Summary

发电<em>德诺</em>抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

Published: October 25, 2016
doi:

Summary

这里我们描述了一种新方法,以产生抗原特异性T细胞受体(TCR)通过配对现有的TCR的TCRα或TCRβ,具有感兴趣的抗原特异性,与外周T细胞受体库的补充hemichain。 从头产生的TCR保留抗原特异性有不同的亲和力。

Abstract

T细胞受体(TCR)在临床上用于指导T细胞的特异性靶向肿瘤免疫疗法的一个有前途的方式。因此,具体的关于各种肿瘤相关抗原的克隆的TCR一直是许多研究的目标。以引发有效的T细胞应答,T细胞受体必须认识到与最佳亲和力的靶抗原。然而,这种克隆的TCR一直是一个挑战,许多可用的TCR具有对同源抗原次优的亲和力。在这个协议中,我们描述了克隆使用现有的TCR通过利用hemichain为中心从头高亲和力的抗原特异性的TCR的方法。众所周知,对于某些的TCR,每个TCRα或TCRβhemichain不相等,以抗原识别贡献,显性hemichain被作为中心hemichain提及。我们已经表明,通过配对与反链从原来的反链不同的中心hemichain,我们能够保持抗原小号pecificity,而调节其作用强度为同源抗原。因此,一个给定的TCR的治疗潜力可通过优化中心和计数器hemichains之间的配对的提高。

Introduction

T细胞受体(TCR)是由T淋巴细胞表达异二聚体适应性免疫受体,一个TCRα和TCRβ链组成。它们通过V(D)J基因片段,其产生能识别的HLA /肽复合物的几乎无限的构型的高度多样化剧目体重排产生的。在临床上,改造为表达克隆型的TCR特异性的肿瘤相关抗原的T细胞在多种癌症1已经证明效力。但是,克隆用于此目的的许多的TCR缺乏感兴趣的抗原,这限制了它们的治疗应用足够的亲和力。

在这里,我们描述了通过利用链为中心克服这种限制对于现有的TCR的方法。已经报道,一个TCR hemichain可以发挥识别靶抗原2的更主要的作用,这里称为为中心。晶体结构分析表明,一个中心一个TCR的hemichain可能占大多数的MHC /肽复合物3,4的足迹。使用这一概念,我们先前已经证明了SIG35αTCRα可以与TCRβ链的多样剧目配对,并保持反应性对抗由HLA-A2 5提出的MART1 27-35肽。类似的结果与TAK1 TCR,其中该中心TCRβhemichain配对与各种TCRα链和维持反应对由HLA-A24 6提出的WT1 235-243肽得到。既MART1和WT1是肿瘤相关抗原。链为中心也被用于研究CD1d的限制不变的自然杀伤(的iNKT)的TCR的抗原识别,通过配对不同Vβ11TCRβ链7人的iNKT的TCR的不变Vα24-Jα18(Vα24i)TCRα链。

在任何情况下,我们能够通过跨产生的TCR的从头剧目ducing的中心的TCR hemichain到外周血T细胞,其中该引入hemichain与内源TCRα或TCRβ计数器链配对。在本质上,中心hemichain作为可用于确定适当的反链,其一起使用时形成保持目的抗原的特异性的TCR,但在亲和力改变一个诱饵。从这些新的全集,我们能够克隆型的TCR与抗相比预先存在的TCR对靶抗原改进相互作用强度隔离。因此,我们认为这种方法将加快确定最佳的TCR为临床应用的管道。

Protocol

1.准备逆转录病毒构建利益编码TCR Hemichain 线性PMX矢量,以允许在随后的步骤的TCR基因的插入。消化用EcoRI和NotI限制性内切酶的质粒DNA在37℃下3小时( 表1)8。 开展1.2%琼脂糖凝胶的消化的质粒的电泳。的约4500个碱基对(BPS)消费税乐队,稀释成30微升无菌水使用市售凝胶提取试剂盒9。 设计5'和3'引物对感兴趣的TCR基因也编码15-20 bps的重叠P…

Representative Results

没有先验知识其中hemichain的是链为中心,在TCRα和TCRβ链应分开克隆,并转到外周血T细胞,将其在HLA-A24 / WT1反应TAK1 TCR的( 图1)的情况下完成的。 TAK1β的转导产生抗原特异性T细胞的显着更高的频率。相反,非中心hemichain转导不会产生从头多聚体阳性T细胞,所看到与TAK1α链( 图1)。在分析过程中,对NGFR +细胞的大门,如果TCR基因?…

Discussion

这种方法的成功应用的第一个要求是实现初级T细胞与感兴趣的hemichain足够的转导效率。根据我们的经验,使用PG13作为包装细胞系和PMX如在人原代T细胞中导入基因的稳定,高效表达逆转录病毒载体的结果的组合。 PG13包装细胞可以是单细胞克隆以选择高滴度包装细胞,以提高转导效率。此外,还需要通过逆转录病毒高转导效率的T细胞的增殖。在所描述的协议中,刺激用可溶性抗CD3单抗OKT3,具有的…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

0.05% Trypsin-EDTA Wisent Bioproducts 325-043-CL
293GPG cells Generated by Ory et al. (ref 8)
Agar Wisent Bioproducts 800-010-CG
Agarose Wisent Bioproducts 800-015-CG
Ampicillin sodium salt Wisent Bioproducts 400-110-IG
Chloroform BioShop CCL402
Deoxynucleotide (dNTP) Solution Mix New England Biolabs N0447L
DMEM, high glucose, pyruvate Life Technologies 11995065
EcoRI New England Biolabs R0101S
EZ-10 Spin Column DNA Gel Extraction Kit BS353
Fetal Bovine Serum Life Technologies 12483020
Ficoll-Paque Plus GE Healthcare 17-1440-02
Filter Corning 431220 0.45 mm pore SFCA membrane
FITC-conjugated anti-human CD271 (NGFR) mAb Biolegend 345104 clone ME20.4
FITC-conjugated anti-human CD3 mAb Biolegend 300440 clone UCHT1
Gentamicin Life Technologies 15750078
Gibson Assembly Master Mix New England Biolabs E2611L used for multi-piece DNA assembly
HLA-A2 pentamer Proimmune depends on antigenic peptide HLA-A2/MART1 multimer used here was purchased from Proimmune
HLA/CD1d monomers NIH Tetramer Core Facility multimerize monomers according to protocol provided by NIH tetramer core facility
Human AB serum Valley Biomedical HP1022
human CD3 microbeads Miltenyi Biotec 130-050-101
IOTest Beta Mark TCR V beta Repertoire Kit Beckman Coulter IM3497
Jurkat 76 cells Generated by Heemskerk et al. (ref 10)
LB Broth Wisent Bioproducts 800-060-LG
LS MACS column Miltenyi Biotec 130-042-401
NEB 5-alpha Competent E. coli New England Biolabs C2987I
NEBuffer 3.1 New England Biolabs B7203S used for EcoRI and NotI digestion
NotI New England Biolabs R0189S
NucleoBond Xtra Midi Macherey-Nagel 740410 used for plasmid purification
PC5-conjugated anti-human CD8 mAb Beckman Coulter B21205 clone B9.11
PG13 cells ATCC CRL-10686
Phusion HF Buffer Pack New England Biolabs B0518S
Phusion High-Fidelity DNA Polymerase New England Biolabs M0530L
pMX retroviral vector Cell Biolabs RTV-010
polybrene Sigma-Aldrich H-9268
Proleukin (recombinant human interleukin-2) Novartis by Rx only equivalent product can be purchased from Sigma-Aldrich
Purified anti-human CD3 antibody Biolegend 317301 clone OKT3, used for T cell stimulation
RPMI 1640 Life Technologies 11875119
SA-PE Life Technologies S866 used for multimerizing monomers from NIH tetramer core facility
SMARTer RACE 5'/3'  Kit Clontech 634858
Sterile water Wisent Bioproducts 809-115-LL
SuperScript III First-Strand Synthesis System Invitrogen 18080051 for cDNA synthesis
Syringe BD 301604 10 mL, slip tip
Tetracycline hydrochloride Sigma-Aldrich T7660
TransIT-293 Mirus Bio MIR 2700 used to transfect 293GPG cells
TRIzol Reagent Life Technologies 15596026

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Cite This Article
Guo, T., Ochi, T., Nakatsugawa, M., Kagoya, Y., Anczurowski, M., Wang, C., Rahman, M. A., Saso, K., Butler, M. O., Hirano, N. Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain. J. Vis. Exp. (116), e54697, doi:10.3791/54697 (2016).

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