Name: generating de novo antigen-specific human t cell receptors by retroviral transduction of centric hemichain
Uploaded: 2016-10-25T15:00:00
Description: Watch this Scientific Journal Video about Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain at JoVE.com

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. Forberedelse Retroviral Construct Kodning TCR Hemichain of Interest <ol><li> Linearisere pMX vektoren for at tillade indsættelsen af ​​en TCR-gen i efterfølgende trin. Fordøje plasmid-DNA med EcoRI og NotI restriktionsenzymer ved 37 ° C i 3 timer (tabel 1) 8. </li><li> Udfør elektroforese af det fordøjede plasmid på 1,2% agarosegel. Excise band på ca. 4500 basepar (bps), og der elueres i 30 pi sterilt vand anvendelse af kommercielt tilgængelige gel udvinding kits 9.</su...

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Den første forudsætning for en vellykket anvendelse af denne metode er at opnå tilstrækkelig transduktionseffektivitet af primære T-celler med hemichain af interesse. Det er vores erfaring, at kombinationen af ​​anvendelse af PG13 som pakningscellelinie og pMX som retrovirale vektor resulterer i stabil og effektiv ekspression af det indførte gen i humane primære T-celler. PG13 pakkeceller kan være enkelt-celle klones at selektere for høj titer pakkeceller for at forbedre transduktionseffektivitet. Endvidere...

T-cellereceptorer (TCR&#39;er) er heterodimere adaptive immune receptorer udtrykt af T-lymfocytter, bestående af et TCRα og TCRβ kæde. De genereres via somatisk omlejring af V (D) J-gensegmenter, som frembringer et meget varieret repertoire stand til at genkende næsten ubegrænsede konfigurationer af HLA / peptid-komplekser. Klinisk har T-celler manipuleret til at udtrykke klonotypisk TCR&#39;er er specifikt for tumorassocierede antigener viste virkning i en række forskellige cancere 1. Men mange TCR&#39;er klonede til dette formål mangler tilstrækkelig affinitet for antigenet af interesse, som begrænser deres terapeutiske anvendelse. Her beskriver vi en metode til at overvinde denne begrænsning for eksisterende TCR&#39;er ved at udnytte kæde-centricity. Det er blevet rapporteret, at en TCR hemichain kunne spille en mere dominerende rolle i erkendelse af målantigenet 2, her betegnet centricity. Crystal strukturelle analyser har vist, at en centrerethemichain af en TCR kunne redegøre for størstedelen af fodaftryk på MHC / peptid-kompleks 3,4. Ved hjælp af dette koncept, har vi tidligere vist, at SIG35α TCRα kan parre med en varieret repertoire af TCRβ kæder og vedligeholde reaktivitet mod MART1 27-35 peptid præsenteret af HLA-A2 5. Lignende resultater blev opnået med TAK1 TCR, hvor den centriske TCRβ hemichain parret med forskellige TCRα kæder og opretholdt reaktivitet for WT1 235-243 peptidet præsenteres af HLA-A24 6. Både MART1 og WT1 er tumor-associerede antigener. Kæde-centricity blev også anvendt til at studere antigen anerkendelse af CD1d-begrænsede invariante naturlige dræberceller (iNKT) TCR, ved at parre den invariante Vα24-Jα18 (Vα24i) TCRα kæde af menneskelige iNKT TCR&#39;er med forskellige Vβ11 TCRβ kæder 7. I alle tilfælde var vi i stand til at generere en de novo repertoire af TCR&#39;er af transducere den centriske TCR hemichain til perifert blod T-celler, hvor den indførte hemichain parret med den endogene TCRα eller TCRβ counter-kæder. I det væsentlige centreret hemichain tjener som en lokkemad, der kan anvendes til at identificere de passende counter-kæder, som, når parret sammen danner TCR&#39;er der opretholder antigenspecificitet af interesse, men varierende affinitet. Ud fra disse nye repertoirer, var vi i stand til at isolere klonotypisk TCR&#39;er med forbedret interaktion styrke mod målantigenet sammenlignet med allerede eksisterende TCR&#39;er. Derfor mener vi, denne metode vil fremskynde rørledningen identificere optimale TCR&#39;er for klinisk anvendelse.

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		<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-cellereceptorer (TCR&#39;er) anvendes klinisk til at dirigere specificiteten af ​​T-celler til at målrette tumorer som et lovende modalitet af immunterapi. Derfor har kloning TCR&#39;er specifikke for forskellige tumorassocierede antigener været målet for mange undersøgelser. At fremkalde en effektiv T-celle respons, skal TCR genkender målantigenet med optimal affinitet. Imidlertid har kloning sådan TCR&#39;er været en udfordring og mange tilgængelige TCR&#39;er besidder suboptimal affinitet for dertil hørende antigen. I denne protokol, beskriver vi en metode til at klone de novo høj affinitet antigen-specifikke TCR&#39;er bruger eksisterende TCR&#39;er ved at udnytte hemichain centricity. Det er kendt, at for nogle TCR&#39;er, behøver hver TCRα eller TCRβ hemichain ikke bidrager ligeligt til antigengenkendelse, og den dominerende hemichain omtales som den centriske hemichain. Vi har vist, at ved parring af centric hemichain med counter-kæder afviger fra den oprindelige counter-kæde, er vi i stand til at opretholde antigenet specificity, mens modulere dens interaktion styrke for den beslægtede antigen. Således kan det terapeutiske potentiale af en given TCR forbedres ved at optimere parring mellem centreret og counter hemichains.

Uden forudgående kendskab til hvilke hemichain er kæde-centreret, det TCRα og TCRβ kæde skal separat klonet og transduceres til perifere blod T-celler, som blev gjort i tilfældet med HLA-A24 / WT1 reaktiv TAK1 TCR (figur 1). Transduktion af TAK1β gav et mærkbart højere frekvens af antigenspecifikke T-celler. Omvendt ville transduktion af en ikke-centreret hemichain ikke give de novo multimer positive T-celler, som set med TAK1α kæde (figur 1)....

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

Heri beskriver vi en hidtil ukendt fremgangsmåde til at generere antigenspecifikke T-celle-receptorer (TCR&#39;er) ved parring af TCRα eller TCRβ af en eksisterende TCR, der besidder den antigen-specificitet af interesse, med komplementær hemichain af den perifere T-celle receptor repertoire. De novo genererede TCR&#39;er bevarer antigen-specificitet med varierende affinitet.

Generering<em&gt; De Novo</em&gt; Antigenspecifikke humane T-celle receptorer ved retroviral transduktion af 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, ...

Research

JoVE Journal

Immunology and Infection

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

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 ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Vurdering af Innate Sensing af HIV-1 inficerede CD4<sup&gt; +</sup&gt; T-celler ved plasmacytoide dendritiske celler ved hjælp af en<em&gt; Ex vivo</em&gt; Co-dyrkningssystem.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Kunstig antigen-præsenterende celler (KaAPC) til effektiv<em&gt; In Vitro</em&gt; Udtømning af humane antigen-specifikke T-celler

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Whole-dyr Imaging og Flowcytometrisk Teknikker til analyse af antigen-specifikke CD8 + T-celle responser efter Nanopartikel Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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

Retroviral transduktion af knoglemarven til at generere T-celle receptor Retrogenic Mus

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Retroviral transduktion af Helper T-celler som en genetisk tilgang til at studere Mekanismer Controlling deres Differentiering og funktion

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Udvikling af Stem Cell-afledte antigen-specifikke regulatoriske T celler mod Autoimmunitet

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay til påvisning af Antigen-specifikke T-celle cytolytisk funktion ved hjælp af en Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Generation af diskriminerende menneskelige monoklonale antistoffer fra sjældne Antigen-specifikke B celler cirkulerer i blodet

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Billedbehandling protokol for analysen af Diffusion og Cluster størrelse af membranreceptorer af Fluorescens mikroskopi

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...