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. Preparazione retrovirale Construct Codifica TCR Hemichain di interesse <ol><li> Linearizzare PMX vettoriale per consentire l&#39;inserimento di un gene TCR nei passaggi successivi. Digerire il DNA plasmide con EcoRI e NotI enzimi di restrizione a 37 ° C per 3 ore (Tabella 1) 8. </li><li> Effettuare elettroforesi del plasmide digerito su 1,2% gel di agarosio. Banda accise di circa 4.500 paia di basi (bps), ed eluire in 30 ml di acqua sterile utilizzando disponibili in commercio kit di es...

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Il primo requisito per l&#39;applicazione di successo di questo metodo sta ottenendo sufficiente efficienza di trasduzione delle cellule T primarie con il hemichain di interesse. Nella nostra esperienza, la combinazione di utilizzare PG13 come linea cellulare di packaging e PMX come risultati vettore retrovirale in espressione stabile ed efficiente del gene introdotto nelle cellule T primarie umane. cellule packaging PG13 possono essere unicellulare clonato per selezionare le celle per imballaggio alto titolo per miglio...

recettori delle cellule T (TCR) sono eterodimeriche recettori immunitarie adattative espressi dai linfociti T, composto da una catena TCRα e TCRβ. Essi sono generati mediante riarrangiamento somatica di V (D) J segmenti genici, che produce un repertorio altamente diversificata capace di riconoscere configurazioni praticamente illimitata di complessi HLA / peptide. Clinicamente, cellule T ingegnerizzate per esprimere clonotypic specifico TCR per antigeni tumore-associati hanno dimostrato l&#39;efficacia in una varietà di tumori 1. Tuttavia, molti TCR clonati per questo scopo non hanno sufficiente affinità per l&#39;antigene di interesse, che limitano la loro applicazione terapeutica. Qui, descriviamo un metodo per superare questo limite per TCR esistenti sfruttando catena di centralità. E &#39;stato riportato che un TCR hemichain potrebbe svolgere un ruolo più dominante in riconoscimento del target dell&#39;antigene 2, qui chiamato centralità. analisi strutturali cristallo hanno dimostrato che uno centrichemichain di un TCR potrebbe spiegare la maggioranza della impronta sulla MHC / peptide complesso 3,4. Usando questo concetto, abbiamo precedentemente dimostrato che l&#39;SIG35α TCRα può accoppiare con un repertorio vario di catene TCRβ e mantenere la reattività contro il peptide MART1 27-35 presentato da HLA-A2 5. Risultati simili sono stati ottenuti con l&#39;TAK1 TCR, dove il centric TCRβ hemichain accoppiato con varie catene TCRα e mantenuto reattività per il peptide WT1 235-243 presentati da HLA-A24 6. Sia MART1 e WT1 sono antigeni associati al tumore. Catena-Centricity è stato applicato anche per studiare il riconoscimento dell&#39;antigene di TCR invarianti CD1d-ristrette natural killer (iNKT), accoppiando la catena invariante Vα24-Jα18 (Vα24i) TCRα di TCR iNKT umani con diverse catene Vβ11 TCRβ 7. In tutti i casi, siamo stati in grado di generare un repertorio de novo di TCR da transdurre il centrica TCR hemichain alle cellule T del sangue periferico, dove il hemichain introdotto accoppiato con il TCRα endogena o TCRβ contro-catene. In sostanza, il hemichain centric serve come esca che può essere utilizzato per identificare le opportune contromisure catene, che quando accoppiati insieme formano TCR che mantengono la specificità antigene di interesse, ma variano in affinità. Da questi nuovi repertori, siamo stati in grado di isolare TCR clonotypic con resistenza migliorata interazione contro l&#39;antigene bersaglio rispetto al TCR preesistenti. Pertanto, riteniamo che questo metodo accelererà la pipeline di identificare TCR ottimali per l&#39;applicazione clinica.

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			<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>
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			<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>
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			<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>
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		<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

recettori delle cellule T (TCR) sono utilizzati clinicamente per dirigere la specificità delle cellule T a colpire i tumori come modalità promettente di immunoterapia. Pertanto, TCR clonazione specifici per diversi antigeni associati al tumore è stato l&#39;obiettivo di molti studi. Per suscitare una risposta delle cellule T efficace, il TCR deve riconoscere l&#39;antigene bersaglio con affinità ottimale. Tuttavia, la clonazione come TCR è stata una sfida e molti disponibili TCR possedere affinità sub-ottimale per l&#39;antigene cognate. In questo protocollo, si descrive un metodo di clonazione de novo ad alta affinità TCR antigene-specifiche che utilizzano TCR esistenti sfruttando hemichain centralità. È noto che per alcuni TCR, ogni TCRα o TCRβ hemichain non contribuiscono ugualmente al riconoscimento dell&#39;antigene, e il hemichain dominante è indicato come il hemichain centric. Abbiamo dimostrato che accoppiando il hemichain centrica con contro-catene differenti dal contatore-catena originale, siamo in grado di mantenere l&#39;antigene specificity, mentre modulando la sua forza di interazione per l&#39;antigene cognate. Pertanto, il potenziale terapeutico di un dato TCR può essere migliorata ottimizzando l&#39;accoppiamento tra le centric e contro hemichains.

Senza la conoscenza preventiva di cui hemichain è a catena-centrica, la catena TCRα e TCRβ dovrebbe essere clonato e trasduzione di cellule T del sangue periferico, che è stato fatto nel caso di HLA-A24 / WT1 reattiva TAK1 TCR (Figura 1) separatamente. La trasduzione di TAK1β prodotto una frequenza notevolmente più elevata di cellule T antigene-specifiche. Al contrario, la trasduzione di un hemichain non-centric non voleva cedere de novo cellule multimero ...

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

Qui si descrive un nuovo metodo per generare recettori delle cellule T antigene-specifiche (TCR) accoppiando la TCRα o TCRβ di un TCR esistente, che possiede l&#39;antigene-specificità di interesse, con hemichain complementare del T periferico recettore delle cellule repertorio. I TCR de novo generati conservano antigene-specificità con vari affinità.

Generazione<em&gt; De Novo</em&gt; Antigene-specifici T umana recettori cellulari di trasduzione retrovirale di 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

Trasduzione retrovirale di recettori delle cellule T nel topo le cellule T

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 ...

Ricerca

Immunologia e infezioni

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.

Valutare l&#39;innato rilevamento dell&#39;infezione da HIV-1 CD4 infetti<sup&gt; +</sup&gt; Cellule T dalle cellule dendritiche plasmacitoidi Utilizzando un<em&gt; Ex vivo</em&gt; Sistema di co-coltura.

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 artificiale cellule presentanti l&#39;antigene (KaAPC) per una efficiente<em&gt; In Vitro</em&gt; L&#39;esaurimento delle Umana antigene-specifiche cellule T

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

Imaging Whole-animale e di citometria a flusso tecniche di analisi di Antigen-specific CD8 + cellule T Risposte dopo nanoparticelle vaccinazione

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

Trasduzione retrovirale di cellule progenitrici del midollo osseo per generare cellule T del recettore Retrogenic Mice

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

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

Trasduzione retrovirale di cellule T helper come un approccio genetico per studiare meccanismi che controllano la loro differenziazione e funzione

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

Lo sviluppo di cellule staminali derivate antigene-specifiche cellule T regolatorie contro autoimmunità

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 Test per la determinazione della funzione citolitica di cellule T antigene-specifiche utilizzando un modello di vaccinazione

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

Generazione di anticorpi monoclonali umani discriminante da rari B antigene-specifiche cellule circolanti nel sangue

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

Elaborazione di protocollo per l'analisi della diffusione e dimensione dei Cluster di recettori di membrana mediante microscopia a fluorescenza

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