Name: cell-based flow cytometry assay to measure cytotoxic activity
Uploaded: 2013-12-17T13:45:00
Description: Watch this Scientific Journal Video about Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity at JoVE.com

This work was supported by the Office of Tourism, Trade, and Economic Development of Florida.

1. Preparation of Effector CD8+ T Cells
<ol>
	<li>Thaw autologous cryopreserved PBMCs (2-3 vials of 50&#160;x 106 cells) by transferring the cryovial from liquid nitrogen to a 37 &#176;C water bath.</li>
	<li>Wash the cells by filling the tube to 50 ml with complete RPMI (4 mM L-glutamine, and 100 U/ml penicillin and streptomycin, supplemented with 10% FBS). Count PBMCs and resuspend cells at a concentration of 5 x 106/ml in complete RPMI.</li>
	<li>Add specific peptide (5 &#181;g/ml) and IL-2 (10...

<ol>
	<li>Appay, V., Van Lier, R. A. W., Sallusto, F., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cytometry. 73, 975-983 (2008).</li><li>Betts, M. R., Harari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+and+function+of+protective+T+cell+immune+responses+in+HIV.">Phenotype and function of protective T cell immune responses in HIV.</a> Curr. Opin. HIV AIDS. 3, 349-355 (2008).</li><li>Roder, J. C., 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=A+new+immunodeficiency+disorder+in+humans+involving+NK+cells.">A new immunodeficiency disorder in humans involving NK cells.</a> Nature. 284, 553-555 (1980).</li><li>Barber, D. L., Wherry, E. J., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+rapid+in+vivo+killing+by+memory+CD8+T+cells.">Cutting edge: rapid in vivo killing by memory CD8 T cells.</a> J. Immunol. 171, 27-31 (2003).</li><li>Byers, A. M., Kemball, C. C., Moser, J. M., Lukacher, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+rapid+in+vivo+CTL+activity+by+polyoma+virus-specific+effector+and+memory+CD8++T+cells.">Cutting edge: rapid in vivo CTL activity by polyoma virus-specific effector and memory CD8+ T cells.</a> J. Immunol. 171, 17-21 (2003).</li><li>Devevre, E., Romero, P., Mahnke, Y. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LiveCount+Assay:+concomitant+measurement+of+cytolytic+activity+and+phenotypic+characterisation+of+CD8(+)+T-cells+by+flow+cytometry.">LiveCount Assay: concomitant measurement of cytolytic activity and phenotypic characterisation of CD8(+) T-cells by flow cytometry.</a> J. Immunol. Methods. 311, 31-46 (2006).</li><li>Altman, J. D., 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=Phenotypic+analysis+of+antigen-specific+T+lymphocytes.">Phenotypic analysis of antigen-specific T lymphocytes.</a> Science. 274, 94-96 (1996).</li><li>Mbitikon-Kobo, F. M., Bonneville, M., Sekaly, R. P., Trautmann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+measurement+of+the+cytotoxic+capacity+of+human+primary+antigen-specific+CD8+T+cells.">Ex vivo measurement of the cytotoxic capacity of human primary antigen-specific CD8 T cells.</a> J. Immunol. Methods. 375, 252-257 (2012).</li><li>Bryant, J., Day, R., Whiteside, T. L., Herberman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculation+of+lytic+units+for+the+expression+of+cell-mediated+cytotoxicity.">Calculation of lytic units for the expression of cell-mediated cytotoxicity.</a> J. Immunol. Methods. 146, 91-103 (1992).</li><li>Varadarajan, 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=A+high-throughput+single-cell+analysis+of+human+CD8(+)+T+cell+functions+reveals+discordance+for+cytokine+secretion+and+cytolysis.">A high-throughput single-cell analysis of human CD8(+) T cell functions reveals discordance for cytokine secretion and cytolysis.</a> J. Clin. Invest. 121, 4322-4331 (2011).</li><li>Trautmann, L., 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=Profound+metabolic,+functional,+and+cytolytic+differences+characterize+HIV-specific+CD8+T+cells+in+primary+and+chronic+HIV+infection.">Profound metabolic, functional, and cytolytic differences characterize HIV-specific CD8 T cells in primary and chronic HIV infection.</a> Blood. 120, 3466-3477 (2012).</li><li>Hildemann, S. 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=High+Efficiency+of+Antiviral+CD4(+)+Killer+T+Cells.">High Efficiency of Antiviral CD4(+) Killer T Cells.</a> PLoS One. 8, (2013).</li></ol>

The assay described here allows for the quantification of the principal function of CD8+ T cells: the cytolytic activity. The accurate measurement of this function is of paramount importance when characterizing effector functions of CD8+ T cells, as previous studies reported discordance between this function and the cytokine secretion of antigen-specific CD8+ T cells10,11. Due to the short coculture incubation time (6 hr) and the use of autologous target cells, there is low rate of nonspecific killing of targe...

Cytolytic activity is the major function of CD8+ T cells but is still rarely measured as assays used for this purpose are cumbersome and have been difficult to standardize. Accurate measurement of this function is of paramount importance when characterizing effector functions of CD8+ T cells, as no reliable predictors of effective cell-mediated cytotoxicity have been described yet1,2. Here, we propose a new functional assay to measure the cytotoxic activity of antigen-specific CD8+ T cells on target CD4+ T cells. Several approaches have been developed to provide alternatives to the gold standard, chromium release assay. We present here a cell-based assay that reveals the entire process of killing as it measures the death of live target cells. This method was derived from protocols of flow cytometry-based cytotoxic assays in vivo in mice4,5 and in vitro in humans6. In this protocol, the antigen-specific CD8+ T cells contained in the total CD8+ T cell population are used as effector cells and autologous CD4+ T cells are used as target cells. Effector CD8+ T cells of interest are enumerated using MHCI/peptide tetramers7. Death of target cells is calculated by the ratio between peptide loaded/nonloaded CD4+ T cells. We have previously shown that this method was reproducible, sensitive, specific and did not depend on the number of effector cells within the total CD8+ T cell population8. By enumerating both the number of effector and target cells in the coculture assay, the intrinsic capacity of CD8+ T cells to kill target cells can be calculated and expressed in lytic units9.

<table border="0" cellpadding="0" cellspacing="0" width="826">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">EasySep Human CD4+ T Cell Enrichment Kit</td>
			<td style="width:132px;">Stem Cell</td>
			<td style="width:155px;">19052</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:539px;">EasySep Human CD8+ T Cell Enrichment Kit</td>
			<td>Stem Cell</td>
			<td>19053</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace CFSE Cell Proliferation Kit - For Flow Cytometry</td>
			<td>Invitrogen</td>
			<td>C34554</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Far Red LIVE/DEAD fixable dead cell</td>
			<td>Invitrogen</td>
			<td>L10120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 700 Mouse Anti-Human CD3</td>
			<td>BD</td>
			<td>557943</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 650 Anti-Human CD4 Antibody</td>
			<td>Biolegend</td>
			<td>317435</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP anti-human CD8 Antibody</td>
			<td>Biolegend</td>
			<td>344707</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium 1x</td>
			<td>VWR</td>
			<td>10-040-CV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin 100x Solution</td>
			<td>Invitrogen</td>
			<td>15070063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum - Gold, Heat Inactivated</td>
			<td>PAA</td>
			<td>A15-252</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well plates; Treated; U-bottom; with lid</td>
			<td>BD Falcon</td>
			<td>353077</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 ml sterile polystyrene, individually wrapped reservoirs</td>
			<td>USA Scientific</td>
			<td>2321-2230</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well U-bottom, sterile 2 ml deep well plates</td>
			<td>Thermo Scientific</td>
			<td>275743</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human IL-2 (v126), research grade</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-096</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RoboSep Buffer, separation buffer</td>
			<td>Stem cell</td>
			<td>20104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml Polypropylene Conical Tube</td>
			<td>BD Falcon</td>
			<td>352097</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">14 ml round bottom tube, polypropylene</td>
			<td>BD Falcon</td>
			<td>352059</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">The Big Easy EasySep Magnet</td>
			<td>Stem cell</td>
			<td>18001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS 1x</td>
			<td>VWR</td>
			<td>21-040-CV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well; V-bottom plate with lid</td>
			<td>Fisher</td>
			<td>12-565-481</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde solution, ACS reagent, 37 wt % in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>252549-500ML</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 ml screw cap microtube, conical</td>
			<td>Sarstedt</td>
			<td>72.692.005</td>
		</tr>
	</tbody>
</table>

cell-based flow cytometry assay to measure cytotoxic activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

The schematic in Figure 1 summarizes the assay. Purified CD8+ T cells (effector cells) were resuspended in 450 &#181;l of complete RPMI and serial dilutions were performed by adding 225 &#181;l of CD8+ T cells to the next tube containing 225 &#181;l of media (upper panel). Purified CD4+ T cells (target cells) were counted, split into two tubes and stained with two different concentrations of CFSE (high and low) as described in the protocol. CFSE-low CD4+ T cells were pulsed with the peptide of interest a...

Watch this Scientific Journal Video about Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity at JoVE.com

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

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