This work was supported by grants to Keith L. Knutson from the Susan G. Komen for the Cure Foundation and NIH/NCI 9R01 CA152045 and P50-CA116201.

1. Generation of Short-term Human Antigen-specific CD8 T cell Lines7
<ol>
 <li>Separate peripheral blood mononuclear cells (PBMCs) from the blood of normal healthy HLA-A2 positive donors using Ficoll-paque plus.</li>
 <li>Add 2 ml/well of PBMCs to 6-well plates at 2 x 106 cells/ml.</li>
 <li>Add HLA-A2-binding HER-2/neu p369-377 peptide (amino acid sequence KIFGSLAFL) or HLA-A2-binding influenza p58-66 peptide (GILGFVFTL) directly to the wells at a final concentration of 10 &mu;g/ml and place in an incubator at 37 &deg;C with 5% CO2.</li>
 <li>To help stimulate and expand T cells, add, every 2 days, 250 &mu;l/well fresh media supplemented with human recombinant IL-2 (stock: 50 units/&mu;l) to achieve a final concentration of 50 units/ml in the culture media.</li>
 <li>Re-stimulate the culture cells with 2 x 106 cells/ml irradiated autologous PBMCs pulsed for 2 hours with 10 &mu;g/ml of the same peptides used in Step 1.3 and add these cells to cultures at 1 ml/well one week after the first peptide stimulation.</li>
 <li>Add human recombinant IL-2 and fresh media, as described in Step 1.4, into the existing cultures every 2 days.</li>
 <li>Re-stimulate the cells with peptide and irradiated autologous PBMCs as described in Step 1.5 one week after the second peptide stimulation.</li>
 <li>Prepare to use cells six days after the last re-stimulation at the earliest.</li>
</ol>
2. Preparation of Breast Cancer Cells
<ol>
 <li>Place the xCelligence impedance measuring station (xCelligence station) in an incubator at 37 &deg;C with 5% CO2.</li>
 <li>Harvest human tumor cells (SKBR3, BT20, MCF7 or HCC1419) that are 75% confluent using 0.25% trypsin and centrifugation (1,000 rpm for 5 minutes).</li>
 <li>Count tumor cells using a hemocytometer and reconstitute them in RPMI tumor media (supplemented with 10% fetal bovine serum (FBS) and 500 units/ml IFN-gamma) at a concentration of 7.5 x 103 tumor cells/100 &mu;l.</li>
 <li>Program the xCelligence software by setting up the layout page to determine well layout and by setting up the schedule page to take impedance readings every 5 minutes for a 40 hour period. For the first 18 hours, the xCelligence system will take impedance readings of the tumor cells only.</li>
 <li>Add 100 &mu;l of RPMI tumor media to the xCelligence E-plates and perform a background reading by measuring impedance in the absence of cells.</li>
 <li>Add tumor cells to the E-plates at 100 &mu;l per well and place in the xCelligence station.</li>
 <li>Press the start button on the xCelligence software to begin taking impedance readings of the tumor cells, which immediately start adhering to the E-plates.</li>
</ol>
3. Co-culture of Tumor with CD8 T cells
<ol>
 <li>Once the tumor cells have attached and doubled to 1.5 x 104 tumor cells per well (approximately 18 hours after initially being plated), harvest T cells prepared in Section 1 by gentle scraping and agitation</li>
 <li>Centrifuge at 1,000 rpm for 5 minutes and count T cells using a hemocytometer and reconstitute them in RPMI media with 10% FBS at various ratios, starting at a maximum ratio of 1 tumor cell to 40 T cells and dilute 2-fold until a ratio of 1 tumor cell to 1.25 T cells is reached. For example, there are 1.5 x 104 tumor cells per well. To achieve a 1:40 ratio, add 6 x 105 T cells per well and dilute the T cells 2-fold until a ratio of 1.5 x 104 tumor cells to 1.875 x 104 T cells per well (1:1.25 ratio) is achieved.</li>
 <li>Pause the xCelligence station and remove the E-plate.</li>
 <li>Remove the media in the wells with a pipet and add 200 &mu;l of media alone or the appropriate ratio of T cells to the wells.</li>
 <li>Place the E-plate back in the xCelligence station and continue the xCelligence software program so impedance readings are taken every 5 minutes for approximately 20 hours after T cell addition.</li>
 <li>Normalize the results at the end of the assay.</li>
</ol>
4. Chromium Release Assay (CRA)7
<ol>
 <li>Follow institutional radiation safety procedures when working with 51Cr and 51Cr-labeled target cells. Always use appropriate shielding to reduce radiation exposure.</li>
 <li>Pulse tumor cells for 2 hours at 1 x 106 tumor cells/ml with 10 &mu;l/ml fresh 51Cr (0.005 Ci/ml).</li>
 <li>Wash tumor cells in 10 ml of RPMI media with 10% FBS and centrifuge at 1,000 rpm for 5 minutes. Add tumor cells to a 96-well round-bottom plate at 1 x 105 tumor cells in 100 &mu;l /well.</li>
 <li>Add T cells at various ratios, starting at 1 tumor cell to 40 T cells and dilute 2-fold until a ratio of 1 tumor cell to 1.25 T cells is achieved. For example, there are 1 x 105 tumor cells per well. To achieve a 1:40 ratio, add 4 x 106 T cells per well and dilute the T cells 2-fold until a ratio of 1 x 105 tumor cells to 1.25 x 105 T cells per well (1:1.25 ratio) is achieved.</li>
 <li>Add spontaneous and maximum tumor cell controls to the plate. In order to calculate the spontaneous release of 51Cr from the tumor cells, add six wells each containing 1 x 105 tumor cells in 100 &mu;l to 100 &mu;l of RPMI media with 10% FBS. To calculate maximum release of 51Cr from the tumor cells, add six wells each containing 1 x 105 tumor cells in 100 &mu;l to 100 &mu;l 0.1% Triton X-100 in PBS, which lyses all cells.</li>
 <li>Incubate the plates for 5 hours at 37 &deg;C with 5% CO2.</li>
 <li>After incubation, remove 50 &mu;l of the supernatant from each well and add it to a Luma plate.</li>
 <li>Dry the plates overnight and measure CPM using a Gamma Counter.</li>
</ol>
5. Percent Lysis Calculation
<ol>
 <li>Impedance assay: Take the impedance reading, reported as the cell index (CI), at various time points and use the following formula to determine percent lysis: (CI tumor only - (CI tumor + T cells)) / (CI tumor only) *100.</li>
 <li>CRA: Use the following formula to determine percent lysis: (CPM experimental - CPM spontaneous) / (CPM maximum - CPM spontaneous)*100.</li>
</ol>
6. Representative Results
T cells alone do not increase impedance
T cells, in general, do not adhere to most solid surfaces and do not require adherence for activation and proliferation. Therefore, it was hypothesized that T cells should not contribute to impedance on the xCelligence E-plates. To test this, wells were loaded with either SKBR3 breast cancer cells or freshly purified human CD8 T cells. Impedance was measured over the course of 40 hours and a representative example demonstrating essentially little effect of T cells is shown in Figure 1A. In contrast, it could be argued that T cells may diminish background impedance, albeit only slightly. Despite that, co-culture revealed that the process of adding T cells at 18 hours following initiation of impedance measurements resulted in an impedance disruption (Figure 1B). Other studies revealed that disruptions were due in part to handling since the diminution in signal could be reduced by ensuring that temperature of the T cell-containing solution was the same as the interior of the incubator.
Reductions in impedance mediated by T cells are dose-dependent.
The utility of an impedance-based assay to compare samples for cytotoxic T cell activity requires the ability to distinguish between different concentrations of antigen-specific T cells. To test whether this is possible, SKBR3 cells were co-cultured with varying concentrations of T cells derived from a HER-2/neu p369-specific T cell line. As shown in Figure 2A, reductions in impedance were dependent on the dose of T cells. Figure 2B shows that the cell indices at 10 hours follow a simple second order polynomial over the range of T cells. Thus, the xCelligence station monitors CD8 T cell mediated death of SKBR3 tumors in real time as a drop in cell index.
The impedance-based assay detects antigen-specific T cells
To determine if reductions in impedance of SKBR3, which is a HER-2/neu and HLA-A2 positive breast cancer tumor cell line, by HER-2/neu p369-specific T cells were antigen specific, separate wells containing SKBR3 were also incubated with T cells derived from an influenza (FLU)-specific T cell line. The FLU specific-T cells served as a non-specific control, being HLA-A2 positive, but not having any ability to recognize HER-2/neu. As shown in Figure 3, there is a significant difference between the lytic activity of HER-2/neu p369-specific T cells as compared to FLU-specific T cells (p&lt;0.0001). In some cases, T cells not specific for tumor (e.g. FLU-specific or anti-CD3/anti-CD28 stimulated) demonstrated a low level of lytic activity (Figures 3A-B). T cells can kill in one of two ways, either non-specifically through Fas:Fas ligand interactions or antigen-specifically through the release of granzymes and perforins. To further corroborate the notion that HER-2/neu-specific T cells were killing in an antigen-specific fashion, we incorporated a blocking antibody to Fas ligand. As shown in Figure 3B, the antibody did not reduce the lytic activity of the HER-2/neu p369-specific T cells. The antibody could inhibit interactions between Fas and Fas Ligand, as demonstrated with T cells that were non-specifically activated with anti-CD3/CD28 beads. These results suggest that when tumor-specific T cells are tested with non-tumor-specific T cells, Fas ligand blockade may be needed to reduce non-TCR mediated interactions to a minimum. Alternatively, when using short-term cultured T cells lines in which only a fraction of the T cells are specific for the antigen used for ex vivo generation, the possibility of MHC mismatch-mediated T cell activation could occur leading to non-specific lysis. In this case, the addition of antibodies that block the irrelevant HLAs may be warranted. To determine if the p369-specific T cells were killing tumor cells in an HLA class I dependent manner, either anti-HLA-class I antibody or isotype control antibody were added to the co-cultures. As shown in Figure 3C, lysis by p369-specific T cells was suppressed by inclusion of antibody demonstrating HLA class I restriction. Lastly, since the development of this assay was largely done using SKBR3, it was important to ascertain if other adherent HLA-A2 and HER-2/neu expressing tumor cells could be killed by p369-specific T cells. Thus, T cells were prepared and added at a 1:5 ratio to the HLA-A2 and HER-2/neu-expressing tumor cells, SKBR3, MCF-7, and HCC1419 cells. The HLA-A2 negative tumor cell line, BT20 was used as a negative control. As shown in Figure 3D, all of these additional tumor cells, except BT20 were also killed as assessed by impedance. Thus, the method appears to be useful for multiple target adherent tumor cells.
The impedance-based assay is more sensitive than the standard chromium release assay in detecting cytotoxic T cell activity
The chromium (51Cr) release assay (CRA) that measures cytolytic activity has long been the gold standard by which to monitor CD8 T cell responses. In this assay, target cells (e.g. tumor cells) are labeled with 51Cr. In most cases, healthy target cells can retain the majority of the radiolabel over the course of the assay. CD8 T cells are added to the target cells, usually at varying concentrations, and killing of the target cells is detected by release of the 51Cr into the medium. Aside from the fact that it uses hazardous radiation, two major problems with the CRA are a lack of sensitivity and that the assay typically requires large amounts of CD8 T cells, limiting its utility. To determine if the impedance-based assay was more sensitive than the CRA, HER-2/neu p369-specific T cells were generated in vitro and applied, in varying amounts, to either E-plates containing unlabeled tumor cells or standard polypropylene plates containing 51Cr-labeled target cells. Measurements, either impedance or free chromium, were measured at 5 hours following T cell addition. Figure 4, a representative example, shows the impedance assay outperforms the chromium release.
The intra-assay variation of the impedance-based assay is typically below 20%.
Intra-assay variation was examined, since wider use of this assay will be largely determined by its reproducibly and variation (Figure 5). Two p369-specific T cell lines were generated independently 30 days apart and added in triplicate over a range of tumor cell to T cell ratios. The inset graph in Figure 5 shows that the lines were largely equivalent in terms of lysing SKBR3 cells. The % coefficient of variation was calculated at each cell ratio and plotted. As shown in Figure 5, between 1:40 and 1:5 ratios the % coefficients of variation (CV) were below 15%. At the 1:2.5 and 1:1.25, however, the CVs were high or unpredictable. Because of extensive biologic variation, it is not possible to measure inter-assay variability with primary T cell lines.
<img src="/files/ftp_upload/3683/3683fig1.jpg" alt="Figure 1"/>
 Figure 1. Impedance needs to be normalized following the addition of the T cells. Panel A: demonstrates that tumor cells (red line) and not T cells (blue line) are responsible for the impedance. Shown are the impedance tracings over 40 hours for either tumor or T cells alone. Similar results were obtained from a second experiment. Note that the signal perturbation at approximately 20 hours represents the point of addition of T cells to the wells not containing tumor cells. Panel B: shows that impedance measurements are transiently disrupted with the addition of T cells to tumor cells. This requires normalization at some time point following the addition of T cells (See point of normalization marked in graph). Traces are composed from duplicate data points. The trace for tumor cells alone is shown in red. The other traces represent tumor cells co-cultured with HER-2/neu p369-specific T cells (Blue: ratio of 1.25 T cells/tumor cell, Green: ratio of 40 T cells/tumor cell). Each trace is calculated from duplicate data points collected every 5 minutes through the duration of the culture. Results are representative of 3 separate experiments. <a href="https://www.jove.com/files/ftp_upload/3683/3683fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/3683/3683fig2.jpg" alt="Figure 2"/>
 Figure 2. Reduction in impedance mediated by T cells is dose dependent. Panel A: Shown are the impedance traces of SKBR3 tumor cells incubated alone or with varying concentrations of tumor-specific T cells. Each trace is calculated from triplicate data points collected every 5 minutes through the duration of the culture. Results are representative of 3 separate experiments. Panel B: Shown are the cell indices at 10 hours across all T cell/tumor cell concentrations. The dashed line represents the cell index for tumor cells alone. Each symbol is the mean (&plusmn;s.e.m.) of triplicate determinations. Results are representative of 3 separate experiments. <a href="https://www.jove.com/files/ftp_upload/3683/3683fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/3683/3683fig3.jpg" alt="Figure 3"/>
 Figure 3. The impedance based assay detects antigen-specific cytotoxic T cell responses. Panel A shows the level of lysis of SKBR3 cells by HER-2/neu p369-specific T cells (Red) or FLU-specific T cells (Blue) at 5, 10, and 15 hours following the co-culture. Each data point is the mean (&plusmn;s.e.m.) of triplicate measurements. Results are representative of 3 separate experiments. Panel B shows lysis of SKBR3 by p369-specific T cells or non-specifically activated T cells, demonstrating that antigen-specific lysis is not due to Fas-Fas ligand interactions. Black bars represent co-cultures that contained a Fas ligand antibody. Each bar is the mean (&plusmn;s.e.m.) lysis in triplicate at 5 hours following the addition of T cells. Results are representative of 3 separate experiments. Panel C shows lysis 10 hours after T cell addition of SKBR3 by p369-specific T cells is HLA-class I dependent. Either HLA-class I blocking or isotype control was added during co-culture. Each bar is the mean (&plusmn;s.e.m.) lysis in triplicate. This experiment was repeated twice with similar results. Panel D shows % lysis 10 hours after T cell addition of several HLA-A2+, HER-2/neu-expressing tumor cell lines by p369-specific T cells. BT20, a HLA-A2-negative breast cancer cell line was used as a negative control. Each spot represents a unique experiment calculated from triplicate determinations. <a href="https://www.jove.com/files/ftp_upload/3683/3683fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img src="/files/ftp_upload/3683/3683fig4.jpg" alt="Figure 4"/> 
Figure 4. The impedance based assay is more sensitive than the chromium release assay for the detection of tumor antigen-specific T cells. Shown are % lysis of SKBR3 in response to varying concentrations of p369-specific T cells as measured at 5 hours following tumor and T cell mixing using both the impedance-based assay and the chromium release assay. Each symbol is the mean (&plusmn;s.e.m) of three replicates. Results are representative of 3 separate experiments.
<img src="/files/ftp_upload/3683/3683fig5.jpg" alt="Figure 5"/> 
Figure 5. % intra-assay coefficients of variation of the impedance-based T cell cytotoxicity assay. Shown are mean (&plusmn;SD) % intra-assay coefficients of variation in lysis of SKBR3 over a range of concentrations of antigen-specific T cells. Each symbol is the mean of three replicates. Numbers shown in the graph are the mean % CVs. Inset panel shows the mean (&plusmn;s.e.m, n=3) % lysis of SKBR3, at varying concentrations of p369-377-specific T cells. Two independently generated p369-specific T cell lines using T cells from two separate donors are shown.

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Production and Free Access of this video-article is sponsored by Roche Diagnostics.

CD8 T cells are important to the adaptive immune response, being able to directly kill infected or malignant cells4. While there are several ways to measure the numbers of antigen-specific CD8 T cells (e.g. ELIspot, tetramer, flow cytometry), often it is helpful to know whether these cells are functionally cytotoxic3. The most recognizable assay for assessing cytotoxic function is the CRA, which is considered the gold standard assay4. Major limitations of the CRA are (1) the requirement of radioactivity, (2) lack of sensitivity, (3) lack of mechanistic information, (4) requirement for large amounts of cells, and (5) laborious set up. Thus, over the past decade, there has been interest in adopting new strategies to minimize these limitations. Newer approaches include those that measure caspase release4, BLT esterase activity5, and surface expression of CD1076. The present manuscript suggests that the impedance-based assay, performed on the Roche xCelligence system, may also be useful as an alternative to the CRA. The ability to adapt the impedance-based approach rests on the observation that T cells, which do not adhere tightly to surfaces, do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. The labor required for the use of the impedance assay system is expected to be significantly less primarily due to the elimination of the regulatory concerns for the use of radioactivity. The only notable drawback of the impedance-based system is that an outlay of $50,000 to $60,000 is required for purchase of the xCelligence unit. However, the xCelligence unit eliminates the need for a radiation counter and is far more versatile, being useful for assessing cellular response to drugs and migration in addition to T cell cytotoxicity 8.
The observations that the diminution in tumor cell impedance is dose-dependent, as depicted in Figure 2, with a reasonably steep slope following a simple secondary quadratic function suggests that the assay could be used in limiting dilution assays (LDAs) to estimate the frequency of cytotoxic cells9. One advantage with the impedance-based approach is that the number of T cells and tumor cells required is less than the CRA, making LDA potentially more feasible. For example, in the CRA, at a 1:40 ratio, 1 x 105 tumor cells per well are plated which requires 4 x 106 total T cells per well. However, in the impedance-based assay, only 1.5 x 104 tumor cells per well are needed, which consumes only 6 x 105 total T cells per well. Thus, the impedance-based assay uses 85% less T cells and tumor cells. This reduction suggests that the impedance-based system could be adapted to immunologic monitoring in human clinical trials where the volumes of blood draws may be limited. Also, while the CRA is limited to a single time point, the impedance-based assay collects continuous real-time data, allowing the user to compare multiple lysis time points on the same experiment with minimal additional labor and cells. Frequency calculations could potentially be combined with other strategies that do not measure function such as tetramer analyses to establish the ratio of functional cytotoxic T cells to total antigen-specific CD8 T cells10. One of the most appealing features of the impedance-based approach is that, unlike most other assays, cytotoxicity can be monitored in real time, potentially making it easier to study cellular interactions with the use of inhibitors or antibodies. Another is that the method may be suitable for tumor cells that resist labeling. Lastly, since the non-killed tumor cells are not labeled and remain sterile, further harvest and study of the T cell-resistant cells is possible.
In summary, while both systems provide tumor killing data in response to specific T cells, the impedance-based assay is more sensitive than the CRA and provides a more flexible platform.

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Ficoll-Paque Plus </td>
 <td>GE Healthcare </td>
 <td>17-1440-03 </td>
 <td>Used to isolate PBMCs from blood </td>
 </tr>
 <tr>
 <td>RPMI 1640 </td>
 <td>Cellgro </td>
 <td>10-040-CV </td>
 <td>Cell culture media </td>
 </tr>
 <tr>
 <td>FBS </td>
 <td>Denville Scientific </td>
 <td>FB5001-H </td>
 <td>Cell culture media </td>
 </tr>
 <tr>
 <td>Human IL-2 </td>
 <td>eBioscience </td>
 <td>14-8029-81 </td>
 <td>For stimulating growth of T cells </td>
 </tr>
 <tr>
 <td>Human Interferon gamma </td>
 <td>Cell Sciences </td>
 <td>CR2026 </td>
 <td>For stimulating MHC expression in tumor cells </td>
 </tr>
 <tr>
 <td>HER-2/neu p369-377 peptide </td>
 <td>Mayo Clinic peptide synthesis core </td>
 <td>amino acid sequence KIFGSLAFL </td>
 <td>Binds to HLA-A2 </td>
 </tr>
 <tr>
 <td>Influenza p58-66 peptide </td>
 <td>Mayo Clinic peptide synthesis core </td>
 <td>amino acid sequence GILGFVFTL </td>
 <td>Binds to HLA-A2 </td>
 </tr>
 <tr>
 <td>Human CD3/CD28 Dynabeads </td>
 <td>Invitrogen </td>
 <td>111-61D </td>
 <td>For non-specific T cell activation and expansion </td>
 </tr>
 <tr>
 <td>Chromium-51 </td>
 <td>MP Biomedicals </td>
 <td>62015 </td>
 <td>CRA labeling reagent </td>
 </tr>
 <tr>
 <td>E-plate </td>
 <td>Roche Applied Science </td>
 <td>05469830001 </td>
 <td>For xCelligence impedance unit </td>
 </tr>
 <tr>
 <td>Luma plate 96 </td>
 <td>Perkin Elmer </td>
 <td>6005630 </td>
 <td>For measuring radioactivity in the Top Count NXT. </td>
 </tr>
 <tr>
 <td>xCelligence RTCA DP Station </td>
 <td>Roche Applied Science </td>
 <td>RTCA DP </td>
 <td>Impedance unit </td>
 </tr>
 <tr>
 <td>Top Count NXT </td>
 <td>Perkin Elmer </td>
 <td>C990601 </td>
 <td>Scintillation &amp; Luminescence Counter </td>
 </tr>
 <tr>
 <td>Fas Ligand antibody</td>
 <td>BD Biosciences</td>
 <td>556372</td>
 <td>For blocking Fas-Fas ligand interactions</td>
 </tr>
 <tr>
 <td>HLA-A2 BB7.2 antibody</td>
 <td>BD Biosciences</td>
 <td>551230</td>
 <td>For blocking HLA class I</td>
 </tr>
 <tr>
 <td>Mouse IgG2b, &kappa;</td>
 <td>BD Biosciences</td>
 <td>555740</td>
 <td>Isotype control for HLA class I</td>
 </tr>
 <tr>
 <td>SKBR3 tumor line</td>
 <td>ATCC</td>
 <td>HTB-30</td>
 <td>Adenocarcinoma</td>
 </tr>
 <tr>
 <td>MCF7 tumor line</td>
 <td>ATCC</td>
 <td>HTB-22</td>
 <td>Adenocarcinoma</td>
 </tr>
 <tr>
 <td>HCC1419 tumor line</td>
 <td>ATCC</td>
 <td>CRL-2326</td>
 <td>Primary ductal carcinoma</td>
 </tr>
 <tr>
 <td>BT20 tumor line</td>
 <td>ATCC</td>
 <td>HTB-19</td>
 <td>Carcinoma</td>
 </tr>
 </tbody>
</table>

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.

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

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 ...

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Immunology and Infection

24.9K Views.  College of Medicine, Mayo Clinic. 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.

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

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.

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 ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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 ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...