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>
	<li>Germain, R. N. <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+development+and+the+CD4-CD8+lineage+decision.">T-cell development and the CD4-CD8 lineage decision.</a> Nat Rev Immunol. 2 (5), 309-322 (2002).</li><li>Singer, A., Adoro, S., Park, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+fate+and+intense+debate:+myths,+models+and+mechanisms+of+CD4-+versus+CD8-lineage+choice.">Lineage fate and intense debate: myths, models and mechanisms of CD4- versus CD8-lineage choice.</a> Nat Rev Immunol. 8 (10), 788-801 (2008).</li><li>Tubo, N. 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 ...

This method can help answer key questions in the immunology field, such as the TCR alpha and beta repertoire of an antigen's specific population, as well as the in vivo functionality of human TCRs. The main advantages of this technique are the relatively high efficiency of the PCR reaction as well as its compatibility with subsequent TCR cloning. This protocol uses a two-step PCR to amplify the TCR alpha and beta chains from a single cell without prior knowledge of the TCR sequence.The first PCR is a multiplex reaction using a pool of alpha or beta variable region-specific primers in conjunction with a primer specific for the constant region. The second PCR is a nested reaction that primes off of the adapter sequence incorporated at the five prime end of the PCR product as well as an internal constant region sequence. These primers also incorporate unique restriction sites into the five prime and three prime ends of the PCR product to facilitate subsequent cloning.Once cloned into the template vector, the encoded TCR sequence is chimeric, consisting of human variable regions with mirroring constant regions. The chimeric TCR construct allows for interaction between the TCR and the mirroring CD3 signaling complex, making this TCR cloning protocol compatible with subsequent functional studies in mirroring cell lines and in humanized mouse models. Set up for the single cell sorting by preparing the reverse transcription reagents in a clean, template-free area.Generally individuals new to the single-cell PCR method will struggle because of the low levels of template. On the other hand, the sensitivity of the multiplex nested PCR is sensitive to low levels of template contamination. Therefore, it is necessary to thoroughly clean the areas in which you will be setting up the PCR reactions.A mixture of three reverse primers specific for the constant region of the alpha and beta chains are used in this reaction. Prepare the reverse transcription master-mix by combining reagents in a pipetting reservoir. To account for pipetting error, make enough reaction mixture for ten extra wells.Use a multichannel to pipette six microliters per well into a 96-well PCR plate. Cover the plate loosely with a seal and keep the plate on ice. Prepare T cells for single cell sort by staining the cells with antibodies to cell surface markers.Sort T cells at once cell per well while leaving the twelve row empty, which will serve as a negative control. Seal the plate with adhesive film and spin down at 1000g for five minutes at four degrees Celsius. Immediately run the reverse transcription reaction.If possible, run the first PCR reaction the same day. To run the multiplex reaction, separately mix the V alpha and V beta PCR master mixes in pipetting reservoirs. Keep the plate with cDNA on ice or on a cold block.Transfer 22.5 microliters of TCR beta reaction into a new 96-well piece PCR plate. Then use a multichannel to transfer 2.5 microliters of cDNA into the PCR plate. Use a multichannel to pipette 22.5 microliters of TCR alpha reaction directly into the plate containing the remainder of the cDNA.Seal the plates with adhesive, gently vortex, and briefly spin down. Run the first PCR reaction. Plates can be stored at 20 degrees Celsius.The next day, prepare the second nested PCR reactions. Using a multichannel, pipette the master mixes for the TCR alpha and TCR beta reactions at 22.5 microliters per well into two new 96-well PCR plates. Add the multiplex PCR reaction template at 2.5 microliters per well.Seal the plates, gently vortex, and briefly spin down. The remaining first PCR reaction should be sealed and stored at 20 degrees Celsius to serve as a source of backup template. Perform the second PCR reaction.After the second nested PCR reaction is complete, run out five microliters of the reaction on a 1%agarose gel. Include the positive and negative control wells. The expected band should run at around 500 base pairs in size.Purify the remainder of the second reaction, twenty microliters, using a 96-well format PCR purification kit and perform Sanger sequencing. The obtained TCR sequences can be analyzed using online available immunogenetic software. The sequence information can be used to assess the repertoire and clonality of the T-cell population.Paired TCR alpha and beta sequences can be used in further functional analysis. Perform consecutive ligations of TCR beta and TCR alpha into the template vector. The template TCR beta and TCR alpha are replaced with the PCR amplified chains.In the first ligation, the template TCR beta is replaced with the PCR amplified beta chain. After successful insertion of TCR beta has been verified by test-digest insequencing, replace the template TCR alpha with the PCR amplified alpha chain. HEK293T cells are used to test successful TCR chain pairing and cell surface expression.HEK293T cells are co-transfected with the TCR vector marked by the ametrine fluorescent reporter and a vector encoding the CD3 complex marked by GFP. Surface expression of the chimeric TCR is verified by flow cytometry by staining the cells with anti-mouse CD3 antibody. The level of surface CD3 expression in cells transfected with the chimeric TCR construct should be comparable to those transfected with the fully mirroring control template vector.In summary, we have described an efficient PCR-based protocol for the identification and cloning of paired TCR chains from single cells. We anticipate that this will be valuable tool to study human T-cell responses in autoimmunity, tumor surveillance, and pathogen elimination. The main advantage of the PCR protocol described here is its compatibility with subsequent in vitro and in vivo studies.

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Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

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 ...

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 ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...