The author would like to thank the NIH Tetramer Core Facility for tetramer production. The author would also like to thank E.D. O&#8217;Connor and K.E. Marquez at the UCSD Human Embryonic Stem Cell Core Facility flow cytometry laboratory for assistance in cell sorting. This work was funded by National Institutes of Health grant K08AI085039 (G.P.M.).

HINWEIS: Dieses Protokoll erfordert die Verwendung von peripheren Blutproben von Probanden. Alle Forschung mit menschlichen Probanden sollten überprüft und durch eine Menschen Studies Institutional Review Board genehmigt, um die Einhaltung der Deklaration von Helsinki (2013) und dem Health Insurance Portability and Accountability Act von 1996 zu gewährleisten. 1. Isolierung von T-Zellen aus Vollblut <ol><li> Vor dem Start, warm das Dichtegradienten-Medium auf Raumtemperatur. Aliquot 4 ml Dichtegradienten-Medium in 2-4 sterile konische 15 ml-Zentrifugenröhrchen (1 Röhrchen pro 10 ml Gesamtvolumen verdünnt Blut verwendet werden). </li><li> Erhalten 10-20 ml Blut in 1-2 Natrium-Heparin sprühbeschichteten (grün-top) venösen Blutröhrchen. Sammeln Humanproben unter der Aufsicht von ausgebildeten Personen und gemäß einem Institutional Review Board genehmigten Protokoll. </li><li> Wischen Sie die Außenseite der Rohre mit 70% Ethanol. Die Spitzen des fi vorsichtig entfernengefüllt Sammelröhrchen und dekantiert die Blut in ein steriles 50 ml-Zentrifugenröhrchen. </li><li> 10 ml sterile phosphatgepufferte Kochsalzlösung (PBS) zu jeder Blutsammelröhrchen. Sammeln Sie die PBS, fügen dem dekantiert Vollblut, und vorsichtig mischen. </li><li> In 25 ul humanen T-Zell Enrichment Cocktail / 2 ml Gesamtvolumen. Inkubieren bei Raumtemperatur für 20 min. </li><li> Verwendung einer 10 ml Pipette Schicht bis zu 10 ml der 1: 1 verdünnten Blut vorsichtig auf der Oberseite der Dichtegradienten-Medium. Achten Sie darauf, nicht die Oberfläche des Dichtegradientenmedium stören. </li><li> Zentrifuge Schicht Blut und Dichtegradienten-Medium bei 1200 × g für 20 min bei 20 o C. </li><li> Die Röhrchen aus der Zentrifuge, dabei die Schnittstelle zwischen dem Dichtegradienten-Medium und der Schicht aus Leukozyten an der Schnittstelle zwischen der Dichtegradienten-Medium und verdünnte Plasma nicht zu stören. Mit einem 5 ml Pipette vorsichtig sammeln die Leukozyten-Schicht und in ein neues steriles 50 ml konischen Wannee. </li><li> In PBS, um das Volumen des gesammelten PBL auf 50 ml zu bringen und vorsichtig mischen. </li><li> Zentrifuge bei 600 × g für 5 min bei 20 o C. Dekantieren. </li><li> Resuspendieren pelletierten Zellen in 10 ml sterile Durchflusszytometrie Sortierpuffer (sterilfiltriert PBS, enthaltend 1% BSA). Probe 10 &amp; mgr; l der Zellsuspension 1:10 verdünnen Trypanblau (90 ul addieren), um Zellen mit einer Zählkammer zählen (erwartete Ausbeute werden 1 - 4 x 10 6 Zellen / Röhrchen Vollblut). <ol><li> Entfernen 1 ml Aliquot, Transfer zum 5-ml-Tube und auf Eis zu halten für die Analyse von T-Zellen nicht durch Tetramer beschriftet. Halten Zellsuspension auf Eis. </li></ol></li></ol> 2. Magnetische Anreicherung von Alloantigen-spezifischen T-Zellen <ol><li> Verdünnte Alloantigen pMHC Tetramer bis 1: 100 in sterilem Sortierpuffer. </li><li> Zentrifuge Zellsuspension (9 ml von Schritt 1.11) bei 600 × g für 5 min bei 20 o C. Dekantieren. </li><li> In 50 ul alloantigen pMHC Tetramer verdünnt, um pelletiertZellen (bis zu 10 7 Zellen). Mischen leicht Pipettieren. Transfer zu einem sterilen 5 ml-Tube. Inkubation für 30 min bei Raumtemperatur. </li><li> 2 ml Art Puffer. Zentrifuge bei 600 × g für 5 min bei 20 o C. Dekantieren. </li><li> Zellen in 100 ul Sortierpuffer. In 10 ul Biotin Selection Cocktail und Inkubation für 15 min bei Raumtemperatur. </li><li> In 5 ul Magnetische Nanopartikel und Inkubation für 10 min bei Raumtemperatur. </li><li> In 2,5 ml Art Puffer und vorsichtig mischen. Entfernen 100 ul Aliquot, Transfer zum 5-ml-Tube und auf Eis zu halten für die Analyse der Voranreicherungszellen. </li><li> Legen Sie die 5 ml Röhrchen mit der Zellsuspension in der Zellseparation Magnet. Die Kappe sollte locker oben auf dem Rohr sein. Inkubation für 5 min bei Raumtemperatur. </li><li> Entfernen Sie vorsichtig die Kappe von der Tube. Halten Sie die Röhre und Magnet zusammen, dekantiert man die Tubeninhalt in ein frisches 5-ml-Tube. Tippen Sie nicht oder schütteln die Tubeninhalt zuentfernt die letzten Tropfen Flüssigkeit. </li><li> Entfernen Sie den Schlauch vom Magnet. 2 ml kaltem Sortierpuffer und vorsichtig mischen. Probe 10 ul Zellsuspension verdünnt 1: 2 (in den 10 ul) Trypanblau um Zellen zu zählen mit einer Zählkammer. </li></ol> 3. Herstellung von T-Zellen für die Einzelzell-Durchflusszytometrie Zellsortierung <ol><li> In kalten Sortierpuffer in alle Röhrchen von Zellen (Tetramer unmarkierten, Tetramer-markierten un-angereichert und Tetramer-markierten angereicherten), um die Lautstärke zu 3 ml zu bringen. </li><li> Zentrifuge bei 600 × g für 5 min bei 4 ° C dekantiert Puffer in Zellen zu gewinnen. </li><li> Resuspendieren pelletierten Zellen in 25 ul kalten Sortierpuffer. In 5 ul menschlichen Fc-Block. Inkubieren auf Eis für 20 min. </li><li> Bereiten Antikörper für die Durchflusszytometrie Sortierung. Mischungs 15 ul Sortierpuffer, 15 &amp; mgr; l anti-CD5, 15 &amp; mgr; l Anti-CD14, und 15 &amp; mgr; l Anti-CD19. </li><li> Mit 20 &amp; mgr; l der Antikörpermischung in jedes Röhrchen der Zellen. Inkubieren auf Eis für 20 min. </li><li> 2 ml kaltem Sortierpuffer in jedes Röhrchen. </li><li> Zentrifuge bei 600 × g für 5 min bei 4 ° C dekantiert Puffer in Zellen zu gewinnen. </li><li> Die Zellen in einer Konzentration von 1 -2 x 10 6 Zellen / ml in Sortierpuffer. </li><li> Aliquots von 100 ul des humanen T-Zellkulturmedium (Iscove DMEM mit 2 mM Glutamax, 10 mM HEPES, 50 ug / ml Gentamycin, 50 &amp; mgr; M 2-Mercaptoethanol, 10% hitzeinaktiviertem humanem AB-Serum und 2,5 ng / ml rekombinanten menschlichen ergänzt IL-2) zu jedem Well einer 96-Well-Rundboden-Zellkulturplatte. Halten Sie auf dem Eis. </li></ol> 4. Isolierung von Tetramer-markierten T-Zellen durch Einzel-Zell-Durchflusszytometrie Sortierung <ol><li> Richten Durchflusszytometrie Gating Parameter alloantigen-pMHC Tetramer markierten T-Zellen (Abbildung 2.a) zu identifizieren. Verwenden Sie die Voranreiche Tetramer markierten Fraktion feststellen Gating Strategien zur Dubletten, Tor auf Lymphozyten zu eliminieren, auszuschließen CD19 exprimieren B-Zellen und CD14exprimierenden Monozyten und T-Zellen zu identifizieren, indem CD5 Ausdruck. Verwenden die Probe nicht mit Tetramer als Fluoreszenz minus einer Steuer markiert die Gating für die Identifizierung von Tetramer bindende T-Zellen zu etablieren (mit 0% von CD5 + CD14 - CD19 - Zellen aus der Probe nicht durch Tetramer markierten innerhalb dieses Tor fällt) . HINWEIS: Gating-Strategien, die nicht ausreichend stringent werden in Isolation von nicht-T-Zellen oder T-Zellen, die nicht antigenspezifisch sind, ergeben, sind, während zu restriktiv Gating Strategien wird die Zahl der T-Zellen isoliert reduzieren. </li><li> Programm den Plattenparameter für die einzelne Zelle sortieren. Richten Sie die Sortierer 1 CD5 + CD14 platzieren - CD19 - Tetramer + Zelle in jede Vertiefung. Richten Sie die eingerichtet, um ein Negativkontrolle (keine Zellen in den gut sortierten) und 1 positive Kontrollvertiefung enthalten (100 CD5 + CD14 - CD19 - Tetramer - Zellen) Platte in jeder Reihe (Abbildung 2.B </strong&gt;). </li><li> Mit dem etablierten Durchflusszytometrie Gating-Strategie und Plattenaufbau isolieren einzelnen Tetramer bindenden T-Zellen direkt in den 96-Well-Platte mit einem durchflusszytometrischen Zellsortierer. </li></ol> 5. Kultur und Ausbau der Alloantigen-spezifischen T-Zellklonen <ol><li> Nach Abschluss der Zellsortierung, Zentrifugenplatte bei 600 × g für 5 min bei 20 o C. Ort Kulturplatte bei 37 ° C in einer 6% CO 2 -Inkubator. </li><li> Vortex stimulierenden anti-CD3 / anti-CD28 Microbeads für 30 Sekunden, um Perlen zu resuspendieren. </li><li> Berechnen Sie Volumen von Aktivator Perlen für Stimulation der T-Zellen erforderlich gesammelt (0,5 ul Aktivator Mikroperlen / Vertiefung). Übertragen berechnete Volumen der Stimulator-Perlen in eine sterile 5 ml Tube. 1 ml Human-T-Zellkulturmedium und Wirbel. </li><li> Zeigen Rohr mit Mikroperlen-Suspension in Magnet. Die Kappe sollte locker oben auf dem Rohr sein. Inkubation für 2 min bei Raumtemperatur. </li><li> Entfernen Sie vorsichtigdie Kappe von der Röhre. Halten Sie die Röhre und Magnet zusammen, dekantieren Tubeninhalt in ein frisches 5-ml-Tube. Nicht tippen oder bewegen Tubeninhalt. </li><li> Entfernen Sie den Schlauch vom Magnet. Hinzufügen menschlichen T-Zellkulturmedium (100 &amp; mgr; l Medium / 0,5 ul Mikroperlen zunächst hinzugefügt). </li><li> Aliquots von 100 ul Aktivator Perlensuspension in jede Vertiefung der 96-Well-Platte. Inkubieren Kulturplatte bei 37 ° C in einer 6% CO 2 -Inkubator. </li><li> Überwachen Sie das Zellwachstum täglich durch mikroskopische Untersuchung. 7 Tage-Kultur (Abbildung 3.a) - kleine Cluster von proliferierenden Zellen nach 5 mikroskopisch beobachtet werden. HINWEIS: Visualisierung der T-Zellkulturen zu diesem Zeitpunkt kann schwierig sein, und die Abwesenheit von beobachtbaren Zellcluster in diesem Stadium nicht notwendigerweise indikativ für einen Mangel an wachsenden T-Zellen sein. </li><li> 14 Tage nach der Zellisolierung, Futterkulturen durch vorsichtiges Entfernen 100 ul Medium weg von der Spitze der Kultur und mit 100 μ ersetzen; L frische humane T-Zellkulturmedium. Weiter Inkubieren Kulturplatte bei 37 ° C in einer 6% CO 2 -Inkubator. </li><li> Überwachung des Zellwachstums täglich durch mikroskopische Untersuchung und Auswertung der Farbe des Kulturmediums. 3 Tage nach dem Mediumwechsel - große Zellhaufen sollte mikroskopisch 2 sichtbar sein. Makroskopisch sollte Zellpellets während der 14 Tage nach dem Mediumwechsel sichtbar. </li><li> Mit 28 Tagen nach Zellisolierung, identifizieren wachsenden Klone durch mikroskopische Untersuchung. Übertragen die 200 &amp; mgr; l Volumen der wachstums positive 96-Well-Plattenkulturen auf einzelne Vertiefungen einer 48-Well-Gewebekulturplatten, die 200 &amp; mgr; l menschlichen T-Zell-Kulturmedium. </li><li> Füge 100 &amp; mgr; l, enthaltend 12,5 ul stimulierenden anti-CD3 / anti-CD28-Mikroperlen (hergestellt wie in Abschnitt 5.2 beschrieben - 5.6). Inkubieren Kulturplatte bei 37 ° C in einer 6% CO 2 -Inkubator. </li><li> Überwachen Kulturwachstum täglich durch mikroskopische exPrüfung und Bewertung der Farbe von dem Kulturmedium. Wenn T Zellkultur erreicht&gt; 2x10 6 / ml (in der Regel 3-5 Tage nach der erneuten Stimulation, kann von dem Tag, an dem das Medium beginnt geschätzt werden Drehen strohgelb), Transfer Kultur zu einer Vertiefung einer 24-Well-Gewebekulturplatte und fügen Sie 500 ul menschliche T-Zell-Medium. </li><li> Bei 10-14 Tagen folgen Restimulation, sammeln 200 ul T-Zellkultur zu alloantigen Spezifität durch Strömungs beurteilen Zytometrie Analyse pMHC Tetramer Bindung (unter Verwendung der in den Abschnitten 3 und 4.1 beschrieben Kennzeichnung und Gating-Strategie). </li></ol> 6. Langfristige Wieder Stimulation und Kultur von T-Zellklonen <ol><li> Ständig neu zu stimulieren und zu erweitern klonalen T-Zell-Kulturen, die mit der gewünschten Spezifität kann alle 14 Tage. Sammeln T-Zellkulturen in eine sterile 5 ml Tube am Tag 14 nach der letzten Anti-CD3 / CD28 Mikrobead Stimulation. Kombinieren mehrerer Vertiefungen der gleichen T-Zell-Klon in einem einzigen Rohr, bis zu einem Volumen von 2,5 ml. </li&gt; <li> In mittleren bis das Endvolumen auf 2,5 ml zu bringen und vorsichtig mischen. </li><li> Legen Sie die 5 ml Röhrchen mit dem T-Zellkultur in die Zellseparation Magneten an alten Mikroperlen aus der Kultur zu entfernen. Die Kappe sollte locker oben auf dem Rohr sein. Inkubation für 5 min bei Raumtemperatur. </li><li> Entfernen Sie vorsichtig die Kappe von der Tube. Halten Sie die Röhre und Magnet zusammen, dekantiert die Zellsuspension in ein frisches 5-ml-Tube. Nicht tippen oder bewegen Tubeninhalt, um die letzten Tropfen Flüssigkeit zu entfernen. </li><li> Hinzufügen Medium, um das Endvolumen auf 4 ml zu bringen. Probe 10 ul, um Zellen mit einer Zählkammer zählen. </li><li> Erholen T-Zellen durch Zentrifugation bei 600 × g für 5 min bei Raumtemperatur. </li><li> Dekantieren. Resuspendieren T-Zellen bei 10 6 Zellen / ml menschlichen T-Zell-Kulturmedium. 1 ml Aliquots in jede Vertiefung einer 24-Well-Gewebekulturplatte. </li><li> Wieder stimulieren T-Zellen durch Zugabe von 100 &amp; mgr; l Medium, enthaltend 12,5 ul stimulierenden anti-CD3 / aNTI-CD28 Microbeads (- 5,6 wie in den Abschnitten 5.2 beschrieben). Inkubieren bei 37 ° C in einer 6% CO 2 -Inkubator. </li></ol>

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The authors declare no competing financial interests.

T cell alloreactivity is a long-studied and clinically-relevant phenomenon. The robust proliferative and effector responses of T cells to allogeneic stimulation has enabled extensive analyses of human T cell responses in vitro through relatively straightforward mixed lymphocyte reactions of peripheral blood T cells against inactivated allogeneic cells. However, these primary alloreactive T cell responses are oligoclonal, comprised of a large number of individual T cells responding to specific alloantigens. This ...

T-Lymphozyten sind kritische Komponenten des adaptiven Immunsystems. T-Zellen sind nicht nur direkt Vermittlung schützende Immunantworten gegen Pathogene durch eine Vielzahl von Effektor-Mechanismen, sondern auch aktiv Aufrechterhalten immunologischen Selbsttoleranz und Lenken der Antworten von anderen Zellen des Immunsystems verantwortlich. Diese Funktionen werden durch eine Reihe von integrierten Signale, einschließlich T-Zell-Rezeptor (TCR), Ligation, Zytokine und Chemokine und Metaboliten 1 gerichtet. Dieser Signale ist von besonderer Bedeutung der TCR, da sie die charakteristische Besonderheit, die die Rolle der T-Zellen in der adaptiven Immunität definiert. Ein TCR wechselwirkt mit linearen Peptids durch MHC-Antigene (HLA menschliche Ortholog) Moleküle (pMHC-Komplexe) in eine hochspezifische und empfindliche Weise dargestellt, um die Signale, die T-Zell-Effektor-Funktion zu initiieren. Die biochemischen Parameter des TCR Wechselwirkungen mit pMHC Liganden bieten nicht nur die Spezifität für TZellaktivierung, sondern auch über eine qualitative Wirkung auf nachfolgende T-Zell-Funktion 2. So studieren T-Zell-Funktion erfordert häufig die Prüfung der Antworten der klonalen T-Zellen mit definierten Antigenspezifität. Das menschliche T-Zell-Kompartiment, die ungefähr 10 12 αβ T-Zellen, enthält schätzungsweise 7. Oktober - 8. Oktober deutliche αβTCRs 3-4. Diese vielfältigen Repertoire bietet Gelegenheit für die Anerkennung der Vielzahl von Peptiden, die von potenziellen Krankheitserregern, die eine T-Zell-Antwort für eine schützende Immunität erfordern würden. Es wird geschätzt, daß die Frequenz von T-Zellen reagieren auf eine gegebene Fremdantigen durch Selbst MHC präsentiert wird, ist in der Größenordnung von 10 -4 - 10 -7 in der ohne vorherige Immunantwort auf dieses Antigen 5. Die naive T-Zell-Repertoire von Thymus Auswahl geformt, um die Fähigkeit zur Selbst-MHC-Antigene erkennen und präsentieren Peptide Grenze reagiert werdenduktivität gegen Selbstpeptidantigene, die Maximierung der potenziellen Nutzen für die Vermittlung eine schützende Immunität 2. Unter Verstoß gegen diese entwickelt Reaktivität, eine relativ große Frequenz, 10 -3 jedoch - 10 -4, der T-Zellen aus immunologisch naive Individuen reagieren auf die Stimulation mit allogenen Zellen, erkennen sowohl die fremden MHC-Moleküle sowie die endogene Peptide präsentieren sie 6. Die Erkennung von allogenen pMHC Liganden ist strukturell ähnlich der Erkennung von fremden Antigenen durch Selbst-MHC präsentiert; Die TCR macht kritischen biochemischen Wechselwirkungen sowohl mit dem allogenen MHC-Molekül als auch die Peptid 7 dargestellt. Die robuste Natur der Reaktion der T-Zellen allogene Stimulierung resultiert aus der Vielfalt der pMHC-Komplexen auf der Oberfläche einer allogenen Zellen 8 vorhanden. Es wird geschätzt, dass jedes MHC präsentiert etwa 2 x 10 4 unterschiedlichen endogenen Peptidantigene 9. Diese breadth der Antwort auf allogene Stimulation ist ein wesentlicher Aspekt der klinisch relevanten Pathologie, wie etwa Transplantatabstoßung oder Graft-versus-Host-Krankheit (GVHD), von T-Zell-Alloreaktivität resultieren. Untersuchung der menschlichen T-Zellantworten alloreaktiven traditionell durch Prüfung polyklonalen Reaktionen von naiven T-Zellen nach Stimulation mit allogenen Zellen verlassen. Wiederholte Stimulierung mit dem gleichen allogenen Zelllinie zusammen mit Grenzverdünnungsanalyse ist in der Lage klonalen T-Zellen mit definierten Erkennung von allogenen HLA 10. Jedoch ist dieser Ansatz problematisch für die Prüfung Antworten auf individuelle allogenen pMHC-Liganden, da die großen und vielfältigen Repertoire von endogenen pMHC-Komplexe für eine gegebene allogenen HLA vorliegenden stimuliert ein breites Repertoire an T-Zellen. Diese Massenpopulation Stimulation und Grenzverdünnungs Ansatz Screening einer großen Anzahl von Klonen erforderlich T-Zellen mit der gewünschten Reacti isolierenvität gegen einen einzelnen pMHC Liganden. Zusätzlich ist die Häufigkeit von T-Zellen reagieren auf eine einzelne allogenen pMHC Ligand unter naive T-Zell-Populationen, die ein Hindernis für die effiziente Erzeugung von menschlichen T-Zell-Klone, die auf ein gegebenes Antigen präsentiert relativ gering. Identifizierung und Isolierung von Antigen-spezifischen T-Zellen von polyklonalen Populationen wurden durch die Entwicklung von Fluorophor-markierten pMHC Multimere 11 aktiviert. Dieser Ansatz nutzt spezifische Peptidantigene in rekombinanten löslichen biotinylierten MHC-Moleküle, die durch Bindung an Streptavidin-markierten Fluorophor markiert werden geladen. Multimerisierung pMHC erhöht die Gier, Kompensieren des intrinsisch niedrigen (uM) Affinität der TCR für lösliche pMHC Liganden. Markierten Zellen identifiziert und mittels Durchflusszytometrie getrennt werden. Jedoch ist dieser Ansatz noch von der niedrigen Frequenz von Antigen-spezifischen T-Zellen unter naive T-Zell-Populationen, die in der Regel begrenztum Größenordnungen geringer als die Grenze der genaue Identifizierung und Quantifizierung der meisten Durchflusszytometer. Um diese Einschränkung zu beheben, wurde ein Verfahren zum pMHC Tetramer Kennzeichnung und anschließende magnetische Perle Bereicherung Tetramer-markierten Zellen wurden 12 entwickelt. Dieses Verfahren hat eine zuverlässige Erkennung, Zählung und Isolierung des Niederfrequenz-Antigen-spezifischen T-Zellen nachgewiesen. Dieses Protokoll beschreibt ein wirksames Protokoll für die Herstellung von menschlichen T-Zell-Klone, die spezifisch auf einzelne allogenen pMHC-Liganden reagieren. Das Protokoll gilt pMHC (HLA) Multimer Kennzeichnung und Anreicherung für die Isolierung von Alloantigen-spezifischer menschlicher T-Zellen mit Durchflusszytometrie und Zellsortierung einem robusten Verfahren zur in vitro Kultur von humanen T-Zellen, die Produktion von T-Zell-Klonen, die aus Einzelzellen sortiert (Freigabe Übersicht in Figur 1).

<table border="0" cellpadding="0" cellspacing="0" width="907">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name of Reagent/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:292px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Sodium heparin venous blood collection tube 16 x 100 mm</td>
			<td style="width:223px;">Becton, Dickenson and Company</td>
			<td style="width:119px;">366480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Lymphoprep</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">7801</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:273px;">Rosette Sep Human T Cell Enrichment Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">15061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Dulbecco&#39;s PBS, 1x without Ca or Mg</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bovine serum albumin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E6635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Fluorophore-labeled pMHC tetramer</td>
			<td style="width:223px;">NIH Tetramer Facility</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Biotin Selection Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Selection magnet</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:273px;">TruStain FcX Human Fc blocking solution</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">422301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD5 PE-Cy7 (clone UCHT2)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">300621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD14 FITC (clone HCD14)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">325603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD19 FITC (clone HIB19)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">302205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Iscove&#39;s DMEM, without b-ME or L-glutamine</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">15-016-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-Mercaptoethanol&#160;</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin sulfate (50 mg/ml)</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">GT-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human AB serum, male donor</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">HS-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2</td>
			<td style="width:223px;">Peprotech</td>
			<td style="width:119px;">AF 200-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Dynabeads Human T-Activator CD3/CD28</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">11131D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Media</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Cell sorting buffer</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">PBS, pH 7.4</td>
			<td style="width:223px;">1 L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BSA</td>
			<td style="width:223px;">10g</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA (0.5 M)</td>
			<td style="width:223px;">2 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human T Cell Culture Medium</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Iscove&#39;s DMEM</td>
			<td style="width:223px;">351.6 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Heat-inactivated human AB serum</td>
			<td style="width:223px;">40 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES (1 M)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax (100 x)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin (50 mg/ml)</td>
			<td style="width:223px;">0.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-mercaptoethanol (14.3 M)</td>
			<td style="width:223px;">1.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2 (1 mg/ml)</td>
			<td style="width:223px;">1 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Dieses Protokoll beschreibt die Erzeugung klonaler menschlichen T-Zellkulturen mit Alloantigen Spezifität über eine Magnetperle Anreicherung und Einzelzell Durchflusszytometrie Sortierstrategie festgelegt. Abbildung 1 gibt einen Überblick über den Prozess. <img alt="Figur 1" src="/files/ftp_upload/52257/52257fig1highres.jpg" /> Figur 1: Protokoll Sicht Das hier beschriebene Protokoll stellt ein zuverlässiges Ve...

Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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 ...

generation-of-human-alloantigen-specific-t-cells-from-peripheral-blood

Research

JoVE Journal

Immunology and Infection

12.9K Aufrufe.  University of California, San Diego. 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.

Serie: Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Induktion von Alloantigen-spezifische Anergie in humanen peripheren mononukleären Zellen durch Alloantigen Stimulation mit Co-stimulatorische Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

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

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

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

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansion 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 ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generation der induzierte regulatorische T-Zellen aus primären humanen naiven und Gedächtnis-T-Zellen

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Herstellung von menschlichen Monozyten abgeleiteten Dendritischen Zellen aus Vollblut

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Generation von diskriminierendem menschlichen monoklonalen Antikörpern aus seltenen Antigen-spezifische B-Zellen im Blut zirkulierenden

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

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 ...