Name: Retroviral Transduction of T-cell Receptors in Mouse T-cells (Video) | JoVE Journal
Uploaded: 2010-10-22T17:35:00.000+00:00
Duration: 10 min 14 s
Description: Learn retroviral transduction of T-cell receptors in mouse T-cells for immunology research. This protocol enables efficient generation of antigen-specific T-cells using flow cytometry validation.

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5 minutes

This work was supported by grants from NIH (5U01CA137070) and the American Cancer Society (RSG-09-070-01-LIB), a Cancer Research Institute Investigator Award (M.K.), an American Heart Association pre-doctoral fellowship (K.M.) and a Spanish Ministry of Education and Science Fellowship (A.P.G).
We would like the thank Dr. Nicholas Restifo and Dr. Zhiya Yu (NIH/NCI) for helpful suggestions to the protocol.

1. Prepare Retroviral Construct
<ol>
 <li>Sub-clone the T-cell receptor (TCR) gene of interest into a retroviral vector (Figure 1, example vectors pMSG7, pMIGII5,8, pMXs from Cellbiolabs). The TCR α and β chain gene should be on the same vector under the control of the same promoter to ensure equal expression. If the TCR of interest is human, the human constant domains need to be replaced by mouse constant domains9. Our observation is that full length human TCRs do not stably express on mouse T-cells.</li>
 <li>Prepare high quality plasmid DNA using Qiagen Maxi Prep Kit or similar product. The concentration of the plasmid should be at around 1 μg/μL.</li>
</ol>
2. Transfection and T-cell Isolation
Day -1 (Plate packaging cells)
<ol>
 <li>Dislodge Platinum-E (Plat-E, Cellbiolabs) retroviral packaging cells with trypsin-EDTA and neutralize with DMEM media.</li>
 <li>Centrifuge the cells at 1000 x g for 5 min. Aspirate the supernatant and resuspend the cell pellet in DMEM media.</li>
 <li>Determine the cell number and dilute the cells at 0.6x106/mL. Plate 10 mL of cell suspension in a 10mm poly-lysine coated tissue culture plate and grow overnight in a cell incubator at 37 °C, 5% CO2.</li>
</ol>
Day 0 (Transfection)
<ol start="4">
 <li>The next morning, examine the cells under a light microscope. The cells should be approximately 80% confluent.</li>
 <li>Gently remove the media from the plate. Wash the cells once with 1x PBS and add 10 mL pre-warmed, fresh DMEM media without Penicillin-Streptomycin (Pen/Strep). 
 (Note: Pen/Strep can interfere with the transfection)</li>
 <li>Prepare transfection complex. 
 
 Prepare mix A and B as following: 
 
 <table border="0">
 <tbody>
 <tr>
 <td>Mix A: Plasmid DNA</td>
 <td>9 μg</td>
 </tr> 
 <tr>
 <td>pCL-Eco helper plasmid</td>
 <td>6.3 μg</td>
 </tr>
 <tr>
 <td>OptiMEM</td>
 <td>1.5 mL</td>
 </tr>
 <tr>
 <td colspan="2"> </td>
 </tr>
 <tr>
 <td>Mix B: Lipopfectime 2000</td>
 <td>60 μL</td>
 </tr>
 <tr>
 <td>OptiMEM</td>
 <td>1.5 mL</td>
 </tr>
 </tbody>
 </table>
 
 Incubate A and B separately for 5 minutes at room temperature 
 
 Mix A and B together gently and incubate for 20 minutes at room temperature to form transfection complex.</li>
 <li>Slowly drip the mixture (total 3 mL) into the Plat-E cells. Gently rock the plate back and forth to distribute the transfection mixture evenly.</li>
 <li>Incubate cells at 37 °C/5% CO2 in a cell incubator.</li>
 <li>After 6-8 hours, replace medium with 10 mL of fresh DMEM medium (with Pen/Strep) for viral production.</li>
</ol>
Coat plate with antibodies
<ol start="10">
 <li>Mouse T-cells need to be activated before viral transduction. There are a variety ways to activate T-cells. Here we use plate bound anti-CD3e and anti-CD28 antibodies. Prepare an antibody mixture of 1 μg/mL of anti-CD3e and 2 μg/mL of anti-CD28 in sterile PBS.</li>
 <li>Dispense 250 μL of the antibody mixture to each well of a 24-well tissue culture plate. The plate can be coated overnight at 4 °C or 2 hours at 37 °C.</li>
</ol>
Preparation of single-cell suspension from mouse spleen
<ol start="12">
 <li>Harvest mouse spleen and transfer to RPMI medium.</li>
 <li>Place the spleen in a cell strainer. Mash the spleen with a syringe plunger into a 5 cm tissue culture plate. Rinse cells off the cell strainer with sterile PBS.</li>
 <li>Centrifuge 1000 xg, 5 min.</li>
 <li>Discard supernatant. Resuspend cell pellet in ACK lysing buffer (2 mL per spleen). Incubate 2 to 3 min at room temperature. Add RPMI medium up to 20 mL and centrifuge 1000 xg for 5 min.</li>
 <li>Discard supernatant. Resuspend cell pellet in sterile PBS. Determine cell number and centrifuge 1000 xg for 5 min at 4 °C. Proceed to the isolation of T-cell.</li>
</ol>
Isolation and activation of mouse T-cells
<ol start="17">
 <li>Magnetically label the cells according to the product manual (CD8+ T-cell isolation kit from Miltenyi Biotec).</li>
 <li>Place a LS column (Miltenyi Biotec) in the magnetic field. Apply labeled cell suspension onto the column. Collect flow-through (enriched mouse CD8+ T-cells). Wash the column three times with 3 mL buffer according to the product manual and combine with previous flow-through.</li>
 <li>Stain the cells with anti-CD3e and anti-CD8a antibodies and analyze by flow cytometry (Figure 2a). For a typical separation, ~8-10% of total cells can be isolated. ~90% of the isolated cells are CD8+ T-cells.</li>
 <li>Centrifuge the cells at 1000 xg, 5 min. Discard the supernatant. Resuspend the cell pellet in RPMI medium with recombinant human IL-2 (rhIL2, 20 ng/mL) at 1x106 /mL.</li>
 <li>Just before adding the cells, remove antibody solution from the plate (prepared previously in 2.11). Rinse each well with sterile PBS and remove PBS.</li>
 <li>Add 1 mL of the cell suspension to each well. Put the plate at 37°C/5% CO2 in a cell incubator.</li>
</ol>
3. (Day 2 and Day 3) Infection of Activated T-cells
<ol>
 <li>After 2 days of virus production, the medium in the Plat-E cell plate should become yellow. Transfer viral supernatant to a 15 mL conical tube. Add 10 mL of fresh DMEM medium to the plate for further viral production (Optional).</li>
 <li>Centrifuge virus supernatant at 1000 xg for 5 min to remove cells debris. Carefully transfer virus supernatant to a new tube. Leave some liquid at the bottom of the tube and do not disturb the cells debris.</li>
 <li>Collect activated T-cells from the plate into a 50 mL conical tube. Determine the cell number. Save some cells into a separate tube to be used as negative (un-transduced) control. Centrifuge at 1000 xg for 5 min and discard supernatant.</li>
 <li>Resuspend the cell pellet in virus supernatant at 106 cells per 1 mL with 20 ng/mL rhIL2 and 10 μg/mL of protamine sulfate. Add 1 mL of cell suspension to each well of a 24 well plate. Resuspend the cells in the control tube in RPMI medium at the same cell density with the same amount of rhIL2 and protamine sulfate. Add the cells to the same plate.</li>
 <li>Wrap the plate with plastic film. Centrifuge 90 minutes at 2000 xg, at 32 °C with no brake.</li>
 <li>After the centrifugation, remove the plastic film. Add 1 mL of fresh RPMI medium with 20 ng/mL rhIL2 to each well. Put the plate back to the incubator.</li>
 <li>(Optional) Higher expression level of TCR can be achieved by collecting the virus supernatant and repeating the infection procedure on day 3.</li>
</ol>
4. Expansion of Cells and Evaluation of Transduction Efficiency
<ol>
 <li>Examine the transduced T-cells daily. Split the cells at 1:3 ratios when necessary in RPMI medium rhIL2. Do not let the cells overgrow (medium turns yellow).</li>
 <li>At day 6 or day 7, stain the cells with antibody and MHC tetramer specific for the TCR to evaluate the expression of TCR on T-cell surface. Use the un-transduced T-cells as control (Figure 2c).</li>
 <li>The transduced T-cells are ready for further downstream applications.</li>
</ol>
5. Representative Results
It is often advantageous to purify T-cell subsets before transduction although splenocytes can be transduced without purification. High purity T-cell subsets can be obtained using commercial magnetic beads (Figure 2a).
To evaluate the expression level of TCRs on T-cells, antibodies specific for TCR alpha or beta chains can be used. Typically 30% to 80% of the cells can express transduced TCR (Figure 2b) depending on the TCR genes and virus titers. MHC tetramer specific for the TCR can also be used to further verify functional expression of TCRs (Figure 2c).
<img src="/files/ftp_upload/2307/2307fig1.jpg" alt="Gene expression diagram with LTR, Ψ signal, peptide linker, showing DNA sequence elements." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Example of T-cell receptor gene construct sub-cloned into a retroviral vector. Full length alpha and beta chain genes are linked by a self-cleavable 2A peptide linker8.
<img src="/files/ftp_upload/2307/2307fig2.jpg" alt="Flow cytometry graphs showing T-cell analysis with anti-CD8, anti-Vbeta, anti-CD3 antibodies." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Example of successful isolation and transduction of mouse T-cells. (a) CD8 T-cells were isolated from splenocytes using CD8a+ T Cell Isolation Kit (Miltenyi Biotec) and stained with anti-CD3e and anti-CD8a antibodies. Isolated CD8 T-cells were transduced with R6C12 TCR specific for human gp100:209 -2174 peptide and stained with anti-human Vbeta 8 antibody (b) and HLA-A2kb gp100 tetramer (c).

<ol><li>Krogsgaard, M., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+T+cells+%27see%27+antigen%5BTitle%5D)+AND+%22Nat.+Immunol%22%5BJournal%5D)">How T cells 'see' antigen.</a> Nat. Immunol. 6, 239-245 (2005).</li>
<li>Berg, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigen%2FMHC-specific+T+cells+are+preferentially+exported+from+the+thymus+in+the+presence+of+their+MHC+ligand%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand.</a> Cell. 58, 1035-1046 (1989).</li>
<li>Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T+cell+receptor+antagonist+peptides+induce+positive+selection%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li>
<li>Yu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Poor+immunogenicity+of+a+self%2Ftumor+antigen+derives+from+peptide-MHC-I+instability+and+is+independent+of+tolerance%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.</a> J. Clin. Invest. 114, 551-559 (2004).</li>
<li>Holst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+T-cell+receptor+retrogenic+mice%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Generation of T-cell receptor retrogenic mice.</a> Nat Protoc. 1, 406-417 (2006).</li>
<li>Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+regression+in+patients+after+transfer+of+genetically+engineered+lymphocytes%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Cancer regression in patients after transfer of genetically engineered lymphocytes.</a> Science. 314, 126-129 (2006).</li>
<li>Hawley, R. G., Lieu, F. H., Fong, A. Z., Hawley, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Versatile+retroviral+vectors+for+potential+use+in+gene+therapy%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">Versatile retroviral vectors for potential use in gene therapy.</a> Gene Ther. 1, 136-138 (1994).</li>
<li>Szymczak, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correction+of+multi-gene+deficiency+in+vivo+using+a+single+%27self-cleaving%27+2A+peptide-based+retroviral+vector%5BTitle%5D)+AND+%22Nat.+Biotechnol%22%5BJournal%5D)">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat. Biotechnol. 22, 589-594 (2004).</li>
<li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+%28TCR%29+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR%2FCD3+stability%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66, 8878-8886 (2006).</li>
<li>Letourneur, F., Malissen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Derivation+of+a+T+cell+hybridoma+variant+deprived+of+functional+T+cell+receptor+alpha+and+beta+chain+transcripts+reveals+a+nonfunctional+alpha-mRNA+of+BW5147+origin%5BTitle%5D)+AND+%22Eur.+J.+Immunol%22%5BJournal%5D)">Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin.</a> Eur. J. Immunol. 19, 2269-2274 (1989).</li>
<li>Bendle, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lethal+graft-versus-host+disease+in+mouse+models+of+T+cell+receptor+gene+therapy%5BTitle%5D)+AND+%22Nat.+Med%22%5BJournal%5D)">Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.</a> Nat. Med. 16, 565-570 (2010).</li>
</ol>

No conflicts of interest declared.

Several steps are critical to achieve optimal results. Plat-E packaging cell line should be kept in healthy condition. If necessary, the cells can be passaged a few times in DMEM medium with 1 &mu;g/mL puromycin, 10 &mu;g/ml blasticidin to prevent loss of retroviral structure protein expression. On the day of transfection, the cells should be ~80% confluent. Too few or too many cells may reduce the virus titer. The virus quality can be checked by testing on cell lines that can be easily transduced. Since TCRs require CD3 complexes to be expressed on the cell surface, we routinely use 58&alpha;-/&beta;- hybridoma cells10(a cell line that does not express endogenous TCR &alpha; or &beta; chain, but does express CD3 complex). We obtain nearly 100% transduction efficiency of hybridoma cells when transducing cells with 1 mL of virus supernatant. Finally, the T-cells need to be activated and proliferating because retrovirus can only infect actively dividing cells.
The method presented here for producing antigen-specific T-cells has several limitations. First, it is difficult to reach 100% transduction efficiency for primary mouse T-cells. A portion of the cells do not express the TCR of interest. Second, the transduced TCR &alpha; and &beta; chains can mispair with endogenous TCRs9, which may reduce expression level of the TCR of interest. In addition, the mispaired TCRs may cause autoimmunity in vivo11. Finally, there is only a limited time frame the transduced T-cells can be used. After around one week, the cells undergo apoptosis unless being re-stimulated. However, cells may become exhausted and lose functions with repetitive re-stimulations.

<table><tbody><tr><td>CD8a+ T Cell Isolation Kit II</td><td>Miltenyi Biotec</td><td>130-095-236</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>Cell Strainer</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>1x PBS (no calcium or magnesium)</td><td>Invitrogen</td><td>10010-049</td><td></td></tr><tr><td>Ack lysing buffer</td><td>Invitrogen</td><td>A10492-01</td><td></td></tr><tr><td>Recombinant human IL2</td><td>PeproTech Inc</td><td>200-02</td><td></td></tr><tr><td>Anti-CD3e antibody</td><td>BD Biosciences</td><td>553057</td><td></td></tr><tr><td>Anti-CD28 antibody</td><td>BD Biosciences</td><td>553294</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-019</td><td></td></tr><tr><td>Plat-E Packaging cell line</td><td>Cell Biolabs</td><td>RV-101</td><td></td></tr><tr><td>OptiMEM medium</td><td>Invitrogen</td><td>31985-062</td><td></td></tr><tr><td>10cm Poly-lysine coated plates</td><td>BD Biosciences</td><td>356469</td><td></td></tr><tr><td>pCL-Eco helper plasmid</td><td>Imgenex</td><td>10045P</td><td></td></tr><tr><td>Protamine sulfate</td><td>APP Pharmaceuticals</td><td>22905</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>12491-023</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH3008803</td><td></td></tr><tr><td>Penicillin-Streptomycin Liquid</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>Sodium Pyruvate Solution</td><td>Invitrogen</td><td>11360-070</td><td></td></tr><tr><td>Non-Essential Amino Acids Solution</td><td>Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>RPMI</td><td>Invitrogen</td><td>12633-020</td><td></td></tr><tr><td>&amp;#946;- Mercaph&amp;#8211;thanol</td><td>Invitrogen</td><td>21985-023</td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Media:&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;DMEM medium (DMEM +10% heat inactivated FBS + Pen/Strep + L-Glu+ Sodium Pyruvate + Non-essential amino acid).&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;DMEM medium without Pen/Strep (Same as DMEM medium, but without adding Pen/Strep).&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;RPMI medium (RPMI +10% heat inactivated FBS + L-Glu + Non-essential amino acid + &amp;#946;- Mercaph&amp;#8211;thanol + Pen/Strep+ Sodium Pyruvate).&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;DMEM medium (DMEM +10% heat inactivated FBS + Pen/Strep + L-Glu+ Sodium Pyruvate + Non-essential amino acid).&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;DMEM medium without Pen/Strep (Same as DMEM medium, but without adding Pen/Strep).&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;RPMI medium (RPMI +10% heat inactivated FBS + L-Glu + Non-essential amino acid + &amp;#946;- Mercaph&amp;#8211;thanol + Pen/Strep+ Sodium Pyruvate).&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;DMEM medium without Pen/Strep (Same as DMEM medium, but without adding Pen/Strep).&lt;/p&gt;&lt;p class=&quot;jove_content&quot;&gt;RPMI medium (RPMI +10% heat inactivated FBS + L-Glu + Non-essential amino acid + &amp;#946;- Mercaph&amp;#8211;thanol + Pen/Strep+ Sodium Pyruvate).&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;RPMI medium (RPMI +10% heat inactivated FBS + L-Glu + Non-essential amino acid + &amp;#946;- Mercaph&amp;#8211;thanol + Pen/Strep+ Sodium Pyruvate).&lt;/p&gt;</td></tr></tbody></table>

retroviral transduction of t-cell receptors in mouse t-cells

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 ...

The aim of this procedure is to express functional antigen specific T-cell receptors on mouse T cells. First, transfect a retroviral producing cell line with plasmid containing the TCR gene of interest to package TCR expressing retrovirus. Next, isolate and purify T cells from mouse spleens infect these purified T cells with the retrovirus produced in the first step.Finally, expand the transduced T cells to express and obtain appreciable levels of receptor. Ultimately, results can be obtained that show the expression of functional antigen specific TCRs on mouse T cells through flow cytometry. Hi, my name is Michelle Crow Guard.I'm at the NYU Cancer Institute at NYU Medical School. Today we're gonna show you how to transduce primary T-cell, the people who will be showing you how to do this will be shiong, a postdoc in the lab, Kaleena Melek, a graduate student in the lab, and Arian Perez Garcia, a postdoc in the lab. The main advantage of this technique over existing method like transgenic mice is that it is much less time consuming.This method can help answer key immunological questions such as which TCR can give better response against antigens. This technique can be applied for adoptive T-cell transfer therapy, which has been shown to be promising for the treatment of cancer patients and consists in the in vitro modification of human T-cells To ensure equal expression of the T-cell receptor alpha and beta chains. Subclone the gene of interest into the same retroviral vector under control of the same promoter.Prepare high quality plasma DNA using the Kyogen Maxi prep kit and make a stock of one microgram per microliter. Remove media from the plate with platinum E retroviral packaging cells and wash the plate once with one XPBS. Dislodge the cells by adding trypsin EDTA and incubate at 37 degrees Celsius for a few minutes.Neutralize the dislodged cells with DMEM media and transfer to a falcon tube. Next centrifuge the cells at 1000 times G for five minutes. Aspirate the supinate and resuspend the cell pellet in DMEM media.Determine the cell number and dilute the cells at 0.6 times 10 to the six per milliliter plate. 10 milliliters of cell suspension in a 10 millimeter polylysine coated tissue culture plate and grow overnight in a cell incubator at 37 degrees Celsius in 5%carbon dioxide. The next morning, examine the cells under a light microscope to verify approximately 80%co fluency.Gently remove the media from the plate. Wash the cells once with one XPBS and add 10 milliliters of prewarm fresh DMEM media without penicillin or streptomycin. For the lip mean transfection complex, prepare two mixes and opti ME M1 of DNA and the other of lipectomy reagent equilibriate separately for five minutes at room temperature.Then mix together gently and incubate for 20 minutes. At room temperature, slowly drip the mixture into the platinum E cells. Gently rock the plate back and forth to distribute the transfection mixture evenly.Then incubate cells at 37 degrees Celsius and 5%carbon dioxide in a cell incubator. After six to eight hours, replace medium with 10 milliliters of fresh DMEM medium for viral production. Harvest a mouse spleen and transfer to RPMI medium.Place the spleen in a cell strainer. Mash the spleen with a syringe plunger into a five centimeter tissue culture plate. Rinse cells off the cell strainer with sterile PBS in order to harvest the cells centrifuge at 1000 times G for five minutes.Discard the sup natant. Continue by Resus suspending the cell pellet in a CK lysing buffer. Adding two milliliters per spleen incubate for two minutes.At room temperature. Add RPMI medium up to 20 milliliters and centrifuge at 1000 times G for five minutes. Finally, resuspend the cell pellet in sterile PBS.Determine the cell number and centrifuge at 1000 times G for five minutes at four degrees Celsius. In order to activate the mouse T cells before viral transduction coat plates with activating antibodies, namely anti CD three epsilon and anti CD 28. Prepare an antibody mixture of one microgram per milliliter of anti CD three epsilon and two micrograms per milliliter of anti CD 28 in sterile PBS dispense 250 microliters of the antibody mixture into each well of the 24 well tissue culture plate and incubate for two hours at 37 degrees Celsius.Magnetically label the spleen cells according to the product manual. Place an LS column in the magnetic field. Apply labeled cell suspension onto the column.Collect flow through of enriched mouse CD eight plus T cells. Wash the column three times with three milliliters of buffer and combined with the previous blow through. Stain the cells with anti CD three epsilon and anti CD eight alpha antibodies and analyzed flow cytometry.Next, centrifuge the cells at 1000 times G for five minutes and resuspend the cell pelle to 1 million cells per milliliter in RPMI medium with recombinant human IL two. Remove the antibody solution from the activation plate. Rinse each well with sterile PBS.Add one milliliter of the cell suspension to each well and place the plate at 37 degrees Celsius. 5%carbon dioxide in a cell incubator. After two days of virus production, the medium in the platinum ESE cell plate should become yellow.Transfer the viral supinate to a 15 milliliter conical tube to remove cell debris. Centrifuge the virus S supernatant at 1000 times G for five minutes. Carefully transfer the virus supinate to a new tube.Leave some liquid at the bottom of the tube and do not disturb the cells debris. Collect activated T cells from the plate into a 50 millimeter conical tube. Determine the cell number.Save some cells in a separate tube to be used as a negative control centrifuge at 1000 times G for five minutes and discard the supine natant. Then resuspend the cell pellet in virus supine. Natant at 10 to the six cells per milliliter with 20 nanograms per milliliter of recombinant human, interleukin two and 10 micrograms per milliliter of protein sulfate.Add one milliliter of cell suspension to each well of the 24 well plate for the control tube.Resus. Suspend the cells in RPMI medium at the same cell density with the same amount of recombinant human, interleukin two and protein sulfate. Add the cells to the same plate.Wrap the plate with plastic film centrifuge for 90 minutes at 2000 times G at 32 degrees Celsius with no break. After the centrifugation, remove the plastic film. Add one milliliter of fresh RPMI medium with 20 nanograms per milliliter of recombinant human interleukin two to each, well put the plate back into the incubator.It is important to examine the transduced T cells daily. Split the cells at one to three ratios when necessary. Do not let the cells overgrow or let the medium turn yellow at day six or day seven.Stain the cells with antibody and MHC tetramer specific for the TCR to evaluate the expression of TCR on T-cell surface. Use the UNSD T cells as a control. These transduced T cells are now ready for further downstream applications.Prior to viral transduction. It is advantageous to obtain high purity T-cell subsets using commercial magnetic beads to evaluate the expression level of TCRs on T cells. Antibody specific for TCR alpha or beta chains can be used.Typically, 30%to 80%of the cells can express transduced TCR depending on the TCR genes and virus titers. MHC Tetramer specific for the TCR can also be used to further verify functional expression of TCRs. Once mastered, this technique can be done in one week if performed properly.While attempting this procedure, it is important to remember to keep the cells in healthy condition Following this procedure. Other methods like cytotoxicity acid or cytokine release assays can be performed in order to answer additional questions like the specificity and the sensitivity of transduce. The TCRs.After watching this video, you should be an expert in transducing your favorite TCR into primary T cells. So see you in the lab.

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Research

JoVE Journal

Immunology and Infection

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

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.

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

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.

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

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

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.

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

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.

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

We outline a 9-day protocol for the transduction and expansion of rhesus macaque peripheral blood mononuclear cells which yields cells with excellent co-expression of the genes of interest in sufficient number for infusion studies of cell efficacy.

Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

Learn retroviral transduction of T-cell receptors in mouse T-cells for immunology research. This protocol enables efficient generation of antigen-specific T-cells using flow cytometry validation.

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

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