Supported in part by NIH R01 CA121258, R01 CA125244, R01 CA154256, NIH/NCATS UCLA CTSI Grant Number UL1TR000124, USAMRMC W81XWH-08-1-0734, and the Joan S. Holmes Memorial Research Fund. MJH and GCO received supported from the Joan S. Holmes Memorial Postdoctoral Fellowship at UCLA. The CSM device was obtained from Creative Scientific Methods: www.creative-sci.com. The lentiviral vector was received from the UCLA Vector Core, which is supported by CURE/P30 DK041301.

1. Slide Preparation
<ol>
	<li>Insert Cell Sedimentation Manifold slides into sterilization pouches and seal with autoclave tape. Face the Teflon-coated side of the slide the paper-side of the pouch to avoid plastic deposits on the wells.</li>
	<li>Autoclave pouches for 15 min at 121 &#176;C.</li>
	<li>Remove slide from sterilization pouch inside a biosafety cabinet and place in a sterile 150 x 15 mm sterile Petri dish. Up to four slides can fit per dish. Place a 35 x 10 mm Petri dish next to the slides. Add 2-3 ml of sterile H2O to provide humidification.</li>
	<li>Pre-coat slides with poly-D-lysine at 100 &#181;g/ml by using enough volume to cover the entire well. After one hr at RT, aspirate the solution and rinse the surface of the well twice by pipetting 1x PBS over the surface of the wells.</li>
	<li>Optionally, add fibronectin or other ECM components to ensure stronger tumor cell adherence. Add fibronectin at 5 &#181;g/ml in enough volume that the wells will not dry within a short period of time at RT.
	<ol>
		<li>After 1 hr, aspirate the leftover solution and wash twice by pipetting up and down with 1x PBS.</li>
	</ol>
	</li>
	<li>Keep PBS on the wells until tumor is ready for plating. Use slides the same day.</li>
	<li>Harvest a confluent flask of the desired adherent tumor cell type. Count viable cells using Trypan Blue dye exclusion by light microscopy and resuspend at 5 x 106 cells/ml in adequately-buffered growth medium&#8212;for example, Dulbecco&#8217;s minimal essential medium (DMEM) with 10% fetal bovine serum (FBS), L-glutamine and sodium pyruvate.</li>
	<li>If fluorescent imaging of the adherent cell population is desired, label the adherent cell population with a vital fluorescent dye or a cell proliferation dye by following the protocols provided by the manufacturer.</li>
	<li>Smoothly pipette 10 &#956;l of complete medium into each well of the slide taking care to avoid creating bubbles in the wells, as these can prevent uniform tumor cell adherence.</li>
	<li>Add 2.5-5.0 x 104 cells (5-10 &#956;l of cell suspension) to the medium in each well (Figure 1A). Adjust the optimal number depending on tumor cell type and the number of days of growth desired. Larger tumor cells or fast growing cells may require fewer cells initially. Bring well volume up to 40 &#956;l by adding medium and pipette up and down to ensure an even distribution of cells.</li>
	<li>After all wells are seeded, allow the tumor cells to adhere at RT, then place the lid on the 150 x 15 mm Petri dish and carefully move the dish to a 37 &#176;C/5% CO2 humidified incubator for at least 24 hr.</li>
	<li>Change the culture medium daily. Carefully pipette a portion of the spent medium from the monolayer and replace with fresh complete medium. 
	NOTE: Because of evaporation, more volume will need to be added than was taken out. Wells will be ready for assay when an even, confluent layer of cells is present. This is typically 1-3 days after initial seeding.</li>
	<li>Wash monolayer gently once with PBS as described in step 1.4 and either proceed to step 2 if monolayers are desired, or to step 1.13 below to extract ECM proteins from the monolayer.</li>
	<li>Make a 0.5% Triton-X (v/v) solution in complete medium and add 30 &#956;l to each well. Let monolayer digest in hood for 2 min, then wash twice with complete medium by pipetting as described above. 
	NOTE: The ECM layer may be visible by light microscopy (Figure 2). Use slides right away, or keep in complete medium in the humidified CO2 incubator for 1-2 days. Do not allow wells to dry out.</li>
</ol>
2. Sedimentation of Non-adherent T Cells onto Slide
<ol>
	<li>If fluorescent imaging of the non-adherent cell population is desired, label the non-adherent cell populations with a vital fluorescent dye, such as CFSE, Cell Tracker Red CMPTX, or eFluor 670 by following the protocols provided by the manufacturer at least 1 hr in advance of the assay. Alternatively, manipulate cells to express fluorescent proteins encoded by viral vectors.</li>
	<li>Autoclave the cell sedimentation manifold in an autoclave pouch as described in 1.2 above.</li>
	<li>Remove the humidified chamber containing Teflon coated slides from the incubator and place in biological safety cabinet.</li>
	<li>Wash wells with 1x PBS by pipetting up and down, then add 45 &#956;l of complete medium containing at least 10% serum.</li>
	<li>Remove the manifold from the sterilization envelope and carefully slide in over the wells until the &#8216;hook&#8217; at the end of the manifold touches the bottom of the slide (Figure 1B).</li>
	<li>Ensure that the culture medium is visible in each of the channels. If none is seen, take off the manifold and add more medium to the corresponding well, then replace and check again.</li>
	<li>Repeat step 2.5 for each channel of the manifold, or for as many wells as desired.</li>
	<li>Count non-adherent cells and resuspend at no less than 1.0-2.0 x 105/&#956;l. Draw up 1 &#956;l of cells and slowly pipette them into the channel (Figure 1C). Do this for each well desired.</li>
	<li>Leave the entire cell loaded apparatus, i.e., manifold and slide, inside the bio-hood for 20-30 min&#160;to allow the cells in the channel to settle.</li>
	<li>Remove the manifold by touching both ends and gently lifting it straight up from the slide so as to not disturb the cells focally seeded in the center of the wells (Figure 1D).</li>
	<li>Inspect each well to ensure that the non-adherent cells have been successfully seeded at the center of the well staying within the circumference of the manifold channel. The non-adherent cells will ideally slightly adhere to the monolayer or ECM and to each other, therefore gently moving the slide around will not cause the cell pellet to disperse. Do not jostle the slide.</li>
</ol>
3. Fluorescence Microscopy
<ol>
	<li>With appropriate experimental equipment, perform the imaging after sedimentation is confirmed. Ideally, use a microscope that is equipped with a CO2 and humidity chamber. Image at low power, i.e., 4X or 10X, to allow for multiple images to be taken of a larger area so that the entirety of the well can be seen.</li>
	<li>Acquire digital images using image capture software. Perform brightfield and/or multi-channel fluorescence imaging.</li>
	<li>If desired, set up a time-lapse to record images in different channels over a given area, or keep the slide humidified and in a 37 &#176;C/5% CO2 incubator and manually take out to image at the time points desired (Figure 1E &#38; F, recommended for longer time points).</li>
</ol>
4. Analysis and Display
<ol>
	<li>Using image editing software, merge light and fluorescent images into one layer so multiple cell types and markers can be seen in the same image. Drag and drop the image files for each individual color into the program and, when prompted, select the &#8216;Add Layer&#8217; option to create an image file with multiple layers.
	<ol>
		<li>At higher magnification, take, align, and stitch together images across the well digitally. Align files by looking at conserved regions between two images and overlaying one image on top of the other until a complete picture is generated.</li>
	</ol>
	</li>
	<li>Add micron bars to images during acquisition using the capture software to determine distance individual cells have migrated.</li>
	<li>To determine the cell migration at various times, make surface intensity fluorescence maps with image software, i.e., ImageJ, to quantify fluorescent T cell aggregation or spread over time.
	<ol>
		<li>To generate surface plots, take a layered image generated in step 4.1 and under the Layers window, uncheck all layers except the ones corresponding to the channel desired. For instance, if a surface plot is desired to visualize EMD expression, only the &#8216;green&#8217; layers corresponding to the filter used for this marker should be visible.</li>
		<li>Flatten the image and save it in a file format recognized (e.g., .png) and load the image into the program. To generate the surface plot, change the image type to 8-bit, change the brightness/contrast as needed to reduce background, and select the &#8216;Analyze/Surface Plot&#8217; option. The images in Figure 5 were generated by checking the &#8216;Shade&#8217;, &#8216;Draw Axis&#8217;, &#8216;One Polygon Per Line&#8217;, and &#8216;Smooth&#8217; checkboxes.</li>
		<li>Alternatively, take measurements using the radius or diameter from the focal cell deposit at time zero and compare to the cells at the outermost point at different times. The diameter of the Teflon wells is 6.0 mm.</li>
	</ol>
	</li>
</ol>

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A., Giese, 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+role+of+extracellular+matrix+in+human+astrocytoma+migration+and+proliferation+studied+in+a+microliter+scale+assay.">The role of extracellular matrix in human astrocytoma migration and proliferation studied in a microliter scale assay.</a> Clin Exp Metastasis. 12 (6), 405-415 (1994).</li><li>Osawa, H., Smith, C. A., Ra, Y. S., Kongkham, P., Rutka, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+membrane+cytoskeleton+cross-linker+ezrin+in+medulloblastoma+cells.">The role of the membrane cytoskeleton cross-linker ezrin in medulloblastoma cells.</a> Neuro-oncology. 11 (4), 381-393 (2009).</li><li>Berens, M. E., Beaudry, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radial+monolayer+cell+migration+assay.">Radial monolayer cell migration assay.</a> Methods Mol Med. 88, 219-224 (2004).</li><li>Giese, A., Rief, M. D., Loo, M. A., Berens, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+human+astrocytoma+migration.">Determinants of human astrocytoma migration.</a> Cancer Res. 54 (14), 3897-3904 (1994).</li><li>Vlodavsky, I., Levi, A., Lax, I., Fuks, Z., Schlessinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cell+attachment+and+morphological+differentiation+in+a+pheochromocytoma+cell+line+and+embryonal+sensory+cells+by+the+extracellular+matrix.">Induction of cell attachment and morphological differentiation in a pheochromocytoma cell line and embryonal sensory cells by the extracellular matrix.</a> Dev Biol. 93 (2), 285-300 (1982).</li><li>Hulkower, K. I., Herber, R. 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The authors declare they have no competing financial interests.

Tumor cells in a single cell suspension were pipetted into the wells of a Teflon-masked slide. The cells were allowed to adhere and then formed monolayers in a humidified 5% CO2, 37&#176;C incubator (Figure 1A). Established monolayers or ECM proteins derived from the monolayer could be harvested for these assays (Figure 1B). Effector T lymphocytes labeled with vital fluorescent dyes or transduced with vectors coding for EMD were seeded into the center of the well using a CSM. ...

The Radial Monolayer Cell Migration assay was originally developed to measure the infiltrative properties of adherent tumor cells1-4 on slides coated with extracellular matrix (ECM) proteins5-7 or with individual ECM components, such as fibronectin or laminin1,2. The technique involved seeding a single cell suspension of tumor cells in the center of wells using a stainless steel cell sedimentation manifold (CSM). After sedimentation, the tumor cells would adhere to the bottom of the well and the change in the diameter of the initial cell population over time was used to establish a rate of horizontal motility. The Radial Monolayer Cell Migration assay provided a visual advantage over other existing methods that employed transwell plates to assay the in vitro migratory capabilities of cells; these assays are non-conducive to imaging8. As well, it also provided a great amount of freedom in choosing the timepoints when migration is assessed, with no limit on the number of timepoints a researcher could choose to image after sedimentation.
Because the ability to migrate is an important functionality for non-adherent cells, especially in the area of immunotherapy or where they may be used as delivery vehicles for viral vectors, we adapted the use of the CSM to evaluate the migration of non-adherent cell types on tumor cell monolayers, in addition to ECM proteins. The added benefit of microscopically visualizing the migration of non-adherent cells on viable tumor cell monolayers, on complex ECM isolated from the tumor, or on individual ECM components makes this assay versatile. Assays that employ wells coated with a single extracellular protein do not reflect accurately the ECM tissue substrate or tumor the cells would migrate through in vivo.
Here, we used alloreactive cytotoxic T lymphocytes (alloCTL), sensitized to major histocompatibility complex (MHC) proteins using one-way mixed lymphocyte tumor cell reactions (MLTR) or mixed lymphocyte reactions (MLR)9, as our representative non-adherent cell type. We tested cells of both human and murine origin. When migration was measured on tumor monolayers, the tumor cells employed were either partially relevant targets, displaying some of the same MHC proteins found on the cell population used to sensitize the effectors, or fully relevant targets, with a full set of MHC molecules that the effectors had been sensitized towards. In some experiments, we used fluorescent CellTracker Red CMPTX or cell proliferation dye eFluor 670 to differentiate between effector and target cells. We also used transduction with viral vectors encoding for fluorescent proteins as an additional way to visualize the cells. For certain assays, we transduced the alloCTL with retroviral replicating vectors (RRV) coding for Emerald Green (EMD) fluorescent protein10,11; for others, tumor cells were transduced with lentiviral vectors coding for mStrawberry.
The alloCTL were seeded through a channel of the manifold into the center of either tumor cell monolayers or ECM harvested from tumor cell monolayers. Adherent and non-adherent cell interactions were visualized by light and/or by fluorescence microscopy over time. Disruption in the tumor cell monolayer at low power, or tumor cells with fragmented nuclei at high power were indicators of cell injury by lysis and apoptosis, respectively. We digitally created surface intensity fluorescent maps showing the migration of non-adherent fluorescing T cells over the monolayer cultures. We also noted the cytotoxicity engendered to the adherent glioma cell monolayer after cluster formation of the overlaid non-adherent alloCTL. As well, horizontal transduction of RRV-EMD from the alloCTL to the glioma monolayer was observed.

<table border="0" cellpadding="0" cellspacing="0" width="920">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:239px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:312px;">Teflon-masked microscope slides</td>
			<td style="width:203px;">Creative Scientific Methods</td>
			<td style="width:239px;">CSM002</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:312px;">Cell Sedimentation Manifold</td>
			<td style="width:203px;">Creative Scientific Methods</td>
			<td style="width:239px;">CSM001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Petri Dish, 150mm</td>
			<td style="width:203px;">Corning</td>
			<td style="width:239px;">430597</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Petri Dish, 35mm</td>
			<td style="width:203px;">Corning</td>
			<td style="width:239px;">430588</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">21-040</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">20 &#956;L Pipetman</td>
			<td style="width:203px;">Gilson</td>
			<td style="width:239px;">F123600</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">200 &#956;L Pipetman</td>
			<td style="width:203px;">Gilson</td>
			<td style="width:239px;">F123601</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">200 &#956;L pipette tips</td>
			<td style="width:203px;">VWR</td>
			<td style="width:239px;">89003-060</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Distilled, deionized water (sterile)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-055</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Trypsin</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-054-CL</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Celltracker Red CMPTX</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:239px;">C34552</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">TrypLE Express (optional)</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:239px;">12604</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Tumor cell culture media (e.g. DMEM)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">10-013</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">AIM-V serum-free media</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:239px;">12055-083</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Fetal Bovine Serum</td>
			<td style="width:203px;">Omega Scientific</td>
			<td style="width:239px;">FB-02</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Inverted microscope</td>
			<td style="width:203px;"></td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">SPOT Advanced</td>
			<td style="width:203px;">Diagnostic Instruments</td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Poly-D-Lysine</td>
			<td style="width:203px;">Millipore</td>
			<td style="width:239px;">A-003-E</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Fibronectin</td>
			<td style="width:203px;">BD</td>
			<td style="width:239px;">354008</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">31/2 x 51/4 Autoclave Pouches</td>
			<td style="width:203px;">Crosstex</td>
			<td style="width:239px;">SCXS2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Trypan Blue</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-900-CI</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Cell Proliferation eFluor 670</td>
			<td style="width:203px;">eBioscience</td>
			<td style="width:239px;">65-0840-85</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">ImageJ</td>
			<td style="width:203px;">NIH</td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Viral vectors encoding for fluorescent proteins can be used in addition to, or instead of, fluorescent dye. Viral transduction should be performed in advance of the motility assay. Both adherent and non-adherent cell types can be differentially-labeled. The protocol for transduction will depend on the type of vector employed. Here, we transduced the alloCTL in Figures 3, 5 and 6 with RRV-EMD at least two days in advance of the assay using the protocol described12</s...

Watch this Scientific Journal Video about Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes at JoVE.com

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

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 ...

radial-mobility-cytotoxic-function-retroviral-replicating-vector

Research

JoVE Journal

Immunology and Infection

7.7K vistas.  UCLA David Geffen School of Medicine. 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. 

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

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 ...

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 ...

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 ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...