This work was supported by the R01AI118694 NIH grant to Michael R. Betts, which includes sub-award 566950 to Yuri Sykulev. We thank the Sidney Kimmel Cancer Center Bioimaging Shared Resource for their excellent support.

This study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants, and blood and lymph node samples were acquired with the approval of the Institutional Review Board at the University of Pennsylvania (IRB#809316, IRB# 815056). All human subjects were adults. Cord blood samples were kindly provided by Labor and Delivery of the Department of Obstetrics &#38; Gynecology at Thomas Jefferson University. All samples were de-identified.
<p class="jove_title"...

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+theta+regulates+stability+of+the+peripheral+adhesion+ring+junction+and+contributes+to+the+sensitivity+of+target+cell+lysis+by+CTL.">Protein kinase C theta regulates stability of the peripheral adhesion ring junction and contributes to the sensitivity of target cell lysis by CTL.</a> The Journal of Immunology. 181, 4815-4824 (2008).</li><li>Beal, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+early+T+cell+receptor+signaling+regulate+the+pathway+of+lytic+granule+delivery+to+the+secretory+domain.">Kinetics of early T cell receptor signaling regulate the pathway of lytic granule delivery to the secretory domain.</a> Immunity. 31, 632-642 (2009).</li><li>Dustin, M. L., Starr, T., Varma, R., Thomas, V. 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The authors have nothing to disclose.

The novel technique described here utilizes similar reagents required to build planar bilayers in the conventional flow cell5 and can be successfully applied to perform the imaging of primary human T cell&#8211;bilayer interfaces3,4,15. The technique offers a significant reduction in the fluorescent molecules usage and requires 10&#8211;20x fewer T cells as compared to a flow cell system...

Scientific knowledge of the structural features of T-cell immune synapses and their link to the functional activity of T cells has been generated primarily from the study of cell lines and clones derived from PB. To what degree these findings relate to primary T cells obtained from blood or human lymphoid tissues remains unclear, as the synaptic interfaces of T cells residing in lymphoid and other tissues have not been analyzed thus far. Importantly, emerging data suggest that tissue-resident and lymphoid-organ-derived T cells may have significant differences in their phenotype and functional activity compared to those in PB6,7. This has further solidified the need to better understand the features of the T-cell synaptic interface in primary human T cells.
To this end, we have developed a novel mini-scale approach exploiting lipid bilayers built into multichannel flow slides enabling us to perform the imaging of T-cell/bilayer interfaces with less than 105 primary T cells isolated from human PB and LN. This novel technique allows the study of the biophysical properties of primary human T-cell synaptic interfaces in order to better model and understand in vivo cell-cell interactions.

<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

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.

First, we compared the structure of the synaptic interface formed by activated cord-blood-derived CD8+ T cells exposed to lipid bilayers built either in traditional large-scale flow cell systems (see the Table of Materials for details)1,2,3,4&#160;or in multichannel flow slides. The bilayers contained fluorescent-labeled anti-CD3 and ...

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

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.

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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Immunology and Infection

6.3K visualizações.  Thomas Jefferson University. 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 n...