笔者想感谢杜利瑞建设pCAGAdDU载体和奥利弗·戈尔卡提供的固定协议。

1。感兴趣的基因的克隆到入门载体<ol><li>感兴趣的基因的克隆到一个条目载体。 PCR扩增的基因，然后通过平末端连接到一个拓扑异构酶耦合向量（ 例如载体pENTR / D-TOPO）或限制性内切酶介导的克隆可以用于此过程。 </li></ol> 2。感兴趣的基因转移到的pCAGAdDu目标向量<ol><li>从输入矢量的兴趣基因转移到目标向量由LR重组（ 例如网关LR克隆酶II酶混合物）。这将创建的腺病毒表达载体（图1）。 </li><li> 10微克线性化的腺病毒表达载体中的PacI位限制性内切酶消化，沉淀DNA，并重新悬浮在水中的浓度为3微克每100微升。线性化会释放病毒的反向重复序列（ITR），复制和包壳的V所必需的到病毒颗粒中的iral DNA。 </li></ol> 3。生成的主要病毒裂解液<ol><li>种子1×10 5 HEK293A细胞在2ml的细胞培养基（DMEM，10％胎牛血清，5％PenStrep）以及一个6孔板中于室温孵育6 - 14小时，在37℃下在10％的CO 2培养箱，让他们坚持。然后，细胞应在约50％汇合。 </li><li>脂质体：转让6微升jetPEI转染试剂50μM的氯化钠，涡简要成94微升。的混合溶液添加至100μl，线性化的腺病毒载体在涡旋和15 - 30分钟，在室温下孵育当前转染混合物。 </li></ol>执行以下步骤，在适当的生物安全条件，腺病毒感染！ <ol start="3"><li> HEK293A细胞以及含有分配溶液的溶液，并于室温孵育在37℃和10％的CO 2。当使用荧光标记，评估transfe的CTION效率视觉后12 - 36小时，使用倒置荧光显微镜。添加0.5毫升的新鲜培养基，每3天。 </li><li>细胞病变效应（CPE），这是放大和圆润的细胞开始分离领域具有光或荧光显微镜检查，每2 - 3天。这是表示高效的病毒的产生。一旦发生更广泛的区域CPE（图3），它会需要24 - 72小时之前，所有的细胞被感染。 </li><li>当所有的细胞出现CPE，但前整体支队细胞时，细胞分离轻柔吹打和转让细胞上清（SN，3 - 5毫升），15毫升聚苯乙烯管。 </li><li>冻结细胞在干冰上15 - 20分钟的SN和快速解冻，在37℃后，破裂的细胞。重复冷冻和解冻周期（F / TC）两次以上。保持主病毒裂解液在冰上，在一天之内的使用，或冻结，在-80℃下长期存储。任何的其它F/ TC会降低病毒滴度30 - 50％。 </li></ol> 4。病毒扩增<ol><li>种子和HEK293A细胞成长14厘米的组织培养皿90％汇合。 </li><li>感染细胞的主要病毒裂解液（1.5 - 2.5毫升）的一半，室温孵育至少36小时。几乎所有的细胞被感染，然后，通过荧光显微镜观察，可确定。对于已经放大（ 即较为集中）的病毒库存再度放大，感染HEK293A感染复数（MOI）为50（见6.3）。 </li><li>当所有的人都显示CPE，但支队前，应收获细胞。高效的病毒产量达到这种状态是在感染后48小时内。 （如果需要长达一个星期，考虑另一轮扩增通过增加用于4.2中描述感染腺病毒库存量。） </li><li>轻柔吹打和转移细胞和SN 50毫升聚苯乙烯管分离细胞。自旋细胞以300×g，10分钟，在4℃下</li><li>取出的SN和悬浮颗粒介质或SN（ 约 1毫升）在合适的音量。 </li><li>执行3 F / TC破碎细胞并在4℃下以800×g离心15分钟脱掉的SN包含该病毒颗粒（ 即浓缩的病毒溶胞产物），和病毒裂解物等分将其存储于-80℃。 </li></ol> 5。病毒滴度测定<ol><li>种子10 5 A549细胞，每孔为5个孔的12孔板，1毫升培养基，让细胞附着6小时。 </li><li>使用1微升浓腺病毒（冰解冻）进行连续稀释介质（1:5000 1:10000 1:50000 1:100000），每加10微升。留一井未受感染的调整浇注流式细胞。 </li><li> 36小时后，脱下的SN，用PBS及分离细胞（ 如胰蛋白酶）。对于生物危害的预防措施，建议修复C在100μl的PBS中的4％多聚甲醛在室温和10分钟，用PBS洗涤一次，细胞l。 </li><li>执行一个感染标记表达的流式细胞仪分析。剧情μl病毒裂解应用于对感染的细胞的绝对数量（图4）。确定感染的线性范围内，并从标准曲线中计算出每毫升未稀释的病毒滴度在用x = 1,000微升的线性范围。 </li></ol> 6。 T细胞病毒感染<ol><li>隔离天真/静息CD4 + T细胞DO11.10 TG，TGCARΔ1小鼠使用MACS（幼稚的CD4 + T细胞分离试剂盒II）或FACS分类（CD4 + CD25-CD62L + CD44-）。 </li><li>对于小规模的实验中，吸液管适当体积的病毒的溶胞产物，实现到一个良好的96孔圆底板的MOI为50。 </li><li>最多可添加4×10 5个T细胞，在最终体积为50μl感染T细胞的培养基（RPMI1640，10％胎牛血清，5％PenStrep，5％NaPyruvate，1×NEAA，1个MEM本质维生素，1个L-谷氨酰胺，1:25万巯基乙醇，10毫米HEPES）。 例如： MOI为50，应使用3×10 5 T细胞感染; 病毒滴度为3×10 9毫升-1 病毒量= MOI x厚的细胞数/病毒滴度= 50×（3×10 5）/（3×10 9毫升-1）=0.005毫升</li></ol> 注：较大的细胞数量，规模达宽松杯（可达3毫升，每管）165微升每10 6幼稚T细胞的感染量在聚苯乙烯管使用MOI为50的感染。 <ol start="4"><li> ，在5％CO 2培养箱中 ，在37℃下孵育90分钟。 </li><li>降速细胞在300 XG在室温下5分钟，起飞SN，在200μLPBS重悬。再次离心，脱下SN。 </li></ol> （可选：细胞可能被再次洗涤更有效地清除病毒） <ol start="6"><li>悬浮细胞加入200μlT细胞介质无刺激抗体和无IL-2或其它细胞因子，休息40小时，在37℃下，在5％CO 2培养箱中 ，以允许激活之前的感兴趣的基因的表达。 </li></ol> 7。 T细胞活化和极化<ol><li>移液管取一定体积的抗-CD3和抗CD28耦合等于成一个小的试剂杯的细胞数（ 例如，4×10 5）的有孔玻璃珠，加入10倍体积的PBS，并把它们放在一个磁铁2分钟。起飞上清，重悬珠200微升偏光介质（调节性T细胞培养基+ 1 ng / ml的TGF-β，100 U / ml的IL-2）。 </li></ol> 注意：使用涂有抗CD28和抗CD3抗体的组织培养皿的细胞，也可以激活，或者在一个DO11.10 T细胞受体特异性的方式，使用脉冲照射的BALB / c小鼠的脾细胞与卵清蛋白的323-339肽抗原。 <ol start="2"><li>离心机e为休息的细胞和以前一样，取下SN和重悬细胞，在200微升的偏振介质中含有抗-CD3和抗CD28抗体偶联有孔玻璃珠。不改变培养基，在5％CO 2培养箱中在37℃下孵育72小时。 </li></ol> 8。 T细胞固定和染色流式细胞仪<ol><li>洗涤细胞：细胞离心5分钟，在室温下在300 XG，脱下SN，在200μLPBS重悬。再次离心，脱下SN。执行以下相应的洗涤步骤。 </li><li>在100μl可固定死细胞染色溶液重悬细胞，并温育30分钟，在4℃。 </li><li>洗涤细胞，重新悬浮于100μlPBS中，加入100微升4％多聚甲醛的PBS中孵育15分钟，在RT。 </li><li>洗涤细胞，重悬在200μl冰冷的70％甲醇的PBS，并在冰上孵育30分钟。 </li></ol> 注：细胞可以治疗无生物危害的预防措施，从现在开始！<ol start="5"><li>准备60μL主混合60微升PBS + 10微克/毫升块（FC-抗FCR3阻断非特异性结合）。洗涤细胞，重悬在40μl的PBS +抗FCR3，并在室温孵育15分钟。 </li><li>加入20μlPBS +抗FCR3含有1微克PE耦合抗Foxp3抗体，拌匀和孵化，在4℃过夜。 </li><li>在PBS中洗涤细胞两次，流式细胞仪分析细胞。 </li></ol>

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作者宣称没有利益冲突。

病毒生成和滴定为了获得最佳的转染结果，线性化载体的质量和数量的出现最重要的。我们并没有观察到初级裂解生产的负面影响，从最初的文化繁茂，因为感染会迅速进行一次高效的病毒生产发生。然而，HEK293A细胞的病毒产量可受长的插入效率下降。一些的开放阅读框架，实际上发现干扰病毒生产。这些病毒的股票通常可以通过几轮的扩增获救，只有少数的开放阅读框被认?...

调节性T细胞是维持免疫耐受，抑制过度的免疫反应的关键。调节性T细胞抑制T细胞活化的旁观者。因此，调节性T细胞的消融导致致命的自身免疫和自我毁灭的驱动由活化的T细胞1。调节性T细胞在胸腺中发展CD4单阳性前体的阴性选择过程中，但他们也可以从幼稚后，低剂量的抗原刺激的CD4 + T细胞具有次优的协同刺激1,2分化的周围。胸腺调节性T细胞似乎抑制组织对自身抗原的自身免疫性疾病，而周边有牵连的调节性T细胞在肠道或肺部提供宽容。这些诱导Tregs的有效阻止T细胞活化后在粘膜外来抗原的识别，包括环境抗原的食物和空气，共生细菌，过敏原3,4。此外，调节性T细胞是关键，建立孕产妇胎儿肽的耐受性和预先发泄移植物抗宿主病。与此同时，调节性T细胞介导不良影响肿瘤细胞的免疫监视7,8衰减。调节性T细胞的共同特征，是指定的子集转录因子Foxp3，叉头域的转录因子，是必要的，足以赋予Treg细胞功能9,10的表达。一些信号转导通路，可诱导Foxp3的表达是众所周知的。然而，分子的过程控制，调节，或调节调节性T细胞分化的T细胞受体触发的了解较少。 调节性T细胞可以非常有效地在体外诱导幼稚的CD4 + T细胞与抗-CD3和抗CD28抗体的存在下，TGF-β和IL-2 11通过刺激。由于新兴的调节性T细胞在体内的功能，促进调节性T细胞分化的分子操纵承担巨大的潜力，未来的therapi上课，例如，治疗哮喘，克罗恩病，移植11,12。相反，治疗调制阻止Treg细胞分化的分子可能在未来的肿瘤患者的综合治疗方法中受益。 一直在体外诱导分化实验的描述都与T细胞亚群分化的分子变化。在此刻，实验尝试搜索或基因产物的屏幕控制T细胞分化异位基因表达的最常用的方法失败，在幼稚T细胞的事实而受到阻碍。例如，电穿孔，逆转录病毒转导是唯一有效的活化的T细胞中。与此相反的最初预期的慢病毒转导，这是通常在静息细胞中有效，需要预活化幼稚T细胞的细胞因子是13。此外，在电穿孔过程中的cDNA或mRNA的转移涉及质膜的去极化，其本身赋予T细胞活化的功能，甚至可能动员的Ca 2 +信号 并且激活NFAT蛋白（未发表的观察和参考文献14）。类似地，对于逆转录病毒转导，幼稚T细胞被激活为18 - 40小时。在这段时间内，在细胞分裂过程中，核膜破裂的发生，并允许为随后的基因组整合的逆转录病毒载体15。因此，这些方法是不能够解决早期的分子调控初始T细胞与抗原相遇，这是辅助性T细胞分化的决定性阶段。 腺病毒转导是已知的，赋予短暂异位基因表达的人类细胞类型中表达的人柯萨奇腺病毒受体（CAR）的一些。进行没有要求细胞活化或细胞周期进程。表面表达的CAR是必不可少的有效的病毒附件和内在，发现转基因表达的截断版本CARΔ1的T细胞特异性启动子下使小鼠胸腺细胞和T细胞受到腺病毒感染16。重要的是，转基因不改变胸腺细胞发育或在体外分化幼稚的CD4 T细胞分成不同的子集（数据未显示;文献17）。以前使用的腺病毒介导的转导T细胞过表达17,18和击倒方法19,20。的转基因T细胞可被纯化从市售的DO11.10的TG;：CARΔ1的TG（Taconic公司，Inc。和参考文献17）。重要的是，腺病毒转导允许在幼稚T细胞中感兴趣的基因的高表达而诱导激活的明显迹象。 T细胞保持天真（CD44 低 ，CD62L 高 ），休息（CD25 - ，CD69 - ）后infecti和可以被激活和调节性T细胞分化成类似的非感染的细胞。 腺病毒介导的生产可以实现HEK293A细胞转染后，腺病毒载体（图1）。这些质粒通常含有E1和E3基因删除，以使重组病毒无法复制的21人5型腺病毒基因组。 HEK293A细胞补充不足，因为他们已经通过稳定整合剪切腺病毒22永生复制。由于腺病毒载体是大的（〜40 kb的），因此不适合于传统的限制性内切酶介导的克隆，我们采用的网关系统。感兴趣的基因的最初克隆到一个较小的输入矢量，从它可以很容易地转移到腺病毒的目标向量通过拉姆达重组反应（LR）23。我们构建了pCAGAdDu载体相结合的的CAG启动（鸡肌动蛋白启动子和CMV增强）的表达盒含有LR网站侧翼原核CCDB选择标记24。此表达盒的一个内部核糖体进入位点（IRES）元件，允许共表达真核感染标记增强型绿色荧光蛋白（EGFP），这是含有牛生长激素多聚（A）信号序列融合到融合。我们选择的CAG顺式调控序列，由于原型的CMV启动子被认为是高度活化相关的，因此不利于在幼稚T细胞的基因表达。 在这里，我们提供了一个协议，用于高效在体外调节性T细胞的分化和幼稚的CD4 T细胞转导方法，无需激活（图2）。该方法使异位基因表达或击倒前面的CD4 T细胞分化的幼稚状态。它允许测试一个overexpresse的效果d基因的兴趣在早期信号事件在初始TCR刺激，直到T细胞亚群的承诺。我们的验证实验也提供了基础，建立类似的腺病毒应用程序在其他T细胞亚群，如Th1细胞和Th2，TH9，Th17细胞，TH22，或TFH细胞的分化。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>pENTR/D-TOPO Cloning Kit</td>
			<td>Invitrogen</td>
			<td>K240020</td>
		</tr>
		<tr>
			<td>Gateway LR Clonase II Enzyme mix</td>
			<td>Invitrogen</td>
			<td>11791020</td>
		</tr>
		<tr>
			<td>PacI</td>
			<td>New England Biolabs</td>
			<td>R057S</td>
		</tr>
		<tr>
			<td>jetPEI</td>
			<td>Polyplus-transfection</td>
			<td>101-10N</td>
		</tr>
		<tr>
			<td>HEK293A Cell Line</td>
			<td>Invitrogen</td>
			<td>R705-07</td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td>CCL-185</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>BD Falcon</td>
			<td>353046</td>
		</tr>
		<tr>
			<td>14 cm tissue culture dish</td>
			<td>Nunc</td>
			<td>168381</td>
		</tr>
		<tr>
			<td>BALB/cJ-Tg(DO11.10)10Dlo Tg(CAR&Delta;1)1Jdgr</td>
			<td>Taconic Farms</td>
			<td>Model Nr. 4285</td>
		</tr>
		<tr>
			<td>Naive CD4+ T Cell Isolation Kit II</td>
			<td>Miltenyi Biotech</td>
			<td>130-094-131</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41966-052</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>PAN Biotech</td>
			<td>1502-P110704</td>
		</tr>
		<tr>
			<td>PenStrep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Lonza</td>
			<td>BE12-167F</td>
		</tr>
		<tr>
			<td>NaPyruvate</td>
			<td>Lonza</td>
			<td>BE13-115E</td>
		</tr>
		<tr>
			<td>NEAA 100x</td>
			<td>Lonza</td>
			<td>BE13-114E</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution (1 M)</td>
			<td>Invitrogen</td>
			<td>15630-056</td>
		</tr>
		<tr>
			<td>&beta;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M-7522</td>
		</tr>
		<tr>
			<td>MEM Essential vitamin mixture (100x)</td>
			<td>Lonza</td>
			<td>13-607C</td>
		</tr>
		<tr>
			<td>Dynabeads Mouse T-Activator CD3/CD28 for Cell Expansion and Activation</td>
			<td>Invitrogen</td>
			<td>114-56D</td>
		</tr>
		<tr>
			<td>Recombinant Human TGF-beta 1</td>
			<td>R&amp;D Systems</td>
			<td>240B</td>
		</tr>
		<tr>
			<td>Proleukin S (18 x 106IE)</td>
			<td>Novartis</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit</td>
			<td>Invitrogen</td>
			<td>L-23105</td>
		</tr>
		<tr>
			<td>Mouse BD Fc BlockT</td>
			<td>BD Pharmingen</td>
			<td>553141</td>
		</tr>
		<tr>
			<td>Anti-Mouse/Rat Foxp3 PE</td>
			<td>eBioscience</td>
			<td>12-5773-82</td>
		</tr>
		<tr>
			<td>Mmu-miR-155 TaqMan MicroRNA Assay</td>
			<td>Roche Applied Biosystems</td>
			<td>4427975</td>
		</tr>
		<tr>
			<td>LightCycler 480 Probes Master</td>
			<td>Roche Applied Biosystems</td>
			<td>04902343001</td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit</td>
			<td>Roche Applied Biosystems</td>
			<td>4366596</td>
		</tr>
	</tbody>
</table>

adenoviral transduction of naive cd4 t cells to study treg differentiation

调节性T细胞（Treg细胞）是必不可少的，提供自我以及某些外来抗原的免疫耐受。调节性T细胞可产生从幼稚的CD4 T细胞在体外与T细胞受体和TGF-β和IL-2的存在下的共刺激。承担巨大的潜力，未来的治疗，但是，分子和信号通路，控制分化在很大程度上是未知。 原发性T细胞可以通过操纵异位基因表达，但常用的方法没有针对前最重要的幼稚状态的T细胞抗原识别初级。在这里，我们提供了一个协议异位基因表达在幼稚的CD4 T细胞在体外诱导调节性T细胞的分化之前。它适用于复制缺陷型腺病毒转导和解释它的产生和生产。腺病毒可以占用大量插入（最多7 KB），并可以配备促销员实现高瞬态曝光过度在T细胞ression。它有效传导天真的小鼠T细胞，如果他们表达的转基因柯萨奇腺病毒受体（CAR）。重要的是，感染后T细胞仍然幼稚（CD44，CD62L 的高点 ）和休息（CD25 - CD69 - ），并且可以被激活和调节性T细胞分化成类似的非感染的细胞。因此，该方法从一开始使CD4 + T细胞分化的操纵。确保异位基因表达已经到位，当早期信号事件的初始TCR刺激诱导细胞的变化，最终导致Treg细胞分化。

病毒生产新一代的病毒滴度高，在初级生产病毒或病毒扩增HEK293A细胞收获的时机是至关重要的。代表荧光和相位与病毒生产的视觉迹象的对比度的图像如图3所示。 CPE观察转染的HEK293A细胞与控制pCAGAdDu向量没有插入后10天。 CPE的特点是外观方面扩大和圆形细胞开始分离，并表现出高表达的感染标记GFP。 CPE的程度是表示高效的病毒的产生。细胞培养板，显示类似CPE可...

Watch this Scientific Journal Video about Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation at JoVE.com

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

腺病毒基因转移到幼稚的CD4 T细胞与柯萨奇腺病毒受体的转基因表达，使调节性T细胞分化的分子分析<em&gt;在体外</em&gt;。

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 ...

adenoviral-transduction-naive-cd4-t-cells-to-study-treg

Research

JoVE Journal

Immunology and Infection

15.7K 次观看.  Helmholtz Zentrum München. 腺病毒基因转移到幼稚的CD4 T细胞与柯萨奇腺病毒受体的转基因表达，使调节性T细胞分化的分子分析在体外。

视频: Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

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

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 ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

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

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

骨髓祖细胞的逆转录病毒转导到生成T细胞受体Retrogenic小鼠

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

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

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

辅助性T细胞作为一种遗传方法来研究逆转录病毒转导机制控制其分化和功能

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

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 ...

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played by T cells in an immune response. This protocol describes the in vitro differentiation of bone marrow (BM) progenitors to obtain granulocyte macrophage colony-stimulating factor (GM-CSF) derived-dendritic cells (DCs). The protocol also describes the adoptive transfer of ovalbumin peptide (OVAp)-loaded GM-CSF-derived DCs and na&#239;ve CD4 T cells from OTII transgenic mice in order to analyze the in vivo activation, proliferation, and Th1 differentiation of the transferred CD4 T cells. This protocol circumvents the limitation of purely in vivo methods imposed by the inability to specifically manipulate or select the studied cell population. Moreover, this protocol allows studies in an in vivo environment, thus avoiding alterations to functional factors that may occur in vitro and including the influence of cell types and other factors only found in intact organs. The protocol is a useful tool for generating changes in DCs and T cells that modify adaptive immune responses, potentially providing important results to understand the origin or development of numerous immune associated diseases.

An &lt;em&gt;In Vivo&lt;/em&gt; Mouse Model to Measure Na&amp;#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

Here, we present a protocol for the in vivo determination of na&#239;ve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

小鼠骨髓基质树突状细胞诱导 CD4 T 细胞活化、增殖和 Th1 分化的体外模型研究

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

破译急性 LCMV 感染中的早期滤泡辅助性 T 细胞定型术

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

作者聚焦：阐明急性 LCMV 挑战中 TFH 细胞分化的途径

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

在急性 LCMV 感染小鼠中获得病毒特异性滤泡辅助性 CD4 + T 细胞的早期分化

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, ...