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 &alpha; and &beta; 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 &mu;g/&mu;L.</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 &deg;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 &mu;g</td>
 </tr> 
 <tr>
 <td>pCL-Eco helper plasmid</td>
 <td>6.3 &mu;g</td>
 </tr>
 <tr>
 <td>OptiMEM</td>
 <td>1.5 mL</td>
 </tr>
 <tr>
 <td colspan="2">&nbsp;</td>
 </tr>
 <tr>
 <td>Mix B: Lipopfectime 2000</td>
 <td>60 &mu;L</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 &deg;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 &mu;g/mL of anti-CD3e and 2 &mu;g/mL of anti-CD28 in sterile PBS.</li>
 <li>Dispense 250 &mu;L of the antibody mixture to each well of a 24-well tissue culture plate. The plate can be coated overnight at 4 &deg;C or 2 hours at 37 &deg;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 &deg;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&deg;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 &mu;g/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 &deg;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="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="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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+T+cells+'see'+antigen.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen/MHC-specific+T+cells+are+preferentially+exported+from+the+thymus+in+the+presence+of+their+MHC+ligand.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+immunogenicity+of+a+self/tumor+antigen+derives+from+peptide-MHC-I+instability+and+is+independent+of+tolerance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T-cell+receptor+retrogenic+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+regression+in+patients+after+transfer+of+genetically+engineered+lymphocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+retroviral+vectors+for+potential+use+in+gene+therapy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+graft-versus-host+disease+in+mouse+models+of+T+cell+receptor+gene+therapy.">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.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CD8a+ T Cell Isolation Kit II</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>130-095-236</td>
<td>&nbsp;</td>
<tr>
<td>LS Columns</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>130-042-401</td>
<td>&nbsp;</td>
<tr>
<td>Cell Strainer</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-771-2</td>
<td>&nbsp;</td>
<tr>
<td>1x PBS (no calcium or magnesium)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10010-049</td>
<td>&nbsp;</td>
<tr>
<td>Ack lysing buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A10492-01</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant human IL2</td>
<td>&nbsp;</td>
<td>Peprotech</td>
<td>200-02</td>
<td>&nbsp;</td>
<tr>
<td>Anti-CD3e antibody</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>553057</td>
<td>&nbsp;</td>
<tr>
<td>Anti-CD28 antibody</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>553294</td>
<td>&nbsp;</td>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11668-019</td>
<td>&nbsp;</td>
<tr>
<td>Plat-E Packaging cell line</td>
<td>&nbsp;</td>
<td>Cell Biolabs</td>
<td>RV-101</td>
<td>&nbsp;</td>
<tr>
<td>OptiMEM medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31985-062</td>
<td>&nbsp;</td>
<tr>
<td>10cm Poly-lysine coated plates</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>356469</td>
<td>&nbsp;</td>
<tr>
<td>pCL-Eco helper plasmid</td>
<td>&nbsp;</td>
<td>Imgenex</td>
<td>10045P</td>
<td>&nbsp;</td>
<tr>
<td>Protamine sulfate</td>
<td>&nbsp;</td>
<td>APP Pharmaceuticals</td>
<td>22905</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12491-023</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH3008803</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin Liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>35050-061</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Pyruvate Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11360-070</td>
<td>&nbsp;</td>
<tr>
<td>Non-Essential Amino Acids Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12633-020</td>
<td>&nbsp;</td>
<tr>
<td>&beta;- Mercaphoethanol</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21985-023</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			Media:
DMEM medium (DMEM +10% heat inactivated FBS + Pen/Strep + L-Glu+ Sodium Pyruvate + Non-essential amino acid).
DMEM medium without Pen/Strep (Same as DMEM medium, but without adding Pen/Strep).
RPMI medium (RPMI +10% heat inactivated FBS + L-Glu + Non-essential amino acid + &beta;- Mercaphoethanol + Pen/Strep+ Sodium Pyruvate).		</div>

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.

Watch this Scientific Journal Video about Retroviral Transduction of T-cell Receptors in Mouse T-cells at JoVE.com

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

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 ...

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

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JoVE Journal

Immunology and Infection

34.7K Views.  New York University School of Medicine. Мы представляем протокол производить антиген-специфические Т-мышь клеток с использованием ретровирусной трансдукции

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

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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 ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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 ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...