Name: streamlined single cell tcr isolation and generation of retroviral vectors for in vitro and in vivo expression of human tcrs
Uploaded: 2017-09-10T13:00:00
Description: Watch this Scientific Journal Video about Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs at JoVE.com

This study was funded by JDRF 1-FAC-2014-243-A-N, ADA 7-14-JF-07, NIH 5 P30 DK079638-05 PILOT PJ, and The Robert and Janice McNair Foundation.
We thank Sandra Pena and Andrene McDonald for patient recruitment, Samuel Blum for technical assistance, Dr. George Makedonas for the gift of antibodies and control DNA used for PCR optimization.

1. Identify the T cell population of interest.
NOTE: Antigen specific proliferation in combination with cell division dye (like Carboxyfluorescein succinimidyl ester, CFSE) can be used to isolate T cells based on their proliferation in response to antigenic stimulation. If starting from PBMCs, a 7-day in vitro expansion should be sufficient to identify an antigen specific CFSE low population12.
<ol>
	<li>Mix PBMCs 1:1 with MHC-matched feeder cells, and label ...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+naive+CD4++T+cells+from+a+diverse+repertoire+produce+different+effector+cell+types+during+infection.">Single naive CD4+ T cells from a diverse repertoire produce different effector cell types during infection.</a> Cell. 153 (4), 785-796 (2013).</li><li>Sprouse, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+expression+of+human+TCRs+in+retrogenic+mice.">Rapid identification and expression of human TCRs in retrogenic mice.</a> J Immunol Methods. , (2016).</li><li>Lennon, G. P., et al. <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+islet+accumulation+in+type+1+diabetes+is+a+tightly+regulated,+cell-autonomous+event.">T cell islet accumulation in type 1 diabetes is a tightly regulated, cell-autonomous event.</a> Immunity. 31 (4), 643-653 (2009).</li><li>Dash, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+analysis+of+TCRalpha+and+TCRbeta+chains+at+the+single-cell+level+in+mice.">Paired analysis of TCRalpha and TCRbeta chains at the single-cell level in mice.</a> J Clin Invest. 121 (1), 288-295 (2011).</li><li>Kobayashi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+cloning+and+expression+system+yields+and+validates+TCRs+from+blood+lymphocytes+of+patients+with+cancer+within+10+days.">A new cloning and expression system yields and validates TCRs from blood lymphocytes of patients with cancer within 10 days.</a> Nat Med. 19 (11), 1542-1546 (2013).</li><li>Szymczak, A. L., et al. <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 (5), 589-594 (2004).</li><li>Szymczak, A. L., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+2A+peptide-based+strategies+in+the+design+of+multicistronic+vectors.">Development of 2A peptide-based strategies in the design of multicistronic vectors.</a> Expert Opin Biol Ther. 5 (5), 627-638 (2005).</li><li>Bettini, M. L., Bettini, M., Vignali, D. 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+retrogenic+mice:+a+rapid,+flexible+alternative+to+T-cell+receptor+transgenic+mice.">T-cell receptor retrogenic mice: a rapid, flexible alternative to T-cell receptor transgenic mice.</a> Immunology. 136 (3), 265-272 (2012).</li><li>Holst, J., Vignali, K. M., Burton, A. R., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+T-cell+selection+in+vivo+using+T+cell-receptor+retrogenic+mice.">Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice.</a> Nat Methods. 3 (3), 191-197 (2006).</li><li>Mannering, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J Immunol Methods. 283 (1-2), 173-183 (2003).</li><li>James, E. A., LaFond, R., Durinovic-Bello, I., Kwok, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen+specific+CD4++T+cells+using+MHC+class+II+tetramers.">Visualizing antigen specific CD4+ T cells using MHC class II tetramers.</a> J Vis Exp. (25), (2009).</li><li>Corthay, A., Nandakumar, K. S., Holmdahl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+percentage+of+peripheral+T+cells+with+two+different+T+cell+receptor+alpha-chains+and+of+their+potential+role+in+autoimmunity.">Evaluation of the percentage of peripheral T cells with two different T cell receptor alpha-chains and of their potential role in autoimmunity.</a> J Autoimmun. 16 (4), 423-429 (2001).</li><li>Lee, T., Shevchenko, I., Sprouse, M. L., Bettini, M., Bettini, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+Transduction+of+Bone+Marrow+Progenitor+Cells+to+Generate+T-cell+Receptor+Retrogenic+Mice.">Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice.</a> J Vis Exp. (113), (2016).</li><li>Bettini, M. L., Bettini, M., Nakayama, M., Guy, C. S., Vignali, D. A. <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:+improved+retroviral-mediated+stem+cell+gene+transfer.">Generation of T cell receptor-retrogenic mice: improved retroviral-mediated stem cell gene transfer.</a> Nat Protoc. 8 (10), 1837-1840 (2013).</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 (17), 8878-8886 (2006).</li></ol>

In the current protocol, we describe an efficient method for single cell TCR amplification and subsequent sub-cloning of paired TCR alpha and beta chains into a template retroviral expression vector. Although, several single cell PCR protocols have been developed, none so far have been compatible with immediate sub-cloning into an expression vector. In most cases, a partial sequence encompassing the highly variable CDR3 regions is amplified, with enough sequence to extrapolate the variable region. This smaller PCR produc...

The T cell receptor (TCR) dictates T cell fate decision during development, steady state/homeostasis, and antigenic stimulation in periphery1,2,3. Recent expansion of deep sequencing technologies has uncovered a previously underappreciated TCR diversity within antigen specific T cell responses. TCR diversity suggests a potential for functionally broad T cell responses. In order to integrate the TCR sequence repertoire analysis with TCR functional assays, the sequencing approaches should be designed to be compatible with experimental systems and in vivo models utilized for subsequent functional analysis of select TCRs. We have developed an efficient approach for human TCR sequence isolation and streamlined sub-cloning into a chimeric human/mouse TCR template vector compatible with TCR expression in HLA-humanized mice4. Isolation of corresponding alpha and beta chains of heterodimeric TCRs requires PCR amplification of both chains from a single cell. Although, several single-cell TCR cloning protocols have been developed and utilized, none so far have been easily compatible with high-throughput streamlined direct cloning of unknown V&#945;/V&#946; TCRs into retroviral vectors necessary for re-expression in vivo4,5,6,7. Previous studies have utilized two major approaches, either to selectively amplify a limited portion of the TCR sufficient to extrapolate the sequence, or to amplify the entire TCR sequence4,5,6,7. Downstream functional analysis of TCR sequences obtained via the first approach requires costly in silico assembly and de novo construction of the TCR. While the second approach provides the complete TCR sequence, the human constant region is not compatible with in vivo expression of the cloned TCRs in mouse models. Our approach is specifically designed to be compatible with in vivo functional analysis of TCRs in mouse models. We developed an efficient and streamlined single cell PCR protocol that allows for direct sub-cloning of PCR fragments into the template expression vector.
Our approach utilizes a highly sensitive multiplex-nested PCR reaction that is performed in two steps. In the first multiplex reaction step a pool of 40 primers specific for all the V-beta chains and a pool of 44 primers specific for all the V-alpha chains are used to amplify the TCR-alpha or TCR-beta without the prior knowledge of the sequence (Table 1). The forward primer has an adaptor sequence, which is incorporated into the 5&#39; of the PCR product. The reverse primer is based on the constant region of the TCR. We have identified unique restriction sites that are absent from human variable or junction TCR regions, and incorporated these into newly redesigned TCR&#945; and TCR&#946; primers (Figure 1). In the second nested reaction, a primer specific for the adaptor sequence and a nested reverse primer within the constant region are used to further amplify the TCR chains with increased specificity (Table 1, Figure 1). After single cell isolation, two rounds of PCR (first reaction with a pool of both V&#945; and V&#946; primers, and second with adaptor primers) result in a PCR product that can be directly sub-cloned into the template retroviral vector. The final TCR construct will encode, in a single open reading frame (ORF), human variable regions combined with mouse constant regions connected by the &#39;self-cleaving&#39; protein sequence, P2A (hV&#945;-mC&#945;-P2A-hV&#946;-mC&#946;)8. The P2A sequence has been used in multiple systems, and has been extensively tested specifically for the expression of TCRs8,9,10,11. Although, after translation most of the P2A sequence remains attached to the C-terminus of the alpha chain connected by a flexible linker, while the beta chain signal sequence has an additional proline, this modification has no detrimental effect on TCR function. The mouse constant region is used in the construct instead of human to avoid potential altered interactions with downstream signaling components when re-expressed in mouse cells. The single ORF will result in stoichiometric separate expression of alpha and beta chains9,11. The current protocol is based on reagents and approaches that are widely available, and is designed to be performed in a streamlined and efficient fashion. Although we have specifically used this technique to assay TCRs from self-reactive T cells implicated in autoimmune diabetes, we anticipate that this protocol can be widely applicable for identification and functional assessment of human TCRs specific for autoimmune epitopes, cancer epitopes, or responses to pathogens and vaccines.

<table border="0" cellpadding="0" cellspacing="0" width="815">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">CFSE&#160;</td>
			<td style="width:148px;">eBioscience&#160;</td>
			<td style="width:116px;">65-0850-84</td>
			<td style="width:223px;">5 &#956;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human CD4 (OKT4) BV421</td>
			<td>Biolegend&#160;</td>
			<td>317433</td>
			<td>2 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human CD3 (OKT3) PerCP/Cy5.5</td>
			<td>Biolegend&#160;</td>
			<td>317336</td>
			<td>2 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse CD3 AF647</td>
			<td>Biolegend&#160;</td>
			<td>100322</td>
			<td>2.5 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant RNAse inhibitor biotech grd 2.5KU&#160;&#160;</td>
			<td>VWR</td>
			<td>97068-158</td>
			<td>0.7 U</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Applied Biosystems High capaity cDNA RT kit&#160;</td>
			<td>Invirtogen&#160;</td>
			<td>4368814</td>
			<td>9 U (RT enzyme)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NF-H2O: Nuclease-free water 1000mL</td>
			<td>Invirtogen&#160;</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gotaq DNA polymerase 2500U</td>
			<td>VWR</td>
			<td>PAM3008</td>
			<td>1 U</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 Well Half Skirt PCR Plate</td>
			<td>Phenix</td>
			<td>MPX-96M2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MicroAmp Clear Adhesive Film&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>4306311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure Agarose&#160;</td>
			<td>Invirtogen&#160;</td>
			<td>15510-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SnaBI</td>
			<td>New England Biolabs&#160;</td>
			<td>R0130</td>
			<td>15 U (insert) 30 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SacII</td>
			<td>New England Biolabs&#160;</td>
			<td>R0157</td>
			<td>40 U (insert) 80 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MfeI</td>
			<td>New England Biolabs&#160;</td>
			<td>R3589S</td>
			<td>40 U (insert) 80 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BstBI</td>
			<td>New England Biolabs&#160;</td>
			<td>R0519</td>
			<td>40 U (insert) 80 U (vector)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calf Intestinal Phosphatase (CIP)</td>
			<td>New England Biolabs&#160;</td>
			<td>M0290S</td>
			<td>10 U</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quick ligase&#160;</td>
			<td>New England Biolabs&#160;</td>
			<td>M2200S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Plus minipreps DNA purification systems&#160;</td>
			<td>Promega&#160;</td>
			<td>A1460</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Plus midipreps DNA purification systems&#160;</td>
			<td>Promega&#160;</td>
			<td>A7640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA clean &#38; concentrator-25 kit</td>
			<td>Genesee Scientific&#160;</td>
			<td>11-304C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZR-96 DNA clean &#38; concentrator-5 kit</td>
			<td>Genesee Scientific&#160;</td>
			<td>11-306A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick gel extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Subcloning Efficiency DH5a Competent Cells&#160;</td>
			<td>ThermoFisher&#160;</td>
			<td>18265017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transit LT-1 Reagent 1mL (transfection reagent)</td>
			<td>Mirus&#160;</td>
			<td>MIR2300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACS Aria II (sorter)</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Fortessa (flow cytometer)</td>
			<td>BD&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The efficiency of the multiplex-nested PCR reaction is checked in step 3.2.7 (Figure 2) by running out 5&#181;L of the second reaction on an agarose gel. On average the efficiency of TCR-beta amplification is expected to be around 80%, while the efficiency of the TCR-alpha reaction is usually lower, at around 50%4. Only paired TCR-alpha and TCR-beta chains can be used for TCR expression; however, all PCR products could be sequenced to ...

Watch this Scientific Journal Video about Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs at JoVE.com

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.

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 ...

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