1MG </td><td></td></tr><tr><td> ionomycine </td><td> Sigma </td><td> 13909-1ML </td><td></td></tr><tr><td> IntraPrep </td><td> BECKMAN COULTER </td><td> A07803 </td><td></td></tr><tr><td> anti-humain CD3-FITC ou PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07746 FITC A07749 PE/Cy5 </td><td></td></tr><tr><td> anti-CD4 humain DPE </td><td> BECKMAN COULTER </td><td> 6604727 </td><td></td></tr><tr><td> anti-humain CD8-PE/Cy5 </td><td> BECKMAN COULTER </td><td> 6607011 </td><td></td></tr><tr><td> anti-humain CD14-PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07765 </td><td></td></tr><tr><td> anti-humain CD19-PE </td><td> BECKMAN COULTER </td><td> A07769 </td><td></td></tr><tr><td> anti-humain CD45-ECD </td><td> BECKMAN COULTER </td><td> A07784 </td><td></td></tr><tr><td> anti-humain CD56-PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07789 </td><td></td></tr><tr><td> anti-humain TCRap-PE </td><td> BECKMAN COULTER </td><td> A39499 </td><td></td></tr><tr><td> TCR anti-humaine-FITC Vy9 </td><td> BECKMAN COULTER </td><td> IM1463 </td><td></td></tr><tr><td> anti-humain CD27-PE/Cy5 </td><td> BECKMAN COULTER </td><td> 6607107 </td><td></td></tr><tr><td> anti-humain CD45RA-DPE </td><td> BECKMAN COULTER </td><td> IM2711 </td><td></td></tr><tr><td> anti-humain CD69-PE </td><td> BD </td><td> 555531 </td><td></td></tr><tr><td> anti-humain NKG2D-PE </td><td> BECKMAN COULTER </td><td> A08934 </td><td></td></tr><tr><td> Anti-humal IFN-PE </td><td> BECKMAN COULTER </td><td> IM2717U </td><td></td></tr><tr><td> Souris IgG1 isotype contrôle-PE </td><td> BECKMAN COULTER </td><td> A07796 </td><td></td></tr><tr><td> Souris IgG1 isotype contrôle-ECD ou PE/Cy5 </td><td> BECKMAN COULTER </td><td> A07797 A07798 </td><td></td></tr></tbody></table>

expansion of human peripheral blood  &gamma;&delta; t cells using zoledronate

L&#39;homme des cellules T γδ peuvent reconnaître et répondre à une grande variété de stress induit par les antigènes, développant ainsi une large activité innée anti-tumorale et anti-infectieux. 1 La majorité des cellules T γδ dans le sang périphérique ont le récepteur des cellules T Vγ9Vδ2. Ces cellules reconnaissent un antigène dans un complexe d&#39;histocompatibilité majeure manière indépendante et de développer de solides cytolytique et Th1-like fonctions effectrices. 1 Par conséquent, γδ lymphocytes T sont des cellules effectrices candidat intéressant pour l&#39;immunothérapie du cancer. Vγ9Vδ2 cellules T de répondre à phosphoantigènes tel que (E)-4-hydroxy-3-méthyl-but-2-ényle pyrophosphate (HMBPP), ce qui est synthétisée dans la bactérie via la biosynthèse des isoprénoïdes; 2 et isopentényl pyrophosphate (IPP), qui est produite dans les cellules eucaryotes par la voie du mévalonate. 3 Dans l&#39;état physiologique, la génération des IPP dans la cellule non transformée n&#39;est pas suffisant pour l&#39;activation des cellules T γδ. Le dérèglement de voie du mévalonate dans les cellules tumorales conduit à l&#39;accumulation des IPP et γδ lymphocytes T d&#39;activation. 3 Parce aminobisphosphonates (comme le pamidronate ou zolédronate) inhibent farnésyl pyrophosphate synthase (FPPS), l&#39;enzyme agissant en aval de la PIP dans la voie du mévalonate, les taux intracellulaires de IPP et sensitibity aux cellules T γδ reconnaissance peut être thérapeutique augmenté de aminobisphosphonates. IPP accumulation est moins efficace dans les cellules nontransfomred que des cellules tumorales avec une concentration pharmacologiquement pertinentes de aminobisphosphonates, qui nous permettent d&#39;immunothérapie pour le cancer en activant les cellules T γδ avec aminobisphosphonates 4. Fait intéressant, IPP s&#39;accumule dans les monocytes lors PBMC sont traitées avec aminobisphosphonates, en raison de l&#39;efficacité l&#39;absorption des médicaments par ces cellules. 5 monocytes qui s&#39;accumulent IPP deviennent cellules présentatrices d&#39;antigène et stimuler Vγ9Vδ2 cellules T dans le sang périphérique. 6 Basé sur ces mécanismes, nous avons développé une technique à grande échelle l&#39;expansion des cultures de cellules T γδ utilisant zolédronate et l&#39;interleukine -2 (IL-2). 7 D&#39;autres méthodes pour l&#39;expansion des cellules T γδ utiliser le pyrophosphate bromhydrine synthétiques phosphoantigènes (BrHPP) 8 ou 2-méthyl-3-butényle-1-pyrophosphate (2M3B1PP) 9. Toutes ces méthodes permettent ex l&#39;expansion in vivo, ce qui entraîne un grand nombre de cellules T γδ pour une utilisation en immunothérapie adoptive. Toutefois, seul le zolédronate est un réactif approuvé par la FDA disponible commercialement. Zolédronate-élargi γδ lymphocytes T CD27 écran - CD45RA - phénotype mémoire effecteurs et leur fonction peut être évaluée par l&#39;IFN-γ test de production 7.

Watch this Scientific Journal Video about Expansion of Human Peripheral Blood  Î³Î´ T Cells using Zoledronate at JoVE.com

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

Une méthode pour développer des cellules T γδ à partir des cellules mononucléées du sang périphérique (PBMC) est décrite. PBMC dérivés des lymphocytes T γδ sont stimulés et étendu en utilisant le zolédronate et l&#39;interleukine-2 (IL-2). L&#39;expansion à grande échelle de cellules T γδ peuvent être appliquées à l&#39;immunothérapie cellulaire autologue de 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 ...

expansion-of-human-peripheral-blood-t-cells-using-zoledronate

Research

JoVE Journal

Immunology and Infection

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

26.3K vues.  University of Tokyo Hospital. Une méthode pour développer des cellules T γδ à partir des cellules mononucléées du sang périphérique (PBMC) est décrite. PBMC dérivés des lymphocytes T γδ sont stimulés et étendu en utilisant le zolédronate et l'interleukine-2 (IL-2). L'expansion à grande échelle de cellules T γδ peuvent être appliquées à l'immunothérapie cellulaire autologue de cancer.

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

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

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

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

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

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

Recherche

Immunologie et infection

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

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

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

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

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

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

Generation of Human 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 ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Isolement et cytométrie de flux Analyse de Human Endocervical Gamma Delta cellules T

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextrane augmente l’efficacité de la Transduction des gènes des cellules NK primaires murines et humaines

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Imagerie haute résolution et ultra-résolution des cellules T à l&#39;aide de madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

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

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

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

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

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

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

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

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

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

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

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

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

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

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

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

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

Collection et cryostockage PBMC pour l’analyse bioénergétique mitochondriale

Cryopreservation and Bioenergetic Evaluation of Human Peripheral Blood Mononuclear Cells

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.

Pleins feux sur l’auteur : Préservation des paramètres bioénergétiques dans les cellules mononucléées du sang périphérique après cryoconservation 

Isolated peripheral blood mononuclear cells can be used for the analysis of immune functions and disorders, metabolic diseases, or mitochondrial functions. In this work, we describe a standardized method for the preparation of PBMCs from whole blood and the subsequent cryopreservation. Cryopreservation makes this time and place independent.

Cryoconservation et évaluation bioénergétique des cellules mononucléées du sang périphérique humain

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases ...