Diese Arbeit wurde durch einen Dan L. Duncan kollaborative Forschungsstipendium (CMB und EJS), der National Heart, Lung, and Blood Institute (US4HL081007), ein Leukemia and Lymphoma Society Clinical Research Scholar Award (CMB), und dem National Cancer Institute unterstützt (RO1 CA06150816; EJS).

Ein. Mononukleären Zellen Isolation (Tag 0) <ol><li> Legen Spitze von weiblichen Luer-Adapter in die Steckdose Port des 20% Anteil an der Nabelschnurblut-Einheit legen Spritze und entfernen Sie das Blut. Die aufgetauten Blut 20 ml RPMI erwärmt in 50 ml Zentrifugenröhrchen. Spülen Nabelschnurblut Beutel mit 5 ml RPMI und Transfer zum gleichen Zentrifugenröhrchen. </li><li> Zentrifuge Zellen für 10 Minuten bei 400 x g beträgt. Absaugen des Überstandes. </li><li> Resuspendieren Zellen in 20 ml warmem RPMI. Schicht von Zellen auf 15 ml Lymphoprep in 50 ml Zentrifugenröhrchen. Zentrifuge 40 Minuten bei 400 x g. </li><li> Ernten die Schnittstelle mit den mononukleären Zellen und waschen in 20 ml RPMI. Zentrifuge für 10 Minuten bei 450 x g. </li><li> Absaugen des Überstandes und waschen in 20 ml RPMI. Zählen von Zellen und Spin für 5 Minuten bei 400 x g. </li><li> Absaugen des Überstandes und resuspendieren Zellen bei 5 x 10 6 mononukleären Zellen / ml CellGenix DC Medien (Serum-frei). Entfernen 1 mL für EBV-LCL-Generation und in 15 mL centrifuge Röhre. </li></ol> 2. Dendritische Zelle Generation (beginnend am Tag 0) <ol><li> Platte 2 ml Zellen (bereits bei 5 x 10 6 Zellen / ml Medium DC) pro Vertiefung in eine Platte mit 6 Vertiefungen. Lassen Sie im Inkubator für 1-2 Stunden. </li><li> Nach 1-2 Stunden, Auswaschen der nicht-adhärenten Zellen durch Waschen 3-4 mal mit PBS. Sammeln Sie die nicht-adhärenten Zellen und einfrieren. 2 ml / Vertiefung des DC Medien mit 1000 U / ml IL-4 und 800 U / ml GM-CSF. </li><li> 3-4 Tage nach DC Initiierung, füllt die IL-4 und GM-CSF durch Zugabe von 100 ul / Vertiefung von Medien mit einer Endkonzentration von 1000U/mL IL-4 und 800 U / ml GM-CSF. </li><li> Am Tag 5-6, Ernte der DC durch Abkratzen der gut mit einer Pipette. Zählen Sie die großen Zellen und Zentrifuge für 5 Minuten bei 400 x g. Aspirate Medien und resuspendieren DC bei 2x10 6 Zellen / ml DC Medien. 0,5 mL / Well einer 24-Well-Platte. </li><li> Transduzieren die DC mit dem Ad5f35pp65 Vektor bei einer MOI pro Zelle von 10 infektiösen Einheiten. Fügen vector zu jedem Well in einer 24-Well-Platte. Inkubieren Zellen für 1,5 Stunden. Nach 1,5 Stunden, 1,5 mL / Well DC Medien mit: 1000 U / mL IL-4, 800 U / ml GM-CSF, 10 ng / mL TNF-alpha, 1 ug / mL PGE-1, 100 ng / mL IL-6, und 10 ng / ml IL-1beta. Diese Zytokine reifen die DCs. </li></ol> 3. Erzeugung von EBV-LCL (beginnend am Tag 0) <ol><li> Zentrifugieren enthaltend die 5 x 10 6 mononukleären Zellen. Fügen Sie 200 ul konzentrierter EBV B95.8 Überstand und 1,8 ml komplettem Medium (RPMI + 10% FBS + 2mM L-Glutamin) enthält Cyclosporin (1 pg / mL). </li><li> Aliquot 100 ul Zellen in 10 Wells einer 96-Well-Platte und in 200 ul 5 Vertiefungen. 100 ul von kompletten media + Cyclosporin zu den Vertiefungen, die nur 100 ul der Zellen. Füllen restlichen Vertiefungen mit sterilem Wasser. </li><li> Führen Sie das LCL wöchentlich und erweitern von einer 96-Well-Platte auf eine 24-Well-Platte nach ~ 2 Wochen. Nach einer weiteren Woche zu einer T25-Flasche übertragen und in der anschließenden Woche Transfer zuma T75-Flasche. In diesem Stadium ist es empfehlenswert, dass Sie Aliquots von LCL einfrieren. Es dauert in der Regel 1 Monat bis eine ausreichende Anzahl von LCL generieren. </li></ol> 4. CTL Initiation - 7 Tage nach Beginn der DCs <ol><li> Ernte DCs und strahlen bei 30 Gy. Waschen Sie 4 x und zählen. Resuspendieren DC in T-Zell-Medium mit humanem Serum (45% RPMI, 45% der CLICK, 10% humanem Serum, 2 mM L-Glutamin) bei 1 x 10 5 DCs / ml. </li><li> Auftauen nichtadhärenten Zellen aus Schritt 2.2. Waschen der Zellen und zu zählen. Resuspendieren bei 2 x 10 6 nicht-adhärenten Zellen / ml in T-Zell-Medium, das humanes Serum. Fügen Sie 10 ng / ml IL-7 und IL-12 und 5 ng / ml IL-15. 1 mL Zellen pro Well in einer 24-Well-Platte. 1 mL DC pro Vertiefung. Füllen Sie leere Vertiefungen von 24-Well-Platte mit sterilem Wasser. </li><li> 7 Tage nach der T-Zell-Initiierung, aufgeteilt Futter und / oder die Zellen mit T-Zellen-enthaltenden Medien Humanserum. </li><li> 9-12 Tage nach T-Zell-Initiation, frieren die T-Zellen, bis die LCL bereit sind. </li><li> Once LCL ausgebaut wurden und in T75-Flaschen passagiert, transduzieren das LCL durch Zentrifugation für 5 Minuten bei 400 x g beträgt. Hinzufügen des Vektors zu dem Zellpellet bei einer MOI von 100 infektiösen Einheiten pro Zelle. Inkubieren für 1,5 Stunden. Nach 1,5 Stunden, in komplettem Medium auf 5 x 10 5 LCL / mL resuspendieren und 2 mL pro Vertiefung einer 24-Well-Platte. 12 </li><li> Nach 2 Tagen ernten die LCL und strahlen bei 40 Gy. Waschen Sie 4 x und zählen. Resuspendieren des LCL in T-Zell-Medien mit menschlichem Serum bei 2,5 x 10 5 Zellen / ml. 1 mL LCL pro Vertiefung einer 24-Well-Platte. Fügen Sie außerdem 5 x 10 6 LCL einer Grex40 Kultur Gerätes sowie 5 ng / mL IL-15. 13 </li><li> Ernten Sie die T-Zellen. Zentrifuge für 5 Minuten bei 400 xg, den Überstand aspirieren und resuspendieren in T-Zell-Medien mit menschlichem Serum bei 1 x 10 6 T-Zellen / mL. Add 5 ng / mL IL-15 und geben Sie 1 ml / Vertiefung von T-Zellen (insgesamt 1 x 10 6 T-Zellen) der LCL, die bereits in den 24-Well-Platte. In der Grex40 bringendas Gesamtvolumen der Medien bis 30 mL. </li><li> Am Tag 3-4 nach der Stimulation, wenn die Zellen konfluent sind dann teilen (in der Platte) 1:1 und frisches Medium mit 50 U / ml IL-2. Wenn sie nicht konfluent sind dann einfach absaugen ½ die Medien und ersetzen mit Medien, die 50 U / ml IL-2. In der Grex40 absaugen die Hälfte der Medien, ersetzen Sie sie durch frische Medien, und 50 U / ml IL-2. </li><li> Am Tag 7, wie in Schritt 4,6 wiederholen, sondern verwenden IL-2 am Tag der Stimulation, nicht IL-15. </li></ol> 5. Repräsentative Ergebnisse Eine schematische Darstellung der GMP-konformen FDA-zertifizierten Fertigungsstätte Protokoll ist in Abbildung 1 dargestellt. Der Vorgang dauert etwa 50 Tage. Typische Methoden zur Erzeugung von virus-spezifischen T-Zellen zu erweitern bereits vorhandene Gedächtnis-T-Zellen, jedoch fehlt Nabelschnurblut Virus-erfahrenen T-Zellen und deshalb müssen wir prime naiven T-Zellen ex vivo. Hierzu verwenden wir dendritischen Zellen als auch die Zytokine IL-7, IL-12 und IL-15,die zum Erzeugen viralen Spezifität. Nach 3 Stimulationen, sollte die Ausbeute über 100x10 6-Zellen sein. Wenn genügend Zellen zur Verfügung stehen, können zusätzliche Stimulationen durchgeführt, bis die gewünschte Anzahl von CTLs zur Verfügung stehen. Die Mehrheit dieser Zellen sollten CD3 + mit einem Gemisch aus CD4 + und CD8 + T-Zellen sein. Es sollte weniger als 15% NK-Zellen (CD3-/CD56 +) und weniger als 1% CD19 + B-Zellen (Abb. 2). Das expandierte CTL sollte das pp65 von CMV, Hexon und Penton erkennt aus Adenovirus, sowie zahlreiche EBV-Antigene, die auf EBV-LCL exprimiert werden. Wenn in einem ELISPOT-Assay getestet, CTL sollte sezernieren mehr IFN-? in Reaktion auf diese Antigene als irrelevant Antigene (Abbildung 3). Die CTL sollte auch lysieren virale Peptid-gepulsten Ziele wie PHA-Blasten. In einem 51 Cr-Release-Assay sollte CTL lysieren LCL, CMVpp65-gepulste und Adenovirus Hexon-und /oder Penton-gepulste Ziele, aber keine Ziele mit irrelevanten Peptide (Abbildung 4) gepulst. <img src="/files/ftp_upload/3627/3627fig1.jpg" alt="Abbildung 1."/> Abbildung 1. Generation von Multi-virus-spezifischen CTL aus Nabelschnurblut. Schematische Darstellung des gesamten Prozesses der CTL Herstellung aus Nabelschnurblut. Zuerst werden die Nabelschnurblut mononukleäre Zellen aus dem 20%-igem Anteil des fraktionierten Nabelschnurblut Gehäuse isoliert. Mit Ausnahme von 5x10 6 Zellen, die für LCL Generation gespeichert werden, werden alle Zellen dann in dendritische Zelle Medien für 1-2 Stunden ausplattiert, an welchem ​​Punkt die nicht-adhärenten Zellen geerntet und eingefroren. Die DC werden dann zugeführt DC Medien mit IL-4 und GM-CSF. Nach 5 Tagen Kultur werden die DC gereift und transduziert mit einem adenoviralen Vektor, der das immundominante CMV pp65. Bei der Initiierung werden diese DC mit den nicht haftenden Zellen sowie den Zytokinen IL-7, IL-12 in Kombination,und IL-15. Bei nachfolgenden Stimulationen wird derselbe adenovirale Vektor verwendet, die EBV-LCL, die als Antigen-präsentierende Zellen verwendet werden transduzieren. IL-15 ist an der zweiten Stimulation und IL-2 danach verwendet. <img src="/files/ftp_upload/3627/3627fig2.jpg" alt="Abbildung 2."/> Abbildung 2. Phänotyp der resultierenden T-Zellen. Gezeigt ist der Prozentsatz an lebenden Zellen in der Lymphozyten-Gate enthalten. CTL sind CD3 + und CD4 + oder CD8 + aber weitgehend CD3-/CD56- und CD19-. <img src="/files/ftp_upload/3627/3627fig3.jpg" alt="Abbildung 3."/> Abbildung 3. T-Zell-Funktionalität. T-Zell-Spezifität wurde durch IFN-γELISPOT getestet. T-Zellen wurden mit überlappenden Peptiden, die die gesamte Protein von Hexon, Pentonbasis, pp65 gepulst und das irrelevante IE-1 Antigens. CTL allein zeigt Medien allein. Autologen LCL bestrahlt wurden und fügte bei 1x10 5 / well. Dargestellt ist die Kassamittelkurs bildende Zelle count der dreifachen Wells. <img src="/files/ftp_upload/3627/3627fig4.jpg" alt="Abbildung 4."/> Abbildung 4. Cytolytische Aktivität von CTL. Die Fähigkeit der resultierenden CTL-Ziele zu exprimierenden viralen Antigenen lysieren wurde in einem 51 Cr-Assay getestet. 51 Cr-markierten autologen PHA-Blasten mit überlappenden Peptide, die die gesamte Antigen oder 51 Cr-markierten autologen LCL wurden kokultiviert mit CTL wurden gepulst. Nach 4 Stunden wurde Gamma-Release auf einem Gamma-Zähler gezählt.

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Die Autoren haben nichts zu offenbaren.

<html> Aktuelle Strategien zur Steuerung virale Infektionen nach CBT soll wirksam sein kann, aber sie sind mit erheblicher Toxizität verbunden sind, sind teuer und nicht verleihen langfristigen Schutz gegen spätere Infektionen. In der Tat kann die Verwendung einiger antiviralen Medikamenten begrenzen die Expansion des Virus-spezifischen T-Zellen, die sonst Schutzschicht. 14 weitere Option ist die Infusion von Donor-derived Virus-spezifischen T-Zellen. Wir und andere haben gezeigt, dass solche T-Zellen sicher...

<table border="1"><tbody><tr><td> Namen Reagenz </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 21870-076 </td></tr><tr><td> DC Medien </td><td> CellGenix </td><td> 20801-0500 </td></tr><tr><td> EHAA (Anweisungen-Medium) </td><td> Irvine Scientific </td><td> 9195 </td></tr><tr><td> Human Serum </td><td> Gemini Bio Produkte </td><td> 100-110 </td></tr><tr><td> Gasdurchlässige Cultureware 18 </td><td> Wilson-Wolf </td><td> 80040S </td></tr><tr><td> IL-2 </td><td> Chiron (TCH Pharmacy) </td><td></td></tr><tr><td> IL-12 </td><td> NCI / CTEP </td><td></td></tr><tr><td> IL-15 </td><td> CellGenix </td><td> 1013-050 </td></tr><tr><td> IL-7 </td><td> R &amp; D </td><td> AFL207 </td></tr><tr><td> IL-1beta </td><td> R &amp; D </td><td> AFL201 </td></tr><tr><td> IL-6 </td><td> ZelleGenix </td><td> 1004-050 </td></tr><tr><td> GM-CSF </td><td> TCH Pharmacy </td><td></td></tr><tr><td> IL-4 </td><td> R &amp; D </td><td> <a href="http://www.rndsystems.com/pdf/AFL204.pdf" target="_blank">AFL204</a> </td></tr><tr><td> TNF-alpha </td><td> R &amp; D </td><td> AFL210 </td></tr><tr><td> Ad5f35pp65 </td><td> BCM CAGT Vector Production Facility </td><td></td></tr><tr><td> Plasma Transfer mit Luer-Adapter-Set </td><td> Charter Medical </td><td> 89-550 66j </td></tr><tr><td> Lymphoprep </td><td> Nycomed </td><td> 1114550 </td></tr></tbody></table>

expanding cytotoxic t lymphocytes from umbilical cord blood that target cytomegalovirus, epstein-barr virus, and adenovirus

Virus-Infektionen nach einer Stammzelltransplantation gehören zu den häufigsten Todesursachen, besonders nach Nabelschnurblut (CB) Transplantation (CBT), wo die CB enthält keine nennenswerte Zahl von Virus-erfahrenen T-Zellen, die den Empfänger vor einer Infektion schützen können 1. - Wir und andere 4 gezeigt, dass Virus-spezifische CTL von seropositiven Spendern erzeugt und an den Empfänger infundiert haben sind sicher und schützend. 5-8 jedoch bis vor kurzem Virus-spezifischen T-Zellen nicht aus Nabelschnurblut erzeugt werden, was wahrscheinlich auf die Abwesenheit von virus-spezifischen Gedächtnis-T-Zellen. In dem Bemühen, besser zu imitieren die in vivo Bedingungen Priming von naiven T-Zellen haben wir ein Verfahren, CB-abgeleitete dendritische Zellen (DC) transduziert mit einem adenoviralen Vektor (Ad5f35pp65), enthaltend die CMV immundominanten pp65 verwendet, daher T-Zell-Spezifität Antreiben Richtung CMV und Adenovirus. 9 Bei der Initiierung, verwenden wir diese marierte DCs sowie CB-abgeleiteten T-Zellen in Gegenwart der Cytokine IL-7, IL-12 und IL-15. 10 In der zweiten Stimulation verwendeten wir EBV-transformierten B-Zellen oder EBV-LCL, die sowohl exprimieren latente und lytische EBV-Antigene. Ad5f35pp65-transduzierten EBV-LCL werden verwendet, um die T-Zellen in Gegenwart von IL-15 bei der zweiten Stimulation zu stimulieren. Nachfolgende Reize verwenden Ad5f35pp65-transduzierten EBV-LCL und IL-2. Von 50x10 6 CB mononukleären Zellen können wir nach oben von 150 x 10 6 Virus-spezifischen T-Zellen, Antigen-gepulste Freisetzung Targets und Zytokinen in Reaktion auf Antigenstimulation lysieren erzeugen. 11. Diese Zellen wurden in einer GMP-gerechte Weise hergestellt unter Verwendung nur die 20%-Anteil an einer fraktionierten Nabelschnurblut Einheit und haben für den klinischen Einsatz übersetzt worden.

Watch this Scientific Journal Video about Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus at JoVE.com

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Hier beschreiben wir die erste Good Manufacturing Practice (GMP)-kompatiblen Verfahren zur Herstellung virus-spezifische zytotoxische T-Lymphozyten (CTL) aus Nabelschnurblut, eine Quelle von überwiegend naiven T-Zellen.

Ausbau zytotoxische T-Lymphozyten aus Nabelschnurblut, dass Target Cytomegalovirus, Epstein-Barr-Virus und Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

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Immunology and Infection

12.7K Aufrufe.  Baylor College of Medicine. Hier beschreiben wir die erste Good Manufacturing Practice (GMP)-kompatiblen Verfahren zur Herstellung virus-spezifische zytotoxische T-Lymphozyten (CTL) aus Nabelschnurblut, eine Quelle von überwiegend naiven T-Zellen.

Serie: Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Herstellung und Verwendung von HIV-1 infizierten primären CD4 + T-Zellen als Zielzellen in der natürlichen Killerzellen Zytotoxische Assays

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Forschung

Immunologie und Infektion

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Methoden zur Beta Cell Death vermittelt durch zytotoxische T-Lymphozyten Beurteilung

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Gründung des Epstein-Barr Virus Growth-transformierten lymphoblastoiden Zelllinien

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

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 ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Isolierung von Precursor B-Zell-Subsets aus Nabelschnurblut

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Isolierung und Differenzierung von Th17 Naive CD4-T-Lymphozyten

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Entwicklung eines IFN-γ ELISPOT-Assay zu beurteilen, Varicella-Zoster-Virus-spezifische Zellvermittelte Immunität nach Nabelschnurblut-Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

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 ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

Radial Mobilität und zytotoxische Funktion der retroviralen Replikation von Vektor transduziert, Nichthaft Alloresponsive T-Lymphozyten

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...