我们感谢提供山中伸弥博士（京都大学）IPS-MEF的NG-20D-17细胞系，支持OT1-2A的达里奥Vignali博士（圣裘德儿童研究医院），博士胡安•pMig II结构卡洛斯·祖尼加Pflucker（免疫学系，多伦多大学）支持OP9-DL1的细胞系，博士，肯特，éVrana（宾夕法尼亚州立大学医学院药理学教研室）为帮助本研究设计。该项目资助下，拨款补助人数从国家癌症研究所，Barsumian信托和黑色素瘤研究基金会（J宋）K18CA151798。

1。细胞培养<ol><li>制备辐照SNL76 / 7（irSNL76 / 7）饲养层细胞培养。 SNL76 / 7细胞一般都保持在10％胎牛血清（FBS），杜尔贝科的改良Eagle培养基（DMEM培养基）媒体。 <ol><li>培养皿或瓶，将涂有0.1％的明胶溶液在37°C，30分钟孵化器前恢复SNL76 / 7细胞从液氮。 </li><li>达到汇合SNL76 / 7细胞时，细胞就会被胰酶消化，离心5分钟在400Ğ和悬浮在新鲜的媒体。 </li><li>悬浮SNL76 / 7细胞，将在5000拉德的剂量照射钴 -60辐照。 另一种方法，SNL76 / 7细胞可代替小鼠胚胎成纤维细胞（MEF）和丝裂霉素灭活可以取代照射。 </li><li>照射后，细胞将在5分钟的400Ğ离心，并在10％二甲基亚砜（DMSO）胎牛血清冻结缓冲区，分装成悬浮冷冻管，并在将来使用液氮保存。 </li></ol></li><li> OP9-DL1的细胞在体外分化的制备。 OP9-DL1的细胞一般将保持在20％胎牛血清的α-最低限度的基本培养基（α-MEM）的媒体。当他们到达汇合细胞将被分割在1:5稀释。 </li><li>的E.G7胸腺瘤细胞的制备。 E.G7胸腺瘤细胞一般将保持在10％FBS的罗斯韦尔公园纪念研究所培养基（RPMI）-1640媒体。当他们到达汇合，细胞将被分割在1:10稀释。 </li><li> IPS和一般维修的TCR转iPS细胞。 <ol><li>培养皿，在37℃，30分钟前播种irSNL76 / 7饲养层细胞或iPS细胞恢复分裂提前一天将涂以0.1％的明胶溶液。 </li><li>为iPS细胞分裂，细胞将胰酶消化，离心400Ğ5分钟和15％FBS DMEM培养基悬浮。 </li><li>胰酶消化的iPS细胞将孵育30分钟，上一个新的培养皿在37°C间孵化器前播种新鲜irSNL76 / 7馈线细胞覆膜菜排除分化的细胞及残渣饲养层细胞上。 </li><li>孵化后，将4×10 6细胞接种在一个100mm的培养皿。 </li></ol></li></ol> （2） 在体外编程<ol><li> 在体外培养系统。 <ol><li> 0天，5X10 4 iPS细胞将一个100mm的文化菜中含有20％胎牛血清的α-MEM的媒体合流OP9-DL1的单层细胞接种。 </li><li>在第3天，文化传媒，将新鲜的改变。 </li><li><ol><li>在第5天，细胞就会被胰酶消化和400Ğ离心5分钟，前30分钟，在37°C间孵化器孵化上一个新的100毫米培养皿。 </li><li>悬浮细胞，将收集和计算，5×10 5细胞将被转移到一个新的文化菜中含有20％胎牛血清的α-MEM的媒体合流OP9-DL1的细胞单层。细胞因子mFlt-3L（终浓度:5毫微克/毫升）将被添加在文化。 </li></ol></li><li><ol><li>在第8天，松散连接的细胞会被轻轻吸取下来。 </li><li>洗OP9-DL1的饲养层，10毫升的PBS更多的时间来得到最大恢复部分分化的iPS细胞。 </li><li>收获后共培养的细胞，将细胞离心5分钟在400Ğ和悬浮于20％胎牛血清的α-MEM的媒体为辅mFlt-3L（5毫微克/毫升）和MIL-7（1毫微克/毫升）。 </li><li>细胞将被转入6孔培养板涂有合流OP9-DL1的细胞。通常从一个100毫米的培养皿中恢复的iPS细胞将被转移到一个6孔板。 </li></ol></li><li><ol><li>从第10天，文化传媒将改变隔日（20％FBS的α-MEM的媒体supplemente与mFlt-3L（5毫微克/毫升）和MIL-7（1毫微克/毫升））。 </li><li>培养板涂有接驳OP9​​-DL1的细胞将在4-6天改变取决于饲养层细胞的生长。 </li></ol></li></ol></li><li> 在体内的iPS细胞部分分化成熟。 <ol><li> iPS细胞共培养第22天，将关闭胰酶消化，离心5分钟，在400Ğ在37℃上一个新的培养皿中培养30分钟。 </li><li>将收集悬浮细胞，通过70微米尼龙过滤器通过排除这可能会导致肺栓塞小鼠的细胞团块和冷PBS洗三次。 </li><li> 1.5×10 7细胞/毫升的浓度，细胞就会悬浮在PBS。 </li><li>细胞将保持在注射前的冰。 </li><li> 四，通过尾静脉注射之前，老鼠将被置于下的红外光，其尾静脉扩张。 </li><li>经过静脉ð转移到4周龄通过尾静脉B6.129S7 RAG1 tm1Mom / J小鼠的ilatation，200μL细胞悬液或3X10 6个细胞，将继。三个星期被允许在部分分化的iPS细胞的体外成熟。 </li></ol></li><li>评价。 <ol><li>分化的细胞和细胞回收率。 图1的形态学变化。 <ol><li> OP9-DL1的细胞共培养不同天，将采取传统的显微镜下活细胞图片。 </li><li>将细胞复苏率计算的基础上，从文化收获的细胞数量。 </li></ol></li><li>流式细胞仪分析表面标志的变化。 图2a。 <ol><li>在不同天的共培养，细胞将被删除从文化胰蛋白酶，用冷PBS洗，然后进行细胞表面染色。 </li><li>在染色机智不同的荧光标记的抗体，细胞就会被封锁在4℃，20分钟的Fc受体阻滞剂24G2。 </li><li> 20分钟后，染色在4°C时，细胞​​就会被冷PBS洗涤流式细胞仪检查前的三倍。 </li></ol></li><li> iPS细胞在体外分化。 图2b激活。 <ol><li>激活检测的前一天，预涂与抗CD3的（终浓度:4μg/ mL的PBS中）24孔板，在4°C过夜。 </li><li>共培养22天，iPS细胞的T细胞将收获从文化，用冷PBS洗前用板涂层的抗CD3和可溶性抗CD28抗体（终浓度为4微克/毫升）刺激。 </li><li>孵化器将进行37℃，5％CO2培养箱40小时，然后Befeldin将被添加到文化，另外4个小时。 </li><li>在年底共培养，细胞就会HAR既得利益，洗净，如上所述的Fc受体阻滞剂阻断。 CD8和TCRVβ链的表面标志，使用荧光标记的抗体将阻止细胞染色。 </li><li>细胞表面染色后，细胞将用4％甲醛固定，并通过使用Biolegend的透套件透。 </li><li>通透性后，将通过使用荧光标记的抗体染色，如IL-2和IFN-γ的细胞内分子。 </li><li>前最后流式细胞仪检测，细胞将在冷PBS洗涤三次，以排除过量的抗体。 </li></ol></li><li>在RAG-/ -小鼠的成熟。 <ol><li>从小鼠，经过三个星期在体内发育，RAG-/ -小鼠会被牺牲，脾和淋巴结肿大，将被删除。 </li><li>将单个细胞通过机械故障处理。将使用ACK裂解液和裂解红细胞mononucleocytes将收集和冷PBS洗两次。 </li><li>洗净后，将被阻止细胞的Fc受体阻滞剂24G2在4℃，20分钟和在阻塞年底，将用不同的荧光标记的抗CD3，抗CD4，抗CD8和反TCRβ抗体染色细胞4°C 20分钟。 </li><li>在染色结束时，细胞就会被冷PBS洗涤，流式细胞仪检查前的三倍。 </li></ol></li></ol></li></ol> 3。 在体内编程<ol><li>代逆转录病毒结构。 <ol><li> MSCV-IRES-DsRed的（MiDR）载体MSCV-IRES-GFP载体的基础上，构造代DsRed基因与绿色荧光蛋白基因。 </li><li> OT-T细胞受体基因的克隆，使OT-I/MiDR建设到在Midr的载体。 </li></ol></li><li>逆转录病毒转导和细胞分选。 <ol><li>开发平台-E的包装细胞ARE中生成假病毒，将用于以下传导。 <ol><li> 3X10 6开发平台-E细胞接种于100毫米培养皿转染前一天。 </li></ol></li><li>第0天，开发平台-E的细胞，将转加时赛，我MiDR使用GeneJamma转染试剂质粒。 </li><li>第1天，1×10 6 iPS细胞将被接种到0.1％的明胶的预涂24孔板。 </li><li><ol><li>第2天，假病毒含有从开发平台-E的文化上清将被收集并通过0.4微米过滤器，以排除潜在的污染物。 </li><li>将于32°ç离心机的条件下进行转1小时5微克/毫升凝聚胺的存在，在1400转。 </li><li>经过离心机的转导，细胞将被放置在32°C时，晚上5％CO2培养箱。 </li></ol></li><li>第3天，重复的第2天TRA如上所述nsduction程序。 6孔板将预涂irSNL76 / 7饲养层细胞，以供将来使用。 </li><li>第4天，转iPS细胞将胰酶消化，离心5分钟为400克，播种覆膜irSNL76 / 7饲养层细胞。 </li><li>在汇合，细胞将被胰酶消化，离心5分钟，在400Ğ和细胞分选处理。将GFP和DsRed的双阳性细胞排序由MoFlo细胞分选仪。排序将irSNL76 / 7饲养层细胞培养细胞，以供将来使用。 </li></ol></li><li>继转移和肿瘤的挑战。 OT-我的TCR转IPS（OT-I/iPS）细胞通常保持在irSNL76 / 7饲养层细胞，如上所述。 <ol><li>继转移的一天，OT-I/iPS细胞是胰酶消化，离心5分钟，在400Ğ悬浮在新鲜媒体。 </li><li> 30分钟，上一个新的培养皿在37°C间孵化器的孵化需要消除分化的细胞和残馈线细胞。 </li><li>悬浮细胞在培养结束时，将收集的400Ğ离心5分钟。 </li><li>将在冰冷的PBS洗涤细胞沉淀三次，将通过在两个清洗之间的尼龙70微米过滤器，以排除细胞团块（2X过滤）通过细胞。 </li><li>洗净后，将细胞计数，并在冷PBS悬浮浓度在1.5×10 7细胞/ ml。 </li><li>细胞将保持在注射前的冰。 </li><li>继转移，将4-6周龄雌性C57BL/6J小鼠。 四，通过尾静脉注射之前，老鼠将被放置在红外光，其尾静脉扩张。 </li><li>静脉扩张后，将200μL细胞悬液或3X10 6个细胞通过尾静脉继转移。六个星期将被允许在体内成熟OT我的TCR转iPS细胞。 </li><li><ol><li> 静脉注射6周后，将4X10 6 E.G7胸腺瘤细胞腹腔接种。 </li><li>从文化E.G7胸腺瘤细胞会有所收获，并在PBS洗三次。 </li><li>在洗年底，细胞将悬浮在冷PBS浓度在8×10 7细胞/ ml。 </li><li>将50μL细胞悬液或4X10 6个细胞注射到腹腔。 </li></ol></li></ol></li><li>评价。 <ol><li> OT我的TCR特性， 在体外转iPS细胞。 <ol><li>红色荧光的荧光显微镜检查，将绿色荧光蛋白双阳性细胞进行常规荧光显微镜下，与不固定的活细胞。 </li><li>将进行PCR和RT-PCR分析基因的整合和表达。 <ol><li>将被隔离单独使用QIAGEN的DNA或从样品细胞的DNA或RNARNA分离试剂盒。 </li><li>将进行PCR和RT-PCR引物特异性识别的TCRVβ5链重组VDJ地区使用。 </li></ol></li></ol></li><li> T细胞的发育和成熟。 图3a。 <ol><li>在第2周，4和6细胞移植后，动物将被处死，脾，淋巴结肿大，将会从动物中删除。 </li><li>单细胞悬液，将通过机械故障。红血细胞将裂解使用的ACK裂解液mononucleocytes的，将被收集和冷PBS洗两次。 </li><li>洗净后，将被阻止细胞的Fc受体阻滞剂24G2在4℃20分钟，并在的阻塞年底，细胞将被aliquotted和不同荧光标记的抗体，在4℃染色20分钟。 </li><li>在染色结束时，细胞就会被冷PBS洗涤，流式细胞仪装载前的三倍。 </li></ol></li><li>肽刺激。 图3b。 <ol><li>动物肿瘤的挑战第50天，将被处死，脾，淋巴结肿大，将会从动物中删除。 </li><li>单细胞悬液，将通过机械故障。红血细胞将裂解使用的ACK裂解液mononucleocytes的，将被收集和冷PBS洗两次。 </li><li> CD8 + T细胞将通过美天旎生物技术的CD8 + T细胞分离试剂盒分离。孤立的CD8 + T细胞，将混在1:10的比例，从天真的C57BL/6J小鼠小鼠隔离，脉冲和0.5μmol/毫升的卵257-264肽为40个小时的照射脾。此后，布雷菲德菌素A将被添加到文化，另外4个小时。 </li><li>在年底共培养，细胞就会有所收获，洗涤，如上所述的Fc受体阻滞剂阻断。 </li><li>将阻止细胞表面标志染色CD8和TCRVβ5链ERS通过使用荧光标记的抗体。 </li><li>细胞表面染色后，细胞将用4％甲醛固定，并通过细胞通透性套件透。 </li><li>通透性后，将通过使用荧光标记的抗体染色，如IL-2和IFN-γ的细胞内分子。 </li><li>前最后流式细胞仪检测，细胞将在冷PBS洗涤三次，以排除过量的抗体。 </li></ol></li><li> 在体内的杀法。 图3C。 <ol><li>从天真的C57BL/6J小鼠的脾细胞将被孤立和羧基琥珀酰亚胺酯（CFSE）标记靶细胞。 </li><li> 5μmol/毫升CFSE标记（ConA 您好细胞）将与10微克/毫升卵257-264肽和0.5μmol/毫升CFSE标记（CFSE标记的卤味细胞）将不会进行脉冲标记细胞的脉冲标记细胞。 </李&gt; <li> 2.5×10 6 CFSE标记的喜细胞加2.5×10 6 CFSE标记劳细胞的混合物将继静脉注射转移到指定的收件人。 </li><li> 16小时后，这些小鼠的脾细胞将被孤立和CFSE标记+细胞将通过流式细胞仪分析。 </li></ol></li><li>腹腔肿瘤细胞计数图4。 小鼠肿瘤的挑战第20天，将被处死，腹腔灌洗，将用冷PBS。腹腔灌洗回收，将肿瘤细胞计数。 </li><li>肿瘤浸润性T细胞识别。 图5。 <ol><li>在晚期肿瘤的挑战，将小鼠牺牲，将来自不同群体的腹腔取出肿瘤。 </li><li>肿瘤将切成小块，一块将被放入离心管放在干冰立即，另一半将被固定在空调的RPMI-1640媒体将来使用甲醛和第三条，将被保留。 </li><li>根据从甲醛固定石蜡包裹的样品一般协议，将进行H＆E染色。 </li><li>将冷冻样品进行免疫荧光染色。 <ol><li>组织在使用前将切片和保存在-20℃。 </li><li>组织切片，将空气中干燥15分钟，15分钟前冷丙酮固定。 </li><li>固定后，该路段将是另一个15分钟前5分钟PBS洗涤干燥的空气。 </li><li>后洗净，在潮湿室的幻灯片，并覆盖30微升3％BSA的PBS 30分钟，以阻断非特异性结合的组织切片。 </li><li>在阻塞年底，涂抹封闭液和50μLPE-抗TCRVα2在3％BSA稀释的抗体和FITC标记的抗卵抗体混合物覆盖切片在PBS。 </li><li>孵育在2个小时的湿室和孵化结束，幻灯片将在冷PBS洗涤三次，并用荧光显微镜检查前一种水性安装媒体上。 </li></ol></li><li>流式细胞仪分析肿瘤浸润性T细胞。 <ol><li>肿瘤会挤压成单细胞悬液，将ACK裂解液裂解红细胞。 </li><li>洗涤和阻断后，细胞将被贴上明确承认CD8 +，TCRVα2和TCR Vβ5分子在细胞表面表达的不同荧光标记的抗体。 </li></ol></li></ol></li><li>小鼠的存活率。 图6。 肿瘤的挑战后，小鼠的生存将受到严格监控。 </li></ol></li></ol> 4。代表结果 CD3和TCRβ被用作标记的T细胞。我们评估，以确定是否与Notch配体DL1的iPS细胞的刺激，可促进T细胞分化表达CD3和TCRβ+的 iPS细胞源性细胞，并进一步分析CD4和CD8的表达，对CD3门+和TCRβ+人口。如下所示，在22天的CD3 +TCRβ+，CD4 - CD8 +单阳性（SP）的T细胞产生iPS细胞在体外 。此外，iPS细胞衍生的SP细胞能产生IL-2和γ-干扰素在体外刺激的iPS细胞衍生板涂层的抗CD3和可溶性抗CD28抗体（ 图2）， T细胞的功能。 继转移到受体小鼠后，大部分的TCR基因转iPS细胞进行分化成CD8 + +淋巴细胞，回应肽刺激体外本身creting IL-2和γ-干扰素（ 图3）。最重要的是，继转移TCR转iPS细胞引发OVA反应的淋巴细胞进入肿瘤组织和肿瘤的挑战（ 图5-6）保护动物的渗透。因此，TCR基因转导的iPS细胞能够分化为功能性抗原特异性淋巴细胞在体内 。 <img src="/files/ftp_upload/3986/3986fig1.jpg" alt="图1"/> 图1。iPS细胞分化的形态。在不同的日子，小鼠iPS细胞共培养与执行部分第9段，DL1的细胞在α-MEM培养基辅以20％FCS和2.2 g / L的碳酸氢钠存在5毫微克/毫升mFlt3L 1毫微克/毫升MIL-7 。 <img src="/files/ftp_upload/3986/3986fig2.jpg" alt="图2"/> 图2 iPS细胞，T细胞的分化。与图1中描述的OP9-DL1的细胞共培养小鼠iPS细胞。对DAY 22，iPS细胞源性细胞的分离和分析。一），CD4 +，CD8 -或CD4 - CD8 +细胞后，门上的CD3 +和TCRβ+人口。 b）细胞在37°C，5％的CO 2刺激板涂层的抗CD3和可溶性抗CD28抗体为5小时。 IL-2和IFN-γ的细胞内染色分析后，门活CD4 - CD8 + T细胞。 <img src="/files/ftp_upload/3986/3986fig3.jpg" alt="图3"/> 图3。抗原特异性CD8 + T细胞的iPS细胞在体内的发展。 OT-TCR基因转导的iPS细胞注入C57BL / 6小鼠第四 。 6到10周后，OVA特异性CD8 +Vβ5+ T细胞的发育。一），CD8 +Vβ5流式细胞仪门后的C +汇集的淋巴结和脾脏细胞进行了分析，D8 +标准人口。乙）IL-2和γ-干扰素生产（暗线，阴影部分表示同型对照）测定细胞内细胞因子染色，CD8 +Vβ5+人口的门后。 C） 在体内的增殖/细胞毒性实验。 CFSE标记喜 （右峰）和的CFSE 劳 （左峰）靶细胞与卵257-264肽和控制脉冲，分别注入小鼠iPS细胞转移或OT-CTL的转移后，有一天，10周后。 <img src="/files/ftp_upload/3986/3986fig4.jpg" alt="图4"/> 图4。继转移OT我的TCR基因转导的iPS细胞，抑制肿瘤生长。 OT-TCR基因转iPS细胞过继转移到C57BL / 6小鼠。一组小鼠OVA反应CD8 + T细胞注射OT-TCŕ转基因小鼠，一组老鼠没有细胞的转移。小鼠后，无论是六个星期或细胞移植后的翌日，受到挑战E. G7肿瘤细胞。第20天，腹腔中的肿瘤细胞进行了列举。 <img src="/files/ftp_upload/3986/3986fig5.jpg" alt="图5"/> 图5。iPS细胞源性抗原特异性CTL渗透到肿瘤组织。肿瘤的挑战后30至35天，肿瘤组织进行了研究，为肿瘤反应T细胞的浸润。一）H＆E染色。炎症细胞浸润在肿瘤组织中（↓）。二）免疫组化染色。 OVA特异性Vα2+淋巴细胞（红色）（绿色）卵表达的肿瘤组织中浸润。 c）从肿瘤组织中的单细胞悬液进行了分析Vα2表达+ +通过流式细胞仪，之后的CD8 +人口浇注Vβ5。 <img srC ="/ files/ftp_upload/3986/3986fig6.jpg"ALT ="图6"/&gt; 图6。继转移的OT-我的TCR基因转导的iPS细胞维持小鼠的存活率。卵TCR基因转导的iPS细胞过继转移到C57BL / 6小鼠受到挑战E.的七国集团肿瘤细胞图所述。 4。 Kaplan-Meier生存曲线（N = 6）50天的小鼠的存活率。

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没有利益冲突的声明。

<html>为使基于疗法， 在体外产生大量高活性银特定的T细胞在体内重新输液是最佳方法。虽然我们在体外培养的方法，给出了iPS细胞，iPS细胞源性细胞的死亡，尤其是在第四个星期，在四个星期内的大量功能性T细胞上升。我们得出这样的结论:从缺口的生存信号的信号介导的DL1的IL-7和FLt3L不足以维持iPS细胞衍生的祖T细胞的生存以及其他存活的因素可能是合作，以调节这些细胞?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> C57BL/6J小鼠</td><td>杰克逊实验室</td><td> 000664 </td></tr><tr><td> B6.129S7 RAG1 tm1Mom / J的 </td><td>杰克逊实验室</td><td> 002216 </td></tr><tr><td>抗CD3抗体（2C11） </td><td>屋宇署Pharmingen公司</td><td> 553058 </td></tr><tr><td>抗CD28（37.51）抗体</td><td>屋宇署Pharmingen公司</td><td> 553295 </td></tr><tr><td>抗CD3抗体（17A2） </td><td> BioLegend </td><td> 100202 </td></tr><tr><td>抗CD4（GK1.5）抗体</td><td> BioLegend </td><td> 100417 </td></tr><tr><td>抗-CD8（53-6.7）抗体</td><td> BioLegend </td><td> 100714 </td></tr><tr><td>抗CD25抗体（3C7） </td><td> BioLegend </td><td> 101912 </td></tr><tr><td>一个TI-CD44的抗体（1M7） </td><td> BioLegend </td><td> 103012 </td></tr><tr><td>抗-CD117的抗体（2B8） </td><td> BioLegend </td><td> 105812 </td></tr><tr><td>抗TCR-β（H57597）抗体</td><td> BioLegend </td><td> 109220 </td></tr><tr><td>抗IL-2（JES6-5H4）抗体</td><td> BioLegend </td><td> 503810 </td></tr><tr><td>抗γ-干扰素（XMG1.2）抗体</td><td> BioLegend </td><td> 505822 </td></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> ABCD1234 </td></tr><tr><td> α-MEM中</td><td> Invitrogen公司</td><td> A10490-01 </td></tr><tr><td>胎牛血清</td><td>胎牛血清</td><td> SH3007.01 </td></tr><tr><td>布雷菲德菌素A </td><td>西格玛</td><td> B7651 </td></tr><tr><td>凝聚胺</td><td>西格玛</td><td> 107689 </td></tr><tr><td> GeneJammer </td><td>综合科学</td><td> 204130 </td></tr><tr><td> RNA试剂盒&lt;/ TD&gt; <td> Qiagen公司</td><td> 74104 </td></tr><tr><td> DNA试剂盒</td><td> Qiagen公司</td><td> 69504 </td></tr><tr><td> CD8分离试剂盒</td><td>美天旎生物技术</td><td> 130-095-236 </td></tr><tr><td> ACK裂解液</td><td>龙沙</td><td> 10-548E </td></tr><tr><td> mFlt-3L </td><td> PeproTech公司</td><td> 250-31L </td></tr><tr><td> MIL-7 </td><td> PeproTech公司</td><td> 217-17 </td></tr><tr><td>明胶</td><td>西格玛</td><td> G9391 </td></tr><tr><td> FITC标记的抗卵抗体</td><td>罗克兰Immunochemicals </td><td> 200-4233 </td></tr><tr><td>通透性缓冲区</td><td> Biolegend </td><td> 421002 </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A7906 </td></tr><tr><td>甲醛</td><td>西格玛</td><td> F8775 </td></tr><tr><td> 0.4微米过滤器</td><td> Millipore公司</td><td></td></tr><tr><td&gt; Moflo细胞分选仪</td><td>岳Cytomation </td><td></td></tr><tr><td>刀魂流式细胞仪</td><td>屋宇署</td><td></td></tr><tr><td> LSR的第二流式细胞仪</td><td>屋宇署</td><td></td></tr><tr><td>鼠标限位</td><td>布伦特里科学</td><td></td></tr></tbody></table>

directed differentiation of induced pluripotent stem cells towards t lymphocytes

抗原特异性CD8 +细胞毒性T淋巴细胞（CTL）的收养细胞转移（ACT）是一个很有前途的治疗各种恶性肿瘤的1。淋巴细胞可以识别肿瘤抗原与T细胞受体（TCR），释放细胞毒素以及细胞因子的相互作用，以杀死肿瘤细胞的恶性细胞。据了解，少分化和中央内存（称为高活性 ）CTL的是为使基于免疫的最佳人口，因为这些淋巴细胞具有较高的增殖潜能，是不易，更分化的细胞凋亡，并有更高的响应能力2-7稳态细胞因子。然而，由于在这种淋巴细胞从患者获得大量的困难，是一个迫切需要找到一种新的方法产生高活性的银特有的成功为基础的ACT疗法的淋巴细胞。 自我再生的干的TCR转导细胞免疫重建具有治疗疾病的治疗8-10潜力。然而，从病人身上获取胚胎干细胞（ESCs）的方法是不可行的。利用造血干细胞（造血干细胞用于治疗目的）虽然已被广泛应用在临床上11-13减少，造血干细胞的分化和增殖能力，造血干细胞难以在体外细胞培养14-16扩大。 iPS细胞技术和基因传递在体外系统的发展能够产生iPS细胞从患者没有任何的手术方法。此外，胚胎干细胞一样，iPS细胞具有无限期的体外增殖能力，已被证明可以分化成造血干细胞。因此，iPS细胞具有更大的潜力，使用胚胎干细胞或造血干细胞相比，在ACT - 基于免疫。 在这里，我们提出了T淋巴细胞的生成方法iPS细胞在体外 cytes，并在促进癌细胞的免疫监视iPS细胞的抗原特异性CTL的体内编程。推动iPS细胞在体外与Notch配体刺激T细胞的分化和TCR基因转导到抗原特异性T细胞分化的iPS细胞在体内 ，从而阻止肿瘤的生长结果。因此，我们证明iPS细胞的抗原特异性T细胞的分化。我们的研究提供了一个潜在的更有效的方法使基于疗法产生抗原特异性CTL和促进疾病的治疗策略的发展。

Watch this Scientific Journal Video about Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes at JoVE.com

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

诱导多能干细胞（iPS）细胞，T淋巴细胞的生成，给出了一个使用基于T细胞免疫治疗的胚胎干细胞的替代方法。该方法表明，利用任<em&gt;在体外</em&gt;或<em&gt;在体内</em&gt;感应系统，iPS细胞能够分化成传统和抗原特异性T淋巴细胞。

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

directed-differentiation-induced-pluripotent-stem-cells-towards-t

Research

JoVE Journal

Immunology and Infection

18.5K 次观看.  Pennsylvania State University College of Medicine. 诱导多能干细胞（iPS）细胞，T淋巴细胞的生成，给出了一个使用基于T细胞免疫治疗的胚胎干细胞的替代方法。该方法表明，利用任在体外或在体内感应系统，iPS细胞能够分化成传统和抗原特异性T淋巴细胞。

视频: Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

科研

免疫与感染

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

T淋巴细胞有丝分裂原诱导芽生的快速定量辨识免疫调节药物

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

人类干细胞衍生的伊拉尔肠细胞单层中M细胞样细胞的诱导分化

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

干细胞衍生病毒Ag特异性T淋巴细胞抑制小鼠HBV复制

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...