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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<li>Hulkower, K. I., Herber, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+migration+and+invasion+assays+as+tools+for+drug+discovery%5BTitle%5D)+AND+%22Pharmaceutics%22%5BJournal%5D)">Cell migration and invasion assays as tools for drug discovery.</a> Pharmaceutics. 3 (1), 107-124 (2011).</li>
<li>Hickey, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implementing+preclinical+study+findings+to+protocol+design%3A+translational+studies+with+alloreactive+CTL+for+gliomas%5BTitle%5D)+AND+%22Am+J+Transl+Res%22%5BJournal%5D)">Implementing preclinical study findings to protocol design: translational studies with alloreactive CTL for gliomas.</a> Am J Transl Res. 4 (1), 114-126 (2012).</li>
<li>Tai, C. K., Wang, W. J., Chen, T. C., Kasahara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-shot%2C+multicycle+suicide+gene+therapy+by+replication-competent+retrovirus+vectors+achieves+long-term+survival+benefit+in+experimental+glioma%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Single-shot, multicycle suicide gene therapy by replication-competent retrovirus vectors achieves long-term survival benefit in experimental glioma.</a> Mol Ther. 12, 842-851 (2005).</li>
<li>Logg, C. R., Baranick, B. T., Lemp, N. A., Kasahara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adaptive+evolution+of+a+tagged+chimeric+gammaretrovirus%3A+identification+of+novel+cis-acting+elements+that+modulate+splicing%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">Adaptive evolution of a tagged chimeric gammaretrovirus: identification of novel cis-acting elements that modulate splicing.</a> J Mol Biol. 369 (5), 1214-1229 (2007).</li>
<li>Koya, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+phases+of+distribution+and+tumor+targeting+by+T+cell+receptor+engineered+lymphocytes+inducing+robust+antitumor+responses%5BTitle%5D)+AND+%22Proc+Nat+Acad+Sci+USA%22%5BJournal%5D)">Kinetic phases of distribution and tumor targeting by T cell receptor engineered lymphocytes inducing robust antitumor responses.</a> Proc Nat Acad Sci USA. 107 (32), 14286-14291 (2010).</li>
<li>Zumwalde, N. A., Domae, E., Mescher, M. F., Shimizu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ICAM-1-dependent+homotypic+aggregates+regulate+CD8+T+cell+effector+function+and+differentiation+during+T+cell+activation%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">ICAM-1-dependent homotypic aggregates regulate CD8 T cell effector function and differentiation during T cell activation.</a> J Immunol. 191 (7), 3681-3693 (2013).</li>
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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><tbody><tr><td>Teflon-masked microscope slides</td><td>Creative Scientific Methods</td><td>CSM002</td><td></td></tr><tr><td>Cell Sedimentation Manifold</td><td>Creative Scientific Methods</td><td>CSM001</td><td></td></tr><tr><td>Petri Dish, 150mm</td><td>Corning</td><td>430597</td><td></td></tr><tr><td>Petri Dish, 35mm</td><td>Corning</td><td>430588</td><td></td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Mediatech</td><td>21-040</td><td></td></tr><tr><td>20 &amp;mu;l Pipetman</td><td>Gilson</td><td>F123600</td><td></td></tr><tr><td>200 &amp;mu;l Pipetman</td><td>Gilson</td><td>F123601</td><td></td></tr><tr><td>200 &amp;mu;l pipette tips</td><td>VWR</td><td>89003-060</td><td></td></tr><tr><td>Distilled, deionized water (sterile)</td><td>Mediatech</td><td>25-055</td><td></td></tr><tr><td>Trypsin</td><td>Mediatech</td><td>25-054-CL</td><td></td></tr><tr><td>Celltracker Red CMPTX</td><td>Invitrogen</td><td>C34552</td><td></td></tr><tr><td>TrypLE Express (optional)</td><td>Gibco</td><td>12604</td><td></td></tr><tr><td>Tumor cell culture media (&lt;em&gt;e.g.&lt;/em&gt; DMEM)</td><td>Mediatech</td><td>10-013</td><td></td></tr><tr><td>AIM-V serum-free media</td><td>Invitrogen</td><td>12055-083</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Omega Scientific</td><td>FB-02</td><td></td></tr><tr><td>Inverted microscope</td><td></td><td></td><td></td></tr><tr><td>SPOT Advanced</td><td>Diagnostic Instruments</td><td></td><td></td></tr><tr><td>Poly-D-Lysine</td><td>Millipore</td><td>A-003-E</td><td></td></tr><tr><td>Fibronectin</td><td>BD</td><td>354008</td><td></td></tr><tr><td>3&lt;sup&gt;1/2 &lt;/sup&gt;x 5&lt;sup&gt;1/4 &lt;/sup&gt;Autoclave Pouches</td><td>Crosstex</td><td>SCXS2</td><td></td></tr><tr><td>Trypan Blue</td><td>Mediatech</td><td>25-900-CI</td><td></td></tr><tr><td>Cell Proliferation eFluor 670</td><td>eBioscience</td><td>65-0840-85</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td></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 ...

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Immunology and Infection

7.7K Views.  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 ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

Biology

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 ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

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 ...

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 ...

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Deciphering Early Follicular Helper T-Cell Commitment in Acute LCMV Infection

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

Author Spotlight: Elucidating the Pathways of TFH Cell Differentiation in Acute LCMV Challenges

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, ...

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

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