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
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).