<p class="jove_content"作者希望感谢他们协助流式细胞仪分析和排序詹妮弗怪和格雷格·鲍曼。这项工作得到了NIH的资助从NCRR 2P20 RR020171和大学由肯塔基启动资金调频; GIE位承认总统的研究生奖学金和肯塔基机遇奖学金的支持。</p

1。分离人外周血单个核细胞（单核细胞从白膜） <ol><li>从医院或附近的血液中心位置，获得一个单位白膜。我们的血液中心提供约40-60毫升单位正常献血者中获得的棕黄色大衣。 </li><li>倒入蒸压500毫升玻璃瓶含无菌PBS稀释白膜血。最终体积的PBS +白膜应该是250毫升。 </li><li>装满十50​​ ml锥形管各20毫升Lymphoprep的解决方案。 </li><li>轻轻覆盖25毫升稀释白膜Lymphoprep解决方案，小心不扰乱Lymphoprep的解决方案的。 </li><li>在室温下30分钟500×Ğ旋转管。确保关闭离心机的制动，以便不打扰淋巴细胞分数。 </li><li>用吸管的Lymphoprep和等离子体介质层之间的接口在收集的外周血单核细胞。吸出，直到约1毫升血浆介质关闭仍然涵盖的的白膜层含有的单核细胞。使用10毫升吸管，单核细胞转移到一个新的50 mL管。 </li><li>用PBS洗涤细胞两次和然后悬浮在50毫升冷的RPMI上血球计数细胞。典型的复苏是8×10 8 - 1×10 9单核细胞。 </li></ol>此过程可缩减为较小的血液量。在PBS稀释的全血样本是1:1。 2。总的CD4 + T细胞的磁负选择，CD4 + CD45RA +幼稚T细胞或CD4 + CD45RO + +记忆T细胞使用EasySep富集试剂盒的单核细胞（干细胞的技术） 要遵循的步骤为1×10 8〜4.25×10 8单核细胞。 <ol><li>重悬单核细胞的终浓度为5×10 7 PBS中，每毫升含有2％FBS和1mM EDTA。细胞移动到一个新的14 mL聚丙烯圆底管。 <ol clasS ="lalpha"&gt; <li> 总人CD4 + T细胞的阴性选择分离:增加人体的CD4 + T细胞富集抗体鸡尾酒（50μL每毫升的外周血），混合，在室温下孵育10分钟。 </li><li> 人类幼稚T细胞的阴性选择分离:添加50μL抗CD45RO的每毫升外周血单个核细胞悬液，混合抗体，孵育15分钟，在室温下。加入浓缩试剂盒（50μL每毫升外周血），混合，在室温10分钟孵育提供的鸡尾酒。 </li><li> 人类的记忆性T细胞的阴性选择分离:添加人类记忆CD4 + T细胞富集抗体鸡尾酒（50μL每毫升的外周血），混合孵育10分钟，在室温下。 </li></ol></li><li>混合磁性粒子，平均分配在整个解决方案。做不涡纳米粒子，从天真套件。 </li><li>添加磁性颗粒（100μL，每毫升的外周血总CD4 +幼稚T细胞的选择，50μL每毫升的外周血记忆性T细胞的选择）。混合轻轻吹打在室温下的2-3倍，并培育幼稚T细胞的选择10分钟或5分钟内存或总的CD4 + T细胞的选择。 </li><li>添加PBS包含2％胎牛血清和1 mM的EDTA带来量高达每管10毫升。混合轻轻吸取前封顶管总的CD4 + T细胞，幼稚和记忆亚群，分别为5年，10或2.5分钟放入银EasySep磁铁的2-3倍。 </li><li>管仍然在EasySep磁铁，注入新的50 mL试管液体隔离感兴趣的细胞。 </li><li>重复步骤2.5和2.6为更好地恢复。 </li></ol> 3。细胞培养条件诱导调节性T细胞<ol><li>步骤1和2，大衣组织培养板前一天先稀释的抗CD3抗体（OKT3治疗克隆）1微克/毫升无菌PBS浓度。 6孔板，加入2毫升的PBS +每口井的抗CD3，12孔板，加1毫升，或24孔板，每孔加500μL的。保持涂层板在4°C，直到使用。 </li><li>准备加入RPMI-1640前用2.06毫米补充Glutamax-I和25毫米的10％热灭活小牛血清（FBS），100 U / mL青霉素，100μg/ mL链霉素和50μMβ-巯基乙醇介质极化HEPES缓冲。接下来，添加2毫微克/毫升TGF-β和5 ng / ml的IL-2。在这里，人们可以添加媒体感兴趣的配体或抑制剂。悬浮细胞从第2步，在2×10 6细胞/毫升的极化介质的浓度。 </li><li>预暖板在37°C和吸PBS的抗CD3涂井前加入细胞。 6孔板，12孔板或24孔板1毫升细胞2毫升细胞，加入4毫升，每孔细胞悬液。填补井空，以减少水分蒸发，用PBS媒体。在37°C / 5％CO 2孵育3天。 </li><li>第3天后电镀，降速为5分钟离心500×Ğ板。不打扰板底部的T细胞，取出一半的媒体和新媒体取代。另外，如果井变得人满为患与细胞（3×10 6 /毫升以上的浓度），平分到另一个抗CD3涂层板的体积和补充新鲜媒体回升到原来体积。两到三个多天在37°C / 5％CO 2孵育。 </li></ol> 4。荧光激活细胞分选（FACS）T细胞群体<ol><li>准备加入0.5％（W / V）牛血清白蛋白（BSA）和2 mM EDTA的无菌PBS洗涤液的流式细胞仪。地方缓冲区在4°C至冷。 </li><li>删除从细胞培养细胞，每孔2毫升的PBS冲洗，以确保完整的细胞恢复。将50毫升聚丙烯管的所有细胞，离心10 500 XG分钟，在4°C。 </li><li>血球计数细胞，以确定细胞密度。 </li><li>地方3-5×10 5细胞每管四个独立的5毫升圆底聚苯乙烯补偿控制管。将50毫升聚丙烯管，不超过35×10 6个细胞管中剩余的细胞。 </li><li>降速细胞在4°C和抽吸介质为5分钟。悬浮细胞在90μL冷水洗每1×10 6细胞的缓冲区排序。悬浮在100μL冷洗涤液的补偿控制管。 </li><li>对细胞进行排序，结合抗CD25-PE，2μL的反CD45RA的PE-Cy5的3μL，1μL，每1×10 6细胞的抗CD127-APC。没有抗体补偿控制管，2μL抗CD25-PE的第二管，抗CD45RA的3μL - PE-Cy5的第三管AND1μL抗CD127-APC第四管。冰上孵育45分钟，在黑暗中所有的管子。 </li><l&gt;冷洗涤液加入10毫升的细胞进行排序和1毫升的补偿控制管。离心5分钟，所有的细胞在500 XG 4°C。 </li><li>吸在1×10 7细胞每毫升洗涤液浓度的缓冲和重悬细胞。通过40微米尼龙细胞过滤器过滤前添加的DNase II的细胞每毫升1.5μL。将细胞要多5 mL圆底聚丙烯管，每管不超过3.5毫升。补偿控制细胞重悬于300μL洗涤液。 </li><li>补偿设置上MoFlo流式细胞仪的PE，APC和PE-Cy5的过滤器，以尽量减少交叉检测。设置门排序iTregs（CD25表达喜 CD127 -/LOW CD45RA的-细胞）5 mL圆底聚苯乙烯管含有1 mL的小牛血清T细胞，幼稚（CD25-CD45RA的+）和内存（CD25表达- - CD45RA的）。 </li></ol> 5。制止含量<ol><li>使suppr加入100的U / ml青霉素，链霉素100微克/毫升，5毫微克/毫升的IL-2和2毫微克/毫升，TGF-β在AIM-V的分裂国家法媒体。 </li><li>每天的检测，净化异的CD4 + T为细胞在步骤1和2表示从白膜。这些都将抑制试验的靶细胞和不含有CD25 + Treg细胞，可以看到在图2 0日。 </li><li>标签细胞CellTrace套件，为制造商的指示，只用1μL5毫米，每毫升的细胞，而不是2μL，原液除外。直接光了，一小瓶CFSE标记添加18μL二甲基亚砜提供由CellTrace套件做一个5毫米的原液。悬浮在预热的PBS + 0.1％（W / V）牛血清白蛋白的靶细胞所需数量（最高为1×10 7）至终浓度为1×10 6细胞/ ml。加入1μL5毫米每毫升细胞CFSE标记，并在37℃水浴中孵育5 minutES。加入含10％胎牛血清5卷完整的，冰冷的RPMI，解渴染色并在冰上孵育5分钟。洗净用冷水完整的RPMI 1重悬细胞两倍以上×10 5细胞每100μL抑制法媒体。 </li><li> Treg细胞抑制督察珠的股票在2×10 7珠/毫升的浓度。颗粒的珠子数量等于每Eppendorf管中的快速离心实验细胞的总数。一次洗珠用RPMI和再沉淀。的RPMI愿望后，重悬珠，使每口井珠适量8μL抑制法媒体。 </li><li>到96圆底培养板，添加CFSE染色的细胞（1×10 5细胞/ ml），督察珠和新鲜抑制试验介质的极化和排序细胞（1×10 5细胞/ ml）到一个理想的目标（CFSE染色）:在200微升的最终体积效应（排序）的比率。所有条件都在tripli设置盖茨。 </li><li>准备加入100μLCFSE染色的细胞，8μL督察珠和1×10新鲜的，未染色的细胞，每孔92μL抑制试验介质的两个控制条件。与上述相同的细胞成分，但没有调节性T细胞督察珠准备第二控制。 </li><li>覆盖在37°C / 5％CO 2板铝箔，并培育了五天。 </li><li>在黑暗中，从每口井的细胞收集在5 mL圆底聚苯乙烯管的移液器和地点。离心5分钟，在500 XG细胞在4°C时，抽吸介质，并从第4步洗涤缓冲液在300冷流式细胞仪悬浮。分析的第一个3×10 4 CFSE标记+代表靶细 ​​胞与细胞Quest软件的直方图从现场淋巴细胞门事件。 </li></ol> 6。代表结果例如，流式细胞仪伪点缀了为期五天的时间，凑地块RSE的监测iTreg分化的基础上共同表达的CD25与FOXP3的相对，CTLA-4和CD45RA的可以看出，在图2。在图3中的直方图显示了一个成功的抑制实验中排序iTregs（CD25表达喜 CD45RA的-细胞CD127 -/LOW）。为期五天的文化，已获得监管/抑制能力是唯一的一个子集， 图4显示了调节性T细胞诱导从一个天真的T细胞池（顶部面板）和记忆T细胞池（底板）后五天文化标准iTreg介质。 CFSE标记的原始细胞染色表明，无论是天真的（右上面板）或记忆T细胞（右下面板）区分只经过几轮细胞分裂到iTregs（最高Foxp3的表达）。 <img alt="图1" src="/files/ftp_upload/3738/3738fig1.jpg" /> 图1。实验过程示意图。外周血ART细胞-通过梯度离心分离出人外周血前磁+ CD25表达的CD4阴性选择E。 5至6天培养后，细胞进行流式细胞仪，并与异CFSE标记靶细胞共同孵育测量抑制活性。 <img alt="图2" src="/files/ftp_upload/3738/3738fig2.jpg" /> 图2纯化的人主要的CD4 + CD25 - T细胞培养iTreg介质。等份细胞被收集后隔离（0天），并在细胞培养1天，3和5，监察CD45RA的，FOXP3，CTLA-4和CD25标记的进展。 iTreg轮廓对应CD45RA的- ，FOXP3 嗨 ，CTLA-4 高和CD25 HI（在图形窗口中高亮显示）。 <img alt="图3" src="/files/ftp_upload/3738/3738fig3.jpg" /> 纯化的CD4 + CFSE标记拉图3。贝莱德细胞（1×10 5 /孔）培养中存在天真排序，内存或iTreg细胞的调节性T细胞抑制督察珠 （3×10 4 /孔）。五天后，细胞收获和染细胞的流式细胞仪检测CFSE标记的文件。 iTreg细胞的存在，完全废除的CD4 + T细胞的增殖。有数字表明CFSE标记的细胞发生分裂的百分比。 <img alt="图4" src="/files/ftp_upload/3738/3738fig4.jpg" /> 图4纯化的人类主要天真（的CD4 + CD25 - CD45RA +）和内存（的CD4 + CD25 - CD38 +）T细胞被染色CFSE和培养iTreg中等。五天后，细胞phenotyped和细胞分裂率估计。 如图2所示，从两个亚群分化的iTreg子集对应CD45RA的- CD25表达喜 FOXP3 高和CTLA-4 高 （后者未显示）。比较CFSE染色型材iTregs（这里的最高表达Foxp3的T细胞）识别在为期五天的文化最增殖细胞。

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripherally+induced+human+Regulatory+T+cells+uncouple+Kv1.3+activation+from+TCR-associated+signaling.">Peripherally induced human Regulatory T cells uncouple Kv1.3 activation from TCR-associated signaling.</a> European Journal of Immunology. , (2011).</li></ol>

作者没有透露。

虽然拥有巨大的调节性T细胞的转移在打击自身免疫排斥反应和其他免疫或炎症介导的疾病，其高效发电和维护稳定的方法治疗的承诺尚未开发。由于只有1-5％的循环人类T细胞是调节性T细胞，他们控制的扩张和分化克服这种缺乏作为实施调节性T细胞进入临床的主要威慑。另一方面，我们从审判13 TGN1412了解到，这是科学和伦理的必要深入了解分子事件，编排实施任何治疗方案之前，人类T细胞命运的决定。用这种方法iTreg分化，导致人口的天真，内存，效应T细胞和iTregs促进从一个单一的人外周血捐助派生的细胞种群之间的基因表达或功能的比较。在我们的手中，排序细胞已相比，在芯片和QRT-PCR分析来衡量遗传印迹的变化，研究关键的信号事件，或在补丁夹紧功能测量离子通道的使用14。 我们的系统提供了额外的好处的能力来操纵的分化过程中的关键环节，围绕一个中心框架和读数。因此，可以预排序的单核细胞，改变细胞的输入人口或包括小分子抑制剂和配体的培养基。也可以转染细胞过度或击倒感兴趣的蛋白质或介绍记者建造。作为一个额外的好处，我们可以改变培养条件，以最大限度地发挥产生iTregs的百分比。总的来说，这个人类T细胞培养与生理意义远远比在小鼠系统创建描绘了非常可靠的实验平台。重要的是，它也可能维持一个直接的治疗价值。事实上，作为MOST电流的调节性T细胞的治疗方法， 在体外或体外的发展或操纵细胞的参与，我们的系统可以被看作是对列入中Treg细胞疗法的护理标准的道路上的重要渠道。这个系统未来的扩展将裁缝调节性T细胞成为激活后，与具体的 ，而不是描述的多克隆激活体内的炎症抗原相遇。

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> lymphoprep </td><td>轴盾</td><td> 1114547 </td><td>保持室温</td></tr><tr><td>冷冻台式离心机</td><td> Eppendorf公司</td><td> 5810R </td><td></td></tr><tr><td>亮线血球</td><td>豪塞尔科学</td><td> 3110 </td><td></td></tr><tr><td> EasySep人类CD4 + T细胞富集试剂盒</td><td>干细胞技术</td><td> 19052 </td><td></td></tr><tr><td> EasySep人类记忆CD4 + T细胞富集试剂盒</td><td>干细胞技术</td><td> 19157 </td><td></td></tr><tr><td> EasySep人类朴素的CD4 + T细胞富集试剂盒</td><td>干细胞技术</td><td> 19155 </td><td></td></tr><tr><td>大易EasySep MAG净</td><td>干细胞技术</td><td> 18001 </td><td>银</td></tr><tr><td> 5 mL圆底聚苯乙烯管</td><td>屋宇署猎鹰</td><td> 352008 </td><td></td></tr><tr><td> 15 mL聚丙烯离心管</td><td>厂商VWR </td><td> 89004-368 </td><td></td></tr><tr><td> 14 mL聚丙烯圆底管</td><td>屋宇署猎鹰</td><td> 352059 </td><td></td></tr><tr><td> 50 mL聚丙烯离心管</td><td>厂商VWR </td><td> 89004-364 </td><td></td></tr><tr><td>人类抗CD3抗体</td><td>生物X手机</td><td> BE0001-2 </td><td>克隆:OKT3治疗</td></tr><tr><td> 24孔细胞聚苯乙烯培养板</td><td>屋宇署猎鹰</td><td> 353047 </td><td></td></tr><tr><td> 96井圆底培养板</td><td>格雷尼尔生物一</td><td> 650180 </td><td></td></tr><tr><td> Glutamax-I和HEPES缓冲的RPMI 1640 </td><td> Gibco公司</td><td> 72400 </td><td></td></tr> &lt;TR&gt; <td>胎牛血清（FBS）， </td><td> Gibco公司</td><td> 16000 </td><td></td></tr><tr><td> β-巯基乙醇</td><td>西格玛</td><td> M7522 </td><td></td></tr><tr><td>重组人转化生长因子-β1 </td><td> eBioscience </td><td> 14-8348-62 </td><td></td></tr><tr><td>重组人IL-2 </td><td> eBioscience </td><td> 14-8029-63 </td><td></td></tr><tr><td>牛血清白蛋白（BSA） </td><td> MP Biomedicals公司</td><td> 810531 </td><td></td></tr><tr><td> 0.5 M的EDTA </td><td> AMRESCO </td><td> E177 </td><td></td></tr><tr><td>鼠抗人CD25 - PE </td><td>美天旎生物技术</td><td> 130-091-024 </td><td>克隆:4E3 </td></tr><tr><td>鼠抗人CD45RA的PE-Cy5的</td><td> eBioscience </td><td> 15-0458-42 </td><td>克隆:HI100 </td></tr><tr><td>鼠抗人CD127-APC </td><td>美天旎生物技术</td><td> 130-094-890 </td><td>克隆:MB15-18C9 </td></tr><tr><td> DNA酶二</td><td>中号带够Biomedicals公司</td><td> 190370 </td><td></td></tr><tr><td> MoFlo流式细胞仪</td><td>贝克曼</td><td></td><td></td></tr><tr><td> FlowJo软件</td><td>树星公司</td><td></td><td></td></tr><tr><td>细胞任务Pro软件</td><td> BD公司</td><td></td><td></td></tr><tr><td>新生牛血清</td><td> Gibco公司</td><td> 16010 </td><td></td></tr><tr><td> L-谷氨酰胺</td><td> Gibco公司</td><td> 25030 </td><td></td></tr><tr><td> AIM-V的</td><td> Gibco公司</td><td> 0870112 </td><td></td></tr><tr><td> CellTrace CFSE标记细胞增殖试剂盒</td><td> Invitrogen公司</td><td> C34554 </td><td></td></tr><tr><td> Treg细胞抑制督察珠</td><td>美天旎生物技术</td><td> 130-092-909 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Gibco公司</td><td> 15140 </td><td></td></tr><tr><td> 40微米尼龙细胞株呃</td><td>屋宇署猎鹰</td><td> 352340 </td><td></td></tr></table>

generation of induced regulatory t cells from primary human na&iuml;ve and memory t cells

免疫的CD4 +调节性T细胞（Tregs）的开发和维护，促进周边的宽容，需要保持大量对人体的自我和共生抗原在免疫稳态。调节性T细胞和炎症常规T细胞之间的平衡扰动可导致免疫病理或癌症。虽然已被证明是4几个根本的分歧在人类与小鼠的调节性T细胞生物学5 1，I型糖尿病，类风湿关节炎和移植物抗宿主病，结肠炎的小鼠模型中的有效调节性T细胞治疗注射迄今排除了临床使用。由于缺乏足够数量，纯度，稳定性和特异性治疗调节性T细胞归巢的需要Treg的人类发展的动力平台上，以优化条件下体外扩增 6。 在这里，我们倒是描述一个从一个单一的人外周血献血，其中可分为四个阶段的分化诱导调节性T细胞（iTregs）分离外周血单个核细胞，CD4 + T细胞磁性选择的方法: 在体外细胞培养和荧光激活T细胞亚群细胞分选（FACS）。由于Treg的签名转录因子叉头框P3（FOXP3）是人类7激活诱导的转录因子，并没有其他存在独特的标记，标记的组合面板必须用于识别与抑制活性的T细胞。 6天培养后，细胞在我们的系统可以被划分成幼稚T细胞，记忆T细胞或基于其相对表达CD25，CD45RA的iTregs。由于内存和幼稚T细胞有不同的极化要求和可塑性8，牛逼成CD45RA +和CD45RO细胞人口的初始预分拣+亚群可以E中检查这些差异。与他人一致，CD25表达喜 CD45RA的- iTregs快递高水平的FOXP3 9，GITR和CTLA-4的第11和CD127 12的水平低。按照每个人口的流式细胞仪，由此产生的细胞可用于在评估相对延缓羧基琥珀酰亚胺酯（CFSE）标记的自体T细胞增殖能力的抑制实验。

Watch this Scientific Journal Video about Generation of Induced Regulatory T Cells from Primary Human NaÃƒÂ¯ve and Memory T Cells at JoVE.com

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

我们描述了从一个单一的人献血的监管，内存和幼稚T细胞产生的一种方法。极化调节性T细胞可以比较多种遗传同质性，这里还详细介绍了包括抑制法与遗传和功能应用中的其他子集。

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 ...

generation-induced-regulatory-t-cells-from-primary-human-nave-memory

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

23.9K 次观看.  University of Kentucky. 我们描述了从一个单一的人献血的监管，内存和幼稚T细胞产生的一种方法。极化调节性T细胞可以比较多种遗传同质性，这里还详细介绍了包括抑制法与遗传和功能应用中的其他子集。

视频: Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells

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

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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 ...

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 ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

表型和慢性淋巴细胞性脉络丛脑膜炎病毒感染小鼠中分离激活调节性T细胞的功能分析

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

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

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

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

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In 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.

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 ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

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

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