Name: generating de novo antigen-specific human t cell receptors by retroviral transduction of centric hemichain
Uploaded: 2016-10-25T15:00:00.000+00:00
Duration: 8 min 48 s
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.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>

<ol>
	<li>Maus, M. V., 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=Adoptive+immunotherapy+for+cancer+or+viruses.">Adoptive immunotherapy for cancer or viruses.</a> Annu. Rev. Immunol. 32, 189-225 (2014).</li><li>Yokosuka, T., , ., 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=Predominant+role+of+T+cell+receptor+(TCR)-alpha+chain+in+forming+preimmune+TCR+repertoire+revealed+by+clonal+TCR+reconstitution+system.">Predominant role of T cell receptor (TCR)-alpha chain in forming preimmune TCR repertoire revealed by clonal TCR reconstitution system.</a> J.Exp.Med. 195, 991-1001 (2002).</li><li>Rudolph, M. G., Stanfield, R. L., Wilson, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+TCRs+bind+MHCs,+peptides,+and+coreceptors.">How TCRs bind MHCs, peptides, and coreceptors.</a> Annu. Rev. Immunol. 24, 419-466 (2006).</li><li>Shimizu, 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=Structure+of+TCR+and+antigen+complexes+at+an+immunodominant+CTL+epitope+in+HIV-1+infection.">Structure of TCR and antigen complexes at an immunodominant CTL epitope in HIV-1 infection.</a> Sci. Rep. 3, 3097 (2013).</li><li>Nakatsugawa, M., 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=Specific+roles+of+each+TCR+hemichain+in+generating+functional+chain-centric+TCR.">Specific roles of each TCR hemichain in generating functional chain-centric TCR.</a> J. Immunol. 194 (7), 3487-3500 (2015).</li><li>Ochi, T., 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=Optimization+of+T-cell+Reactivity+by+Exploiting+TCR+Chain+Centricity+for+the+Purpose+of+Safe+and+Effective+Antitumor+TCR+Gene+Therapy.">Optimization of T-cell Reactivity by Exploiting TCR Chain Centricity for the Purpose of Safe and Effective Antitumor TCR Gene Therapy.</a> Cancer Immunol. Res. 3 (9), 1070-1081 (2015).</li><li>Chamoto, K., 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=CDR3beta+sequence+motifs+regulate+autoreactivity+of+human+invariant+NKT+cell+receptors.">CDR3beta sequence motifs regulate autoreactivity of human invariant NKT cell receptors.</a> J. Autoimmun. 68, 39-51 (2016).</li><li>Grozdanov, P. N., MacDonald, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+plasmid+vectors+expressing+FLAG-tagged+proteins+under+the+regulation+of+human+Elongation+Factor-1&#945;+promoter+using+Gibson+Assembly.">Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human Elongation Factor-1&#945; promoter using Gibson Assembly.</a> J. Vis. Exp. (96), e52235 (2015).</li><li>Beshiri, M. L., 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=Genome-wide+analysis+using+ChIP+to+identify+isoform-specific+gene+targets.">Genome-wide analysis using ChIP to identify isoform-specific gene targets.</a> J. Vis. Exp. (41), e2101 (2010).</li><li>Gibson, D. G., 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=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat. Methods. 6 (5), 343-345 (2009).</li><li>Johnson, D., 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+and+structure+of+the+human+NGF+receptor.">Expression and structure of the human NGF receptor.</a> Cell. 47 (4), 545-554 (1986).</li><li>Kim, J. 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=High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines,+zebrafish+and+mice.">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PloS one. 6, e18556 (2011).</li><li>Yang, S., 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=Development+of+optimal+bicistronic+lentiviral+vectors+facilitates+high-level+TCR+gene+expression+and+robust+tumor+cell+recognition.">Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition.</a> Gene Ther. 15 (21), 1411-1423 (2008).</li><li>Froger, A., Hall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+plasmid+DNA+into+E.+coli+using+the+heat+shock+method.">Transformation of plasmid DNA into E. coli using the heat shock method.</a> J. Vis. Exp. (6), e253 (2007).</li><li>Birnboim, H. C., Doly, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+alkaline+extraction+procedure+for+screening+recombinant+plasmid+DNA.">A rapid alkaline extraction procedure for screening recombinant plasmid DNA.</a> Nucleic Acids Res. 7 (6), 1513-1523 (1979).</li><li>Ory, D. S., Neugeboren, B. A., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stable+human-derived+packaging+cell+line+for+production+of+high+titer+retrovirus/vesicular+stomatitis+virus+G+pseudotypes.">A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.</a> Proc. Natl. Acad. Sci. U.S.A. 93 (21), 11400-11406 (1996).</li><li>Imataki, O., 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=IL-21+can+supplement+suboptimal+Lck-independent+MAPK+activation+in+a+STAT-3-dependent+manner+in+human+CD8(+)+T+cells.">IL-21 can supplement suboptimal Lck-independent MAPK activation in a STAT-3-dependent manner in human CD8(+) T cells.</a> J. Immunol. 188 (4), 1609-1619 (2012).</li><li>Davies, J. K., Barbon, C. M., Voskertchian, A. R., Nadler, L. M., Guinan, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+alloantigen-specific+anergy+in+human+peripheral+blood+mononuclear+cells+by+alloantigen+stimulation+with+co-stimulatory+signal+blockade.">Induction of alloantigen-specific anergy in human peripheral blood mononuclear cells by alloantigen stimulation with co-stimulatory signal blockade.</a> J. Vis. Exp. (49), e2673 (2011).</li><li>Butcher, M. J., Herre, M., Ley, K., Galkina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+of+immune+cells+within+murine+aortas.">Flow cytometry analysis of immune cells within murine aortas.</a> J. Vis. Exp. (53), e2848 (2011).</li><li>Butler, M. O., 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=Establishment+of+antitumor+memory+in+humans+using+in+vitro-educated+CD8++T+cells.">Establishment of antitumor memory in humans using in vitro-educated CD8+ T cells.</a> Sci. Transl. Med. 3 (80), 80ra34 (2011).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal. Biochem. 162 (1), 156-159 (1987).</li><li>Peterson, S. M., Freeman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+isolation+from+embryonic+zebrafish+and+cDNA+synthesis+for+gene+expression+analysis.">RNA isolation from embryonic zebrafish and cDNA synthesis for gene expression analysis.</a> J. Vis. Exp. (30), e1470 (2009).</li><li>Ying, S. Y., Ying, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+DNA+Libraries.">Complementary DNA Libraries.</a> Generation of cDNA Libraries: Methods and Protocols. 221, 1-12 (2003).</li><li>Hirano, 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=Engagement+of+CD83+ligand+induces+prolonged+expansion+of+CD8++T+cells+and+preferential+enrichment+for+antigen+specificity.">Engagement of CD83 ligand induces prolonged expansion of CD8+ T cells and preferential enrichment for antigen specificity.</a> Blood. 107 (4), 1528-1536 (2006).</li><li>Scotto-Lavino, E., Du, G., Frohman, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5'+end+cDNA+amplification+using+classic+RACE.">5' end cDNA amplification using classic RACE.</a> Nat. Protoc. 1 (6), 2555-2562 (2006).</li><li>Heemskerk, M. 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rhIL-2的高浓度沿，提供了必要的激活信号，以诱导细胞分裂。单核细胞所必需的可溶OKT3的刺激能力，可能通过与Fc受体的结合。因此，散装的PBMC，并没有纯化的T细胞，需要这个特定的刺激方案。 ŧ细胞应培养在RPMI补加人类血清的最佳实验的结果。这里描述的所有其它细胞可以是在胎牛血清存在下培养。 第二，表达细胞从头生成抗原特异性的TCR必须是可检测的。对于某些TCR hemichains，如TAK1β， 从头的TCR表达细胞与抗原冲激的抗原呈递细胞（APC）刺激后仅检测到。因此，如果不为中心hemichain转导后立即检测抗原特异性的TCR，他们可能是由的APCs膨胀后可检测的。注意非中心hemichain的转导甚至后的APC抗原特异性膨胀不会产生从头抗原特异性T细胞，与TAK1αhemichain（ 图1）可见。在我们的研究中，HLA多聚体用于检测的，然而，这可能对某些的TCR，特别是限制性TCR HLA类II可用秒。另一种方法是检测T细胞的功能反应。 Hemichain转导的T细胞能够与感兴趣的抗原脉冲的APC刺激。车辆脉冲的APC和未转导的T细胞可以被用作对照。反应性T细胞可通过的表面活化标记如CD107a，CD154，或CD137上调来检测。 最后，为了验证克隆的反链做实际上识别目的抗原时与中心hemichain配对是重要的。这是特别重要的，当中心hemichain是TCRβ和反向链是TCRα，因为等位基因排斥对TCRα基因是漏水，据估计该外设αβT细胞的显著一部分表达一个以上的TCRα链28。反应可以用多聚体染色或测量与装甲运兵车呈现同源抗原刺激后功能反应得到证实。 一limitati这种技术的是，并非每一个TCR将组成一个中心hemichain，因为这两个TCRα和TCRβ贡献同等抗原识别在某些情况下3。因此，这样的非中心hemichains转导外周T细胞可能不会产生从头抗原特异性的TCR。 尽管如此，这种方法代表经常规方法克隆的TCR，其中两个TCRα和TCRβ基因必须被克隆并正确配对必须确保29,30的概念的进步。在我们的技术中，hemichains之间配对已经不再是一个主要关注的问题，并且只有TCR链的需要被克隆，鉴于其他固定之一。除了HLA I类或CD1d分子-限制性TCR，所描述的方法也可以应用到对TCR基因治疗的HLA II类限制性TCR的分离。

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><tbody><tr><td>0.05% Trypsin-EDTA</td><td>Wisent Bioproducts</td><td>325-043-CL</td><td></td></tr><tr><td>293GPG cells</td><td>Generated by Ory &lt;em&gt;et al.&lt;/em&gt; (ref 8)</td><td></td><td></td></tr><tr><td>Agar</td><td>Wisent Bioproducts</td><td>800-010-CG</td><td></td></tr><tr><td>Agarose</td><td>Wisent Bioproducts</td><td>800-015-CG</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Wisent Bioproducts</td><td>400-110-IG</td><td></td></tr><tr><td>Chloroform</td><td>BioShop</td><td>CCL402</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) Solution Mix</td><td>New England Biolabs</td><td>N0447L</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Life Technologies</td><td>11995065</td><td></td></tr><tr><td>EcoRI</td><td>New England Biolabs</td><td>R0101S</td><td></td></tr><tr><td>EZ-10 Spin Column DNA Gel Extraction Kit</td><td>BS353</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>12483020</td><td></td></tr><tr><td>Ficoll-Paque Plus</td><td>GE Healthcare</td><td>17-1440-02</td><td></td></tr><tr><td>Filter</td><td>Corning</td><td>431220</td><td>0.45 mm pore SFCA membrane</td></tr><tr><td>FITC-conjugated anti-human CD271 (NGFR) mAb</td><td>Biolegend</td><td>345104</td><td>clone ME20.4</td></tr><tr><td>FITC-conjugated anti-human CD3 mAb</td><td>Biolegend</td><td>300440</td><td>clone UCHT1</td></tr><tr><td>Gentamicin</td><td>Life Technologies</td><td>15750078</td><td></td></tr><tr><td>Gibson Assembly Master Mix</td><td>New England Biolabs</td><td>E2611L</td><td>used for multi-piece DNA assembly</td></tr><tr><td>HLA-A2 pentamer</td><td>Proimmune</td><td>depends on antigenic peptide</td><td>HLA-A2/MART1 multimer used here was purchased from Proimmune</td></tr><tr><td>HLA/CD1d monomers</td><td>NIH Tetramer Core Facility</td><td></td><td>multimerize monomers according to protocol provided by NIH tetramer core facility</td></tr><tr><td>Human AB serum</td><td>Valley Biomedical</td><td>HP1022</td><td></td></tr><tr><td>human CD3 microbeads</td><td>Miltenyi Biotec</td><td>130-050-101</td><td></td></tr><tr><td>IOTest Beta Mark TCR V beta Repertoire Kit</td><td>Beckman Coulter</td><td>IM3497</td><td></td></tr><tr><td>Jurkat 76 cells</td><td>Generated by Heemskerk &lt;em&gt;et al.&lt;/em&gt; (ref 10)</td><td></td><td></td></tr><tr><td>LB Broth</td><td>Wisent Bioproducts</td><td>800-060-LG</td><td></td></tr><tr><td>LS MACS column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>NEB 5-alpha Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>New England Biolabs</td><td>C2987I</td><td></td></tr><tr><td>NEBuffer 3.1</td><td>New England Biolabs</td><td>B7203S</td><td>used for EcoRI and NotI digestion</td></tr><tr><td>NotI</td><td>New England Biolabs</td><td>R0189S</td><td></td></tr><tr><td>NucleoBond Xtra Midi</td><td>Macherey-Nagel</td><td>740410</td><td>used for plasmid purification</td></tr><tr><td>PC5-conjugated anti-human CD8 mAb</td><td>Beckman Coulter</td><td>B21205</td><td>clone B9.11</td></tr><tr><td>PG13 cells</td><td>ATCC</td><td>CRL-10686</td><td></td></tr><tr><td>Phusion HF Buffer Pack</td><td>New England Biolabs</td><td>B0518S</td><td></td></tr><tr><td>Phusion&amp;nbsp;High-Fidelity DNA Polymerase</td><td>New England Biolabs</td><td>M0530L</td><td></td></tr><tr><td>pMX retroviral vector</td><td>Cell Biolabs</td><td>RTV-010</td><td></td></tr><tr><td>polybrene</td><td>Sigma-Aldrich</td><td>H-9268</td><td></td></tr><tr><td>Proleukin (recombinant human interleukin-2)</td><td>Novartis</td><td>by Rx only</td><td>equivalent product can be purchased from Sigma-Aldrich</td></tr><tr><td>Purified anti-human CD3 antibody</td><td>Biolegend</td><td>317301</td><td>clone OKT3, used for T cell stimulation</td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>11875119</td><td></td></tr><tr><td>SA-PE</td><td>Life Technologies</td><td>S866</td><td>used for multimerizing monomers from NIH tetramer core facility</td></tr><tr><td>SMARTer RACE 5&#039;/3&#039;&amp;nbsp; Kit</td><td>Clontech</td><td>634858</td><td></td></tr><tr><td>Sterile water</td><td>Wisent Bioproducts</td><td>809-115-LL</td><td></td></tr><tr><td>SuperScript III First-Strand Synthesis System</td><td>Invitrogen</td><td>18080051</td><td>for cDNA synthesis</td></tr><tr><td>Syringe</td><td>BD</td><td>301604</td><td>10 ml, slip tip</td></tr><tr><td>Tetracycline hydrochloride</td><td>Sigma-Aldrich</td><td>T7660</td><td></td></tr><tr><td>TransIT-293</td><td>Mirus Bio</td><td>MIR 2700</td><td>used to transfect 293GPG cells</td></tr><tr><td>TRIzol Reagent</td><td>Life Technologies</td><td>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基因被标记为具体分析转导的细胞（ 图1 - 2）。另一方面，单中心hemichain可以，如果它被称为是占主导地位的被转导，例如SIG35α和不变Vα24TCRα链（ 图2和3）。特异于各自COGN T细胞吃抗原在一个中心TCRαhemichain转导的频率增加。 （ 图2 - 3）。总之，这些数据表明，引入一个中心TCRα或TCRβhemichain的外周血T细胞可以产生与所关注的抗原特异性从头的TCR。 抗原特异性群可通过流动辅助细胞分选进行排序，并作为在协议部分4克隆型T细胞受体的反链可以在稳定表达中心hemichain一个T细胞系单独重构描述TCR hemichains可以克隆。 76 Jurkat细胞转染，表达新克隆的TAK1β或SIG35α中心hemichain导的T细胞的独特的TCR，具备了相应的同源抗原不同的亲和力，如HLA /肽多聚体染色5,6确定。具体来说，我们能够隔离从头 TCRα反链Ť当与TAK1β配对帽与由WT1抗原复合物比亲TAK1αβ配对或较低或较高的强度（ 图4）进行染色。同样，SIG35α搭配独特的反链公认的HLA-A2 / MART1中度至高度的亲和力（ 图5）。 SIG35α克隆独立一个TCRαβ异二聚体5，因此对于这个hemichain亲配对是用于比较的不可用。重要的是，这些数据表明，有可能通过配对与各种反链的中心hemichain调制对靶抗原的亲和力。 <img alt="图1" src="/files/ftp_upload/54697/54697fig1.jpg" /> 图1:TAK1β作为 HLA-A24 / WT1反应TAK1 TCR的HLA限制性TCRβ中心hemichainTCRα或TCRβhemichains 的一个例子是具有标记截断神经生长因子RECEptor（ΔNGFR），并单独转导到末梢血的T细胞。转染子与PC5缀合的抗CD8（133倍稀释）和FITC缀合的抗NGFR（20倍稀释的）的单克隆抗体（mAb）染色，并表示HLA-A24多聚（5微克/毫升），以下抗原特异性两次刺激。 6捐助者共进行了测试。所有的数据进行门控上NGFR +细胞。图重新打印许可参考6。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54697/54697fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54697/54697fig2.jpg" /> 图2:SIG35α作为HLA限制性TCRα中心hemichain的一例 SIG35αTCRα链具有标记ΔNGFR，或单独的标签，转导到外周血T细胞。转用PC5共染色njugated抗CD8（133倍稀释）和FITC缀合的抗NGFR（20倍稀释）的mAb，并表示HLA-A2多聚（8微克/毫升）。 4捐助者共进行了测试。所有的数据进行门控上NGFR +细胞。图重新印有从基准5（ 版权2015年免疫学家的美国协会，公司 ）的许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54697/54697fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54697/54697fig3.jpg" /> 图3:不变的自然杀伤T（的iNKT）细胞受体Vα24（Vα24i）作为CD1d的限制TCRα中心hemichain的例子 Vα24iTCRα链转外周血T细胞。转染用FITC缀合的抗-CD3（20倍稀释）的mAb染色，并指示CD1d的多聚体（5微克/毫升）。卸载CD1d的莫lecules从HEK293细胞内源性存在未知的自我脂质产生。的iNKT TCR由识别CD1d的呈递α-神经酰胺或其类似物的PBS-57中定义。 4捐助者共进行了测试。图重新打印许可参考7。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54697/54697fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54697/54697fig4.jpg" /> 图4:从TAK1β克隆代表TCRα计数器链转导的T细胞 TCRα克隆T262，A262，A186，A133，和T4，从HLA-A24 / WT1克隆的多聚体阳性TAK1β转导外周T细胞，对Jurkat 76细胞分别重构。表达CD8，与TAK1β链一起。转染子与PC5共轭的抗CD8单克隆抗体（133倍稀释）染色，并表示HLA-A24多聚体（5微克/毫升）。数据代表两个独立的实验。误差棒表示范围。图重新打印许可参考6。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54697/54697fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54697/54697fig5.jpg" /> 图5:从SIG35α克隆代表TCRβ计数器链转导的T细胞 TCRβ克隆413，523，788，1086，794，和830，从HLA-A2 / MART1克隆阳性SIG35α多聚体转导的外周T细胞，并单独重构对Jurkat。 76细胞中表达CD8，与SIG35α链一起。 DMF5是以前克隆的高亲和力的TCR识别HLA-A2 / MART1。转染子与PC5共轭的抗CD8单克隆抗体（133倍稀释）染色，并表示HLA-A2多聚（2微克/立方米1）。数据代表两个独立的实验。误差棒表示范围。图重新印有从基准5（ 版权2015年免疫学家的美国协会，公司 ）的许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54697/54697fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54697/54697table1.jpg" /> 表1:。试剂和各卷均会显示安装程序PMX矢量消化 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54697/54697table1large.jpg" target="_blank">请点击这里查看此表的放大版本。</a> <img alt="表2" src="/files/ftp_upload/54697/54697table2.jpg" /> 表2:设置为第一 轮5&#39;RACËPCR检测试剂和各自的体积，PCR设置和引物序列显示， <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54697/54697table2large.jpg" target="_blank">请点击这里查看此表的放大版本。</a> <img alt="表3" src="/files/ftp_upload/54697/54697table3.jpg" /> 表3:设置第二 轮5&#39;RACE PCR试剂和各自的体积，PCR设置和引物序列显示。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54697/54697table3large.jpg" target="_blank">请点击此处查看该表的放大版本。</a>

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, ...

generating-de-novo-antigen-specific-human-t-cell-receptors-retroviral

Nanyang Technological University

Research

JoVE Journal

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

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

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 ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.

简化的单细胞 TCR 分离和逆转录病毒载体的生成, 用于体外和体内表达人类 TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

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 ...

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

单链 mhc 技术在人 cd8+ t 细胞活化中的相关研究

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

<meta charset="utf8"></head><body>用于免疫调节疗法的人 CAR Treg 的工程化和扩增

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

<meta charset="utf8"></head><body>人嵌合抗原受体调节性 T 细胞的产生

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

人嵌合抗原受体调节性 T 细胞的产生

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

发电

摘要

探索更多视频

此视频中的章节