Name: assessment of the synaptic interface of primary human t cells from peripheral blood and lymphoid tissue
Uploaded: 2018-07-30T15:00:00
Description: Watch this Scientific Journal Video about Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue at JoVE.com

Diese Arbeit wurde unterstützt durch die R01AI118694 NIH Grant, Michael R. Betts, worunter Sub Award 566950, Yuri Sykulev. Wir danken der Sidney Kimmel Cancer Center Bioimaging geteilt Ressource für ihre hervorragende Unterstützung.

Diese Studie wurde in Übereinstimmung mit der Deklaration von Helsinki. Schriftliche Einwilligung wurde von allen Teilnehmern, und Blut-und Lymphknoten wurden mit Zustimmung des Institutional Review Board an der University of Pennsylvania (IRB #809316, IRB # 815056) erworben. Alle Probanden waren Erwachsene. Schnur Blutproben wurden freundlicherweise zur Verfügung gestellt von wehen und der Geburt von der Abteilung für Geburtshilfe und Gynäkologie an der Thomas Jefferson University. Alle Proben wurden anonymisierte.<...

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Die neuartige Technik, die hier beschriebenen nutzt ähnliche Reagenzien erforderlich, um planare Bilayer in den herkömmlichen Flow Zelle5 bauen und kann erfolgreich eingesetzt werden, um die Darstellung der primären menschlichen T-Zell-Bilayer Schnittstellen3,4 durchführen ,15. Die Technik bietet eine deutliche Reduzierung der fluoreszierende Moleküle Nutzung und erfordert 10 – 20 x weniger T-Zell...

Wissenschaftlicher Erkenntnisse über die strukturellen Merkmale des T-Zell-immun-Synapsen und ihre Verknüpfung, die funktionelle Aktivität der T-Zellen erzeugt worden ist, in erster Linie aus der Studie von Zelllinien und Klone von PB abgeleitet. Inwieweit diese Ergebnisse beziehen sich auf primäre T-Zellen entnommen Blut oder lymphatischen Gewebe bleibt unklar, wie die synaptischen Schnittstellen von T-Zellen, die ihren Wohnsitz in den lymphatischen und anderen Geweben bisher nicht analysiert wurden. Wichtig ist, neue Daten deuten darauf hin, dass Gewebe-Resident und lymphatischen Organ abgeleitet T-Zellen möglicherweise erhebliche Unterschiede in ihren Phänotyp und funktionelle Aktivität im Vergleich zu denen in PB6,7. Dies hat die Notwendigkeit, besser zu verstehen, die Funktionen der T-Zell-synaptischen Schnittstelle in primären menschlichen T-Zellen weiter verfestigt.
Zu diesem Zweck entwickelten wir eine neuartige Mini-Ansatzes Ausnutzung Lipid Bilayer eingebaut Multichannel-Fluss Folien ermöglichen die Darstellung der T-Zelle/Bilayer Schnittstellen mit weniger als 105 primäre T-Zellen aus menschlichen PB und LN isoliert ausführen Diese neuartige Technik ermöglicht die Studie die biophysikalischen Eigenschaften des primären menschlichen T-Zellen synaptischen Schnittstellen um besser zu modellieren und in Vivo Zell-Zell-Interaktionen zu verstehen.

<table>
	<tbody>
		<tr>
			<td>CD4 T cell isolation kit, human</td>
			<td>Miltenyl Biotec</td>
			<td>130-096-533</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8 T cells Isolation Kit, human</td>
			<td>Miltenyl Biotec</td>
			<td>130-096-495</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti Polar Lipids</td>
			<td>850375C</td>
			<td></td>
		</tr>
		<tr>
			<td>DOGS NTA</td>
			<td>Avanti Polar Lipids</td>
			<td>790528C</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinyl Cap PE</td>
			<td>Avanti Polar Lipids</td>
			<td>870273C</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin</td>
			<td>Octapharma USA</td>
			<td>NDC 68982-643-01</td>
			<td></td>
		</tr>
		<tr>
			<td>sticky-Slide VI 0.4</td>
			<td>ibidi</td>
			<td>80608</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips for sticky-Slides</td>
			<td>ibidi</td>
			<td>10812</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioptech FCS2 Chamber</td>
			<td>Bioptech</td>
			<td>060319-2-03</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD3 antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>16-0037-81</td>
			<td>OKT3 clone, hybridoma cells are available from ATCC</td>
		</tr>
		<tr>
			<td>anti- CD28 antibody</td>
			<td>Genetex</td>
			<td>GTX14664</td>
			<td>9.3 clone</td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma</td>
			<td>C5890</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-PEO4-NHS</td>
			<td>Thermo Fisher Scientific</td>
			<td>21329</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 protein labeling kit with column for labeled protein purification</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10235</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 protein labeling kit with column for labeled protein purification</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10238</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Cy5 NHS Ester</td>
			<td>GE Life Science</td>
			<td>PA15101</td>
			<td></td>
		</tr>
		<tr>
			<td>pMT/V5-His A, B, C Drosophila Expression Vectors</td>
			<td>Thermo Fisher Scientific</td>
			<td>V412020</td>
			<td></td>
		</tr>
		<tr>
			<td>pcopneo, G418 Drosophila expression vector for positive selection</td>
			<td>ATCC</td>
			<td>37409</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum free Drosophial media Insect-XPRESS</td>
			<td>Lonza</td>
			<td>12-730Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridoma YN1/1.7.4</td>
			<td>ATCC</td>
			<td>CRL1878</td>
			<td>The hybridoma secrets antibody against ICAM-1.</td>
		</tr>
		<tr>
			<td>Cyanogen bromide-activated-Sepharose 4B</td>
			<td>Sigma-Aldrich</td>
			<td>C9142</td>
			<td>Utilized for preparation of Sepharose with covelently bound anti-ICAM antibody.</td>
		</tr>
		<tr>
			<td>MasterFlex tangential flow concentrator</td>
			<td>Cole-Parmer</td>
			<td>77601-60 7592-40</td>
			<td>Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant</td>
		</tr>
		<tr>
			<td>Centramate Lab Tangential Flow Systems</td>
			<td>Pall Laboratory</td>
			<td>FS002K10 OS010T12 FS005K10</td>
			<td>Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant</td>
		</tr>
		<tr>
			<td>Ni-NTA Agarose</td>
			<td>QIAGEN</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing</td>
			<td>Spectra/Por</td>
			<td>131384</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain from papaya latex</td>
			<td>Sigma</td>
			<td>P3125</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-human antibody against CD107a</td>
			<td>BD Bioscences</td>
			<td>555798</td>
			<td>Clone H4A3</td>
		</tr>
		<tr>
			<td>Ansell Natural Blue Gloves</td>
			<td>Fisher Scientific</td>
			<td>19-014-539</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Polypropylene Scissor-Type Forceps</td>
			<td>Thermo Fisher Scientific</td>
			<td>6320-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>ProZyme</td>
			<td>SA10</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Nikon TiE inverted microscope equipped with PFS for long-term image stability control, 60x oil objectives, 4 lasers with excitation lines at 405, 458, 488, 514, 561, and 640 nm, 2 GaAsP detectors and 2 high sensitivity PMTs, DIC transmitted light, Programmable X,Y,Z stage for multiple positions and stitching of large areas, time lapse functions, Tokai-Hit temperature and CO2-controlled chamber for live imaging, and anti-vibration isolation table</td>
		</tr>
		<tr>
			<td>TIRF microscope</td>
			<td>Andor</td>
			<td></td>
			<td>Andor Revolution XD system equipped with Nikon TIRF-E illuminator, Lasers with 405,488,561 and 640 lines, DIC transmitted light, Yokogawa CSU-X1 spinning disk head for confocal imaging, 100/1.49 NA objective, Andor iXon X3 EM-CCD camera, objective heater, and a piezoelectric motorized stage with Perfect Focus System (PFS)</td>
		</tr>
		<tr>
			<td>MetaMorph Premier Image Analysis Software</td>
			<td>Molecular devices</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of the synaptic interface of primary human t cells from peripheral blood and lymphoid tissue

Das gegenwärtige Verständnis der Dynamik und strukturellen Merkmale der T-Zellen synaptischen Schnittstellen wurde weitgehend bestimmt durch den Einsatz von Glas-gestützte planar Bilayer und in-Vitro-abgeleiteten T-Zell-Klone oder Linien1,2 ,3,4. Wie diese Erkenntnisse für die primären menschlichen T-Zellen aus Blut isoliert oder lymphatischen gelten Gewebe ist nicht bekannt, teilweise erhebliche Schwierigkeiten, eine ausreichende Anzahl von Zellen für die Analyse5. Hier begegnen wir dies durch die Entwicklung einer Technik, die Nutzung von Folien, Multichannel-Fluss um planare Lipid Bilayer mit Aktivierung und Adhäsion Moleküle bauen. Die geringe Höhe der Fluss Folien fördert schnelle Zelle Sedimentation, um Zelle: Bilayer Anlage, wodurch Forscher studieren die Dynamik der synaptischen Schnittstelle Bildung und die Kinetik der Granulat-Version synchronisieren. Wir wenden diesen Ansatz zur Analyse der synaptischen Schnittstelle von so wenig wie 104 bis 105 primäre kryokonservierten T-Zellen isoliert von Lymphknoten (LN) und peripherem Blut (PB). Die Ergebnisse zeigen, dass die neuartige planar Lipid Bilayer Technik die Studie die biophysikalischen Eigenschaften des primären menschlichen T-Zellen aus Blut und Gewebe im Zusammenhang mit Gesundheit und Krankheit abgeleitet ermöglicht.

Zunächst verglichen wir die Struktur der synaptischen Schnittstelle gebildet von aktivierten CD8 Schnur Blut abgeleitet+ T Zellen, Lipid Bilayer entweder in traditionellen großen Fluss gebaut Zellsysteme (siehe die Tabelle der Materialien für Details)1 ,2,3,4 oder im Multi-Channel-Flow-Folien. Die Bilayer enthalten Leuchtstoff-mar...

Watch this Scientific Journal Video about Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue at JoVE.com

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

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

Das Protokoll beschreibt eine Technik, um die Fähigkeit der primären polyklonale menschlichen T-Zellen synaptische Schnittstellen mit planaren Lipid Bilayer bilden zu studieren. Wir verwenden diese Technik, um die differenzielle Synapse Bildung Fähigkeit der menschlichen primäre T Zellen aus Lymphknoten und peripheren Blut zu zeigen.

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

The approach that we're gonna be talking today about has been derived to compare properties and functional activities of tissue-derived and peripheral blood T cells. The significance of the topic is exemplified by the fact that peripheral blood T cells represent only 20%of the total T cells in a body. Being able to study a small number of tissue-derived T cells is very essential, and that's exactly what we have achieved.The main advantage of this technique is that it requires smaller number of cells and smaller volumes of reagents than traditional techniques. Begin by using polypropylene scissor-type forceps to submerge glass coverslips in freshly-prepared acidic piranha solution for 25 to 30 minutes. At the end of the wash, rinse the coverslips seven times in a fresh volume of ultra-pure water for each wash.Then set the wet coverslips aside to let the remaining water roll off the clean glass. When the coverslips are dry, aliquot two microliters of the final liposome mixture precisely in the center of a self-adhesive slide channel within a sterile flow hood. And immediately but carefully align one clean and dry coverslip with the slide to allow the coverslip to be gently lowered onto the sticky side of the slide.And use the outer ring of the polypropylene scissor-type forceps to apply a gentle pressure to the peripheral contact of the coverslip with the slide, making sure that the slip is tightly attached to the slide to prevent leakage. Then turn the slide over, and use a permanent marker to draw four dots around the bilayer on the external side of the slide assembly. Before the first injection, designate one port of the channel as the entry port, and the other as the exit port, maintaining this designation throughout the experiment.To avoid bubble formation, insert the end of the pipet tip directly into the entry port until you feel the rubber seal of the slide channel being penetrated with the tip. Slowly fill the channel with 50 microliters of warm assay buffer. Inject 200 microliters of nickel(II)chloride in Casein blocking solution into the entry port.And remove 50 microliters of the buffer from the exit port. After a 45-minute incubation, remove the excess Casein solution from the exit port. Inject 100 microliters of an ICAM-1 in streptavidin solution into the entry port for a 45-minute incubation at room temperature.At the end of the incubation, remove the excess protein solution from the exit port. And wash the bilayer two times with 100 microliters of assay buffer per wash. Then label the bilayers with 100 microliters of fluoro-4-conjugated anti-CD3 antibody for 45 minutes at room temperature.Followed by two washes in 100 microliters of assay buffer per wash as demonstrated. To image T cell bilayer interactions, place the slide on the 37-degree-Celsius heated stage of a total internal reflection fluorescence, or TIRF microscope. And adjust the stage using the ink marks to center the bilayer onto the 100-power objective.For granule release imaging, add fluorescence-conjugated anti-CD107A antibody FAB fragments to the CD4 T cell suspension. And resuspend the labeled cells in 50 microliters of assay buffer for injection into the entry port of the slide channel. Then select the appropriate number of experimental fields, and record images of each field once every minute for 30 minutes after the injection.In this representative experiment, no differences between CD8 T cells that formed classical immunological synapses on the lipid bilayers built in either the flow cell or the multi-channel flow slide were observed. Upon the lipid bilayer interaction, CD4 T cells established mature immune synapses, but either do or do not release granules, release granules without the formation of mature synapses, or neither form synapses nor release granules. Of note, peripheral blood mononuclear cell-derived CD4 T cells are able to begin releasing granules almost twice as quickly as can lymph node-derived CD4 cells.While attempting this procedure, it is important to maintain constant temperature and pipet slowly so that there are no bubbles formed. In addition to our capabilities to investigate a T cell bilayer interface, this approach would also allow us to perform an intercellular staining of the T cells that touched the bilayer. Which would significantly facilitate an amount of information that can be used to describe particular T cells derived from either lymphoid tissue or other tissues.Don't forget that working with piranha solution is extremely hazardous, and it is crucial that you wear proper PPE while attempting this procedure.

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

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

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

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Isolation von Leukozyten aus dem menschlichen Mutter und Fötus-Schnittstelle

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

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

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

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

Analysis of Human Natural Killer Cell Metabolism

Analyse des menschlichen natürlichen Killerzellstoffwechsels

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...

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

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 ...