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.准备逆转录病毒构建利益编码TCR Hemichain <ol><li>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。 </li><li>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。 </li><li>设计5&#39;和3&#39;引物对感兴趣的TCR基因也编码15-20 bps的重叠PMX矢量，分别为8的EcoRI和NotI消化位点。 </li><li>扩增TCR基因引物。如步骤1.2中所述进行的约1,000个基点的PCR产物和洗脱带电泳。 </li><li>克隆的TCR基因导入消化的载体由每个线性片段与可商购的质粒组合装配主混合试剂，并在50℃温育1小时。 注:参见制造商的协议对于每个组件的相对体积。这种装配方法是基于最初由Gibson等 10中所述的技术。 </li><li> （可选的）标记的TCR基因标为转导的细胞与人神经生长因子受体的截短形式（ΔNGFR，氨基酸1-277）11由弗林蛋白酶切割位点，丝氨酸-甘氨酸接头分开，和图2A序列12,13。设计引物编码序列重叠片段末端，并按照步骤1.3-1.5描述组装质粒。 注意:这些序列可参考11-13找到。 </li><li>变换化学感受态大肠杆菌具有下列制造商的协议14组装质粒。在琼脂平板上的种子转化的细菌（20毫克/毫升LB培养基（LB），15毫克/毫升琼脂和1微克/毫升氨苄青霉素），在37°C孵育18小时。 </l我&gt; <li>培养单个菌落在50毫升LB培养基（20毫克/毫升的LB和1微克/毫升氨苄青霉素），用于在振荡培养箱16小时，在37℃和200转每分钟。 </li><li>净化用以下制造商的协议市售中提试剂盒的质粒。质粒稀释到大约1微克/μL的浓度。 注:质粒纯化协议基于碱性提取方法15。 </li></ol> 2.生成稳定的包装细胞系注:两个293GPG和PG13细胞贴壁。在Dulbecco氏改良Eagle培养基（DMEM）中培养的细胞在补充有10％胎牛血清（FCS）和50μg/ ml庆大霉素。培养293GPG细胞转染前1微克/毫升四环素。孵育细胞在37℃，5％的CO 2的所有步骤之间。 <ol><li>转染293GPG包装细胞中，在步骤1.9，USI得到的hemichain编码矢量的T75烧瓶16培养纳克市售的转染试剂依照制造商的方案1 7。注:转染细胞293GPG在50-60％汇合。 </li><li>对于293GPG细胞吸中，加入10毫升新鲜DMEM培养基后1天转染。 </li><li>嘉实步2.2转增培养基注射器，并通过0.45微米的过滤器后第2天瞬时产生病毒转染的细胞293GPG。 注:病毒可以立即使用或储存于-80℃以备将来使用。 </li><li>培养1×10 5个 PG13细胞的T25烧瓶中。算使用血球细胞。一天后，吸培养基，并添加1.5 293GPG源性病毒的溶液从步骤2.3和1.5毫升DMEM培养基中，以8微克/毫升聚凝胺的沿。 </li><li> 4天每天一次在步骤2.4所描述的改变介质，以建立编码目的TCR hemichain稳定PG13包装细胞系制备逆转录病毒。 </li><li>吸媒体和替换为PG13细胞系进行进一步培养上一次感染后24小时的新鲜DMEM培养基。 注:冻结或子培养细胞用0.05％胰蛋白酶-EDTA溶液脱离。 </li><li>至如在步骤2.33天接种细胞后描述产生从转导PG13细胞系的病毒，培养2×10 6个细胞在T75用10ml DMEM培养基烧瓶中，收获病毒。 注:病毒立即最好使用，但可存储在-80℃达两个月。 </li></ol> 3.激活和人类T细胞的转导注意:人的样品，获得并根据机构伦理委员会批准的方案使用。罗斯韦尔园区纪念研究所培养原代人细胞补充有10％人血清，而不是FCS和50μg/ ml庆大霉素（RPMI）培养基。孵育细胞在37℃，5％的CO 2的所有步骤之间。 <ol><li>分离人外周血单核细胞（PBMC）通过密度依照制造商的协议18梯度分离。 </li><li>活化T细胞以诱导对逆转录病毒感染所需的增殖。每孔培养2×10 7个新鲜或解冻的PBMC在6孔板，在5毫升与100IU / ml重组人白介素-2（的rhIL-2）和50ng / ml的抗CD3单克隆抗体的RPMI培养基中的（克隆OKT3）。 </li><li>三天后的刺激，在350 XG吹打，离心收集T细胞4分钟。弃上清和种子每孔0.5-1×10 6个 T细胞再悬浮于1ml PG13病毒从步骤2.7 24孔板，1补充有200单位/毫升的rhIL-2的RPMI培养基中。在1000 xg离心离心机板和32℃下1小时。 </li><li> 24小时后，收集T细胞吹打，离心350 XG 4分钟。丢弃上清，重悬细胞于1ml PG13病毒从步骤2.7和1补充有200单位/毫升的rhIL-2的RPMI培养基中。重复此步骤，总共6侵染的离子。 注:感染数量应根据由包装细胞系产生的病毒的滴度进行优化。如果需要的话，通路T细胞每天除去细胞的20-30％，以防止过度生长。 </li><li>最后在感染后24小时，收集T细胞吹打，离心350 XG 4分钟。丢弃与100IU / ml的重组人IL-2的用于进一步培养RPMI培养基上清，重悬细胞。 </li><li>步骤3.5，染色的T细胞在4℃，15分钟，4℃，30分钟，然后加入抗人CD3的HLA多聚和共受体的单克隆抗体（mAb）后2-3天。通过流式细胞仪分析。使用多聚体无关和/或细胞未转染为阴性对照（ 见图1 - 3）19。 </li><li>如果所引入的中心hemichain是TCRα链，使用市售的Vβ特异性抗体面板通过流式细胞仪20分析的从头多聚阳性细胞的Vβ的使用。 </li></ol> 4.克隆德诺 TCR反hemichains <ol><li>排序从头多聚积极步骤3.6流辅助细胞分选的人口。 </li><li>从使用酸性硫氰酸胍-苯酚-氯仿提取方法21,2 2排序的T细胞分离RNA。 注:RNA可被储存在-80℃，但应使用尽快生成的cDNA。 </li><li>使用以下制造商的协议23,24市售逆转录酶-PCR试剂盒从提取的RNA合成cDNA文库。 </li><li>如果克隆TCRβ反链，设计Vβ基因和TCRβ恒定区特异性的引物，如在步骤3.7确定的，并克隆全长TCRβ步骤1.3-1.9中描述的基因作为。请参阅步骤4.5，如果反链TCRα，否则继续执行步骤5.1。 </li><li>市售Vα特异性抗体有限，因而Vα基因的使用不能通过流式细胞仪来确定。通过cDNA末端（RACE）25 5&#39;-快速扩增克隆TCRα反链，使用市售5&#39;RACE包6。 <ol><li>合成从在步骤4.2中提取的RNA下列制造商的协议5&#39;RACE兼容的cDNA。 </li><li>如表2所示进行第一轮PCR。 </li><li>进行PCR产物的电泳和洗脱的约1,100个基点带，如在步骤1.2中描述。 </li><li>如表3中所述，使用第一轮PCR产物作为模板进行第二轮PCR。 注:下表3所示的引物序列被设计用于克隆到EcoRI和NotI消化PMX载体。 </li><li>进行PCR产物的电泳和洗脱的大约1000个基点带，如在步骤1.2中描述。 </li><li>克隆TCRα基因插入EcoRI和NotI消化PMX载体和普里FY质粒如步骤1.5-1.9中描述。 </li></ol></li></ol> 5.重建新的抗原特异性TCR克隆注:培养的Jurkat 76细胞和随后的细胞系在RPMI培养基中补充有10％FCS和50μg/ ml的庆大霉素。孵育细胞在37℃，5％的CO 2的所有步骤之间。 <ol><li>转导的Jurkat 76细胞26（或同等学历TCR - / -人类T细胞系）用在步骤2.3生产293GPG病毒中心TCR hemichain。对Jurkat细胞76，种子，每孔5×10 4个细胞在24孔板用1ml病毒和1ml RPMI培养基中。在1000 xg离心离心机板和32℃下1小时。 </li><li>感染后24小时，收集hemichain转导的Jurkat 76细胞吹打，离心350 XG 4分钟。弃上清，重悬在进一步培养新鲜RPMI培养基。 </li><li>净化转导细胞如果hemichain分子是5.2步后2-3天标记，通过染色ING与荧光团共轭抗神经生长因子受体单克隆抗体其次是流动，协助或磁辅助细胞分选（可选）27。 </li><li>以下步骤2.1至2.3，生产293GPG病毒编码TCR反链从步骤4.4或4.5克隆。 </li><li>要完全重建的TCR，转导T细胞系稳定表达使用病毒从步骤5.4步骤5.1-5.3产生的TCR为中心hemichain。按照步骤5.1-5.2描述执行传导。 </li><li>净化使用抗CD3磁辅助细胞分选CD3 +转以下制造商的协议，步5.5 27后2-3天。 </li><li>为了验证抗原特异性，染色表达与各种反链配对的中心的TCR hemichain组成克隆型的TCR，用抗CD3和/或共受体的mAb，和HLA多聚的转染子，3-5天后CD3的纯化。分析通过流式细胞术（ 图4 - 5）细胞19。 </li></ol>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redirection+of+antileukemic+reactivity+of+peripheral+T+lymphocytes+using+gene+transfer+of+minor+histocompatibility+antigen+HA-2-specific+T-cell+receptor+complexes+expressing+a+conserved+alpha+joining+region.">Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region.</a> Blood. 102 (10), 3530-3540 (2003).</li><li>Yan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+cell+sorting+and+flow+cytometry+sorting+methods+for+the+isolation+and+function+analysis+of+mouse+CD4++CD25++Treg+cells.">Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+ CD25+ Treg cells.</a> J. Zhejiang Univ. Sci. B. 10, 928-932 (2009).</li><li>Padovan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+two+T+cell+receptor+alpha+chains:+dual+receptor+T+cells.">Expression of two T cell receptor alpha chains: dual receptor T cells.</a> Science. 262 (5132), 422-424 (1993).</li><li>Johnson, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=transfer+of+tumor-reactive+TCR+confers+both+high+avidity+and+tumor+reactivity+to+nonreactive+peripheral+blood+mononuclear+cells+and+tumor-infiltrating+lymphocytes.">transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes.</a> J. Immunol. 177 (9), 6548-6559 (2006).</li><li>Chinnasamy, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+TCR+targeting+the+HLA-A*0201-restricted+epitope+of+MAGE-A3+recognizes+multiple+epitopes+of+the+MAGE-A+antigen+superfamily+in+several+types+of+cancer.">A TCR targeting the HLA-A*0201-restricted epitope of MAGE-A3 recognizes multiple epitopes of the MAGE-A antigen superfamily in several types of cancer.</a> J. Immunol. 186 (2), 685-696 (2011).</li></ol>

The University Health Network has filed a patent related to this methodology on which N.H., M.N., and T.O. are named as inventors. The other authors have no financial conflicts of interest.

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...

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。

<table border="0" cellpadding="0" cellspacing="0" width="702">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<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。

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?...

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

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。

发电<em&gt;德诺</em&gt;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

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

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Immunology and Infection

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

8.4K 次观看.  University of Toronto. 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。 

视频: 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 presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

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 ...

科研

免疫与感染

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

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

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

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

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

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

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

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

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

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

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

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

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

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

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

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

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

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

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

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

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

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

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

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

用荧光显微镜分析膜受体扩散和簇簇的图像处理协议

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