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. 레트로 바이러스가 관심의 인코딩 TCR Hemichain를 구축 준비 <ol><li> 후속 단계에서 TCR 유전자의 삽입을 허용하도록 PMX 벡터 선형화. 3 시간 동안 37 ° C (표 1)에 제한 효소 EcoRI과 NotI을 제한 효소로 플라스미드 DNA를 소화 8. </li><li> 1.2 % 아가 로스 겔에 소화 플라스미드의 전기 영동을 수행하십시오. 소비세 약 4,500 염기쌍 (BPS)의 밴드 및 용출은 30 ㎕의 멸균 수에 시판 겔 추출 키...

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이 방법을 성공적으로 적용하는 첫 번째 요건은 관심 hemichain 일차 T 세포의 충분한 전달 효율을 달성한다. 경험 인간 T 세포 주에서 도입 유전자의 안정하고 효율적인 발현 레트로 바이러스 벡터의 결과로서 패키징 세포주와 PMX로서 PG13를 사용하는 조합. PG13 패키징 세포는 형질 도입 효율을 향상시키는 고역가 포장 셀 선택 클로닝 단일 셀 수있다. 또한, T 세포 증식은 또한 레트로 바이러스에 의?...

T 세포 수용체 (TCR도)는 TCRα 및 TCRβ 쇄로 이루어지는 T 림프구에 의해 발현 이종 적응 면역 수용체이다. 그들은 HLA / 펩타이드 복합체의 거의 무한대의 구성을 인식 할 수있는 매우 다양한 레퍼토리를 생산 V (D) J 유전자 세그먼트의 체세포 재 배열을 통해 생성된다. 임상 종양 관련 항원에 대한 clonotypic TCR도의 특정을 발현하도록 유전자 조작 된 T 세포는 암 (1)의 다양한 효능을 증명하고있다. 그러나, 이러한 목적을 위해 많은 클론 TCR도 그들의 치료 학적 적용을 제한 관심있는 항원에 충분한 친 화성이 부족하다. 여기서는 체인 중심성을 이용하여 기존의 TCR도이 제한을 극복하는 방법을 설명한다. 그것은 하나의 TCR의 hemichain는 표적 항원이 인식에서 더 지배적 인 역할을 할 수 있다고보고되고있다, 여기 중심성 불린다. 크리스탈 구조 분석은 하나의 중심을 보여 주었다TCR의 hemichain는 3,4 복잡 MHC / 펩타이드의 공간의 대부분을 설명 할 수있다. 이 개념을 사용하여, 우리는 이전에 SIG35α TCRα가 TCRβ 체인의 다양한 레퍼토리와 페어링 및 HLA-A2 (5)에 의해 제시된 MART1 27-35 펩타이드에 대한 반응성을 유지할 수 있음을 증명하고있다. 유사한 결과는 중심 TCRβ의 hemichain 다양한 TCRα 사슬과 페어링 및 HLA-A24 (6)에 의해 제공되는 WT1 펩티드 235-243 반응성을 유지 TAK1 TCR로 얻었다. MART1 및 WT1 모두 종양 관련 항원이다. 체인 중심성은 다른 Vβ11 TCRβ 체인 (7) 인간하여 iNKT TCR도의 불변 Vα24-Jα18 (Vα24i) TCRα 체인을 페어링하여, CD1d 제한 불변 자연 살해 (인 iNKT) TCR도의 항원 인식을 연구하기 위해 적용되었다. 모든 경우에서는 트랜스로 TCR도의 드 노보 레퍼토리를 생성 할 수 있었다도입 hemichain이 내생 TCRα 또는 TCRβ 카운터 사슬와 결합 말초 혈액 T 세포에 중심 TCR의 hemichain를으로 줄. 본질적으로, 중심 hemichain 함께 쌍으로 관심의 항원 특이성을 유지 TCR도 형성하면서도 친 화성 변화 적절한 이의 사슬을 식별하는데 사용될 수있는 미끼로서 기능한다. 이러한 신규 레퍼토리에서, 우리는 기존에 비하여 TCR도 표적 항원에 대해 향상된 상호 작용 강도 clonotypic TCR도를 분리 할 수 ​​있었다. 따라서, 우리는이 방법이 임상 응용 프로그램에 대한 최적의 TCR도를 식별하는 파이프 라인을 가속화 할 전망이다.

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			<td height="44" style="height:44px;width:216px;">0.05% Trypsin-EDTA</td>
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			<td height="44" style="height:44px;width:216px;">293GPG cells</td>
			<td style="width:180px;">Generated by Ory et al. (ref 8)</td>
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			<td height="44" style="height:44px;width:216px;">Agar</td>
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			<td height="44" style="height:44px;width:216px;">Deoxynucleotide (dNTP) Solution Mix</td>
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			<td height="44" style="height:44px;width:216px;">Filter</td>
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			<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 세포 수용체 (TCR도)의 면역 요법의 유망 양상으로 종양 타겟팅 T 세포의 특이성을 직접 임상 사용된다. 따라서, 다양한 종양 관련 항원에 대한 특정 복제 TCR도 많은 연구의 목표였다. 효과적인 T 세포 반응을 유도하기 위해, TCR 최적 친화력 표적 항원을 인식해야한다. 그러나, 복제 등의 TCR도는 도전과 많은 사용할 수 TCR도하고 동족 항원에 대한 차선의 친 화성을 가지고있다. 이 프로토콜에서는, 우리는 hemichain의 중심성을 이용하여 기존 TCR도를 사용하여 드 노보 선호도가 높은 항원 특이 TCR도를 복제하는 방법을 설명합니다. 이는 일부 TCR도, 각 TCRα 또는 TCRβ hemichain의 항원 인식에 동일하게 기여하지 않는 것으로 알려져 있으며, 지배적 hemichain는 중심 hemichain로 지칭된다. 우리는 원래 반 체인에서 다른 반 체인으로 중심 hemichain 페어링하여, 우리는 항원의를 유지할 수 있음을 보여 주었다pecificity, 동족 항원 상호 작용의 강도를 조절하면서. 따라서, 주어진 TCR의 치료 가능성은 중심과 대향 hemichains 사이의 페어링을 최적화함으로써 개선 될 수있다.

사전 지식없이하는 hemichain의 TCRα 및 TCRβ 체인은 별도 복제 및 HLA-A24 / WT1 반응 TAK1 TCR (그림 1)의 경우에 이루어졌다 말초 혈액 T 세포에 형질 도입해야 체인 중심이다. TAK1β의 전달은 항원 특이 적 T 세포의 현저히 더 높은 주파수를 수득 하였다. 반대로, 비 중심 hemichain의 전달은 드 노보 합체 양성 T 세포 TAK1α 체인 것과 같이 (도 1)를 산출하...

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

여기서 우리는, 기존 TCR의 TCRα 또는 TCRβ 페어링 말초 T 세포 수용체 레퍼토리 상보 hemichain으로 관심 항원 특이성을 보유하여 항원 특이 적 T 세포 수용체 (TCR도)을 생성하는 신규 한 방법을 설명한다. 드 노보 생성 TCR도 선호도 변화와 항원 특이성을 유지한다.

생성<em&gt; 드 노보</em&gt; 중심 Hemichain의 레트로 바이러스 형질 도입에 의해 항원 특이 적 인간 T 세포 수용체

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

마우스 T - 세포에서 T - 세포 수용체의 Retroviral 전달

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 ...

연구

면역학 및 감염병학

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.

HIV-1 감염 CD4의 타고난 감지 평가<sup&gt; +</sup&gt;를 사용하여 형질 수지상 세포에 의한 T 세포<em&gt; 예 생체 내</em&gt; 공동 문화 시스템.

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

효율적인위한 킬러 인공 항원 제시 세포 (KaAPC)<em&gt; 체외</em&gt; 인간의 항원 특이 T 세포의 고갈

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

나노 입자 백신 접종 후 분석 항원 특이 CD8 + T 세포 반응에 대한 전체 동물 이미징 및 유세포 기술

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

골수 전구 세포의 레트로 바이러스 형질 도입은 T 세포 수용체 Retrogenic 마우스를 생성하는

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

공부하는 유전 적 접근으로 헬퍼 T 세포의 레트로 바이러스 형질 도입은 메커니즘 자신의 차별화 및 기능 제어

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

자가 면역 대해 줄기 세포 유래의 항원 특이 적 조절 T 세포의 발달

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

Vivo에서 예방 접종 모델을 사용 하는 항 원 특정 T-셀 Cytolytic 함수의 검색에 대 한 분석 결과

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

희귀 한 항 원 특정 B 세포 혈액 순환에서 구별 인간 단일 클론 항 체의 생성

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

확산의 분석 및 형광 현미경 검사 법에 의해 수용 체 막의 클러스터 크기에 대 한 프로토콜을 처리 하는 이미지

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