Name: assessment of the synaptic interface of primary human t cells from peripheral blood and lymphoid tissue
Uploaded: 2018-07-30T15:00:00.000+00:00
Duration: 6 min 27 s
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

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.
1. Isolation of CD4+ T Cells for an Image Analysis
<ol>
	<li>Thaw a 1 mL aliquot containing 107 frozen peripheral blood mononuclear cells (PBMCs) or lymph node mononuclear cells (LNMCs) from collected samples. In a sterile hood, add the thawed cells to 9 mL of RPMI supplemented with penicillin/streptomycin and glutamine.
	<ol>
		<li>Centrifugate the cells for 10 min at 300 x g at 4 &#176;C, aspirate the supernatant, and resuspend the cells in 5 mL of supplemented RPMI containing 10% FBS (complete medium). Incubate the cells overnight in a CO2 incubator at 37 &#176;C.</li>
	</ol>
	</li>
	<li>The next day, purify CD4+ T cells by negative immunomagnetic sorting using a commercially available kit according to the manufacturer&#39;s instruction.</li>
	<li>To measure the number of freshly purified CD4+ T cells, mix 5 &#181;L of the cell suspension with an equal volume of a trypan blue solution. Load a hemocytometer with a cell-trypan blue mixture and count the live cells inside the 5 sections of the hemocytometer.</li>
	<li>Take the average of the cell number and determine the number of cells in the original cell suspension: the number of cells/1 mL = the average count x 2 x 104. If the total number of isolated cells is too small, use the cells as is without counting.</li>
	<li>Centrifuge the cells at 300 x g for 10 min and resuspend them in an assay buffer (20 mM HEPES, pH 7.4, 137 mM NaCl, 2 mM Na2HPO4, 5 mM D-glucose, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, and 1% human serum albumin) at 105 cells/50 &#181;L or less and keep the cells at 4 &#176;C (for 1&#8211;2 h) until ready to use in the experiments.</li>
	<li>Dispose of all biological waste according to the relevant institutional guidelines.</li>
	<li>If desired as a control cell population, prepare activated CD8 T cells from cord blood PBMC, place 107 cells in 5 mL of complete medium in a T25 culture flask covered with a mixture of anti-CD3 and anti-CD28 antibodies at 10 &#181;g/mL and 1 &#181;g/mL, respectively.</li>
	<li>The next day, remove the activated cord blood cells from the flask, wash them 1x with fresh complete medium and expand the cells in the presence of recombinant IL-2 (100 U/mL) for 2 weeks.</li>
	<li>Purify the cord-blood CD8+ T cells by negative immunomagnetic sorting using the commercially available kit according to the manufacturer&#39;s instruction. Count the cells and exchange the media to the assay buffer as described in steps 1.3&#8211;1.6 for LN and PB CD8+ T cells.</li>
</ol>
2. Components for the Preparation of the Planar Lipid Bilayers
<ol>
	<li>Prepare 3 kind of liposomes as described elsewhere5: (a) 0.4 mM DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) liposomes, (b) 0.4 mM DOPC liposomes containing 33 mol% DOGS-NTA (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (ammonium salt)) lipids, (c) 0.4 mM DOPC liposomes containing 4 mol% Biotinyl-Cap-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt)).</li>
	<li>Prepare 5% casein solution as previously described5.
	<ol>
		<li>Dissolve 5 g of casein powder in 100 mL of ultrapure water and add 350 &#181;L of 10 M sodium hydroxide. Stir everything on a regular magnetic stirrer at a slow speed according to the available scale, at room temperature for 2 h, and then overnight at 4 &#176;C. Adjust the pH to 7.3 and ultracentrifuge the solution for 2 h at 100,000 x g at 4 &#176;C. Filter the supernatant with a 0.22 &#181;m sterile filter and store the solution in aliquots at -80 &#176;C. 
		CAUTION: Sodium hydroxide solution can cause chemical burns and may induce permanent blindness upon contact with eyes. Use rubber gloves, safety clothing, and eye protection when handling this chemical or its solutions.</li>
	</ol>
	</li>
	<li>Label anti-CD3 antibody with biotin to produce mono-bionylated antibody molecules with a previously described approach8.
	<ol>
		<li>Prepare a solution of Biotin-PEO4-NHS in dimethyl sulfoxide (DMSO) at 0.1 mg/mL. Add 3.7 &#181;L of the Biotin-PEO4-NHS solution to 1 mg of antibody in 0.5 mL of phosphate-buffered saline (PBS) containing 100 mM sodium bicarbonate.</li>
		<li>Incubate the mixture for 2 h at room temperature. Prepare a solution of Alexa Fluor 488 NHS ester at 10 mg/mL in DMSO. Add the Alexa Fluor 488 NHS ester solution to the antibody labeled by Biotin-PEO4-NHS at a 10-fold molar excess.</li>
		<li>Incubate the mixture for 1 h at room temperature with slow stirring on a regular magnetic stirrer. Separate the unbound dye using size-exclusion chromatography.</li>
		<li>Determine the antibody concentration by measuring the optical density of the antibody solution at 280 nm (A280). Measure the optical density of the labeled antibodies at 577 nm (A577).</li>
		<li>Determine the dye-to-antibody ratio using the following equation: 
		<img alt="Equation" src="/files/ftp_upload/58143/58143eq1.jpg" /> 
		NOTE: Find additional details in the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Express a recombinant soluble ICAM-1 protein in a Drosophila expression system as described previously3,4,9,10.
	<ol>
		<li>Clone, with cDNA encoding, the ectodomain of ICAM-1 into Drosophila expression vector pMT/V5-His with an inducible metallothionein promoter to produce a recombinant protein appended with a His6 tag on the C-terminal end.</li>
		<li>Co-transfect S2 cells with the resulting ICAM-1 containing plasmid and a G418 expression vector. Select stable transfectants using Schneider&#39;s Drosophila Media supplemented with 10% fetal calf serum (FCS) and 0.5 mg/mL of G418 for 3 weeks. Expand the cells in serum-free insect medium and induce a protein expression with 0.5 mM CuSO4 for 3 d.</li>
		<li>Concentrate the culture supernatant 10x and dialyze against PBS via a tangential flow concentrator as previously described11.</li>
		<li>Apply the concentrated culture supernatant to a column containing Sepharose with a covalently immobilized anti-ICAM-1 monoclonal antibody and elute the bound ICAM-1 with a 50 mM glycine buffer, pH 3.0. Immediately neutralize the eluted ICAM-1 protein with a 2 M Tris buffer, pH 8.0.</li>
		<li>Dialyze the eluted material against PBS, pH 8.0 and add the dialyzed material to a column containing Ni-NTA agarose. Elute soluble ICAM-1 with 200 mM imidazole, pH 8.0. Dialyze the eluted material against a PBS buffer, pH 8.0.</li>
		<li>Label the purified ICAM-1 with Cy5 NHS ester according to the manufacturer&#39;s instruction. 
		NOTE: The best final dye-to-protein ratio is 1:1.</li>
	</ol>
	</li>
	<li>Produce Fab fragments from an anti-CD107a antibody by papain digestion and purify the Fab fragments by ion exchange chromatography as described previously3. Label the Fab fragments with Alexa Fluor 568 NHS ester according to the manufacturer&#39;s instruction.</li>
</ol>
3. Formation of Glass-supported Planar Lipid Bilayers
<ol>
	<li>Prepare a fresh acidic piranha solution by mixing 140 mL of concentrated sulfuric acid and 60 mL of 30% hydrogen peroxide. Wash the glass coverslips for the flow slides by soaking them in the acid piranha solution for 30 min. Hold the glass coverslip with polypropylene scissor-type forceps. 
	CAUTION: Piranha solution is an extremely strong oxidizer. Remember to wear safety glasses or goggles or a full-face shield together with thick rubber gloves at all times while handling the solution. Only work with piranha solution under a fume hood. Avoid heating, transporting, or shaking it at any time during use, as it may explode. Collect the piranha waste into a glass bottle with a lead-containing hole. Contact the Institutional Safety Committee about proper waste utilization.</li>
	<li>Rinse the washed coverslips 7x with ultrapure water by transferring them sequentially into beakers containing fresh water. Set the wet coverslips aside to let the remaining water roll off the clean glass, leaving the dry glass behind.
	<ol>
		<li>Alternatively, use a pipette tip attached to a vacuum pump to carefully remove the remaining water droplets from the coverslips.</li>
	</ol>
	</li>
	<li>In the sterile hood, perform dilutions of various lipids to produce the liposome mix for making bilayers. First, combine 37 &#181;L of DOPC liposomes and 3 &#181;L of Biotinyl-Cap-PE liposomes. Second, mix 14 &#181;L of DOPC liposomes and 15 &#181;L of DOGS-NTA liposomes. Third, add 1 &#181;L of the first mix to 29 &#181;L of the second mix to fabricate the final liposome mixture.</li>
	<li>In the sterile hood, set up a workspace with dry coverslips close by. Aliquot 2 &#181;L of the final liposome mixture (see step 3.3) precisely in the center of a self-adhesive slide channel. Immediately and very precisely align a clean and dry coverslip with the slide and gently lower the coverslip on the sticky side of the slide.
	<ol>
		<li>If preparing more than one slide, work on one slide at a time since the liposome mixture evaporates rapidly. Turn the slide over and use the outer ring of 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 preclude leakage. 
		NOTE: Do not press against the channels of the slide to avoid breaking or cracking the coverslip.</li>
		<li>Turn the slide over again and mark the position of the formed bilayer, which looks like a drop between the coverslip and the channel slide, by drawing 4 dots with a permanent marker around the bilayer on the external slide side of the assembly.</li>
	</ol>
	</li>
	<li>Before the first injection of a liquid into the channel, designate one port of the channel as the entry port and the other as the exit port and maintain this designation throughout the experiment.</li>
	<li>To avoid forming bubbles, insert the end of the pipet tip directly into the entry port of the channel.Slowly fill the channels of the slide with 50 &#181;L of warm (at least room temperature) assay buffer (see step 1.5 for the buffer composition).</li>
	<li>Prepare a 0.5 M nickel(II) chloride solution. Thaw a 2 mL aliquot of casein solution in a water bath at 37 &#176;C for 30 min and supplement it with a nickel chloride solution at a final concentration of 200 &#181;M.</li>
	<li>Wash the bilayers by first injecting 100 &#181;L of the casein solution in the entry port of the channel and then immediately removing 100 &#181;L out of the exit port on the slide by pipetting. Block the bilayers with the same solution by injecting 100 &#181;L of the casein solution into the entry port of each channel and incubating the slide for 45 min at room temperature.</li>
	<li>Thaw aliquots of Cy5-ICAM-1-His6 and streptavidin proteins. Combine the proteins in the assay buffer at the final concentration of 2 &#181;g/mL each. Centrifuge the solution for 30 min at 20,000 x g and 4 &#176;C to remove any aggregates.</li>
	<li>Remove the rest of the blocking solution from the exit port of the slide channel by pipetting. Inject 100 &#181;L of the solution containing ICAM-1 and streptavidin into the entry port.</li>
	<li>Incubate the slide for 45 min at room temperature. Remove any excess of the protein solution from the exit port. Wash the bilayer 2x by first injecting 100 &#181;L of the assay buffer in the entry port of the channel and then immediately removing 100 &#181;L out of the exit port.</li>
	<li>Dilute an Alexa-Fluor-488-labelled anti-CD3 antibody with the assay buffer to a final concentration of 2 &#181;g/mL. Inject 100 &#181;L of the antibody solution into the entry port of the slide and incubate it for 45 min at room temperature. Remove any excess of the protein solution from the exit port. Wash the bilayer 2x with 100 &#181;L of the assay buffer as in step 3.11.</li>
</ol>
4. Imaging of the T Cells Interaction with the Planar Bilayer
<ol>
	<li>Preheat the stage and the objective of a confocal or total internal reflection fluorescence (TIRF) microscope until the temperature is equilibrated at 37 &#176;C. Set up the slide with the bilayer(s) on the heated stage. Move the stage to an appropriate position according to the ink marks and focus on the bilayer employing fluorescence of Cy-labelled ICAM-1 molecules.
	<ol>
		<li>Use a 61X objective for the confocal microscope, or a 100X objective for the TIRF microscope, with appropriate filter settings.</li>
	</ol>
	</li>
	<li>For granule release imaging by TIRF microscopy, add Alexa-Fluor-568-labeled anti-CD107a antibody Fab fragments to the cell suspension at a final concentration of 4 &#181;g/mL before injecting the cells into the entry ports.
	<ol>
		<li>Resuspend the prepared CD4+ T cells isolated from LN or PB or cord blood and inject 50 &#181;L of the cell suspension into the entry port of the slide channel containing the bilayer.</li>
	</ol>
	</li>
	<li>Choose the desired numbers of fields and record images of each field 1x every 2 min for 30 min after the injection.</li>
	<li>Exploit bright-field, reflected light, and fluorescent channels (Alexa 488 and Cy5) of the confocal microscope to acquire the images. Use TIRF mode for Alexa-Fluor-488 and Alexa-Fluor-568 fluorescence and widefield for Cy5 fluorescence, as well as bright-field imaging, on the TIRF microscope.</li>
</ol>
5. Image Analysis
<ol>
	<li>Analyze the acquired images using appropriate software. Observe cell morphology in the transmitted light images and exclude clustered and visibly damaged or apoptotic cells from the analysis. Include in the analysis only those cells that productively interact with the bilayer surface (i.e., cells accumulating Alexa-Fluor-488 fluorescence (anti-CD3 antibodies) at the interface).</li>
	<li>Determine the size of the cell adhesion area at 20 min after the initiation of the cell-bilayer interaction. 
	NOTE: The adhesion area is the dark area developed at the cell-bilayer interface on the interference reflection microscopy (IRM) images.</li>
	<li>Observe any accumulation of Cy5-ICAM-1 fluorescence and a formation of the ring junction by segregated Cy-ICAM-1 molecules at the cell-bilayer interface. If accumulated ICAM-1 molecules formed an adhesion ring junction on at least two consecutive images, designate such cells as cells developing a peripheral supramolecular activating cluster (pSMAC)12.</li>
	<li>Evaluate the granule release by measuring the Alexa-Fluor-568 fluorescence intensity at the T cell-bilayer interface over the background fluorescence outside the contact area in close proximity to the cell. Designate cells with a ratio of Alexa-Fluor-568 signal-to-background of at least 1.3 as degranulating cells.</li>
</ol>

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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 CO&lt;sub&gt;2&lt;/sub&gt;-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 ...

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JoVE Journal

Immunology and Infection

6.4K Views.  Thomas Jefferson University. 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.

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

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Genetics

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&#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.

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

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Bioengineering

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.

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

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

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

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Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

Imaging the Human Immunological Synapse

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses