该项目资助，一部分，根据卫生（R01AI121180，R21AI109239和K18CA151798），美国糖尿病协会全国学院助学金（1-16 IBS-281），和健康的宾夕法尼亚部门（烟草和解基金） JS

所有的动物实验是由医学动物保健委员会的宾夕法尼亚州立大学学院（IACUC协议＃45470）批准，并遵守协会的实验动物的评估和认可的准则进行。 1.干细胞培养<ol><li>孵育10cm皿在10阶毫升0.1N％明胶的为至少30分钟，在37℃（恒温箱）涂覆该板。 </li><li>从盘和板3×10 6除去明胶照射SNL76 / 7细胞，并使用10％的DMEM培养基（10％FBS和1％青霉素链霉素）中于37℃（孵化）孵育一天。 </li><li>从板和培养解冻iPSCs的过度使用的iPSC介质SNL76 / 7细胞饲养层移除介质（含15％胎牛血清，0.1毫摩尔/升的非必需氨基酸，1毫摩尔/升L-谷氨酰胺的DMEM培养基，并0.1毫摩尔/升β巯基乙醇）9。在小鼠iPS-MEF-NG-20D-17锂电池NE，其从通过的Oct3 / 4，Sox2的，Klf4和c-Myc的的逆转录病毒转染小鼠胚胎成纤维细胞诱导的，由山中伸弥博士（研究所再生医科学，京都大学，京都，日本）获得的。 </li><li>改变在第3天的介质与新鲜介质和在显微镜下观察的iPSC集落。 注意:这些菌落小，有光泽集群或圆形细胞呈现出荧光显微镜下GFP表达一个明确的划分。 </li></ol> 2. 在特定的Ag-iPSC的调节性T细胞的体外分化<ol><li>生成的构建体在逆转录病毒转导由亚克隆对OT-II T细胞受体基因和FoxP3的使用与自我裂解肽2A连结的MIDR质粒10使MIDR-TCRα-2A-TCRβ-2A-FoxP3的构建体。 </li><li>执行使用开发平台E细胞作为包装细胞系的iPSC的逆转录病毒转导9。 </li><li>在第0天，种子OP9-DL1-DL4-IA B细胞通过使用含有20％FCS和为2.2g / L碳酸氢钠。板每10cm培养皿1×10 6个细胞的OP-9培养基（α-MEM介质10 4细胞/ cm 2的た最小密度。 </li><li>第3天，当OP9-DL1-DL4-IA B细胞成为80-90％汇合，删除媒体和种子0.5 - 1×10 5 iPS细胞在OP9-DL1-DL4 - IA B细胞OP-9介质。 注意:此确立有关OP9-DL1-DL4-IA B细胞的iPSC的共培养，并被认为是分化9的第0天。 </li><li>在第5天，通过抽吸除去从10cm皿的媒体，用10ml 1×PBS中洗细胞，并吸出PBS中。添加4毫升0.25％胰蛋白酶并在37℃下孵育10分钟。在400 XG另一个8毫升的iPSC媒体的添加到细胞，重悬浮它们，离心机在室温下5分钟。 <ol><li>吸出上清液并重新悬浮细胞在10ml的iPSC介质。这些孵育重悬细胞上新的10 cm培养皿并返回到培养箱30分钟。 注意:删除OP9-DL1-DL4-IA b饲养层细胞，并保持差异化的iPSCs漂浮在介质中。维持OP9-DL1-DL4-IA B细胞不断实现80 -进一步共培养90％汇合。 </li><li> 30分钟后，收集漂浮细胞，通过70微米的细胞滤网过滤它们，并用血细胞计数器计数细胞。 </li></ol></li><li>种子5×10的iPSC的5到新鲜的80 -在OP9媒体OP9-DL1-DL4-IA B细胞90％汇合。补充用mFlt-3L的培养基中于5毫微克/毫升的最终浓度。 </li><li>在第8天，通过与来自使用10毫升吸管皿本身的媒体洗涤该板收集部分分化的iPSC。使用另一个5毫升OP-9介质的菜通过仔细的，有力的移液轻轻冲洗掉半贴壁细胞，以免在培养皿底部打破OP9单层。重复用10ml的PBS洗涤收获所有半广告赫伦特分化细胞。 <ol><li>在400 XG结合两种洗涤，离心，在室温下5分钟，在10含有mFlt-3L（5毫微克/毫升）和MIL-7（1纳克/毫升）OP9媒体毫升重悬浮，并通过70过滤微米的细胞过滤。 </li></ol></li><li>转移细胞到含80 6孔培养板- 90％汇合OP9-DL1-DL4-IA 的B细胞，如在步骤1.1。传递细胞从一个10cm平皿收获到6孔板的一个孔中。 </li><li>在第10天，改变半媒体从细胞到补充有mFlt-3L（5毫微克/毫升）和MIL-7（1纳克/毫升）的新鲜OP9介质。重复此步骤，每两天。 </li><li>每4-6天，这取决于生长，再种子微分的iPSC上板与OP9-DL1-DL4-IA 的B细胞的新鲜层，如在步骤1.1中描述。 </li></ol> 3. 在体外 Treg细胞的分化和成熟的评价<ol><li> iPS细胞分化的形态变化。 <ol><li>莫nitor与OP9-DL1-DL4-IA B细胞的iPSC的共培养每天通过常规亮场显微镜（20X）下观察活细胞。第5天，观察与中胚层样的特点，如扁平细胞集落。到第8天，观察细胞的小而圆簇，它代表分化细胞。 </li><li>使用台盼蓝排除法计数细胞来检测活细胞的数量和百分比。计算细胞存活率作为活细胞的血细胞计数器的四个网格内的细胞的总数除以数。如果电池占用台盼蓝，认为他们死的或不能存活的。记录从培养收获的活细胞的数量。 </li></ol></li><li>流式细胞仪iPS细胞分化的分析。 <ol><li>天5，7，11，15，19，21，和共培养28除去用0.25％胰蛋白酶的细胞如在步骤25。 </li><li>之前用不同的荧光标记的抗体的表面染色，incubatë1×10 6个细胞用100μl，在4℃，20分钟，在PBS中（最终浓度为10μg/ ml）稀释的Fc阻断2.4G2的，以防止抗体的结合在细胞表面上的Fc受体。 </li><li>天5，7，11，15，19，21，和28，流式细胞仪使用1×10 6个细胞用于流具有不同荧光标记的抗体来检测细胞表面标记物，包括CD3，TCRβ，CD4，CD8，CD25，和CTLA4。 </li><li>以下的Fc块，洗涤细胞用10ml的PBS和染色用不同荧光染料标记的抗体进行20分钟，在4℃，然后执行与15色流式细胞分析流式细胞仪。使用抗体的0.200微克每于100μlPBS中稀释10 6个细胞。 </li><li>在第28天，分析细胞通过流式细胞仪11和栅极上用CD4和CD8荧光标记染色的 CD4 + CD8 +细胞;分析TCRVα2，TCRVβ5，CD25，CTLA表达-4，和FoxP3的（ 图1）。对于FoxP3的染色，修复细胞，它们permealize，然后进行抗体染色11。 </li></ol></li><li> iPS细胞在体外抗原刺激。 <ol><li>上共培养，收获iPSC的调节性T细胞从通过收集漂浮细胞培养第28天。 Trypsinize用0.25％胰蛋白酶的细胞的其余部分（如在步骤2.5）和重新悬浮的细胞在8ml的iPSC介质。离心机在室温下以400 xg离心5分钟，吸出介质，并再次重新悬浮细胞在10ml的介质。 <ol><li>孵育在一个新的10cm平皿重悬浮的细胞在37℃下30分钟，收集漂浮细胞。清洗细胞用冷PBS。 </li></ol></li><li>在4℃小鼠在200μl的媒体为5μM卵清蛋白肽（OVA 323-339），用于在一个96孔板11 30分钟- / -从C57BL / 6 RAG1脾脏孵育3×10 6个脾细胞。 </li><li>混合调节性T细胞与OVA 323-339 </子&gt; -pulsed 在1脾:4的比例（0.75用×10 6的Tregs）。在CO 2培养箱中进行40小时在37℃。在过去的4小时，加入4微升稀释布雷菲德菌素A的到培养基（实际浓度1000倍，在1×培养基稀释）。 </li><li>收获的细胞用0.25％胰蛋白酶（如在步骤2.5），洗净，用10％NaN 3的和0.5M EDTA溶液（FACS缓冲液），块用100μl稀释（终浓度为10μg/ ml）的Fc阻断2.4G2，和染色对下述表面标志物，CD4，CD8，TCRVα2和TCRVβ5如步骤3.2.3-3.2.4。 </li><li>固定在PBS中的4％低聚甲醛溶液中的细胞，并用100μl1×透化缓冲液的细胞表面染色后通透。 注意！多聚甲醛是过敏，致癌和有毒的。 </li><li>染色用荧光标记的TGF-β和在黑暗中20分钟的IL-10的细胞。使用0.200微克抗体/ 10 6个细胞dilutED 100微升的PBS。检测胞内产生的TGF-β和IL-10与15色流式细胞仪（ 图2）。 </li><li>清洗细胞三次前的流式细胞仪分析11冷FACS缓冲液。 </li></ol></li><li> 在抗原特异性iPSC的调节性T细胞的体内持久性。 <ol><li>后共同培养的上OP9-DL1-DL4-IAβ细胞8天，转移源自iPSC的预调节性T细胞（Thy1.2），为在经由步骤3.3.1进入祢1.1同类系小鼠（4-6周龄）尾静脉注射无需麻醉。之前执行所述尾静脉注射，扩张尾静脉用红外线灯进行5分钟。尾静脉扩张后，放置在一个限制器鼠标和继转移3×10 6个细胞重悬浮于200μl的PBS通过尾静脉注射。 </li><li>在调节性T细胞转移后六个星期，通过安乐死的 CO 2窒息的小鼠颈椎脱位。确保英里CE都没有了呼吸。通过切割使用剪刀和镊子腹膜的外皮，并轻轻拉回来，以暴露内壳衬里腹膜腔打开腹腔。隔离脾注入你的1.1同类小鼠的外周淋巴结。 </li><li>收集来自使用机械破碎，得到单细胞悬浮液的脾和淋巴结细胞。孵育细胞用5ml ACK裂解缓冲液中5分钟，在RT以裂解红血细胞。收集和洗涤剩余的脾细胞或淋巴细胞用10毫升冷流式细胞仪缓冲的。 </li><li>继洗涤，治疗与在4℃下与Fc受体阻滞剂2.4G2 20分钟的细胞如步骤3.2.2和染色用100μl稀释荧光标记的抗Thy 1.2抗体的产生。 </li><li>洗涤细胞，用10ml FACS缓冲液，并进行流式细胞仪分析11。 </li></ol></li></ol> 4. 在体内成熟和抑制自身免疫性关节炎</p&gt; <ol><li>生成的构建体如在步骤2.1。 </li><li>执行逆转录病毒转导9如在步骤2.2。 </li><li> 在体内分化和关节炎诱导小鼠。 <ol><li>区分的OT-II T细胞受体/ FoxP3的转导的iPSC（OT-II的FoxP3 / iPSCs的），对细胞因子mFlt-3L的存在下OP9-DL1-DL4-IA b基质细胞的FoxP3转导的iPSC和DsRed的转导的iPSC和2.6 - 白细胞介-7 8天的步骤2.1节所述。 </li><li> Trypisinize所有三种细胞从10cm平板，并重新暂停从每个10cm平板的细胞在10ml新鲜的培养基。细胞添加到新鲜的10cm平板，并返回到培养箱30分钟。 30分钟后，收集漂浮的细胞。 </li><li>通过细胞通过70微米的细胞滤网以除去细胞团并使用血细胞计数器计数。调节细胞在冷PBS中的1.5×10 7个细胞/ ml的浓度，并根据需要再进行过滤。保持细胞在冰上，直到过继细胞转移到小鼠体内。 </l我&gt; <li>注入200微升细胞悬浮液（3×10 6个细胞）至4的三个不同的组- 6周龄的雌性C57BL / 6小鼠通过尾静脉如3.4.1中所述</li><li>在细胞转移后第10天，注入100 MBSA微克在弗氏完全佐剂在尾的基部通过用1ml注射器乳化的小鼠。 </li><li>第17天，麻醉用异氟烷蒸发器（根据IACUC准则）小鼠。利用4 - 诱导5％异氟醚和1 - 维护2％。在右膝关节诱导了20微克MBSA的关节内注射关节炎于10μlPBS中的左膝关节和20微克MBSA和100​​μg整体OVA于10μlPBS中。食品和GELPACK可放置在被褥地板关节炎已开发之后。将小鼠安乐死:2/5或垂死的，严重的恶病质和/或持续斜卧（24小时周期）体况评分（BCS）和严重度评分4.在比分4，红斑和肿胀严重涵盖踝，足和数字，或肢体的ankyloses。 </li></ol></li><li>对OT-II TCR / FoxP3的转导的iPS细胞的表征。 <ol><li>使用荧光显微镜（20X）来可视化未定影活DsRed的+ GFP +细胞。 </li><li>检查由双方印迹基因整合和表达和流式细胞仪11。 </li></ol></li><li>调节性T细胞的发育和成熟。 <ol><li>在周2，4和6中的细胞转移后，安乐死的小鼠。在第一阶段2升的 CO 2 -对于安乐死，每个笼子使用1。一旦动物已失去知觉，增加的 CO 2的流率，以4 - 5升/分钟。安乐死的CO 2吸入老鼠。隔离脾和通过切割用剪刀和镊子腹膜的外皮，并轻轻拉回来，以暴露内壳衬里腹膜腔排出淋巴结。 </li><li>收集单细胞悬液˚FROM用细胞过滤网，并在1％的iPSC媒体1ml注射器由机械故障的脾脏和淋巴结。离心含有在400克介质5分钟的锥形管中。重悬在5ml ACK裂解缓冲液中的细胞沉淀以裂解红血细胞。重新离心机以1,000rpm 5分钟。重悬细胞沉淀，并用冷FACS缓冲液洗涤剩余的脾细胞或淋巴细胞。按照步骤3.4.4 - 3.4.5，并进行流式细胞仪分析。 </li></ol></li><li>细胞内染色。 <ol><li>关于天50攻击后，分离脾，并用CO安乐死后沥干小鼠的淋巴结（如在步骤4.5.1）2吸入颈椎脱位。 </li><li>收集来自用细胞过滤网和1ml注射器，机械故障的脾和淋巴结的单细胞悬浮液。在室温下孵育的脾细胞5分钟5毫升的ACK裂解缓冲液以裂解红血细胞。收集和洗涤剩余的小号plenocytes或淋巴细胞用冷FACS缓冲。按照步骤3.2.3 - 3.2.4，但与污渍表面标志CD4，CD8，CD25和TCRVβ5。 </li><li>继续如3.3.5所述修复细胞内染色- 3.3.7和分析活CD4 + CD25 +细胞中胞内细胞因子产生（TGF-β和IL-10）的源自iPSC的调节性T细胞的和栅极。 </li></ol></li><li>在膝盖抗原特异性调节性T细胞的浸润和关节炎的抑制。 <ol><li>天7 -下面的步骤（4.3.6 4.3） - 14关节炎诱导后（按照IACUC准则）用CO安乐死的小鼠2吸入颈椎脱位。从膝盖用电动剪清除头发和手术切除膝盖。 </li><li>通过使在与钝端剪刀后腿的切口移除腿部皮肤和抄近路穿过大腿和向下到脚踝的皮肤。轻轻剥离在腿部和脚部，以暴露肌肉皮肤。去掉从腿部肌肉小心不损坏膝关节。 <ol><li>切开后腿只是骨盆/髋关节上方用锋利的解剖剪刀剪胫骨。 </li></ol></li><li>修正了4％福尔马林膝盖48小时。脱钙使用2.5M的甲酸膝盖;三次漂洗在二甲苯为3分钟，在100％乙醇漂洗两次，在95％的乙醇冲洗两次，在去离子水中漂洗两次2分钟，并在1毫摩尔EDTA脱钙。治疗在20分钟的子沸点温度（90℃）。 </li><li>冷却该固定组织30分钟。用1X PBS冲洗4分钟，并嵌入在石蜡。通过一系列乙醇浴脱水组织以置换水，然后用蜡渗透它们。然后嵌入渗透到组织蜡块。执行对染色纵向和横向切片。准备4微米的部分采用了侧滑切片机。 </li><li>在deparaffi的标准程序后各部分进行免疫荧光染色nization及补液使用二甲苯和乙醇12。 </li><li>通过浸渍在3％的过氧化氢溶液的足够量的滑动为银检索之后3分钟阻断内源性过氧化物酶的活性。块幻灯片中的非特异性的3％BSA在PBS中在室温下在潮湿的腔室60分钟的结合。 </li><li>染色切片用200μl荧光标记TCRVβ5抗体的稀释1:在封闭溶液100。孵育在75室温2小时 - 100％湿润的腔室，并在1×PBS洗涤5次，每次5分钟。 </li><li>染剂用于核染色的幻灯片含DAPI的抗淬灭剂。加约300微升稀释DAPI染色溶液（300nM的在1×PBS中）的盖玻片，使得确定整个盖玻片覆盖。在4℃下储存在黑暗中滑动，直到用荧光显微镜（ 图3）下分析。 </li></ol></li><li>关节炎抑制试验<ol><li>通过使用拨号规卡尺，以建立基线测量关节炎诱导前小鼠的两个膝盖的肿胀。 </li><li>按照步骤（4.3 - 4.3.6）关节炎的诱导和Treg转移。测量膝盖鼠标使用拨号规卡尺后Treg细胞转移。 </li><li>计算膝盖直径增加的百分比:百分比增加=（膝盖直径第1天 - 第0天膝关节直径）/第0天膝关节直径。 </li></ol></li><li>通过组织学在膝盖关节破坏和炎症细胞浸润的测量（ 图4）。 <ol><li>后第7天，关节炎诱导，安乐死如前所述小鼠和由外科手术切除的膝盖。请参阅步骤4.7.1 - 4.7.2。 </li><li>修复膝盖在4％福尔马林，在EDTA脱钙，并在石蜡嵌入。请参阅步骤4.7.3 </li><li>卸下两个膝盖，固定在10％福尔马林，并在Formical-4钙化。嵌入石蜡，部分组织在4微米，并与hematoxyl染色在和伊红（H＆E）或番红O染色（如步骤4.7.7），并观察显微镜下。 </li><li>使用H＆E染色半定量计分系统来评价骨侵蚀（0:无糜烂; 4:扩大糜烂和骨质破坏）。使用番红O染色来评价蛋白多糖的损失，并用半定量计分系统评分（0:无蛋白多糖损失; 3:蛋白聚糖染色完全丧失）。 <ol><li>使用半定量评分系统的得分上H＆E染色的载玻片的炎性细胞浸润（0:无细胞浸润; 4:盛产细胞浸润）。通过采用盲法研究双膝评价分数。 </li></ol></li></ol></li></ol> 5.在与高分辨率微型计算机断层扫描（微CT）系统的膝盖骨损失的测量<ol><li>第10天的后关节炎诱导麻醉，用2％异氟醚的小鼠和用于成像做准备。 </li><li>麦克风的位置e为在腔室朝上的膝盖。 </li><li>使用高分辨率微型CT系统在周围的小鼠的膝盖骨结构的体内成像来获取。 </li><li>执行微CT扫描了2.2毫米的长度，包括鼠标膝关节使用以下参数:10.5微米体素尺寸在55千伏，145微安200毫秒的积分时间，211的图像。 </li><li>进口显微CT图像和捕获额面图像进一步图像处理（体积渲染和转换）（ 图5）之后。 </li></ol>

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The authors have nothing to disclose.

在这个协议中，一个关键的步骤是TCR / FoxP3基因转导的iPSC的体外分化。 在体外 Notch信号传导诱导发展朝向T细胞谱系。分化的iPSC成CD4 + FOXP3 + Treg细胞，我们使用了OP9-DL1 / DL4 / IA 的B细胞，其中高表达MHC II类分子（ⅠAB）的分子。大部分的iPSC的分化成CD4 +细胞。然而，表面TCR表达后，许多分化的前T细胞失去分化并最终死亡的能力。其结果是，在源自iPSC的官能的Treg细胞数四周体外分化后显着减少。为了避免这种情况，加入IL-2，可以提高在这些时间点的细胞存活。一种替代方法，以驱动特定的Ag-预调节性T细胞的成熟和存活被转移已在体外分化为一周到小鼠预先的Treg。这些预调节性T细胞可以共存ntinue分化和体内成熟另三个星期。使用这种方法，可以从iPS细胞，这是nTregs状，并有抑制在小鼠模型的自身免疫性关节炎的能力可以产生功能性抗原特异性的Treg的人口。 为了提高抗原特异性iPSC的调节性T细胞的体内发展的功效，不同的化合物（ 例如 ，视黄酸，gelectin）13,14或抑制细胞因子（ 如 ，TGF-β，IL-10）可在过继转移后使用的预调节性T细胞。除了增加的细胞存活，这相结合的办法可以维护的FoxP3表达14,15和增强抗原特异性iPSC的调节性T细胞的质量。 可能出现在体内的Treg发展了一个潜在的问题是整体的免疫抑制，导致感染或随后的减肥期间的并发症。大量抗原特异性的调节性T细胞在vi发展VO会加重感染。在这种情况下，一个自杀基因，诱导胱天蛋白酶9 （iCasp9）15,16，可掺入的TCR / FoxP3的载体。这种方法允许通过生物惰性小分子二聚剂（AP1903）设定为"关闭"的干细胞衍生的Treg的产生，这将克服这个潜在的问题的喷射除去干细胞衍生的Treg。 一种自银是由来自个体自身免疫病症患宿主的免疫系统识别的典型的蛋白质。这种自银是免疫系统的目标，和相关的T细胞不会被删除。产生自银具体调节性T细胞，使用TCR转导是一个不错的选择。一种自银（ 例如 ，热休克蛋白）特异性TCR可以从外周血单核细胞（PBMC）进行转导到成熟的CD4 + CD25 +调节性T细胞，并且这种方法在临床试验中被利用。或者，如德cribed在这个协议中，使用自Ag的基因转导特异性TCR与FoxP3的和刺激Notch信号传导，干细胞衍生的Treg可以自行的Ag特异性的Treg。 用于利用工程化的Treg的自身免疫性疾病的细胞疗法可能是有用的17。虽然在T细胞的TCR转导已被证明是安全的，是可行的，并适用于临床试验中，仍存在重大的安全问题由于由交叉反应性健康组织18中的自身免疫。此外，使用目前的方法，转基因的Tregs通常具有在体内 19短期持久性。可替代地，用于开发大量单克隆抗原特异性的Treg的一个有希望的来源是干细胞。胚胎干细胞（ESCs）有最好的多能性和自我更新，但它不是从患者获得它们是可行的。虽然很容易从外周血中分离，造血干细胞（HSC）是多有力的干细胞，并且可以在细胞培养物同样可以扩展到胚胎干细胞20。本IPSC的技术已经由不同的转录因子的转前进到更有效地产生从患者的体细胞的PSC的。许多改进方法已经开发了近年来最大限度减少潜在的危险，如免疫原性和致瘤性，产生iPS细胞。比起胚胎干细胞，iPS细胞具有多能性相同和自我更新。其结果是，IPSC的技术可以在显影患者 - 和/或疾病特异性的PSCs提供的优点。使用iPS细胞的培养抗原特异性的Treg可以前进基于细胞的疗法的领域为自身免疫性疾病10,11。

Autoimmune arthritis is a systemic disease characterized by hyperplasia of synovial tissue and progressive destruction of articular cartilage, bone, and ligaments1. The defective generation or function of Tregs in autoimmune arthritis contributes to chronic inflammation and tissue injury because Tregs play a crucial role in preventing the development of auto-reactive immune cells. 
Manipulation of Tregs is an ideal strategy for the development of therapies to suppress inflammation in an Ag-dependent manner. For Treg-based immunotherapy, the specificity of the transferred Tregs is important for the treatment of ongoing autoimmunity2. To exhibit the suppressive activity, Tregs need to migrate and be retained at the afflicted region, which can be directed by the specificity of the TCR for the Ag at that location3. Although polyclonal Tregs may contain a small population containing this Ag specificity from their TCRs, the numbers of these Ag-specific Tregs are usually low. Consequently, cell-based therapies using polyclonal Tregs against autoimmune disorders require adoptive transfers of a large number of Tregs4,5. Because pluripotent stem cells (PSCs) have the ability to develop into any type of cell, Ag-specific PSC-Tregs may prove to be good candidates for Treg-based immunotherapy. Previous studies have shown the successful development of PSC-derived T cells, including Tregs6-8. 
Here, we describe a protocol to develop Ag-specific iPSC-Tregs. We further describe a cell-based therapy of autoimmune arthritis in a murine model using such Tregs. This method is based upon genetically modifying murine iPSCs with Ag-specific TCRs and the transcriptional factor FoxP3. The engineered iPSCs then differentiate into Ag-specific Tregs on the OP9 stromal cells expressing Notch ligands DL1, DL4, and MHC-II (I-Ab) molecules in the presence of cytokines mFlt3L and mIL-7. These Ag-specific iPSC-Tregs can produce suppressive cytokines, such as TGF-&#946; and IL-10, when stimulated with the Ag, and adoptive transfer of such Tregs has the ability to suppress AIA development in a murine model. The described protocol can be used to develop stem cell-derived Ag-specific Tregs for potential therapeutic interventions.

<table><tbody><tr><td>C57BL/6j mice</td><td>Jackson Laboratory</td><td>664</td><td></td></tr><tr><td>B6.129S7 Rag1tm1Mom/J</td><td>Jackson Laboratory</td><td>2216</td><td></td></tr><tr><td>Anti-CD3 (2C11) antibody</td><td>BD Pharmingen</td><td>553058</td><td></td></tr><tr><td>Anti-CD28 (37.51) antibody</td><td>BD Pharmingen</td><td>553295</td><td></td></tr><tr><td>Anti-CD4 (GK1.5) antibody</td><td>Biolegend</td><td>100417</td><td></td></tr><tr><td>Anti-CD8 (53&amp;ndash;6.7) antibody</td><td>Biolegend</td><td>100714</td><td></td></tr><tr><td>Anti-CD25 (3C7) antibody</td><td>Biolegend</td><td>101912</td><td></td></tr><tr><td>Anti-TCR-&amp;beta; (H57597) antibody</td><td>Biolegend</td><td>109220</td><td></td></tr><tr><td>Anti-IL10</td><td>Biolegend</td><td>505010</td><td></td></tr><tr><td>Anti-TGF&amp;beta;</td><td>Biolegend</td><td>141402</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>ABCD1234</td><td></td></tr><tr><td>&amp;alpha;-MEM</td><td>Invitrogen</td><td>A10490-01</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH3007.01</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma</td><td>B7651</td><td></td></tr><tr><td>Polybrene</td><td>Sigma</td><td>107689</td><td></td></tr><tr><td>Genejammer</td><td>Integrated science</td><td>204130</td><td></td></tr><tr><td>ACK Lysis buffer</td><td>Lonza</td><td>10-548E</td><td></td></tr><tr><td>mFlt-3L</td><td>peprotech</td><td>250-31L</td><td></td></tr><tr><td>mIL-7</td><td>peprotech</td><td>217-17</td><td></td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G9391</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148-500G</td><td>Caution: Allergenic, Carcenogenic, Toxic</td></tr><tr><td>Permeabilization buffer</td><td>Biolegend</td><td>421002</td><td></td></tr><tr><td>mBSA</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>Ova albumin</td><td>Avantor</td><td>0440-01</td><td></td></tr><tr><td>CFA</td><td>Difco</td><td>2017014</td><td></td></tr><tr><td>Tailveiner restrainer</td><td>Braintree scientific</td><td>RTV 150-STD</td><td></td></tr></tbody></table>

development of stem cell-derived antigen-specific regulatory t cells against autoimmunity

自身免疫疾病的出现是由于免疫自身耐受丧失。调节性T细胞（Treg细胞）是免疫自身耐受的重要介质。调节性T细胞代表约5 -在小鼠和人类成熟的CD4 + T细胞亚群的10％，与约1 -这些Treg细胞在外周血中循环的2％。诱导多能干细胞（iPS细胞）可以分化成官能的Treg，其具有用于自身免疫性疾病的基于细胞的疗法的电位。这里，我们目前开发iPS细胞从抗原（银）特异性调节性T细胞（ 即 iPSC的调节性T细胞）的方法。该方法是基于结合转录因子FoxP3的和Ag特异性T细胞受体（TCR）插入的iPSC，然后分化上表达的Notch OP9基质细胞配体三角状（DL）1和DL4。 下列体外分化，所述iPSC的的Treg表达CD4，CD8，CD3，CD25，FoxP3的，和Ag-特异性TCR并能够对于Ag刺激作出响应。该方法已成功地应用于自身免疫性关节炎的基于细胞的疗法在小鼠模型。这些特定的Ag-iPSC的调节性T细胞的过继转移到银 - 诱导的关节炎（AIA）的-bearing小鼠具有降低关节发炎和肿胀，并防止骨损失的能力。

如这里所示，在第28天，抗原 - 特异性的Treg基本上表达CD3和抗原特异性T细胞受体，两个T细胞标记。该CD3 +TCRVβ5+人口表示CD4。大多数CD3 +TCRVβ5+ CD4 +细胞也表达CD25，CD127，和CTLA-4，这是在天然存在的Ť 暂存器 （nTregs）和在表达FoxP3的异位性T细胞在升高的水平一般表示的。在源自iPSC的细胞中的FoxP3表达与所述Notch配体体外刺激持续即使经过长期的，通过用流式细胞仪（图1）分析细胞内染色所检测。此外，该抗原特异性iPSC的调节性T细胞产生的抑制细胞因子，例如TGF-β和IL-10的， 在体外用抗原脉冲的脾细胞（图2）的刺激时，指示所述iPSC的调节性T细胞具有潜在的抑制活性。荧光显微镜透露<html>更多的FoxP3 +细胞存在于OVA处理的比PBS处理的膝盖;没有FOXP3 +在接到红色荧光蛋白+载体转导的小鼠iPS细胞中存在的膝盖细胞。许多更多的 CD4 +对收到的iPSC小鼠与MIDR-TCR-的FoxP3比MIDR-的FoxP3转膝盖呈现的FoxP3 +TCRVβ5+细胞（图3）。这些观察表明，特定的Ag-iPSC的调节性T细胞迁移到AIA膝盖过继转移到受体小鼠后。传送的源自iPSC的细胞显着减少炎性肿胀的膝盖时，OVA存在，但对控制膝盖被在小鼠模型MBSA只注射（图4）没有影响;减少骨损失在用高分辨率微型CT系统（图5）可视化的细胞转移膝盖。 tp_upload / 54720 / 54720fig1.jpg"/&gt; 图 1: 抗原特异性iPSC的调节性T细胞的分化小鼠iPS细胞转导用的构建体:含有OVA特异性TCR和的FoxP3基因MIDR-TCRα-2A-TCRβ-2A-的FoxP3。基因转导的细胞（红色荧光蛋白+）分别在mFlt3L及mIL-7存在下OP9-DL1-DL4-IA B细胞共培养。（A）中的T形态REGS在第0天，第7，第14和细胞分化对于源自iPSC的细胞对第28天的CD3蛋白表达22（B）的流式细胞仪分析+TCRVβ5+细胞门控为表示（R1）和用于CD4和CD8的表达进行分析，CD25，CD127，CTLA- 4，和Foxp3的门控为CD4 + CD8细胞显示-细胞（R2）。（暗线，阴影部分表示同型对照） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig1large.jpg" target="_blank">点击此处查看LARG</a>呃版本这个数字。 <img alt="图2" src="/files/ftp_upload/54720/54720fig2.jpg" /> 图 2:I N体外 分化抗原特异性的Treg 功能分析小鼠iPSC的调节性T细胞是由脾细胞刺激。（的APC;吨暂存器 /的APCs = 1:4）中，用OVA 323-339肽脉冲。细胞内细胞因子的产生（TGF-β和IL-10）是由流动门控活CD4 + CD25 +细胞后，仪分析。（暗线，阴影部分表示同型对照） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig2large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54720/54720fig3.jpg" /> 图 3:G-特定iPSC的调节性T细胞浸润入膝关节。具体的Ag-iPSC的调节性T细胞过继转移到C57BL / 6小鼠。之后不久关节炎诱导（天7 - 14），膝盖取出并染色免疫组织学（比例尺:20微米）。接受OVA特异性iPSC的调节性T细胞的小鼠有大量TCRVβ5+细胞的膝盖。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig3large.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/54720/54720fig4.jpg" /> 图4: 抗原特异性iPSC的调节性T细胞的过继转移可改善AIA小鼠的小鼠iPS细胞分别与逆转录病毒构建MIDR，MIDR-FoxP3的，或MIDR-TCR-的FoxP3并分别对OP9-DL1共培养/ DL4 /转。 IA b细胞。在第7天，将基因转导的细胞（3×10 6 /小鼠）分别为一doptively转移到该被细胞转移后两周AIA诱导雌性C57BL / 6小鼠。于翌日关节炎诱导，关节炎的严重程度是由膝盖直径测量监控。在膝盖直径（AC）百分点。在膝直径增大是基于注射前膝盖直径为注射第0天的关节炎得分前每只小鼠通过检查以盲方式双膝评价计算;每个膝盖被分配一个分数（0:没有明显的肿胀或变色; 1:可见有或无变色肿胀; 2:中度与变色肿胀; 3:变色严重肿胀）。在各组中，五只小鼠被使用，并且数据代表三次独立的实验。数据被表示为平均值±SD。在第7天（D）的平均得分为两个膝盖从五只小鼠。数据被表示为平均值±来自三个独立实验（SD ** P &lt;0.01，*** P &lt;0.001，双向ANOVA）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig4large.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/54720/54720fig5.jpg" /> 图 5: 抗原特异性iPSC的调节性T细胞迁移至OVA注射膝关节和降低骨丢失 iPSC的调节性T细胞过继转移到C57BL / 6小鼠。 21天内关节炎诱导后，将小鼠麻醉并围绕鼠标膝盖骨架构是由微型CT系统成像。小鼠接收的OT-II T细胞受体/ FoxP3基因转导的鼠相比，控制接收矢量转导的iPSC小鼠iPSC的调节性T细胞表现出骨损失的显著降低。（A）中的扫描用2.2毫米的长度进行的，并且图像被捕捉之后进一步的图像处理（体绘制和改造;比例尺:采用微CT图像的三维重建1毫米）（B）中骨体积膝关节周围进行评价，并计算感兴趣体积内骨体积（比例尺:1毫米） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig5large.jpg" target="_blank">请按此查看该图的放大版本。</a>

Watch this Scientific Journal Video about Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity at JoVE.com

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

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 ...

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Immunology and Infection

8.6K 次观看.  The Pennsylvania State University College of Medicine. 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. 

视频: Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes

My name's Jeff Bluestone. I'm the director of the UCSF Diabetes Center. We're at a laboratory here and oversee the immune tolerance network.My work is involved in basic understanding of how the immune system distinguishes self from non-self and how we can use therapies to try to induce what we call immune tolerance, which is the ability of the immune system being modulated and protect itself from recognizing itself as foreign and attacking things like its own pancreas or brain or joints. That happens in many autoimmune diseases. We're particularly interested in therapies, both drug and cellular therapies that might be used to educate or reeducate the immune system so that we don't block or destroy our own ability To protect our own tissue.So whenever A a T cell comes through the thymus or B cell comes out of the bone marrow, it has to learn to distinguish self from non-self, and this is what we call immune tolerance, which is that ability to tolerate one's own tissue and not destroy it or attack it. What often happens in many disease settings is that the immune system because of environmental and genetic reasons, starts attacking one's own tissue and tolerance is broken and it's that system that we're trying to reestablish with some of our therapies. And the ultimate goal of immune tolerance therapies as opposed to generalized immunosuppression is to try to develop therapies or drugs that will in fact reestablish tolerance without requirement for ongoing therapy.So that today where somebody with a kidney transplant or a liver transplant might require 20 or 30 pills a day to keep the immune system from attacking the, the kidney or pancreas or, or or liver in the future. And we might be able to just provide some kind of therapy that would change the immune system so that it becomes tolerant to the kidney or liver And doesn't reject it. So one of the more Interesting elements of immune tolerance has been to discovery that there are specialized cells in the body whose job it is to protect the organs of the body and and to regulate the immune system so that it doesn't attack those tissues.These so-called regulatory T cells have become a very important and central part of understanding immune tolerance over the last decade or so. In fact, regulatory T cells are often known as suppressor. T cells have been around for 30 years since early discovery and and descriptions by people like Dick Han.Unfortunately, in the eighties, these cells came in some disrepute because of the absence of unique biological or biochemical markers that could distinguish them from other cells and data that was difficult to interpret given our current understanding of how T cells recognize proteins and how the immune system works. But then starting in the mid nineties through seminal work by investigators like Shimon Succi and others, we started to understand both the cellular as well as biochemical basis for regulatory T cells, such that when a transcription factor called Fox P three was discovered in early two thousands, we're able to finally put a specialized tag on these cells and really understand them. And ever since we've really been able to exploit those cells by expanding em, isolating em, and reintroducing em into animals to demonstrate that they are capable of inducing immune tolerance, which is what we are All about.One of the Most important questions that is challenge the field is trying to understand what the mechanism of tregs are, both in immune responses as well as during tolerance induction. And there have been a number of questions related to do the cells act on other T cells that are pathogenic? Do they act on antigen presenting cells or do they make cytokines that ultimately regulate the immune response?Our own studies have suggested based on in vi, in vivo imaging that in fact these cells act directly on the major antigen presenting cell in in the pancreas, and that is a dendritic cell. And we demonstrated that in fact we could label regulatory T cells one color green while the dendritic cells were labeled another color, and show that in fact these regulatory T cells interacted directly with the dendritic cells and by doing so, fundamentally altered the ability of those dendritic cells to activate the pathogenic T cell response. So we think that either directly or through some intermediate cytokine like IL 10 or TGF beta that the tregs educate or change the dendritic cells to make them less able to activate a pathogenic T cell response.Yeah, So one of the more interesting aspects of regulatory T cells has been the notion that in addition to this very specialized subset that exists in the thymus and develops during thymic development, the so-called natural T-Rex that during the course of an immune response, other T cells may in fact turn into regulatory T cells either through direct upregulation of Fox P three due to cytokines like TGF beta, which are known to turn on FOX P three or even other cells which are not Fox P three positive, but take on regulatory T-cell characteristics. Those so-called adaptive tregs are generally very important in in regulating an inflammatory response because they're interacting with the antigens that are present locally in that inflamed tissue. And those adaptive cells are probably critical in maintaining tolerance locally in a targeted tissue, whereas the natural tregs are probably more useful or important in maintaining a, a, a immune homeostasis or generalized suppression of auto reactivity in the whole animal Or patient.So now that we know That these regulatory cells are essential in in maintaining immune homeostasis, we can imagine what would happen if they're disrupted. And in fact, if you destroy Fox P three and either mouse models using knockout technology, or in fact there's a human disease called I pex, the individual animals die in a short period of time of a massive autoimmune disease where each targeted tissue, liver, kidney, heart are in infiltrated with T cells and the tissues are destroyed. So that's the fundamental immuno immune homeostasis that's required that these cells Really protect the individual.One of the important Discoveries with regards to regulatory T cells is the possibility that we might be able to both isolate them and expand them in a specific way to reintroduce into individuals with diseases to bring the immune system back into a homeostatic equilibrium. And in fact, work in our laboratory and others have been able to take these cells out and expand them up to a thousand fold and show that they retain their suppressor activity and can be reintroduced into individual animals to induce an a regulatory response. Where that's been most successful at, at this time has been in treating autoimmune disease and animal models of diabetes, for instance, we can prevent or even reverse diabetes by putting in small numbers of these antigen specific eyelid antigen specific regulatory T cells that we've expanded ex vivo and we hope that we can get into people soon with similar populations that have been expanded in in a similar manner.Now the future hopefully will go beyond autoimmune diseases and into more broad based immunological settings and perhaps the most daunting of which is transplantation. Today we transplant over a half a dozen organs into people, but still are required to give them lifelong immunosuppression to prevent those transplants from being rejected. The hope is that we can take regulatory cells that see the donor tissue from these kidney, liver, pancreas, eyelet transplants and be able to target those regulatory cells to those tissues and prevent the immune system from recognizing those tissues as far and now take this alloimmune response and convert it into a safe self response and prevent rejection without ongoing immunosuppressive Drug therapy.So What we hope is, is that this particular therapy is going to be very appropriate, appropriate for treating people with type one diabetes. Once all of the insulin producing beta cells have been destroyed, the only way we're ultimately gonna be able to treat the disease is by both reconstituting the individual with an eyelet or some insulin producing cell, and at the same time prevent the autoimmune and alloimmune responses from destroying it. So our approach is to take people with long-term diabetes and transplant eyelets at present from cric donors, but in the future, perhaps from stem cells or other sources to actually replace the insulin producing capability, we hope to introduce regulatory T cells into that setting to prevent those eyelets that are coming from foreign sources, from being rejected.As I mentioned, this notion that we can induce tolerance in these individuals will require not just the source of an insulin producing cell, but some combination of drugs and cellular therapies. We hope like regulatory cells to put the individual into a state of tolerance in homeostasis so that these foreign tissues are not recognized and destroyed once introduced into the diabetic patient. Specifically, our approach to actually reconstituting insulin producing beta cells in patients starts out with the kind of experiments we're doing in animal models, which is to take eyelets that have been prepared from a pancreas, remembering that only about 0.5%of all of the cells in the pancreas actually are the eyelets to make insulin to isolate those cells and purify them in a way that we can reintroduce them into a recipient.We reintroduce them either by putting them under the kidney capsule, which is routinely the way we do it in animal models are directly into the liver, which is the way we do it in humans. And once those cells go into that site, either kidney or liver, they then seed that site and start getting the blood and other nutrients that they need in order to survive and make the insulin that then gets circulated to other tissues and organs as needed. And the goal is to obviously, once those cells are in that tissue, to then treat with our various immunotherapeutics to prevent the immune system from attacking the cells Within those tissues.So of course there are a Number of challenges for all novel immunotherapies and tregs are no exception. We need to worry about a number of things as we try to this for a therapeutic. We need to worry about cell numbers at present, that we can only identify small numbers of cells that have the antigen specificity that we need to block both auto and allo immunity.So we'll need to figure out ways to both isolate those cells and expand them to sufficient numbers to treat an individual. In addition, we need to be careful that these cells are truly pure regulatory T cells. We don't wanna make the mistake of putting in cells that have the ability to be pathogenic and attack the foreign tissue as opposed to the regulatory cells which protect.And so understanding the stability and the robustness of these cells and how how they survive in various inflammatory settings will be critical. The third thing we need to worry about is non-specific immunosuppression that could be caused. These regulatory cells are designed to shut down broadly in immune response.And so we don't want to, at the same time we're blocking graft rejection, block the ability of an individual to recognize a bacteria or a virus or cancer cells that might come up, but instead stay targeted and focused on the tissue that we've transplanted into the individual. And then finally, I think the, the other challenge with any cellular therapy is being able to do it robustly and routinely many, many, many times over because each patient is gonna require their own set of cells. This is not a drug you can just take off the shelf.And so we're gonna have to work technologies out that are, use good manufacturing practices that are approved by the food and drug administration that allow us to grow these up the same way every time and be sure that we're working with a, a truly homogeneous and and well-defined population of Cells. When one Thinks about funding for projects like this, it's always a mix between the federal funding that we get through the NIH to support the basic science that's important in moving this forward. And then for us, fortunately, other organizations such as the Juvenile Diabetes Foundation who have been very supportive having raised an enormous amount of money from the community to help in these highly risky projects.And so we've been fortunate in being able to partner with both the NIH and JDRF as well as with pharmaceutical companies on occasion to be able to move these kinds of projects along. So at present, I think the funding is actually adequate and we really hope that we will be able to drive this project forward Effectively. We've been lucky in the diabetes community having such a strong, both federal and non-federal interest in the disease.Of course, that's not inappropriate given the fact that disease is really growing and reaching epidemic proportions, but we hope that the funding that exists will be sufficient for us to move This project. So Like all scientists, we always feel as if we are underfunded and underappreciated when you look at how much money is being spent to, to deal with problems around the world, to deal with war in Iraq, to deal with our, our military, and know that just a couple of days of that funding could support the NIH for a year. It's hard to imagine how we've placed our priorities because we think as scientists that there are a lot of things that we can do to change human health, human, the human state.So we only continue to hope that the advances in the science look in in 50 years. We're not gonna be talking about health insurance, we're not gonna be talking about HMOs and management. We're gonna talk about the amazing discoveries that have been made that are gonna change health in in our country that's gonna change health in the world.And just like the industrial revolution of turn of the last century, that in this century the, the major impact is gonna be on how we've changed diseases, both acute and chronic diseases. And so we hope that the public appreciates that and appreciates our efforts to try to make that kind of Difference.

调节性T细胞治疗移植排斥反应和I型糖尿病的治疗潜力

科研

生物学

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

癌症研究

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&#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 ...

免疫与感染

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

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  ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

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 ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

在自身免疫糖尿病模型中高效生成抗原特异性原小鼠细胞毒性T细胞细胞细胞，用于功能测试

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

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 ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

<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 ...