an in vitro technique to study t cell interaction with supported planar lipid bilayers

An In Vitro Technique to Study T Cell Interaction with Supported Planar Lipid Bilayers

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, and 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 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 pipette 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 and 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 fluorophore-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.

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Universidad San Sebastian

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Immunodiagnostics

Cellular Immunodiagnostics

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
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 instructions.</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 instructions. 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 kinds of liposomes as described elsewhere: (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 biotin) (sodium salt)).</li>
	<li>Prepare 5% casein solution as previously described.
	<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 antibodies with biotin to produce mono-biotinylated antibody molecules with a previously described approach.
	<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 1" src="/files/ftp_upload/21852/21852_Equation1.jpg" style="margin: auto;" /> 
		NOTE: Find additional details in the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Express a recombinant soluble Intercellular Adhesion Molecule 1 (ICAM-1) protein in a Drosophila expression system as described previously.
	<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 geneticin (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 described.</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 (Nickel-Nitriloacetic acid) 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 Cyanine5 N-Hydroxysuccinimide (Cy5 NHS) ester according to the manufacturer&#39;s instructions. 
		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 previously. Label the Fab fragments with Alexa Fluor 568 NHS ester according to the manufacturer&#39;s instructions.</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 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 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 number of fields and record images of each field 1x every 2 minutes 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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>
	</tbody>
</table>

Source: Steblyanko, M., et al. <a href="https://app.jove.com/v/58143">Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue.</a> J. Vis. Exp. (2018).
This video demonstrates an in vitro technique to study immune synapse formation between primary human T cells and supported lipid bilayers (SLB). T cell surface proteins interact with respective ligands immobilized on SLB to form an immune synapse, followed by the movement of lytic granules towards the synapse, which is visualized using fluorescence microscopy.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Isolation of CD4+ T Cells for an ...

Take a flow slide containing a supported lipid bilayer, or SLB.
The SLB is conjugated to specific ligands &#8212; intercellular adhesion molecule 1 or ICAM-1 and anti-CD3&#160; antibodies &#8212; labeled with fluorophores.
Add T cells and fluorophore-tagged anti-CD107a Fab &#8212; the antigen-binding fragment of an antibody against the degranulation marker.
The T cell receptor-CD3 or TCR-CD3 complex binds to the anti-CD3 antibody, triggering the TCR.
Triggering-induced inside-out signaling activates integrins on the surface, which bind to the immobilized ICAM-1.
The conjugation induces cytoskeletal reorganization &#8212; microtubule re-arrangement, and actin polymerization that leads to lateral spreading of the cell on the SLB surface.
The spreading allows more ligand-receptor interactions to form a supramolecular activation cluster or SMAC, creating an immune synapse.
T cell lytic granules move along microtubules and fuse with the plasma membrane, exposing degranulation markers on the cell surface, which bind to the anti-CD107a Fab.
Using a fluorescence microscope, visualize the immune synapse enriched in TCR-CD3 complexes, integrins, and degranulation markers.

35 Aufrufe. Eine In-vitro-Technik zur Untersuchung der T-Zell-Interaktion mit gestützten planaren Lipid-Doppelschichten

Serie: Eine In-vitro-Technik zur Untersuchung der T-Zell-Interaktion mit gestützten planaren Lipid-Doppelschichten - Experiment

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.

Qualitative und Quantitative Analyse der immun Synapse im menschlichen System mit Hilfe von Imaging Durchflusszytometrie

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

Forschung

JoVE Journal

Immunologie und Infektion

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

Visualisierung der Aktin und Mikrotubuli Cytoskeletons an der B-Zelle immun Synapse mit stimulierte Emission Depletion (STED) Mikroskopie

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

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.

Herstellung von perlengestützten Lipiddoppelschichten zur Untersuchung der partikulären Ausscheidung von T-Zell-Immunsynapsen

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

An In Vitro Technique to Study T Cell Interaction with Supported Planar Lipid Bilayers

Transkript

An In Vitro Technique to Study T Cell Interaction with Supported Planar Lipid Bilayers