This work was supported by grants from the National Institutes of Health DK074656 and AI56374 (CEM), as well as the Juvenile Diabetes Research Foundation.

1.	Preparation of cells
<ol>
	<li>Target cell culture: Culture of the mouse beta cell lines, NIT-1 and NIT-4, was a previously described1,2 in DMEM supplemented with 10% FBS, 0.02%BSA, Non-essential Amino Acid, 15mM HEPES, 16.5mM Glocose and Penicillin/Streptomycin. For 51Cr labeling, seed cells in a flat-bottom 96-well culture plate at 5X104 cells/well in 200 &mu;l culture medium. For microscopy experiments, seed cells in a 8-well Lab-Tak II chambered coverglass at 5x104 per chamber in 500 &mu;l culture medium, and allow to grow for 48 hours prior to using in the cytotoxicity experiments.</li> 
	<li>Control T cell line culture: Use the Jurkat cell line (ATCC, CD3+ human lymphocyte cell line) as a negative control. Culture Jurkat cells in RPMI1640 supplemented with 10% FBS, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 10 mM HEPES and 1.0 mM sodium pyruvate.</li> 
</ol>
2.	Collection and activation of effector autoreactive CD8+ T cells
NOD.Cg-Rag1tm1MomTg(TcraAI4)1Dvs/DvsJ [NOD-AI4a] and NOD.Cg-Rag1tm1MomTg(TcrbAI4)1Dvs/DvsJ [NOD-AI4b] were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in our mouse facility. F1 hybrid progeny from matings of NOD-AI4a with NOD-AI4b [NOD-AI4a/b] develop diabetes at 3-5 weeks of age. All mice were housed in specific pathogen-free facilities and approved by the relevant institution&#x2019;s Animal Care and Use Committee.
<ol>
	<li>Euthanize 3-4 week old NOD-AI4a/b mice using CO2. </li>
	<li>Collect spleens from mice. Euthanized mice are prepared with ethanol spray and opened under surgical hood. Spleens are removed and put in ice-cold HBSS buffer immediately.</li>
	<li>Collect spleen cells using a 7 mL glass homogenizer. Two to three spleens each are put in 5 mL of cold HBSS and homogenized with a 7 mL glass homogenizer. Homogenate is collected and centrifuged at 1200rpm for 5 minutes at 4&deg;C using a Sorvall RT7+ centrifuge. Cell pellets are collected.</li>
	<li>Remove red blood cells using hypotonic solution treatment. Pellets are treated with 5 mL hypotonic solution/spleen on ice for 5 minutes, then neutralized by adding an equal volume of HBSS. Collect cells by centrifugation, as above, and resuspend in 50 mL HBSS. Pass the suspended cells through cell strainer and count using a hemocytometer with TrypanBlue staining.</li>
	<li>Activate cells. Resuspend cells at 5x106/mL and activate using a 3 day culture with 0.1 &mu;M AI4 mimotope (amino acid sequence YFIENYLEL) and 25 U/mL IL-2 in RPMI1640 supplemented with 10% FBS, 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 10 mM HEPES and 1.0 mM sodium pyruvate.</li> 
</ol>
3.	51Cr release assay to examine CML
<ol>
<li>Label target cells: Add 51Cr to target cells at 1 &mu;Ci/well and culture for 3 hours, and wash 3 times with culture medium.</li>
<li>Suspend effector cells in RPMI 1640 medium as mentioned in step 2.5 so that the final volume added to each well is 200 &mu;l.</li> 
<li>After the final wash of labeled target cells, add activated effector cells to the target cell cultures at desired effector to target (E:T) ratios. We usually use E:T ratios at 0.4:1, 1:1, 2:1, 5:1, 10:1, 20:1 and 50:1.</li>
<li>Add fresh modified RPMI 1640 medium (see step 2.5) to 6 wells of target cells to act as a spontaneous lysis control.</li>
<li>Co-culture effector and target cells for 16 hours. </li>
<li>Collect supernatant in 6x50 mm Lime Glass tubes</li>
<li>Lyse cells by adding 100&mu;l 2% SDS and pipetting several times gently.</li> 
<li>Collect cell lysate in another set of 6x50 mm Lime Glass tubes.</li> 
<li>Wash wells once with clean H2O and add the wash to the cell lysate tubes.</li>
<li>Count the supernatants and cell lysates using a gamma counter.</li> 
<li>Calculate specific lysis as follows: 
<img src="/files/ftp_upload/2724/2724eq1.jpg" alt="Equation 1" /></li>
</ol>


4.	Staining of cells for live cell imaging

Among most commonly used mitochondrial membrane potential dyes, TMRM exhibits the least mitochondrial toxicity.3 Therefore, we choose TMRM for these live cell imaging studies. 
<ol>
<li>Prepare a 40 mM stock TMRM solution in DMSO and store at -20&deg;C. Make a [25 nM] fresh working solution in phenol red-free DMEM with all the supplements for NIT-1 cell culture 1.</li> 
<li>Stain target NIT-1 cells with TMRM 25 nM for 30 min at 37&deg;C.</li>
<li>Replace the media with fresh media containing 5 nM TMRM to maintain the equilibrium distribution of the dye4.</li> 
<li>Count activated effector AI4 T cells using a hematocytometer.</li>
<li>Stain activated effector AI4 T cells with 1 &mu;l/mL Picogreen for 5 minutes at room temperature and wash with phenol red-free culture medium 3 times.</li>
</ol>
5.	Assessing T lymphocyte-mediated beta cell death using live cell imaging


<ol>
<li>Mount the chambered coverglass with stained NIT cells on the stage of a Zeiss LSM 510 confocal microscope. Remove the tab with a dremmel and gas sterilize the chambered glass with etheline oxide. The stage is equipped with a live-cell imaging chamber that has temperature controlled at 37&deg;C and 5% CO2 with moisture.</li> 
<li>Add activated and stained T cells to designated chambers at different E:T ratios.</li>
<li>Added same number of stained Jurkat cells to the control chambers.</li>
</ol>
6.	Obtaining live cell images
The microscope is equipped with a motorized stage that allowed us to repeatedly acquire images at different locations from the same or different chambers automatically over the time course of the experiment. 
<ol>
	<li>Using a 63x oil objective lens, take images at an interval of every 3 minutes for a total of 300 frames per location.</li> 
	<li>Detect TMRM staining using an excitation wave length of 543 nm and filter set for emission 565-619 nm</li>
	<li>Detect Picogreen staining using an excitation 488 nm and a filter set for emission 500-630 nm. The video was created using Zeiss LSM software.</li>
</ol>
7.	Video conversion for publication
<ol>
	<li>Using the Zeiss LSM software, create an uncompressed movie file in an AVI format. </li>
	<li>To convert the video into the published format, import the video into the software package FIJI (<a href="http://pacific.mpi-cbg.de/wiki/index.php/Fiji">http://pacific.mpi-cbg.de/wiki/index.php/Fiji</a>), an ImageJ (<a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a>) distribution, using the Bioformats plugin from LOCI (<a href="http://www.loci.wisc.edu/software/bio-formats">http://www.loci.wisc.edu/software/bio-formats</a>).</li> 
	<li>Adjust the brightness and contrast settings to balance the green and red signals and the frame rate was set to 2 frames/second using the animation options menu.</li> 
	<li>Crop the video to 512 x 512 pixels and export as an uncompressed AVI file.</li> 
	<li>Open this AVI file in the software Quicktime 7 (Apple computer, Cupertino, CA) and compress and export in the open video file format H264/MPEG-4 at 1200 kbs/second.</li>
</ol>
8. Representative Results:
<ol>
<li>A representative result of 51Cr CML assay. Figure 1 shows a representative result of a CML assay where percent specific lysis (Y axis) increases as the Effector:Target ratio (X axis) increases. Target cells are primary islets isolated from immunodeficient NOD.Rag1-/- mice and labeled with 51Cr. Splenocytes from NOD.AI4&alpha;/&beta; transgenic mice are isolated and activated as described in step 2 above. Targets and effectors were co-cultured for 16 hours. Percent specific lysis is calculated as described in step 3.11 above.</li>
<li>Two representative (positive and negative) results of a T cell-mediated beta cell mitochondrial membrane potential dissipation are presented. Cells are stained and reactions are recorded as described in steps 4, 5 and 6 above, video files are created as in step 7. Video 1: The mouse beta cell line NIT-1 was stained the mitochondrial membrane potential dye TMRM (Red). Activated, autoreactive CD8+ T cell AI4 (Stained green with Picogreen) were co-cultured with these NIT-1 cells at an E:T ratio of 50:1. NIT-1 mitochondrial membrane potential dissipated gradually during a 400-minute duration, but only in the clusters that were interacting with AI4 T cells. 
Video 2: As a control experiment, NIT-1 cells were stained with TMRM and co-cultured with Picogreen stained Jurkat cells at an E:T ratio of 50:1. Jurkat cells are a human T cell line that is MHC mismatched. NIT-1 cells maintained mitochondrial membrane potential throughout the entire duration of the 400 minutes co-incubation period. Jurkat cells did not interact with NIT-1 clusters and therefore, do not induce killing.</li>
</ol>

<img src="/files/ftp_upload/2724/2724fig1.jpg" alt="Figure 1" /> 
Figure 1. Representative result of CML using as target cells primary islets isolated from immunodeficient NOD.Rag1-/- mice and splenocytes from NOD.AI4&alpha;/&beta; transgenic mice. Targets and effectors were co-cultured for 16 hours. Percent specific lysis increases as the Effector:Target ratio increases.
Video 1. Mouse beta cell line NIT-1 was stained the mitochondrial membrane potential dye TMRM (Red). Activated autoreactive CD8+ T cell AI4 (Stained green with Picogreen) were co-cultured with these NIT-1 cells at E:T ratio of 50:1. NIT-1 mitochondrial membrane potential dissipated gradually during a 400-minute duration, but only in the clusters that were interacting with green AI4 T cells. <a href="https://www.jove.com/files/ftp_upload/2724/video.mov">Click here to watch video</a>.
Video 2. NIT-1 cells were stained same as described in Video 1 and co-cultured with Picogreen stained human T cell line Jurkat. NIT-1 cells were able to maintain mitochondrial membrane potential throught out the duration of 400 minutes. Jurkat cells did not interact with NIT-1 clusters and therefore do not induce killing. <a href="https://www.jove.com/files/ftp_upload/2724/Video2.mp4">Click here to watch video</a>.

<ol>
	<li>Hamaguchi, K., Gaskins, H. R., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIT-1,+a+pancreatic+beta-cell+line+established+from+a+transgenic+NOD/Lt+mouse.">NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse.</a> Diabetes. 40, 842-849 (1991).</li><li>Chen, J., Gusdon, A. M., Piganelli, J., Leiter, E. H., Mathews, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mt-Nd2a+modifies+resistance+against+autoimmune+Type+1+diabetes+in+NOD+mice+at+the+level+of+the+pancreatic+beta+cell.">mt-Nd2a modifies resistance against autoimmune Type 1 diabetes in NOD mice at the level of the pancreatic beta cell.</a> Diabetes. , (2010).</li><li>Scaduto, R. C., Grotyohann, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+membrane+potential+using+fluorescent+rhodamine+derivatives.">Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives.</a> Biophys J. 76, 469-477 (1999).</li><li>Lemasters, J. J., Ramshesh, 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=Imaging+of+mitochondrial+polarization+and+depolarization+with+cationic+fluorophores.">Imaging of mitochondrial polarization and depolarization with cationic fluorophores.</a> Methods Cell Biol. 80, 283-295 (2007).</li><li>Abel, M., Krokowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+immune-mediated+(type+1)+diabetes+mellitus:+potential+for+immunotherapy.">Pathophysiology of immune-mediated (type 1) diabetes mellitus: potential for immunotherapy.</a> BioDrugs. 15, 291-301 (2001).</li><li>Mathews, C. E., Graser, R. T., Savinov, A., Serreze, D. V., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+resistance+of+ALR/Lt+mouse+beta+cells+to+autoimmune+destruction:+role+for+beta+cell-expressed+resistance+determinants.">Unusual resistance of ALR/Lt mouse beta cells to autoimmune destruction: role for beta cell-expressed resistance determinants.</a> Proc Natl Acad Sci U S A. , 235-240 (2001).</li><li>Szabadkai, G., Duchen, 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=Mitochondria+mediated+cell+death+in+diabetes.">Mitochondria mediated cell death in diabetes.</a> Apoptosis. 14, 1405-1423 (2009).</li><li>Ly, J. D., Grubb, D. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+membrane+potential+(deltapsi(m))+in+apoptosis;+an+update.">The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update.</a> Apoptosis. 8, 115-128 (2003).</li><li>Dudek, N. L. <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-cells+from+T-cell+receptor+transgenic+NOD8.3+mice+destroy+beta-cells+via+the+perforin+and+Fas+pathways.">Cytotoxic T-cells from T-cell receptor transgenic NOD8.3 mice destroy beta-cells via the perforin and Fas pathways.</a> Diabetes. 55, 2412-2418 (2006).</li><li>Pakala, S. V., Chivetta, M., Kelly, C. B., Katz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+autoimmune+diabetes+the+transition+from+benign+to+pernicious+insulitis+requires+an+islet+cell+response+to+tumor+necrosis+factor+alpha.">In autoimmune diabetes the transition from benign to pernicious insulitis requires an islet cell response to tumor necrosis factor alpha.</a> J Exp Med. 189, 1053-1062 (1999).</li></ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by University of Florida Institutional Animal Care and Use Committee.

Cytotoxic T cell mediated beta cell death is the main pathophysiology of T1D 5. CML assays using 51Cr release allow us to study the degree of effector-target response 6. However, the detailed process and pathways of T cell-mediated beta cell death is still not fully understood. Since mitochondria are critical for beta cell function and death7, we focused on mitochondrial changes during the visual CML. Mitochondrial membrane potential dissipation is an early and irreversible...

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>Cr-51</td>
 <td>Perkin Elmer</td>
 <td>NEZ030500IMC</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>PBS</td>
 <td>Cellgro</td>
 <td>21-040-60</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Tetramethyl Rhodamine Methyl Ester (TMRM)</td>
 <td>Invitrogen</td>
 <td>T668</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Picogreen</td>
 <td>Invitrogen</td>
 <td>P7581</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>AI4 mimotope</td>
 <td>mimotopes.com</td>
 <td>amino acid sequence: YFIENYLEL</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>IL-2</td>
 <td>R &amp; D</td>
 <td>485-MI</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>DMEM</td>
 <td>Invitrogen</td>
 <td>11885</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>FBS</td>
 <td>HyClone</td>
 <td>SH30071.03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Bovine Serum Albumin</td>
 <td>Sigma</td>
 <td>A8412</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>HEPES</td>
 <td>Cellgro</td>
 <td>25-060-Cl</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>MEM Non-Essential Amino Acids</td>
 <td>GIBCO</td>
 <td>11140</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Penicillin/Streptomycin</td>
 <td>Gemini</td>
 <td>400-109</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Phenol red-free DMEM</td>
 <td>Sigma</td>
 <td>D5303</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>CO2 Incubator</td>
 <td>Thermo Fisher</td>
 <td>HeraCell 150i</td>
 <td>Kept sterile for cell culture</td>
 </tr>
 <tr>
 	<td>Biological safety cabinet</td>
 <td>Thermo Fisher</td>
 <td>Forma 1440</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Disposable culture tubes, 6x50 mm Lime Glass</td>
 <td>Fisher</td>
 <td>14-958-A</td>
 <td>&nbsp;</td>
 </tr> 
 <tr>
 	<td>Gamma Counter</td>
 <td>Perkin Elmer</td>
 <td>Wizard 1470 Automatic Gamma Counter</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Confocal microscope </td>
 <td>Zeiss</td>
 <td>LSM 510 Meta Confocal</td>
 <td>With motorized stage and live cell chamber</td>
 </tr>
 <tr>
 	<td>Pipette (1ml, 200&mu;l, 20&mu;l,10&mu;l, 2&mu;l)</td>
 <td>Eppendorf</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Tubes (Amber)</td>
 <td>Fisher</td>
 <td>50819772</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Centrifuge</td>
 <td>Sorvall Legend RT+</td>
 <td>75004377</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Hemacytometer</td>
 <td>Fisher Scientific</td>
 <td>267110</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Upright Microscope</td>
 <td>Zeiss</td>
 <td>Axioskop40</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>NOD.Cg-Rag1tm1Mom Tg(TcraAI4)1Dvs/DvsJ Mice</td>
 <td>The Jackson Laboratory</td>
 <td>004347</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>NOD.Cg-Rag1tm1Mom Tg(TcrbAI4)1Dvs/DvsJ Mice</td>
 <td>The Jackson Laboratory</td>
 <td>004348</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>NOD-AI4&alpha;/ &beta; F1 mice</td>
 <td>N/A</td>
 <td>N/A</td>
 <td>Bred in UF animal facility</td>
 </tr> 
 </tbody>
</table>

methods to assess beta cell death mediated by cytotoxic t lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Watch this Scientific Journal Video about Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes at JoVE.com

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

methods-to-assess-beta-cell-death-mediated-by-cytotoxic-t-lymphocytes

Research

JoVE Journal

Immunology and Infection

13.3K Views.  University of Florida. Клеточный lymphocytotoxicity (ХМЛ), анализы могут быть использованы для тестирования аутореактивных ответы и изучение механизмов смерти клеток в пробирке. Тем не менее, с использованием живых-клеточной конфокальной микроскопии изображений с флуоресцентными красите?...

Video: Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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.

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 ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...