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>

<ol>
	<li>Grakoui, A., 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=The+immunological+synapse:+a+molecular+machine+controlling+T+cell+activation.">The immunological synapse: a molecular machine controlling T cell activation.</a> Science. 285, 221-227 (1999).</li><li>Somersalo, K., 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=Cytotoxic+T+lymphocytes+form+an+antigen-independent+ring+junction.">Cytotoxic T lymphocytes form an antigen-independent ring junction.</a> Journal of Clinical Investigation. 113, 49-57 (2004).</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=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. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+planar+bilayers+for+study+of+the+immunological+synapse.">Supported planar bilayers for study of the immunological synapse.</a> Current Protocols in Immunology. , (2007).</li><li>Reuter, M. A., 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=HIV-Specific+CD8(+)+T+Cells+Exhibit+Reduced+and+Differentially+Regulated+Cytolytic+Activity+in+Lymphoid+Tissue.">HIV-Specific CD8(+) T Cells Exhibit Reduced and Differentially Regulated Cytolytic Activity in Lymphoid Tissue.</a> Cell Reports. 21, 3458-3470 (2017).</li><li>Buggert, 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=Limited+immune+surveillance+in+lymphoid+tissue+by+cytolytic+CD4++T+cells+during+health+and+HIV+disease.">Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease.</a> PLoS Pathogens. 14, e1006973 (2018).</li><li>Carrasco, Y. R., Fleire, S. J., Cameron, T., Dustin, M. L., Batista, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LFA-1/ICAM-1+interaction+lowers+the+threshold+of+B+cell+activation+by+facilitating+B+cell+adhesion+and+synapse+formation.">LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation.</a> Immunity. 20, 589-599 (2004).</li><li>Anikeeva, N., 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=Distinct+role+of+lymphocyte+function-associated+antigen-1+in+mediating+effective+cytolytic+activity+by+cytotoxic+T+lymphocytes.">Distinct role of lymphocyte function-associated antigen-1 in mediating effective cytolytic activity by cytotoxic T lymphocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 6437-6442 (2005).</li><li>Steblyanko, M., Anikeeva, N., Campbell, K. S., Keen, J. H., Sykulev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+Influence+the+Size+and+Dynamics+of+Signaling+Microclusters+in+a+Pyk2-dependent+Manner.">Integrins Influence the Size and Dynamics of Signaling Microclusters in a Pyk2-dependent Manner.</a> The Journal of Biological Chemistry. 290, 11833-11842 (2015).</li><li>Anikeeva, N., Lebedeva, T., Sumaroka, M., Kalams, S. A., Sykulev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+HIV-specific+T-cell+receptor:+expression,+purification+and+analysis+of+the+specificity.">Soluble HIV-specific T-cell receptor: expression, purification and analysis of the specificity.</a> Journal of Immunological Methods. 277, 75-86 (2003).</li><li>Monks, C., Freiberg, B., Kupfer, H., Sciaky, N., Kupfer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+segregation+of+supramolecular+activation+clusters+in+T+cells.">Three-dimensional segregation of supramolecular activation clusters in T cells.</a> Nature. 395, 82-86 (1998).</li><li>Riddell, S. R., Greenberg, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+anti-CD3+and+anti-CD28+monoclonal+antibodies+to+clone+and+expand+human+antigen-specific+T+cells.">The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells.</a> Journal of Immunological Methods. 128, 189-201 (1990).</li><li>Lin, S. J., Yu, J. C., Cheng, P. J., Hsiao, S. S., Kuo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+interleukin-15+on+anti-CD3/anti-CD28+induced+apoptosis+of+umbilical+cord+blood+CD4++T+cells.">Effect of interleukin-15 on anti-CD3/anti-CD28 induced apoptosis of umbilical cord blood CD4+ T cells.</a> European Journal of Haematology. 71, 425-432 (2003).</li><li>Anikeeva, N., Sykulev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+controlling+granule-mediated+cytolytic+activity+of+cytotoxic+T+lymphocytes.">Mechanisms controlling granule-mediated cytolytic activity of cytotoxic T lymphocytes.</a> Immunologic Research. 51, 183-194 (2011).</li><li>Huppa, J. B., 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=TCR-peptide-MHC+interactions+in+situ+show+accelerated+kinetics+and+increased+affinity.">TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.</a> Nature. 463, 963-967 (2010).</li></ol>

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 system5, creating the opportunity to analyze primary human T cells from blood and other tissues.
The number of injected cells used in this study allowed us to image about 50 cells per imaging field with a 60X objective. A larger concentration resulted in cell aggregation, reducing the number of cells suitable for analysis. We could further reduce the number of injected cells 10&#8211;50x, but the lower limit of the cell number depends on cell heterogeneity, on the number of imaging fields, and on the experimental design. Some investigators have previously used bilayers built in an open chamber with a glass bottom that required a similar number of cells for analysis16. However, the closed system described here, with a channel height of 0.1 - 0.4 mm, allows for rapid cell sedimentation to synchronize the cell attachment and analysis of the T-cell response without the risk of liquid evaporation. The sticky slide system further permits the formation of up to three bilayers per channel. Thus, the same cell sample could be used for the analysis of cell behavior on distinct bilayers with a different composition of stimulatory, adhesion, and costimulatory molecules.
Some precautions should be taken during the assembly of the sticky slides and coverslips for a successful bilayer formation. Particularly, the coverslips should be completely dry, as even a small amount of liquid left on the glass surface could result in leakage during the bilayer washing procedure. Importantly, it is essential to even the attached coverslip at the edges of the contact with the outer ring of polypropylene scissor-type forceps to avoid leakage (as described in step 3.4.1). It is also important to avoid any bubbling in the entry ports of the channels. If any bubbles enter a channel, they are almost impossible to remove and ruin the bilayer in the channel. Similarly, it is important to avoid any fast pipetting, since the turbulent flow of liquid may introduce bubbles into the channel and damage the bilayer.
A release of cytolytic granules is detected by the appearance of CD107a at the T cell-bilayer interface. Fab regions of Alexa-Fluor-568-labeled anti-CD107a antibodies are added to CD8 T cells prior to the loading on the bilayers. TIRF microscopy exploits an evanescent wave to illuminate the area about 100 nm above the bilayer in the volume defined as evanescent volume, allowing to increase the vertical resolution of the imaging. Labeled antibody Fabs, which diffuse very rapidly within the evanescent volume at 37 &#176;C, do not generate a detectable fluorescent signal. During the membrane fusion of specialized lysosomes containing lytic molecules with the cell membrane, CD107a membrane protein appears on the T cells contact surface in distinct locations. The Fabs get bound to the CD107s protein at that time. Attached to the CD107a protein, fluorescent-labeled Fab(s) form immobile clusters that generate a bright fluorescence detectable by TIRF microscopy, indicative of T cell degranulation.

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 ICAM-1 at 50 molecules/&#181;m2 and 300 molecules/&#181;m2, respectively. CD8+ T cells derived from human cord blood were activated by a stimulation with plate-bound anti-CD3 and anti-CD28 antibodies13,14. There was no difference between CD8+ T cells that formed classical immunological synapses on the lipid bilayers built in either the flow cell or the multichannel flow slide (Figure 1). All other experiments were performed with lipid bilayers made in the multichannel flow slides.
Next, we examined the ability of PB- and LN-derived human CD4+ T cells to form synaptic interfaces with the planar lipid bilayers and release granules. We exploited TIRF microscopy to maximize the vertical resolution at the T cell-bilayer interface to visualized degranulating cells and to evaluate the kinetics of the granule release. We observed 4 groups of cells for both the LN- and PBMC-derived CD4+ T cells: some T cells established mature immune synapses, but either did or did not release granules, other T cells released the granules without a formation of mature synapses, and still other T cells neither formed synapses nor released the granules (Figure 2)7. The difference between LN- and PBMC-derived CD4+ T cells became apparent when the kinetics of the granule release was evaluated. PBMC-derived CD4+ T cells were able to begin releasing granules almost twice as fast as LN-derived CD4+ cells (Figure 3)7. Overall, the data demonstrate that despite a heterogeneity of LN- and PBMC-derived CD4+ T cells, the latter contained a fraction of T cells capable of rapid degranulation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58143/58143fig1.jpg" /> 
Figure 1: Comparison of the flow slide system and conventional flow chamber. (A) This panel shows the multichannel flow slide allowing an assembly of 6 glass-supported planar lipid bilayers. Each bilayer is connected to the delivery and the exit ports. Less than 1 x 105 cells are sufficient for the analysis. (B) This panel shows the conventional flow chamber system allowing an assembly of 1 glass-supported planar lipid bilayer. 2 x 106 cells are required for the analysis. The other panels show (C and D) representative TIRF microscopy and (E and F) confocal with interference reflection microscopy (IRM) images of the interface developed by activated cord-blood-derived CD8 T cells. The cells were exposed to a lipid bilayer containing ICAM-1 (300 molecules/&#181;m2) and anti-CD3 antibodies (50 molecules/&#181;m2) acquired (C and E) in flow slides or (D and F) in a conventional flow system under similar conditions. The TIRF microscopy and confocal with IRM images are taken using 100X and 60X magnification objectives, respectively. The percentage of T cells that form mature synapses on the bilayers built in either flow slide or conventional flow chamber was very similar but varies between experiments from 75% to 90%. In all images, Cy-5-labelled ICAM-1 molecules are shown in blue; Alexa-Fluor-488-labeled anti-CD3 antibodies are shown in green; Alexa-Fluor-568-labeled anti-CD107a antibodies bound to CD107a are shown in red; IRM images are shown in cyan; and the scale bars are 10 &#181;m in length. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58143/58143fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58143/58143fig2.jpg" /> 
Figure 2: Structure of a T cell&#8211;bilayer interface and the pattern of degranulation by lymph node and peripheral blood mononuclear CD4+ T cells. LNMC- or PBMC-derived cells were sorted to separate CD4+ T cells that were exposed to the bilayers containing 50 molecules/&#181;m2 of anti-CD3 antibodies and 300 molecules/&#181;m2 of ICAM-1 protein. The interface between the cells and the bilayers was imaged by TIRF microscopy. The scale bars are 5 &#181;m. (A) These representative images of a T cell - bilayer interface demonstrate the structure of the T cell&#8211;bilayer interfaces and degranulation pattern. (B) These diagrams show a representation of LNMC- and PBMC-derived CD4+ T cells with a different structure of synaptic interfaces and patterns of granule release. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58143/58143fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58143/58143fig3.jpg" /> 
Figure 3: Dynamic changes of the structure of a T cell&#8211;bilayer interface and the kinetics of a degranulation of lymph node and peripheral blood mononuclear CD4+ T cells. (A) This panel shows time-dependent changes of a T cell&#8211;bilayer interface and the appearance of released granules. The scale bars are 5 &#181;m. (B) This panel shows a quantitation of the kinetics of a granule release by LN-derived CD4+ T cells (closed circles) and PBMC-derived CD4+ T cells (open circles). Each individual circle indicates the time of the first appearance of a detectable granule release by individual cells. The median and IQR (interquartile range) are shown for all scatter plots. Mann-Whitney tests were performed to compare differences between the indicated groups of T cells. * P &#60;0.05, ** P &#60;0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58143/58143fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

assessment-synaptic-interface-primary-human-t-cells-from-peripheral

Nanyang Technological University

Research

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

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

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

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

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

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

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

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

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

Generation of Induced Regulatory T Cells from Primary Human Na&#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 ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

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

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

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

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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 ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

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

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

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

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

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

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

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

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

Analysis of Human Natural Killer Cell Metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

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