This work was supported by NIH grant R01 CA148673 (NH); the Ontario Institute for Cancer Research Clinical Investigator Award IA-039 (NH); BioCanRX Catalyst Grant (NH); The Princess Margaret Cancer Foundation (MOB, NH); Canadian Institutes of Health Research Canada Graduate Scholarship (TG); Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship (TG); Province of Ontario (TG, MA); and Guglietti Fellowship Award (TO). HLA and CD1d monomers were kindly provided by the NIH tetramer core facility.

1. Preparing Retroviral Construct Encoding TCR Hemichain of Interest
<ol>
	<li>Linearize pMX vector to allow the insertion of a TCR gene in subsequent steps. Digest the plasmid DNA with EcoRI and NotI restriction enzymes at 37 &#176;C for 3 hr (Table 1)8.</li>
	<li>Carry out electrophoresis of the digested plasmid on 1.2% agarose gel. Excise band of approximately 4,500 base pairs (bps), and elute in 30 &#956;l sterile water using commercially available gel extraction kits9.</li>
	...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+TCRs+bind+MHCs,+peptides,+and+coreceptors.">How TCRs bind MHCs, peptides, and coreceptors.</a> Annu. Rev. Immunol. 24, 419-466 (2006).</li><li>Shimizu, A., 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=Structure+of+TCR+and+antigen+complexes+at+an+immunodominant+CTL+epitope+in+HIV-1+infection.">Structure of TCR and antigen complexes at an immunodominant CTL epitope in HIV-1 infection.</a> Sci. Rep. 3, 3097 (2013).</li><li>Nakatsugawa, M., 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=Specific+roles+of+each+TCR+hemichain+in+generating+functional+chain-centric+TCR.">Specific roles of each TCR hemichain in generating functional chain-centric TCR.</a> J. Immunol. 194 (7), 3487-3500 (2015).</li><li>Ochi, T., 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=Optimization+of+T-cell+Reactivity+by+Exploiting+TCR+Chain+Centricity+for+the+Purpose+of+Safe+and+Effective+Antitumor+TCR+Gene+Therapy.">Optimization of T-cell Reactivity by Exploiting TCR Chain Centricity for the Purpose of Safe and Effective Antitumor TCR Gene Therapy.</a> Cancer Immunol. Res. 3 (9), 1070-1081 (2015).</li><li>Chamoto, K., 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=CDR3beta+sequence+motifs+regulate+autoreactivity+of+human+invariant+NKT+cell+receptors.">CDR3beta sequence motifs regulate autoreactivity of human invariant NKT cell receptors.</a> J. Autoimmun. 68, 39-51 (2016).</li><li>Grozdanov, P. N., MacDonald, C. C. <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+plasmid+vectors+expressing+FLAG-tagged+proteins+under+the+regulation+of+human+Elongation+Factor-1&#945;+promoter+using+Gibson+Assembly.">Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human Elongation Factor-1&#945; promoter using Gibson Assembly.</a> J. Vis. Exp. (96), e52235 (2015).</li><li>Beshiri, M. 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H., 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=High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines,+zebrafish+and+mice.">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PloS one. 6, e18556 (2011).</li><li>Yang, S., 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=Development+of+optimal+bicistronic+lentiviral+vectors+facilitates+high-level+TCR+gene+expression+and+robust+tumor+cell+recognition.">Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition.</a> Gene Ther. 15 (21), 1411-1423 (2008).</li><li>Froger, A., Hall, J. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+DNA+Libraries.">Complementary DNA Libraries.</a> Generation of cDNA Libraries: Methods and Protocols. 221, 1-12 (2003).</li><li>Hirano, N., 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=Engagement+of+CD83+ligand+induces+prolonged+expansion+of+CD8++T+cells+and+preferential+enrichment+for+antigen+specificity.">Engagement of CD83 ligand induces prolonged expansion of CD8+ T cells and preferential enrichment for antigen specificity.</a> Blood. 107 (4), 1528-1536 (2006).</li><li>Scotto-Lavino, E., Du, G., Frohman, M. 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The first requirement for successful application of this method is achieving sufficient transduction efficiency of primary T cells with the hemichain of interest. In our experience, the combination of using PG13 as packaging cell line and pMX as retroviral vector results in stable and efficient expression of the introduced gene in human primary T cells. PG13 packaging cells can be single-cell cloned to select for high-titer packaging cells to improve transduction efficiency. Furthermore, proliferation of T cells is also ...

T cell receptors (TCRs) are heterodimeric adaptive immune receptors expressed by T lymphocytes, composed of a TCR&#945; and TCR&#946; chain. They are generated via somatic rearrangement of V(D)J gene segments, which produces a highly diverse repertoire capable of recognizing virtually unlimited configurations of HLA/peptide complexes. Clinically, T cells engineered to express clonotypic TCRs specific for tumor-associated antigens have demonstrated efficacy in a variety of cancers1. However, many TCRs cloned for this purpose lack sufficient affinity for the antigen of interest, which limit their therapeutic application.
Here, we describe a method to overcome this limitation for existing TCRs by exploiting chain-centricity. It has been reported that one TCR hemichain could play a more dominant role in recognition of the target antigen2, here termed centricity. Crystal structural analyses have shown that one centric hemichain of a TCR could account for the majority of the footprint on the MHC/peptide complex3,4. Using this concept, we have previously demonstrated that the SIG35&#945; TCR&#945; can pair with a diverse repertoire of TCR&#946; chains and maintain reactivity against the MART127-35 peptide presented by HLA-A25. Similar results were obtained with the TAK1 TCR, where the centric TCR&#946; hemichain paired with various TCR&#945; chains and maintained reactivity for the WT1235-243 peptide presented by HLA-A246. Both MART1 and WT1 are tumor-associated antigens. Chain-centricity was also applied to study antigen recognition of CD1d-restricted invariant natural killer (iNKT) TCRs, by pairing the invariant V&#945;24-J&#945;18 (V&#945;24i) TCR&#945; chain of human iNKT TCRs with different V&#946;11 TCR&#946; chains7.
In all cases, we were able to generate a de novo repertoire of TCRs by transducing the centric TCR hemichain to peripheral blood T cells, where the introduced hemichain paired with the endogenous TCR&#945; or TCR&#946; counter-chains. In essence, the centric hemichain serves as a bait that can be used to identify the appropriate counter-chains, which when paired together form TCRs that maintain the antigen specificity of interest, yet varying in affinity. From these novel repertoires, we were able to isolate clonotypic TCRs with improved interaction strength against the target antigen compared to pre-existing TCRs. Therefore, we believe this method will accelerate the pipeline of identifying optimal TCRs for clinical application.

<table border="0" cellpadding="0" cellspacing="0" width="702">
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		<col />
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	</colgroup>
	<tbody>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">0.05% Trypsin-EDTA</td>
			<td style="width:180px;">Wisent Bioproducts</td>
			<td style="width:139px;">325-043-CL</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">293GPG cells</td>
			<td style="width:180px;">Generated by Ory et al. (ref 8)</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Agar</td>
			<td style="width:180px;">Wisent Bioproducts</td>
			<td style="width:139px;">800-010-CG</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Agarose</td>
			<td style="width:180px;">Wisent Bioproducts</td>
			<td style="width:139px;">800-015-CG</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Ampicillin sodium salt</td>
			<td style="width:180px;">Wisent Bioproducts</td>
			<td style="width:139px;">400-110-IG</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Chloroform</td>
			<td style="width:180px;">BioShop</td>
			<td style="width:139px;">CCL402</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Deoxynucleotide (dNTP) Solution Mix</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">N0447L</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">DMEM, high glucose, pyruvate</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:139px;">11995065</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">EcoRI</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">R0101S</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">EZ-10 Spin Column DNA Gel Extraction Kit</td>
			<td style="width:180px;">BS353</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:139px;">12483020</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Ficoll-Paque Plus</td>
			<td style="width:180px;">GE Healthcare</td>
			<td style="width:139px;">17-1440-02</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Filter</td>
			<td style="width:180px;">Corning</td>
			<td style="width:139px;">431220</td>
			<td>0.45 mm pore SFCA membrane</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">FITC-conjugated anti-human CD271 (NGFR) mAb</td>
			<td style="width:180px;">Biolegend</td>
			<td style="width:139px;">345104</td>
			<td>clone ME20.4</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">FITC-conjugated anti-human CD3 mAb</td>
			<td style="width:180px;">Biolegend</td>
			<td style="width:139px;">300440</td>
			<td>clone UCHT1</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Gentamicin</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:139px;">15750078</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Gibson Assembly Master Mix</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">E2611L</td>
			<td>used for multi-piece DNA assembly</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">HLA-A2 pentamer</td>
			<td style="width:180px;">Proimmune</td>
			<td style="width:139px;">depends on antigenic peptide</td>
			<td>HLA-A2/MART1 multimer used here was purchased from Proimmune</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">HLA/CD1d monomers</td>
			<td style="width:180px;">NIH Tetramer Core Facility</td>
			<td style="width:139px;"></td>
			<td>multimerize monomers according to protocol provided by NIH tetramer core facility</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Human AB serum</td>
			<td style="width:180px;">Valley Biomedical</td>
			<td style="width:139px;">HP1022</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">human CD3 microbeads</td>
			<td style="width:180px;">Miltenyi Biotec</td>
			<td style="width:139px;">130-050-101</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">IOTest Beta Mark TCR V beta Repertoire Kit</td>
			<td style="width:180px;">Beckman Coulter</td>
			<td style="width:139px;">IM3497</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Jurkat 76 cells</td>
			<td style="width:180px;">Generated by Heemskerk et al. (ref 10)</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">LB Broth</td>
			<td style="width:180px;">Wisent Bioproducts</td>
			<td style="width:139px;">800-060-LG</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">LS MACS column</td>
			<td style="width:180px;">Miltenyi Biotec</td>
			<td style="width:139px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">NEB 5-alpha Competent E. coli</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">C2987I</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">NEBuffer 3.1</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">B7203S</td>
			<td>used for EcoRI and NotI digestion</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">NotI</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">R0189S</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">NucleoBond Xtra Midi</td>
			<td style="width:180px;">Macherey-Nagel</td>
			<td style="width:139px;">740410</td>
			<td>used for plasmid purification</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">PC5-conjugated anti-human CD8 mAb</td>
			<td style="width:180px;">Beckman Coulter</td>
			<td style="width:139px;">B21205</td>
			<td>clone B9.11</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">PG13 cells</td>
			<td style="width:180px;">ATCC</td>
			<td style="width:139px;">CRL-10686</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Phusion HF Buffer Pack</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">B0518S</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Phusion&#160;High-Fidelity DNA Polymerase</td>
			<td style="width:180px;">New England Biolabs</td>
			<td style="width:139px;">M0530L</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">pMX retroviral vector</td>
			<td style="width:180px;">Cell Biolabs</td>
			<td style="width:139px;">RTV-010</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">polybrene</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:139px;">H-9268</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Proleukin (recombinant human interleukin-2)</td>
			<td style="width:180px;">Novartis</td>
			<td style="width:139px;">by Rx only</td>
			<td>equivalent product can be purchased from Sigma-Aldrich</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Purified anti-human CD3 antibody</td>
			<td style="width:180px;">Biolegend</td>
			<td style="width:139px;">317301</td>
			<td>clone OKT3, used for T cell stimulation</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">RPMI 1640</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:139px;">11875119</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">SA-PE</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:139px;">S866</td>
			<td>used for multimerizing monomers from NIH tetramer core facility</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">SMARTer RACE 5&#39;/3&#39;&#160; Kit</td>
			<td style="width:180px;">Clontech</td>
			<td style="width:139px;">634858</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Sterile water</td>
			<td style="width:180px;">Wisent Bioproducts</td>
			<td style="width:139px;">809-115-LL</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">SuperScript III First-Strand Synthesis System</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:139px;">18080051</td>
			<td>for cDNA synthesis</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Syringe</td>
			<td style="width:180px;">BD</td>
			<td style="width:139px;">301604</td>
			<td>10 mL, slip tip</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Tetracycline hydrochloride</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:139px;">T7660</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">TransIT-293</td>
			<td style="width:180px;">Mirus Bio</td>
			<td style="width:139px;">MIR 2700</td>
			<td>used to transfect 293GPG cells</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">TRIzol Reagent</td>
			<td style="width:180px;">Life Technologies</td>
			<td style="width:139px;">15596026</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Without prior knowledge of which hemichain is chain-centric, the TCR&#945; and TCR&#946; chain should be separately cloned and transduced to peripheral blood T cells, which was done in the case of HLA-A24/WT1 reactive TAK1 TCR (Figure 1). Transduction of TAK1&#946; yielded a noticeably higher frequency of antigen-specific T cells. Conversely, transduction of a non-centric hemichain would not yield de novo multimer positive T cells, as seen with TAK1&#945; chain (...

Watch this Scientific Journal Video about Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain at JoVE.com

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.

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, ...

The overall goal of this experimental procedure is to generate De Novo T Cell Receptors or TCRS, recognizing a specific antigen of interest. This method can help isolate optimal T Cell Receptors in a field of gene engineered immunotherapy. The main advantage of this technique is only a single alpha or beta hemi-chain needs to be isolated to generate new entrance specific T Cell Receptors.After eluting the TCR, PCR product clone the gene by combining each linear fragment with a commercially available assembly master mixery agent and incubating the mixture at 50 degrees Celsius for one hour. At the end of the incubation, transform chemically confident E Coli with the assembled plasmin following the manufacturers protocol and seed the transformed bacteria onto agar plates for 18 hour incubation at 37 degrees Celsius. The next day, transfer a single bacterial colony into 50 milliliters of lysogeny broth medium for 16 hours in a shaking incubator at 37 degrees celsius and 200 revolutions per minute.Then, using a commercially available Midiprep kit, according to the manufacturer's protocol, purify the plasmin and dilute the DNA to a concentration of around one microgram per microliter for the 293GPG cell transfection. One day after the transfection, replace the transfection medium from the 293GPG cell culture with fresh DMEM medium. Two days after changing the medium, collect the culture medium in a syringe, followed by it's passage through a 0.45 micron filter to collect the virus.Next seed one times ten to the fifth PG-13 cells in a T-25 flask overnight. The next day aspirate the culture medium and add 1.5 milliliters of the 293GPG derived virus, 1.5 milliliters of DMEM medium, and eight micrograms per milliliter of Polygrain. Change the medium once a day for four days to establish a stable PG-13 packaging cell line for producing a retro virus, encoding a TCR hemi-chain of interest.24 hours after the last infection, replace the culture medium with fresh DMEM medium. To harvest the virus from the transduced cell line, culture two times ten to the sixth PG-13 packaging cells in a T-75 flask with 10 milliliters of fresh DMEM medium. And collect the virus three days after seeding, as just demonstrated.For T cell activation, culture two times ten to the seventh PBMCs per well in a six well plate in five milliliters of RPMI medium supplemented with Recombinant Human IL-2 and anti-CD3 monoclonal antibody. Three days post stimulation, harvest the activated T Cells for centrifugation and resuspend 0.5 to one times ten to the six cells in one milliliter of PG-13 virus and one milliliter of RPMI medium. Supplement it with Recombinant Human IL-2 per well, proceeding in a 24 well plate.When all of the cells have been deposited, centrifuge the plate and transfer the plate into a cell cultured incubator. After 24 hours harvest the cells for centrifugation. Then, resuspend the T Cells pellet in one milliliter of PG-13 virus and one milliliter of RPMI by medium supplemented with IL-2 and centrifuge the plate as just demonstrated.24 hours after the last infection, harvest the cells for centrifugation and resuspend the T Cells in RPMI medium supplemented with IL-2 alone. Return the cells to the incubator for two to three more days of culture. Then stain the cells with a HLA multimer for 30 minutes at four degrees Celsius.Followed by anti-human CD3 and clone receptor monoclonal antibodies at four degrees Celsius for 15 minutes. Sort the multimer positive cells by flow cytometry using the appropriate controls. Seed five times ten to the fourth Jurkat cells per well in a 24 well plate in one milliliter of RPMI medium and one milliliter of 293GPG virus per Jurkat cell transfection with the centric TCR hemi-chain of interest.Centrifuge the plate, and place the plate in the cell culture incubator. After 24 hours collect the cells for centrifugation and resuspend the hemi-chain transduced Jurkat 76 cell pellet in fresh RPMI medium for further culture. To fully reconstitute the T Cell Receptor, produce 293GPG virus encoding a TCR counter-chain, cloned from the sorted multimer positive cells.Enter the centric hemi-chain expressing Jurkat 76 cells as just demonstrated. Three to five days post CD3 purification, stain the clonal tipic TCR expressing transfectants with the appropriate HLA multimer and cell surface staining antibodies of interest. And analyze the cells by flow cytometry to verify the antigen specificity.Without prior knowledge of which hemi-chain is chain centric, The TCR alpha and TCR beta chains should be separately clone and transduced to peripheral blood T-cells. As for this HLA A-24 WT1 reactivate TAK1 T Cell Receptor. TAK1 Beta transsection yields a noticeably higher frequency of antigen specific T Cell.While the transduction of a non centric hemi-chain does not yield to know well positive T Cells as observed for the TAK1 alpha chain. Conversely a single centric hemi-chain can be transduced if it is known to be dominant. Further, T Cells specific for CD1d increase in frequency upon transduction of the centric TCR V alpha 24 hemi-chain.Together these data demonstrate that the introduction of a centric TCR alpha or beta hemi-chain into peripheral blood T cells can generate De Novo TCRs with the antigen specificity of interest. Moreover De Novo TCR alpha counter-chains, paired with TAK1 beta, stained with either a lower or higher intensity by the WT1 antigen complex than the parental TAK1 alpha beta pairing. Similarly, SAK-35 alpha paired with unique counter-chains, recognizes HLA A2 MART1 with a moderate to high avidity.While attempting this procedure to obtain the maximal transection efficiency, it is important to maintain primary T Cells under the optimal culture conditions. Following this procedure, novel HRE class 2 restricted entrance specific T Cell Receptors can be cloned by exploiting the chain centricity. After watching this video you should have a good understanding of how to isolate and reconstitute T Cell Receptor genes by molecular cloning and retroviral transduction.Don't forget that working with replication defective retrovirus can potentially be hazardous and that precaution such as wearing gloves and protective garments should always be taken while performing this procedure.

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Research

JoVE Journal

Immunology and Infection

8.4K visualizações.  University of Toronto. The overall goal of this experimental procedure is to generate De Novo T Cell Receptors or TCRS, recognizing a specific antigen of interest. This method can help isolate optimal T Cell Receptors in a field of gene engineered immunotherapy. The main advantage of this technique is only a single alpha or beta hemi-chain needs to be isolated to generate new entrance specific T Cell Receptors.After eluting the TCR, PCR product clone the gene by combining each linear fragment with a commerci...