Name: real time detection of in vitro tumor cell apoptosis induced by cd8+ t cells to study immune suppressive functions of tumor-infiltrating myeloid cells
Uploaded: 2019-01-29T12:30:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells at JoVE

This work was supported by grants from the Wellcome Trust (101067/Z/13/Z (JWP), 109657/Z/15/Z (TK), 615KIT/J22738 (TK), UK), and the MRC (MR/N022556/1 (JWP, TK), UK). NOC and DDS acknowledge support from the National Phenotypic Screening Centre Phenomics Discovery Initiative and Cancer Research UK (NOC)

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The authors have nothing to disclose.

This method is based on two separate co-culture steps: &#160;co-culturing CD8+ T cells with potential suppressor cells, and co-culturing the &#8216;pre-conditioned&#8217; CD8+ T cells with target tumor cells (Figure 1). The first co-culture step is quite similar to that for CD8+ T cell proliferation assays commonly used to determine the effect of suppressor cells on CD8+ T cell function. However, T cell proliferation does not always correlate with their cytotoxicity. For example, we have found that co-culture with M-MDSCs or MAMs increased rather than reduced proliferation of CD8+ T cells in the presence of CD3/CD28 activating antibodies (Supplementary Figure 3), whereas these pre-conditioned CD8+ T cells demonstrated reduced cytotoxicity against target cancer cells (Figure 4, Figure 5, Figure 6). These results highlight the importance of the evaluation of functional activity, evidenced by target cancer cell apoptosis, offered by this CD8+ T cell cytotoxicity assay.
In this assay, we have identified that CD8+ T cells requires approximately 15 h of co-culture in order to induce maximum apoptosis of E0771-LG mouse mammary tumor cells (Figure 5). This delay might be due to the lag between initial contact of effector cells with targets and accompanying immune synapse formation, as well as time required to induce apoptotic signals in targets as measured by activation of caspase-3 (Supplementary Movie 1). We also identified that the number of apoptotic tumor cells reached a plateau after 24 h, which is probably due to the elimination of targets by T cells and/or loss of fluorescent signal from dead cells. This capability to identify the time of peak apoptosis is one major advantage of this assay since determination of an optimal time point is important for appropriate comparisons between different conditions. In our case for example, the difference in cytotoxicity between control CD8+ T cells and MDSC/MAM-educated CD8+ T cells was much larger at 15 - 18 h compared to 72 h (Figure 5), and thus an endpoint experiment using a 72 h incubation period would yield misleading results.
This method also enables visualization of real-time effector-to-target cell interaction, which would provide greater insights into the mechanism underlying limited cytotoxicity of CD8+ T cells pre-incubated with suppressor cells. For example, we observed that CD8+ T cells pre-incubated with M-MDSCs or MAMs encountered and interacted with target tumor cells but did not always induce apoptosis (Supplementary Movie 4, Supplementary Movie 5, Supplementary Movie 6). Although we did not quantify this event, it would be feasible and interesting to quantify and compare the proportion of encounters and their interaction time in correlation with apoptosis induction. Another major advantage is that this method requires a small number of cells (e.g., 1 x 103 of target and 4 x 103 of effector cells per well). In fact, this protocol can be further miniaturized for the 384-well plate format if desired. Therefore, this assay is suitable for high throughput screening and experiments where cell numbers are limited such as in vitro testing using precious cells derived from in vivo or ex vivo samples.
On the other hand, a limitation of the current assay is the presence of significant numbers of dead effector cells in some conditions. In order to increase accuracy in distinguishing apoptosis of target cancer cells from that of effector CD8+ T cells, the nuclei of target cells are labeled and a nuclei size restriction (that excludes effector cells) is applied for data analysis in this assay (Figure 2). However, there are some instances where overlay of aggregates of (green) apoptotic CD8+ T cells onto non-apoptotic target cancer cells, which may confound the results. This limitation could be mitigated by use of a dead cell removal column on effector cells prior to co-culture with target tumor cells, assuming sufficient numbers of effector cells are available. With more complex microscopy systems, it may also be possible to reduce the false positive signal by labeling the effector CD8+ T cells with a fluorophore distinct from the target cell nuclei and the fluorogenic caspase-3 substrate.
So far, this protocol has been utilized to investigate the antigen non-specific activation of CD8+ T cells. Although MDSCs and TAMs in the tumor microenvironment suppress T cell functions through antigen non-specific mechanisms, MDSCs in the peripheral lymphoid tissues suppress T cell responses in an antigen specific manner18. To investigate immune suppressive functions of such cell types, an in vitro proliferation assay using CD8+ T cells from OT-1 transgenic mice is commonly used. In this assay, the OT-1 T cells (expressing ovalbumin (OVA) specific T cell receptor) are co-cultured with suppressor MDSCs in the presence of OVA peptides, which is applicable for the first culture in our cytotoxicity assay (i.e., activation of T cells in the presence or absence of suppressors). It is also feasible to manipulate target cancer cells to express OVA, which can induce antigen-specific cancer cell killing by OT-1 T cells. Therefore, the assay will also enable investigation of the MAM/MDSC-mediated suppression of antigen-specific T cell activation. It is also possible to apply the assay to investigate human cells, as activation antibodies against human CD3 and CD28 are commercially available, and a protocol to isolate human TAMs from clinical samples has been established19.
Collectively, this assay is quite versatile and can be used to examine cytotoxicity of other immune cell types. Currently, in our labs, it is being extended to examine antigen-dependent cell cytotoxicity under various conditions and is also being developed for high throughput screening.

It is known that CD8+ T cells can eliminate tumor cells when they exert their full cytotoxicity. After activation of the T cell receptor (TCR), CD8+ T cells proliferate and differentiate into cytotoxic effector cells. The expanded and activated CD8+ T cells secrete cytotoxic granules, including perforin and granzymes, that are transferred into target cells and initiate various lytic pathways such as caspase-3 mediated apoptosis1. CD8+ T cells can also induce tumor cell apoptosis by activating receptors on target cells, such as receptors for tumor necrosis factor-&#945; (TNF-&#945;), first apoptosis signal ligand (FasL), or TNF-related apoptosis-inducing ligand (TRAIL). Furthermore, the activated CD8+ T cells secrete interferon-&#947; (IFN-&#947;) that can suppress tumor cell proliferation and increase the sensitivity of tumor cells to CD8+ T cells via the up-regulation of FasL receptor1. Given the potential for CD8+ T tumor killing ability, several strategies to boost their cytotoxicity (e.g., checkpoint inhibitors, cancer vaccination, and adoptive transfer of chimeric antigen receptor (CAR) expressing T cells) have been established and shown significant therapeutic effects on certain types of cancer2. However, accumulating evidence suggests that tumor-infiltrating immune cells such as regulatory T cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) can suppress CD8+ T cell functions and restrict efficacy of immunotherapies3,4,5. To improve such immunotherapies, it is important to understand how immune suppressor cells limit CD8+ T cell cytotoxicity. The identification of CD8+ T cell suppression mechanisms as well as druggable targets to overcome it, will require the development and utilization of in vitro assays.
The gold standard method of measuring CD8+ T cell cytotoxicity is the chromium release assay in which the release of the radioactive probe (51Cr), from target cells that are lysed by CD8+ T cells, is determined6. However, this assay has several drawbacks including relatively low sensitivity, high background, inability to detect early apoptotic events, hazardous disposal problems, and limited compatibility with automated liquid handling and detection to support higher throughput applications. Another common method is flow cytometric analyses in which apoptosis of target tumor cells is detected by annexin V binding7. In this assay, it is possible to detect other parameters such as target cell death using propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) and effector cell activation indicated by CD107a or CD69 expression, in addition to the apoptosis in target cells7. However, this assay requires large numbers of suppressor cells compared to the chromium release assay. It also requires the detachment and disaggregation of adherent target cells and this can bias the results. Indeed, the chromium release assay or flow cytometric assay are not commonly used to investigate suppressor cell effects on T cell functions. Instead, the measurement of T-cell proliferation indicated by dilution of a fluorescent dye (e.g., CFSE) pre-loaded into T cells is frequently used to evaluate the inhibition of CD8+ T cell function by suppressor cells. Detection of IFN-&#947; production from cultured T cells is another standard method to evaluate the effects of suppressor cells on T cell activation8,9. However, the results from these assays do not necessarily correlate to the target cell killing ability of CD8+ T cells.
We present here an alternative functional assay to evaluate effects of suppressor cells, particularly macrophages in metastatic tumors, on the cytotoxicity of CD8+ T cells. This method determines cytotoxicity of CD8+ T cells, pre-cultured with or without the suppressor cells in the presence of anti-CD3/CD28 activating antibodies, by detecting tumor cell apoptosis, indicated by fluorescence from a fluorogenic caspase-3 substrate6 using automated time-lapse microscopy (Figure 1). This protocol has several advantages compared to other methods; it requires only a small number of cells, enables detection of adherent tumor cell death with high sensitivity, can image real-time effector-to-target interaction and is amenable to high throughput screening.
In this protocol, metastasis-associated macrophages (MAMs) and their progenitor monocytic-MDSCs (M-MDSCs) isolated from metastatic tumors in mice are used as suppressor cells. In mouse models of metastatic breast cancer, a distinct population of macrophages characterized as F4/80highLy6G-CD11bhighLy6Clow accumulates in the lung containing metastatic tumors. This macrophage population is infrequently found in the normal lung and thus called metastasis-associated macrophages (MAMs)10. In these mouse models, another myeloid cell population, defined as F4/80highLy6G-CD11bhighLy6Chigh, also accumulates predominantly in the metastatic lung where it gives rise to MAMs11. Based on their characteristics, the CD11bhighLy6Chigh MAM progenitor cells might represent M-MDSCs12.

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			<td>Gibco</td>
			<td>25300-054</td>
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			<td>BIO-RAD</td>
			<td>MCA497A647</td>
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			<td>Biolegend</td>
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			<td>Gibco</td>
			<td>41966-029</td>
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		<tr>
			<td>EasySep Mouse CD8+ T Cell Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19853</td>
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			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
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			<td>Biolegend</td>
			<td>100406</td>
			<td>Clone: GK1.5, Lot#: B179194, 0.5 &#956;L/1x10^6 cells</td>
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			<td>Geltrex Ready-to-Use</td>
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			<td>IncuCyte NucLight Red Lentivirus Reagent</td>
			<td>Essen BioScience</td>
			<td>4476</td>
			<td>Lenti viral particules encoding mKate2</td>
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			<td>IncuCyte ZOOM</td>
			<td>Essen BioScience</td>
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			<td>Detector (fluorescence microscope)</td>
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		<tr>
			<td>IncuCyte ZOOM 2018A</td>
			<td>Essen BioScience</td>
			<td></td>
			<td>Analysis software</td>
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		<tr>
			<td>L-Glutamine (100X)</td>
			<td>Gibco</td>
			<td>A2916801</td>
			<td></td>
		</tr>
		<tr>
			<td>Lung Dissociation Kit</td>
			<td>Miltenyi</td>
			<td>130-095-927</td>
			<td>Preparation of single cell suspension from the tumor-bearing lung</td>
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			<td>MycoAlert Mycoplasma Detection Kit</td>
			<td>Lonza</td>
			<td>LT07-318</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acid (100X)</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>NucView488</td>
			<td>Biotium</td>
			<td>10403</td>
			<td>Fluoregenic caspase-3 substrate</td>
		</tr>
		<tr>
			<td>PE anti-mouse Ly6G Antibody</td>
			<td>Biolegend</td>
			<td>127607</td>
			<td>Clone: 1A8, Lot#: B258704, 0.5 &#956;L/1x10^6 cells</td>
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		<tr>
			<td>PE/Cy7 anti-mouse CD11b Antibody</td>
			<td>Biolegend</td>
			<td>101216</td>
			<td>Clone: M1/70, Lot#: B249268, 0.5 &#956;L/1x10^6 cells</td>
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			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Penicillin Streptomycin for primary culture of cells</td>
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		<tr>
			<td>PerCP/Cy5.5 anti-mouse CD45 Antibody</td>
			<td>Biolegend</td>
			<td>103132</td>
			<td>Clone: 30-F11, Lot#: B249564, 0.5 &#956;L/1x10^6 cells</td>
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			<td>Polybrene (Hexadimethrine bromide)</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td></td>
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			<td>Puromycin</td>
			<td>Gibco</td>
			<td>A11138-03</td>
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			<td>RBC Lysis Buffer (10X)</td>
			<td>Biolegend</td>
			<td>420301</td>
			<td></td>
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			<td>Recombinant murine IL-2</td>
			<td>Peprotech</td>
			<td>212-12</td>
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			<td>Sodium pyruvate (100X)</td>
			<td>Gibco</td>
			<td>11360-070</td>
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		<tr>
			<td>TruStain fcX (anti-mouse CD16/32) Antibody</td>
			<td>Biolegend</td>
			<td>101320</td>
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		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>&#160;: Nikon 10X objective (resolution 1.22 &#181;m) 
			&#160;: Green channel excitation: 440 - 480 nm 
			&#160;: Green channel emission: 504 - 544 nm 
			&#160;: Red channel excitation: 565-605 nm 
			&#160;: Red channel emission: 625 - 705 nm</td>
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	</tbody>
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real time detection of in vitro tumor cell apoptosis induced by cd8+ t cells to study immune suppressive functions of tumor-infiltrating myeloid cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

This method is based on simple co-culture of target cancer cells with effector CD8+ T cells that have been pre-cultured with or without suppressor cells in the presence of anti-CD3/CD28 activating antibodies. It detects CD8+ T cell-induced cancer cell apoptosis over time following co-culture, thus enabling evaluation of effects of suppressor cells on cytotoxicity of CD8+ T cells.
Typically, cancer cells increase green fluorescence in their nuclei following activation of a nuclear-targeting caspase biosensor when these cells make contact with CD8+ T cells that are pre-activated by antibodies in the absence of suppressor cells (Figure 3; Supplementary Movie 1). Green fluorescence from the caspase substrate was detectable for at least 15 h after apoptosis was initiated. Some spontaneous apoptosis of effector CD8+ T cells was also observed over time even if these cells were cultured in isolation (Figure 4; Supplementary Movie 2). However, nuclei sizes of CD8+ T cells are smaller than those of cancer cells and thus apoptotic &#39;effector&#39; cells can be excluded from apoptotic &#39;target&#39; cell counts by a size restriction image analysis method (Figure 2 and Figure 4). Although some target cancer cells show a small rounded shape without green fluorescence, this does not affect the analysis as these cells are undergoing mitosis rather than apoptosis (Supplementary Movie 3), and thus are excluded from apoptotic &#39;target&#39; cell counts by a red/green overlap mask (Supplementary Figure 2). Spontaneous apoptosis of target cancer cells is occasionally found even in the single culture (Supplementary Movie 3). However, the co-culture of target cancer cells with pre-activated CD8+ T cells increased tumor cell apoptosis above the levels of spontaneous apoptosis in monoculture of cancer cells (Figure 4). Generally, when using an optimal ratio of target cancer cells to effector cells, a peak in the number of apoptotic target cancer cells can be observed (Figure 5A). This peak is more distinct when the data is expressed as the apoptotic fraction of the target cell population (Figure 5B). In this experiment the basal apoptosis of target tumor cells peaked at 24 h (with apoptotic fraction = 0.08), but CD8+ T cell-induced apoptosis reached a maximum level at 17 h (with apoptotic fraction = 0.66).
We further found that CD8+ T cells pre-incubated with MAMs or M-MDSCs could make contact with target cancer cells but this contact seemed to result in fewer instances of cancer cell apoptosis compared to CD8+ T cells pre-activated without suppressor cells (Figure 3 and Figure 4; Supplementary Movie 4 and Supplementary Movie 5). Although the CD8+ T cells pre-incubated with the myeloid cells occasionally induced cancer cell apoptosis, there was also some proliferation of target cancer cells that have not been stimulated to undergo apoptosis during the time course of the experiment (Supplementary Movie 6). Consistent with these findings, the peak fraction of apoptotic cancer cells cultured with CD8+ T cells pre-incubated with myeloid cells (apoptotic fraction = 0.38 at 23 h for MDSC-E and 0.25 at 20 h for MAM-E) was significantly lower than that of cancer cells cultured with CD8+ T cells which were not pre-incubated with the suppressor cells (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58841/58841fig1.jpg" /> 
Figure 1: A scheme showing the experimental procedure. Na&#239;ve splenic CD8+ T cells are cultured with anti-CD3/CD28 activating antibodies with or without metastasis-associated macrophages (MAMs) or monocytic-myeloid-derived suppressor cells (M-MDSCs). After 4 days, floating CD8+ T cells are collected and co-cultured with target cancer cells in the presence of fluorogenic caspase-3 substrate. Apoptotic cancer cells are detected under real-time fluorescence microscopy. Images shown were acquired using a live cell-imaging platform (refer to Table of Materials). <a href="https://www.jove.com/files/ftp_upload/58841/58841fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58841/58841fig2.jpg" /> 
Figure 2: Identification of apoptotic target cells distinct from apoptotic effector cells. Top row:Image acquisition in the red channel allows the identification of target cell nuclei by target detection mask (pink analysis mask). Middle row: Images acquired in the green channel indicate apoptotic effector and target cells.Asize restricted apoptosis mask (teal analysis mask; greater than 80 &#181;m2) allows single apoptotic effector cells to be excluded from the analysis. Bottom row: composite images merged red and green channels with phase contrast image (left) or red/green overlap mask (right). Identification of co-localized, size-restricted green fluorescence with red fluorescence (yellow analysis mask), allows more accurate detection of apoptotic target nuclei (yellow arrowhead) by excluding aggregates of apoptotic effector cells (white arrowheads). <a href="https://www.jove.com/files/ftp_upload/58841/58841fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58841/58841fig3.jpg" /> 
Figure 3: Interaction between CD8+ T cells and cancer cells. Stills from representative time-lapse movies of target E0771-LG_NLR cells (T) co-cultured with effector CD8+ T cells (E) at 4:1 effector/target ratio. MDSC-E and MAM-E indicate effector cells pre-incubated with M-MDSCs and MAMs respectively. Composite images (including images from phase contrast, red and green channels) are shown. Arrowheads are tracking the same cells through the different fields and time points. Yellow arrowheads: a target that associates with effectors and undergoes apoptosis, white arrowheads: a target that associates with effectors but does not undergo apoptosis. <a href="https://www.jove.com/files/ftp_upload/58841/58841fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58841/58841fig4.jpg" /> 
Figure 4: Detection of apoptotic cancer cells. Representative fields extracted from time-lapse movies at 18 h after imaging. Composite images (left; phase contrast, red and green channels) and images from red channel without (middle) or with (right) red/green overlap mask (yellow) are shown. Yellow dots in the right column represent apoptotic cancer cells. <a href="https://www.jove.com/files/ftp_upload/58841/58841fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58841/58841fig5.jpg" /> 
Figure 5: CD8+ T cell-induced cancer cell apoptosis. (A) Number of apoptotic cancer cells cultured with effector C8+ T cells at different effector to target ratio (E:T). (B) Apoptotic fraction of target cell population. Data are means &#177; SD. Mean area under the curve (AUC) is also shown. Unpaired t-test with Welch&#39;s correction was used to analyze the AUC. *P&#60;0.0001 compared to E:T=0:1. <a href="https://www.jove.com/files/ftp_upload/58841/58841fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58841/58841fig6.jpg" /> 
Figure 6: Effects of tumor-infiltrating myeloid cells on cytotoxicity of CD8+ T cells. (A) Number of apoptotic cancer cells (target: T) cultured with C8+ T cells (effector: E) at 4:1 of E:T ratio. CD8+ T cells were pre-cultured in the absence (black circle) or presence of monocytic-myeloid-derived suppressor cells (MDSC-E: blue circle) or metastasis-associated macrophages (MAM-E: red circle). Data are means &#177; SD. (B) Apoptotic fraction of target cell population. Data are means &#177; SD. Mean AUC is also shown. Unpaired t-test with Welch&#39;s correction was used to analyze the AUC. *P&#60;0.0001 compared to T only, #P&#60;0.0001 compared to E+T. <a href="https://www.jove.com/files/ftp_upload/58841/58841fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1. Gating strategy to isolate suppressor cells from the metastatic lung. (A) Representative dot plots to isolate monocytic myeloid-derived suppressor cells (M-MDSCs) and metastasis-associated macrophages (MAMs). The threshold of Ly6C level to distinguish MAMs (Ly6Clow) and M-MDSCs (Ly6Chigh) is based on that of resident alveolar macrophages (RMAC). (B) Purity of the sorted M-MDSCs (CD45+Ly6G-CD11b+Ly6Chigh) and MAMs (CD45+Ly6G-CD11b+Ly6Clow). <a href="https://www.jove.com/files/ftp_upload/58841/FigureS1_Rvd.pdf">Please click here to download this file.</a>
Supplementary Figure 2. Representative images of mitotic target cells. Stills from representative time-lapse movies of target E0771-LG_NLR cell mono-culture. Top: composite images including images from phase contrast, red and green channels. Bottom: composite images (red and green channels) with red/green overlap mask. <a href="https://www.jove.com/files/ftp_upload/58841/FigureS2_Rvd.pdf">Please click here to download this file.</a>
Supplementary Figure 3. Effects of tumor-infiltrating myeloid cells on proliferation of CD8+ T cells. (A) Representative histograms showing dilution of fluorescent labeling with CFSE in CD8+ T cells. Na&#239;ve splenic CD8+ T cells were isolated as described in Protocol-3 and labeled with 5 &#181;M of CFSE at 37 &#176;C for 15 min. The labeled T cells were cultured in the presence of IL-2 and anti-CD3/CD28 activating antibodies with or without myeloid cells as described in Protocol 4. After 4 days, green fluorescence in T cells was detected by flow cytometer. (B) Division index of CD8+ T cells calculated as previously described17. Data are means &#177; SEM. *P&#60;0.01 compared to control, #P&#60;0.05 compared to &#945;CD3/CD28 Ab. <a href="https://www.jove.com/files/ftp_upload/58841/FigureS3_Rvd.pdf">Please click here to download this file.</a>
Supplementary Movie 1. Movie of Figures 3 and 4; E+T. <a href="https://www.jove.com/files/ftp_upload/58841/MovieS1.mp4">Please click here to download this file.</a>
Supplementary Movie 2. Movie of Figures 3 and 4; Effector (E). <a href="https://www.jove.com/files/ftp_upload/58841/MovieS2.mp4">Please click here to download this file.</a>
Supplementary Movie 3. Movie of Figures 3 and 4; Target (T). <a href="https://www.jove.com/files/ftp_upload/58841/MovieS3.mp4">Please click here to download this file.</a>
Supplementary Movie 4. Movie of Figures 3 and 4; MDSC-E +T. <a href="https://www.jove.com/files/ftp_upload/58841/MovieS4.mp4">Please click here to download this file.</a>
Supplementary Movie 5. Movie of Figures 3 and 4; MAM-E +T. <a href="https://www.jove.com/files/ftp_upload/58841/MovieS5.mp4">Please click here to download this file.</a>
Supplementary Movie 6. Movie of Figures 3 and 4; MAM-E +T (proliferation). <a href="https://www.jove.com/files/ftp_upload/58841/MovieS6.mp4">Please click here to download this file.</a>

Watch this Scientific Journal Video about Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells at JoVE

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

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Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

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