1. Isolierung von PBMC <ol><li> Draw Blut (7,5 bis 8,0 ml) in einen BD Vacutainer CPT Cell Preparation Röhrchen mit Natrium Heparin. Die Röhre enthält eine Natrium-Heparin als Antikoagulans und eine Ficoll-Hypaque Dichte Flüssigkeit, plus ein Polyester-Gel-Schranke, die die beiden Flüssigkeiten voneinander trennt. Zentrifugenröhrchen / Blutprobe bei Raumtemperatur (18 ° C bis 25 ° C) in einer horizontalen Rotor (swing-out Kopf) für 20 min bei 1800 x g. Schalten Zentrifuge bremst ab. </li><li> Nach der Zentrifugation erfolgt die Reihenfolge der Schichten wie folgt (gesehen von oben nach unten): a) Plasma - b) periphere mononukleäre Blutzellen (PBMC) und Thrombozyten - c) Dichte Lösung - d) Polyester Gel - e) Granulozyten - f) roten Blutkörperchen (Abb. 1). </li><li> Sammeln Sie nur einen Bruchteil der Plasmaschicht, so dass 5 bis 10 mm von Plasma oberhalb der Interphase, ohne die Zellschicht. Das Plasma kann für die Kultur (Abschnitt 2.5) verwendet werden. </li><li> Ernte der angereicherten Fraktion (PBMC) in der Interphase mit einer Pipette aufnehmen und in 15 ml konischen Rohr. </li><li> Waschen Sie die PBMC mit 10 ml Phosphat-gepufferter Salzlösung (PBS), durch Invertieren des Röhrchens 5 mal und dann 5 min bei 400 x g zentrifugieren. </li><li> Wiederholen Sie die Waschschritte zweimal, und dann Zellpellet in 5 ml PBS. Bestimmen Sie die Anzahl der Zellen. In der Regel 1,3 x10 6 Zellen sind von 1 ml Vollblut gewonnen. </li><li> Bestimmen Sie die Frequenz und Phänotyp der γδ T-Zellen in PBMC mittels Durchflusszytometrie (Abschnitt 3) (Abb. 2). </li></ol> 2. Expansion von γδ T-Zellen <ol><li> Centrifuge Zellsuspensionen in 15 ml konische Röhrchen für 5 min bei 400 x g bei Raumtemperatur, und entsorgen Sie die Überstände. </li><li> Bereiten Kulturmedium (CM) durch Zugabe von humanem IL-2 (IL-2) und Zoledronat (Zometa), um Endkonzentrationen von 1000 IU / ml und 5 um beträgt. ALyS203 (Cell Science &amp; Technology Institute) oder OpTmizer (Invitrogen) Medien unterstützen eine gute Erweiterung des γδ T-Zellen (weitere Informationen in den Referenzen 6 und 9). Zometa wird in flüssiger Form (4 mg/5-ml Durchstechflasche) zur Verfügung gestellt. Zur Herstellung eines 5 uM Lösung, mit 50 ul von Zometa um 30 ml Kulturmedium. </li><li> Resuspendieren Zellpellet in Kulturmedium und passen zu 1x10 6 Zellen / ml. </li><li> Pipettieren von 1 ml CM mit 1x10 6 Zellen in jedes Well einer 24-Well-Platte. Für großflächige Kulturen, können die Zellen auf 0,5 x 10 6 Zellen / cm ausgesät werden 2 nach den Flächen der Platte Brunnen, Schüssel oder Flasche. </li><li> Add autologem Plasma (Abschnitt 1.3), gepoolten menschlichen AB sera oder FCS, so dass es etwa 10% des Volumens der Kultur (100 ul pro Well einer 24-Well-Platte) ist. Legen Sie die Platten in einem befeuchteten 37 ° C, 5% CO 2-Inkubator für 24-48 Std. </li><li> Pflegen Sie die Kultur mit einer Zelldichte von 0,5-2 x 10 6 Zellen / ml. Fügen Sie frisches Medium mit humanem IL-2 (1000 IU / ml) nur (ohne Zometa) alle 2-3 Tage und Transfer kultivierten Zellen in neue Brunnen oder Flaschen wie nötig, je nach dem Grad der Zellproliferation (Abb. 3). Versorgung Plasma oder Serum, das Medium, so dass die Konzentration im Serum beibehalten werden mindestens 1% betragen. </li><li> Ernten Sie die Zellen am Tag 12-14 und die Frequenz bestimmen, Phänotyp und Funktion der γδ T-Zellen mittels Durchflusszytometrie (siehe unten). </li></ol> 3. Phänotypische Analyse mittels Durchflusszytometrie <ol><li> Transfer-200 ul Proben mit 2 x 10 5 Zellen zur Fluoreszenz-activated cell sorting (FACS) Röhren. </li><li> 2 ml kaltem PBS und zentrifugieren Sie für 5 min bei 400 x g. Dann, das Sediment resuspendieren in 50 ul FACS Puffer (PBS + 1% FCS + 0,1% Natriumazid). Add 5 ul jeder Antikörper an die Proben (die monoklonalen Antikörper sind in Tabelle 1 aufgeführt). </li><li> Inkubieren auf Eis im Dunkeln für 20 min. </li><li> 2 ml FACS-Puffer zu jeder Probe und dann Wirbel. Centrifuge Proben für 5 min bei 400 x g bei 4 ° C. Überstand vorsichtig dekantieren. </li><li> Resuspendieren der Zellen in 300 ul FACS-Puffer und vortexen. Analysieren Sie die Proben an einem Durchflusszytometer (Abb. 2). </li></ol> 4. IFN-γ Produktion Assay 10 <ol><li> Der Tag vor dem Test, bereiten Stimulatorzellen durch Kultivierung von 3-5 x10 5 Daudi Zellen / ml in RPMI 1640-Medium plus 10% FCS (RPMI-10) über Nacht mit Zometa (5 pM) (im Folgenden bezeichnet Z-Daudi). </li><li> Sammeln Z-Daudi und resuspendieren in RPMI-10 bei 2x10 6 Zellen / ml. 100 l von Z-Daudi (2x10 5) in jede Vertiefung einer Rundboden-96-Well-Platte. </li><li> Bereiten γδ T-Zellen bei 2x10 6 Zellen / ml in RPMI-10 mit Brefeldin A bei 20 pg / ml. Transfer 100 ul der γδ T-Zell-Suspension (2x10 5) in jede Vertiefung mit Z-Daudi-Zellen oder in Vertiefungen (100 ul RPMI-10 nur oder RPMI-10 mit 20 ng / ml Phorbol 12-myristat 13-Acetat-Steuerung[PMA] plus 2 pg / ml Ionomycin). </li><li> Mix von Auf-und Abpipettieren mehrmals. Inkubieren für 4 h in einem 37 ° C, 5% CO 2-Inkubator. </li><li> Zentrifugieren Sie die Platte für 5 min bei 400 x g bei 4 ° C und Resuspendieren der Pellets in 200 ul kaltem PBS. </li><li> Übertragen Sie die Proben auf FACS-Röhrchen. 4 ml kaltem PBS und Zentrifugieren für 5 min bei 400 x g bei 4 ° C. </li><li> Resuspendieren der Pellets in 50 ul FACS-Puffer mit FITC-konjugierten Anti-TCRVγ9 (5 ul) und PE/Cy5-conjugated anti-CD3 mAb (2,5 ul). Inkubieren von Licht für 15 min bei Raumtemperatur geschützt. </li><li> 100 l von IntraPrep Reagenz 1 und Inkubation für 15 min bei Raumtemperatur. 4 ml PBS in jedes Röhrchen und zentrifugieren Sie für 5 min bei 400 x g bei Raumtemperatur. </li><li> Entfernen Sie den Überstand durch Absaugen und 100 ul der IntraPrep Reagenz 2. Inkubieren für 5 min bei Raumtemperatur ohne Schütteln. </li><li> Add 5 ul PE-konjugierten anti-IFN-γ mAk in das Reagenzglas. Inkubieren von Licht für 15 min bei Raumtemperatur geschützt. </li><li> 4 ml PBS in jedes Röhrchen und zentrifugieren Sie für 5 min bei 400 x g bei Raumtemperatur. Entfernen Sie den Überstand durch Absaugen und Zellpellet in 0,5 ml FACS-Puffer. </li><li> Analysieren Sie die Zellen durch Durchflusszytometer. Gate auf CD3 + TCRVγ9 + Zellen und untersuchen die Expression von IFN-γ (Abb. 4). </li></ol> 5. Repräsentative Ergebnisse: Es ist wichtig, den Anteil der γδ T-Zellen in PBMC zu Beginn der Kultur bestimmen. Wie in Abb.. 2 A, der Anteil der CD3 + TCRVγ9 + γδ T-Zellen in PBMC lag bei 1,6% am Tag 0. Die dominierende Populationen wurden CD27 + CD45RA + CD27 + naiven oder CD45RA - zentraler Speicher Phänotypen. Wenn γδ T-Zellen effizient stimuliert wurden, bildeten sie sich zu Clustern an den Tagen 3-5 (Abb. 3 A und B). Als Cluster-Bildung verzögert wurde, das Wachstum von anderen Zelltypen, wie zB CD4 + oder CD8 + αβ T-Zellen oder NK-Zellen das Wachstum von γδ T-Zellen (Abb. 3 C und D) zu dominieren. Nach 14 Tagen der Kultur, nahm die Häufigkeit der γδ T-Zellen um mehr als 93,8% der kultivierten Zellen in erfolgreiche γδ T-Zell-Kulturen (Abb. 2 E). Die kultivierten γδ T-Zellen hochreguliert NKG2D und CD69 Expression (Abb. 2 G und H). Sie zeigten CD27 - CD45RA - Effektor-Memory-Phänotyp (Abb. 2 F). Die Funktionen der γδ T-Zellen wurden im Hinblick auf die Zytokin-Produktion und Zytotoxizität untersucht. Die intrazelluläre IFN-γ Färbung zeigte, dass γδ T-Zellen IFN-γ produziert als Reaktion auf PMA / Ionomycin Behandlung oder Z-Daudi-Zellen, die summiert IPP nach Zoledronat-Behandlung (Abb. 4). Diese Ergebnisse zeigen, dass Zoledronat effizient stimulieren und zu erweitern funktionale γδ T-Zellen. <table border="1"><tbody><tr><td> Rohr </td><td> FITC </td><td> PE </td><td> ECD </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> TCRαβ </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 Vγ9 </td><td> TCRαβ </td><td> CD45 </td><td> CD3 </td></tr><tr><td> 5 </td><td> TCR Vγ9 </td><td> NKG2D </td><td></td><td></td></tr><tr><td> 6 </td><td> TCR Vγ9 </td><td> CD69 </td><td></td><td></td></tr><tr><td> 7 </td><td> TCR Vγ9 </td><td> Maus IgG1 </td><td></td><td></td></tr><tr><td> 8 </td><td> TCR Vγ9 </td><td></td><td> CD45RA </td><td> CD27 </td></tr><tr><td> 9 </td><td> TCR Vγ9 </td><td></td><td> Maus IgG1 </td><td> Maus IgG1 </td></tr></tbody></table> Tabelle 1. Monoklonale Antikörper in multicolor Färbung von γδ T-Zellen verwendet. Ein Beispiel für die phänotypische Analyse von γδ T-Zellen in unserem Labor durchgeführt ist in Abb.. 2. <img src="/files/ftp_upload/3182/3182fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Separation von PBMC. Blood (7,5 bis 8,0 ml) wird in eine BD Vacutainer CPT Cell Preparation Röhrchen mit Natrium Heparin gezogen und direkt für 20 min bei 1800 x g zentrifugiert. Nach der Zentrifugation der resultierenden Schichten von oben nach bo gesehenttom: a) Plasma - b) PBMC und Thrombozyten - c) Dichte Lösung - d) Polyester Gel - e) Granulozyten - f) Rote Blutkörperchen. <img src="/files/ftp_upload/3182/3182fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Typische Oberfläche Phänotyp der γδ T-Zellen. PBMC wurden mit Zoledronat und IL-2 für 14 Tage stimuliert. Die Zellen wurden mit FITC-markiertem anti-TCR Vγ9 und PE/Cy5-labeled anti-CD3, um den Ausbau der γδ T-Zellen (A und E) zu überwachen. γδ T-Zellen wurden durch ihre Expression von TCRVγ9 identifiziert und ihre Expression von CD27 und CD45RA (B und F), NKG2D (C und G) oder CD69 (D und H) wurde untersucht. <img src="/files/ftp_upload/3182/3182fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Representative γδ T-Zell-Kulturen. PBMC wurden mit IL-2 (1000 IU / ml) und Zoledronat (5 pM) stimuliert. Vertreter Felder angezeigt werden (inverse Mikroskop IX71 [Olympus] x 200). Cluster und Aggregate von γδ T-Zellen können am Tag 3 (A) und Tag 5 (B), wenn γδ T-Zellen wurden erfolgreich ausgebaut beobachtet werden. Im Gegensatz dazu waren keine Cluster oder Aggregate beobachtet werden, wenn γδ T-Zell-Wachstum war nicht ausreichend (C und D). <img src="/files/ftp_upload/3182/3182fig4.jpg" alt="Abbildung 4"/> Abbildung 4. IFN-γ Produktion. γδ T-Zellen wurden mit RPMI-10 nur (A) oder PMA / Ionomycin (B) oder Z-Daudi (C) für 4 h inkubiert. Zunächst wurde die Oberflächenexpression von TCRVγ9 gefärbt und dann IFN-γ Produktion wurde durch intrazelluläre IFN-γ Färbung untersucht. <img src="/files/ftp_upload/3182/3182fig5.jpg" alt="Abbildung 5"/> Abbildung 5. Kinetik der γδ T-Zell-Kultur. (A) Absolute Zahl der kultivierten Zellen, (B) Anteil der γδ T-Zellen und (C) absolute Anzahl der γδ T-Zellen zu den angegebenen Zeitpunkten.

<ol>
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Die hier vorgestellte Methode ermöglicht eine effiziente Erweiterung der γδ T-Zellen aus PBMC. γδ T-Zellen aktiviert und durch Zoledronat erweitert und IL-2 entwickeln komplette Effektor-Funktionen, durch die Zytokin-Produktion und Zytotoxizität wider. Es wurde berichtet, dass die synthetischen phosphoantigens Bromhydrin Pyrophosphat (BrHPP) und 2-Methyl-3-Butenyl-1-pyrophosphat (2M3B1PP) auch erweitern γδ T-Zellen, jedoch sind sie nicht im Handel erhältlich. Im Gegensatz dazu ist Zoledronat bereits für klinis...

<table border="1"><tbody><tr><th> Reagent Name </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> ZOMETA </td><td> Novartis Pharma K. K </td><td></td><td> Zoledronat </td></tr><tr><td> Proleukin </td><td> Novartis Pharmaceuticals </td><td></td><td> humanem rekombinantem IL-2 </td></tr><tr><td> BD Vacutainer CPT Cell Preparation Röhrchen mit Natrium-Heparin </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-Medium </td><td> Cell 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> Brefeldin A </td><td> Sigma </td><td> B5936-200 &amp;mgr; l </td><td></td></tr><tr><td> Phorbol 12-myristat 13-Acetat (PMA) </td><td> Sigma </td><td> P1585-1MG </td><td></td></tr><tr><td> Ionomycin </td><td> Sigma </td><td> 13909-1ML </td><td></td></tr><tr><td> IntraPrep </td><td> Wichtige Produktinformation </td><td> A07803 </td><td></td></tr><tr><td> anti-human CD3-FITC oder PE/Cy5 </td><td> Wichtige Produktinformation </td><td> A07746 FITC A07749 PE/Cy5 </td><td></td></tr><tr><td> Anti-Human-CD4-ECD </td><td> Wichtige Produktinformation </td><td> 6604727 </td><td></td></tr><tr><td> Anti-Human-CD8-PE/Cy5 </td><td> Wichtige Produktinformation </td><td> 6607011 </td><td></td></tr><tr><td> Anti-Human-CD14-PE/Cy5 </td><td> Wichtige Produktinformation </td><td> A07765 </td><td></td></tr><tr><td> Anti-Human-CD19-PE </td><td> Wichtige Produktinformation </td><td> A07769 </td><td></td></tr><tr><td> Anti-Human-CD45-ECD </td><td> Wichtige Produktinformation </td><td> A07784 </td><td></td></tr><tr><td> Anti-Human-CD56-PE/Cy5 </td><td> Wichtige Produktinformation </td><td> A07789 </td><td></td></tr><tr><td> Anti-Human-TCRαβ-PE </td><td> Wichtige Produktinformation </td><td> A39499 </td><td></td></tr><tr><td> Anti-Human-TCR Vγ9-FITC </td><td> Wichtige Produktinformation </td><td> IM1463 </td><td></td></tr><tr><td> Anti-Human-CD27-PE/Cy5 </td><td> Wichtige Produktinformation </td><td> 6607107 </td><td></td></tr><tr><td> Anti-Human-CD45RA-ECD </td><td> Wichtige Produktinformation </td><td> IM2711 </td><td></td></tr><tr><td> Anti-Human-CD69-PE </td><td> BD </td><td> 555531 </td><td></td></tr><tr><td> Anti-Human-NKG2D-PE </td><td> Wichtige Produktinformation </td><td> A08934 </td><td></td></tr><tr><td> Anti-humal IFNy-PE </td><td> Wichtige Produktinformation </td><td> IM2717U </td><td></td></tr><tr><td> Maus IgG1 Isotyp-Kontroll-PE </td><td> Wichtige Produktinformation </td><td> A07796 </td><td></td></tr><tr><td> Maus IgG1 Isotyp-Kontroll-ECD oder PE/Cy5 </td><td> Wichtige Produktinformation </td><td> A07797 A07798 </td><td></td></tr></tbody></table>

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

Menschliche γδ T-Zellen erkennen und reagieren auf eine Vielzahl von Stress-induzierte Antigene, wodurch die Entwicklung angeborenen breiten Anti-Tumor-und anti-infektiösen Aktivität. 1 Die Mehrheit der γδ T-Zellen im peripheren Blut haben die Vγ9Vδ2 T-Zell-Rezeptor. Diese Zellen erkennen Antigen in einer Haupthistokompatibilitätskomplex-unabhängige Art und Weise und zu entwickeln starke zytolytischer und Th1-like Effektor-Funktionen. 1 Darum sind γδ T-Zellen attraktiven Kandidaten Effektorzellen zur Immuntherapie von Krebs. Vγ9Vδ2 T-Zellen zu phosphoantigens wie reagieren (E)-4-Hydroxy-3-methyl-but-2-enyl-pyrophosphat (HMBPP), die in Bakterien über Isoprenoidbiosynthese synthetisiert wird; 2 und Isopentenylpyrophosphat (IPP), das hergestellt wird in eukaryotischen Zellen durch die Mevalonatweges. 3 In physiologischer Zustand, ist die Erzeugung von IPP in transformierten Zellen nicht ausreichend für die Aktivierung von γδ T-Zellen. Dysregulation des Mevalonat-Weg in Tumorzellen führt zu einer Akkumulation von IPP und γδ T-Zellen-Aktivierung. 3 Weil Aminobisphosphonate (wie Pamidronat oder Zoledronat) hemmen Farnesylpyrophosphat Synthase (FPP), das Enzym wirkt hinter IPP in der Mevalonat-Weg, intrazellulären IPP und sensitibity auf γδ T-Zellen Anerkennung kann therapeutisch durch Aminobisphosphonate erhöht werden. IPP Akkumulation ist weniger effizient in nontransfomred Zellen als Tumorzellen mit einem pharmakologisch relevante Konzentration von Aminobisphosphonate, die es uns ermöglichen Immuntherapie gegen Krebs durch Aktivierung γδ T-Zellen mit Aminobisphosphonate. 4 Interessanterweise, IPP in Monozyten ansammelt, wenn PBMC mit Aminobisphosphonate behandelt werden, weil der effizienten Drogen-Aufnahme durch die Zellen. 5 Monozyten, die IPP zu akkumulieren Antigen-präsentierenden Zellen und stimulieren Vγ9Vδ2 T-Zellen im peripheren Blut. 6 auf diese Mechanismen, entwickelten wir eine Technik, die für eine groß angelegte Ausbau des γδ T-Zell-Kulturen mit Zoledronat und Interleukin -2 (IL-2). 7 Andere Methoden zur Erweiterung der γδ T-Zellen nutzen die synthetische phosphoantigens Bromhydrin Pyrophosphat (BrHPP) 8 oder 2-Methyl-3-Butenyl-1-pyrophosphat (2M3B1PP). 9 Alle diese Methoden ex ermöglichen vivo Expansion, die sich in einer großen Anzahl von γδ T-Zellen für den Einsatz in adoptiven Immuntherapie. Allerdings ist nur Zoledronat eine FDA-Zulassung im Handel erhältlichen Reagenz. Zoledronat-expanded γδ T-Zellen-Display CD27 - CD45RA - Effektor-Memory-Phänotyp und ihre Funktion kann durch IFN-γ Produktion Assay 7 ausgewertet werden.

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

Eine Methode, um γδ T-Zellen aus dem peripheren Blut mononukleären Zellen (PBMC) erweitern wird beschrieben. PBMC-derived γδ T-Zellen stimuliert und erweitert mit Zoledronat und Interleukin-2 (IL-2). Großräumige Ausdehnung der γδ T-Zellen können zur autologen zellulären Immuntherapie von Krebs angewendet werden.

Expansion von humanen peripheren Blut γδ T-Zellen mit Zoledronat

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 Aufrufe.  University of Tokyo Hospital. Eine Methode, um γδ T-Zellen aus dem peripheren Blut mononukleären Zellen (PBMC) erweitern wird beschrieben. PBMC-derived γδ T-Zellen stimuliert und erweitert mit Zoledronat und Interleukin-2 (IL-2). Großräumige Ausdehnung der γδ T-Zellen können zur autologen zellulären Immuntherapie von Krebs angewendet werden.

Serie: 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, Reinigung und Functional Assessment of Human peripheren Blut NK-Zellen

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

Forschung

Immunologie und Infektion

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.

Klinische Anwendung der<em&gt; Dornröschen</em&gt; Und künstliche Antigen präsentierenden Zellen genetisch zu verändern T Zellen aus dem peripheren und Nabelschnurblut

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.

Generation of Human Alloantigen-spezifischen T-Zellen aus peripherem Blut

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.

Isolierung und Analyse über Durchflusszytometrie der Menschen Endozervikale Gamma-Delta-T-Zellen

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.

Dextran erhöht die Effizienz der Lentivirale Transduktion von murinen und menschliche primäre NK-Zellen

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.

Hochmultiplexed, Super-Auflösung Bildgebung von T-Zellen mit 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.

Bewertung der synaptischen Schnittstelle der primären menschlichen T-Zellen aus peripherem Blut und Lymphgewebe

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.

Flotationsbasierte T-Zell-Isolierung, -Aktivierung und -Expansion aus mononukleären Blutproben des menschlichen peripheren Blutes unter Verwendung von Mikrobläschen

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.

Abgrenzung funktioneller Osteoklasten von CD14+ Monozyten aus humanem peripherem Blut

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

PBMC-Sammlung und Kryolagerung für die mitochondriale Bioenergetikanalyse

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.

Autor im Rampenlicht: Konservierung bioenergetischer Parameter in mononukleären Zellen des peripheren Blutes nach Kryokonservierung 

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.

Kryokonservierung und bioenergetische Bewertung von mononukleären Zellen des menschlichen peripheren Blutes

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