Name: qualitative and quantitative analysis of the immune synapse in the human system using imaging flow cytometry
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry at JoVE.com

The work was funded by the German research council (DFG) with grants No. SFB-938-M and SA 393/3-4.

1. Preparation of Pan-leukocytes
<ol>
	<li>Draw 1 mL of peripheral blood from a healthy donor (or patient) in a heparinized syringe. Make sure to have approval by the responsible ethics committee for the blood donation.</li>
	<li>Mix 1 mL of human peripheral blood with 30 mL of ACK lysis buffer (150 mM NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA, pH 7.0) in a 50 mL tube and incubate for 8 min at room temperature.</li>
	<li>Fill the tubes with PBS and centrifuge at 300 x g for 6 min. Aspirate the s...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+engine+in+immunological+synapse.">Actin engine in immunological synapse.</a> Immune Netw. 12 (3), 71-83 (2012).</li><li>Rossy, J., Pageon, S. V., Davis, D. M., Gaus, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+of+the+immunological+synapse.">Super-resolution microscopy of the immunological synapse.</a> Curr Opin Immunol. 25 (3), 307-312 (2013).</li><li>Mace, E. M., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+immunological+synapse+by+dual+color+time-gated+stimulated+emission+depletion+(STED)+nanoscopy.">Visualization of the immunological synapse by dual color time-gated stimulated emission depletion (STED) nanoscopy.</a> J Vis Exp. (85), (2014).</li><li>Comrie, W. A., Babich, A., Burkhardt, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F-actin+flow+drives+affinity+maturation+and+spatial+organization+of+LFA-1+at+the+immunological+synapse.">F-actin flow drives affinity maturation and spatial organization of LFA-1 at the immunological synapse.</a> J Cell Biol. 208 (4), 475-491 (2015).</li><li>Markey, K. A., Gartlan, K. H., Kuns, R. D., MacDonald, K. P., Hill, G. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wiskott-Aldrich+Syndrome:+Immunodeficiency+resulting+from+defective+cell+migration+and+impaired+immunostimulatory+activation.">Wiskott-Aldrich Syndrome: Immunodeficiency resulting from defective cell migration and impaired immunostimulatory activation.</a> Immunobiology. 214 (9-10), 778-790 (2009).</li><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 (5425), 221-227 (1999).</li><li>Dustin, M. L., Depoil, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+T+cell+synapse+from+single+molecule+techniques.">New insights into the T cell synapse from single molecule techniques.</a> Nature reviews. Immunology. 11 (10), 672-684 (2011).</li><li>Mace, E. M., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+views+of+the+human+NK+cell+immunological+synapse:+recent+advances+enabled+by+super-+and+high-resolution+imaging+techniques.">New views of the human NK cell immunological synapse: recent advances enabled by super- and high-resolution imaging techniques.</a> Front Immunol. 3, 421 (2012).</li><li>Yokosuka, T., 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=Newly+generated+T+cell+receptor+microclusters+initiate+and+sustain+T+cell+activation+by+recruitment+of+Zap70+and+SLP-76.">Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.</a> Nat Immunol. 6 (12), 1253-1262 (2005).</li><li>Wabnitz, G. H., 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=Sustained+LFA-1+cluster+formation+in+the+immune+synapse+requires+the+combined+activities+of+L-plastin+and+calmodulin.">Sustained LFA-1 cluster formation in the immune synapse requires the combined activities of L-plastin and calmodulin.</a> European journal of immunology. 40 (9), 2437-2449 (2010).</li><li>Wabnitz, G. H., 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=L-plastin+phosphorylation:+a+novel+target+for+the+immunosuppressive+drug+dexamethasone+in+primary+human+T+cells.">L-plastin phosphorylation: a novel target for the immunosuppressive drug dexamethasone in primary human T cells.</a> Eur J Immunol. 41 (11), 3157-3169 (2011).</li><li>Wabnitz, G. H., Nessmann, A., Kirchgessner, H., Samstag, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=InFlow+microscopy+of+human+leukocytes:+A+tool+for+quantitative+analysis+of+actin+rearrangements+in+the+immune+synapse.">InFlow microscopy of human leukocytes: A tool for quantitative analysis of actin rearrangements in the immune synapse.</a> J Immunol Methods. , (2015).</li><li>Basiji, D., O'Gorman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+flow+cytometry.">Imaging flow cytometry.</a> J Immunol Methods. 423, 1-2 (2015).</li><li>Wabnitz, G. H., Samstag, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+Characterization+of+Human+T-Cell+Immune+Synapses+by+InFlow+Microscopy.">Multiparametric Characterization of Human T-Cell Immune Synapses by InFlow Microscopy.</a> Methods Mol Biol. 1389, 155-166 (2016).</li><li>Balta, E., 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=Qualitative+and+quantitative+analysis+of+PMN/T-cell+interactions+by+InFlow+and+super-resolution+microscopy.">Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution microscopy.</a> Methods. , (2016).</li><li>Hochweller, 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=Dendritic+cells+control+T+cell+tonic+signaling+required+for+responsiveness+to+foreign+antigen.">Dendritic cells control T cell tonic signaling required for responsiveness to foreign antigen.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (13), 5931-5936 (2010).</li><li>Balagopalan, L., Sherman, E., Barr, V. A., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+techniques+for+assaying+lymphocyte+activation+in+action.">Imaging techniques for assaying lymphocyte activation in action.</a> Nat Rev Immunol. 11 (1), 21-33 (2011).</li></ol>

The workflow presented here enables the quantification of immune synapses between human T cells (ex vivo) and APCs. Notably, erythrocyte-lysed pan-leukocytes were used as T-cell sources, making T-cell purification steps dispensable. The B-cell lymphoma cell line Raji served as surrogate APCs. This bears significant advantages, since it allows comparisons between blood donors of the T-cell side of the immune synapse. Furthermore, autologous DCs are hardly available directly from peripheral human blood. The production of m...

T cells are major regulators of the adaptive immune system and are activated through antigenic peptides that are presented in the context of major histocompatibility complexes (MHC). Full T-cell activation requires two signals, the competence signal via the antigen-specific T-cell receptor (TCR)/CD3 complex and the costimulatory signal via accessory receptors. Both signals are generated through the direct interaction of T cells with antigen-presenting cells (APCs). Mature APCs provide the competence signal for T-cell activation through MHC-peptide complexes, and they express costimulatory ligands (e.g., CD80 or CD86) to assure the progression of T-cell activation1. One important function of costimulation is the rearrangement of the actin cytoskeleton2,3,4. The cortical F-actin is relatively static in resting T cells. T-cell stimulation through antigen-bearing APCs leads to a profound rearrangement of the actin cytoskeleton. Actin dynamics (i.e., fast actin polymerization/depolymerization circles) enable the T cells to create forces that are used to transport proteins or organelles, for example. Moreover, the actin cytoskeleton is important for developing a special contact zone between T cells and APCs, called the immune synapse. Due to the importance of the actin cytoskeleton to the immune synapse, it has become essential to develop methods to quantify changes in the actin cytoskeleton of T cells5,6,7,8,9.
By means of actin cytoskeletal aid, surface receptors and signaling proteins are segregated in supramolecular activation clusters (SMACs) within the immune synapse. The stability of the immune synapse is assured by the binding of receptors to F-actin bundles that increase the elasticity of the actin cytoskeleton. Immune synapse formation has been shown to be critical for the generation of the adaptive immune responses. The detrimental effects of a defective immune synapse formation in vivo were first realized in patients suffering from Wiskott Aldrich Syndrome (WAS), a disease in which actin polymerization and, concomitantly, immune synapse formation are disturbed10. WAS patients can suffer from eczema, severe recurrent infections, autoimmune diseases, and melanomas. Despite this finding, it is currently not known whether immune synapse formation differs in the T cells of healthy individuals and patients suffering from immune defects or autoimmune diseases.
Fluorescence microscopy, including confocal, TIRF, and super-resolution microscopy, were used to uncover the architecture of the immune synapse11,12,13,14. The high resolution of these systems and the possibility of performing live-cell imaging enables the collection of exact, spatio-temporal information about the actin cytoskeleton and surface or intracellular proteins in the immune synapse. Many results, however, are based on the analysis of only a few tens of T cells. Moreover, T cells must be purified for these types of fluorescence microscopy. However, for many research questions, the use of unpurified cells rather than the highest-possible resolution is of the utmost importance. This is relevant if T cells from patients are analyzed, since the amount of donated blood is limited and there might be the need to process many samples in parallel.
We established microscopic methods that allow the analysis of the actin cytoskeleton in the immune synapse in the human system15,16,17. These methods are based on imaging flow cytometry, also called In-Flow microscopy18. As a hybrid between multispectral flow cytometry and fluorescence microscopy, imaging flow cytometry has its strengths in analyzing morphological parameters and protein localization in heterogeneous cell populations, such as pan-leukocytes from the peripheral blood. We introduced a methodology that enables us to quantify F-actin in T-cell/APC conjugates of human T cells from whole-blood samples, without the need of time-consuming and costly purification steps17. The technique presented here comprises the whole workflow, from getting the blood sample to the quantification of F-actin in the immune synapse.

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			<td>&#62;Multifuge 3 SR</td>
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			<td>RPMI 1640</td>
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			<td>ReaktiosgefaB</td>
			<td>Sarstedt</td>
			<td>72.699.002</td>
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			<td>Speed Bead</td>
			<td>Amnis</td>
			<td>#400041</td>
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			<td>Minishaker MS1</td>
			<td>IKA Works</td>
			<td>&#160;MS1</td>
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			<td>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>D9542</td>
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			<td>CD3-PeTxR</td>
			<td>Invitrogen</td>
			<td>MHCD0317</td>
			<td>1:30</td>
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			<td>Phalloidin-AF647</td>
			<td>Molecular Probes</td>
			<td>A22287</td>
			<td>1:150</td>
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			<td>IS100</td>
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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.

A major goal of the method described here is the quantification of protein enrichment (e.g., F-actin) in the immune synapse between surrogate APCs (Raji cells) and T cells in unpurified pan-leukocytes taken from low-volume (1 mL) human blood samples. The screenshot in Figure 1 gives an overview of the critical gating strategy of this method. It shows the image gallery on the left and the analysis area on the right (Figure 1). The image gallery sh...

Watch this Scientific Journal Video about Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry at JoVE.com

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

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

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

This method can help to answer key questions in the human immunological field, such as how communication between leukocytes occurs on the molecular and cellular level. The main advantage of this technique is that unpurified human T cells can be analyzed ex vivo in a relatively short time period. Demonstrating this procedure will be Henning Kirchgessner, a technician from our laboratory.Begin by mixing one milliliter of freshly drawn human peripheral blood with 30 milliliters of ACK lysis buffer in a 50-milliliter conical tube. After eight minutes at room temperature, fill the tube with PBS for a centrifugation wash and resuspend the pellet in two milliliters of culture medium for a 60-minute incubation at 37 degrees Celsius. At the end of incubation, add five times 10 to the fifth Staphylococcus aureus enterotoxin B or SEB-loaded or unloaded Raji cells in 500 microliters of culture medium into individual FACS tubes, followed by 650 microliters of pan-leukocytes.Spin down the co-cultures, and resuspend the pellets in 150 microliters of fresh culture medium for a 45-minute incubation at 37 degrees Celsius. Then, gently vortex the cells for 10 seconds at 100 rpm while adding 1.5 milliliters of 1.5%paraformaldehyde. After 10 minutes at room temperature, stop the fixation with one milliliter of PBS supplemented with 1%BSA and collect the cells by centrifugation.Resuspend the pellets in 100 microliters of PBS plus BSA and 0.1%saponin, and add the cell samples to two individual wells of a 96-well, U-bottom plate. After 15 minutes at room temperature, centrifuge the plate and resuspend the pellets in 50 microliters of PBS plus BSA and saponin containing the fluorophore-conjugated antibodies or compounds of interest for a 30-minute incubation in the dark at room temperature. At the end of the incubation, wash the cells three times in 150 microliters of PBS plus BSA and saponin and resuspend the pellets in 60 microliters of PBS.To image the cells by flow cytometry, open the analysis software, check the liquid level, and click Startup in the Instrument menu. Next, click Load, and load the samples into the port when prompted. Under the Illumination tab, change the excitation laser power to the appropriate nanometer lengths.Then, adjust the gates for channels four and 11, and adjust the area for channel one. To evaluate the gating and laser power adjustments, switch the View menu to the respective gate and adjust the gates and laser powers until the cells and cell couples are visible. Then, in the File Acquisition tab, define the sample name and the number of images to acquire and the appropriate gate for CD3 staining and click Acquire to begin the acquisition.To analyze the acquired cell images, open the appropriate analysis software. Following the instructions in the Compensation dropdown, produce a compensation matrix and save the matrix as comp date ctm. Next, open a sample raw image file, and apply the comp date ctm in the window to generate the compensated image and default data analysis files.After opening the compensated image files, convert the images to color mode. Adjust the lookup tables to obtain optimal visible colors in the Image Gallery Properties toolbar. Open the Mask Manager in the Analysis menu, and define the T cells by selecting a 60%Threshold mask for channel five and filling the mask to create the T cell mask.Next, select Valley mask for channel two with a width of three pixels to create the Valley mask, and combine the T cell and Valley masks to create the T cell synapse mask. To calculate the total CD3 expression in T cells, open the Feature Manager and create the feature Intensity, T cell, channel five. To calculate the total amount of F-actin in the T cells, create the feature Intensity, T cells, channel six.To evaluate the CD3 amount in immune synapses, create the feature Intensity, T cell synapse, channel five. To determine the amount of F-actin in the immune synapse, create the feature Intensity, T cell synapse, channel six. Finally, to define the F-actin and CD3 enrichment, calculate the ratios of F-actin or CD3 in the immune synapse and the total expression of the selected protein, respectively.In these graphs, an overview of the critical gating strategy for quantifying the protein enrichment in the immune synapse between the surrogate antigen-presenting cells, or APCs, and the T cells in unpurified pan-leukocytes taken from a low-volume human blood sample is shown. Here, representative images of T-cell-APC conjugates with low levels of CD3 and F-actin within their immune synapses in the absence of SEB are shown, whereas these T-cell-APC conjugates demonstrate a strong CD3 and F-actin enrichment in the presence of SEB. In the absence of superantigen, 15%of the T cells exhibited enrichment of both CD3 and F-actin at the immune synapse, whereas a 29%enrichment is observed in the presence of superantigen.Indeed, in the absence of superantigen, less than 20%of the total F-actin in the cells accumulates at the immune synapse, with over 30%observed at the synapse in the presence of superantigen. Notably, CD3 accumulates at the immune synapse even in the absence of superantigen, although this accumulation is also significantly increased by the addition of superantigen, demonstrating the fitness of this method for quantifying protein accumulation at the T-cell-APC immune synapse. Once mastered, this technique can be completed in six to seven hours if performed properly.While attempting this procedure, it's important to remember to include all relevant controls, including samples without superantigen, as protein enrichment also occurs if APCs without SEB are used. Following this procedure, other methods like super-resolution microscopy can be performed to answer additional questions about the fine structure of the immune synapse. After its development, this technique paved the way for researchers in the field of cellular communications to explore the immune synapse in humans for basic research, clinical trials, and translational science.After watching this video, you should have a good understanding of how to analyze the T-cell-APC connective zone and the immune synapse in human T cells ex vivo. Don't forget that working with primary human materials can be hazardous and that precautions such as wearing gloves or working under biosafety level two conditions should always be taken while performing this procedure.

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Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...