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为是不相容的病毒生产。外观CPE放大病毒感染后48小时内从我们的经验，通常会产生约1×10 9 - 5×10 10的传染性颗粒/毫升病毒裂解。 幼稚T细胞的腺病毒感染腺病毒感染的幼稚T细胞，T细胞的激活状态不会改变之前和之后的刺激。这使得传导系统从最初的TCR刺激T细胞分化研究的一个强大的实验工具。当细胞仍然是幼稚的，并没有显示激活的迹象，我们已经确定了有效的异位表达感兴趣的基因，在感染后40小时的T细胞，在一个时间的&#39;休息&#39;。这意味着随后的T细胞的活化过程中，感兴趣的基因已经过表达和从一开始就可以发挥其T细胞分化的影响。因此，腺病毒转导过电或逆转录病毒转导需要激活和利益表达的基因只能在初次TCR信号15,28具有明显的优势。但是，如果后期分化方面进行分析和感兴趣的基因的表达不需要前初始T细胞活性振动性，幼稚T细胞可能被直接刺激后，腺病毒转导。由于腺病毒的感染是非常有效的，它可以扩展到执行双重感染与表达其他感染的标记，如Thy1.1的或hCD2的腺病毒的两个感兴趣的基因的研究合作活动。后者是抗体表面染色，也可在固定过程中可能绕过标记保存困难。然而，在活化的T细胞，腺病毒感染具有低得多的效率，电穿孔或逆转录病毒转导的方法可能是有利的。腺病毒应用程序的另一个需要注意的是短暂的性格表达。腺病毒作为一种附加体的细胞的核基质附着保持，将稀释的T细胞增殖，从而导致损失的标记基因的表达后五至七天内T细胞活化（数据未显示;文献17）。另外，腺病毒载体转导的细胞readilÝ确认并消除由一个完整的免疫系统， 在体内 ，这限制了它的使用，例如，在小鼠的T细胞过继转移实验。腺病毒系统可能是另一个潜在的应用DO11.10 TG隔离Treg细胞的感染; TGCARΔ1只研究调节性T细胞的功能或沿袭稳定性方面。 验证腺病毒感染细胞的影响腺病毒感染是有效的，几乎没有影响T细胞的活力和调节性T细胞的分化。 MOI为50，我们得到了最好的感染效率没有观察腺病毒感染的T细胞的不利影响。这同样成立，每当系统被施加到其他的细胞培养或分化条件下，一般是通过未经感兴趣的基因的表达，使用空的腺病毒的滴定实验。如果这样的腺病毒感染不影响实验的结果，计息空腺病毒腺病毒基因仍是比较适当揭露利益转移基因的效果。如果不存在的腺病毒感染的影响，所测得的参数也可以在未感染和感染的细胞之间，对同一样品进行比较。 数据分析这里描述的固定方法的分化标志物Foxp3的允许同时分析GFP感染标记，所以可确定一个"相对分化"。这两个群体分析一个样品内有较高的灵敏度比散装人口比较两个样本之间的微妙的影响，因为它需要考虑的小康以及变化。然而，只有固有的感染的细胞的影响将被确认通过该方法，而全效果， 例如分泌的因素所施加的影响，将忽略不计。应该控制在每个相对的分化实验包括控制感染的细胞分化排除感染本身的影响。为了达到最佳效果，一个样本未受感染的细胞内感染几乎相等率应为目的，而感染率较高有利于散装几个样品之间的比较。 总之，使用腺病毒结合体外分化协议的幼稚T细胞的基因转移到探讨Treg细胞分化的分子基础是一个强大的系统。这可允许创造知识，利用所赋予的调节性T细胞在过敏性和自身免疫性疾病的治疗方法对占主导地位的耐受性。

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可收获在24 - 72小时，可以生成一个原始的病毒溶胞产物通过冷冻和解冻循环。系列稀释的病毒裂解物用等MOI（图4），以允许比较感染的病毒滴度可以被确定。的线性范围内，吨他的回归分析方程可以被用来计算每毫升未稀释的病毒裂解物感染性颗粒的数量。在该示例中，在x = 1微升的滴度是30016535微升-1≈3×10 10毫升-1。 腺病毒感染对T细胞的活化和分化的影响评估使用的腺病毒只表示IRES-GFP（图5a）的控制向量组MOI增加感染幼稚的CD4 T细胞对靶细胞的腺病毒转导的影响已被评估。感染效率测定用流式细胞仪分析确定GFP阳性细胞在固定T细胞样品后72小时诱导的Treg细胞分化。 GFP阳性细胞率增加有关教学语言使用的感染，并达到84％以上的感染MOI为50。我们未能取得更高的使用时MOI感染的T细胞的百分比大于50（数据未显示）。尽管INFECTION效率可能会发生变化，取决于感兴趣的基因，通常可以在MOI为50感染的50％以上的细胞。在1和50之间的组MOI细胞存活率没有影响（图5b）。重要的是，幼稚T细胞分化成调节性T细胞的刺激和非感染的细胞（图5c）相比，在被感染的相似。腺病毒转导的一个关键特性是它能够感染幼稚和休息不赋予或需要T细胞活化的T细胞。从分析的标记（CD25，CD69，CD44）的T细胞的活化和样品中的幼稚T细胞（CD62L）的标记带或不带腺病毒感染，休息40小时，在37℃下在5％CO，这是显而易见的2培养箱中 （图6）。的感染和未感染的T细胞之前， 在体外 T细胞的活化与抗-CD3和抗CD28结合了有孔玻璃珠，同样低的 CD25，CD69和CD44 低升:嗷嗷但CD62L 高 ，展示他们的天真和静止状态（图6，上板）。 T细胞活化后，在感染的细胞显示上调活化标志物CD25，CD69和CD62L的下调几乎没有区别未感染的细胞（图6中，下面板）。因此，T细胞的激活状态是没有改变的腺病毒感染。休止期相关联的损失的60〜70％的细胞生长因子或细胞因子的情况下，比10 - 20％的死细胞激活后40小时不休息。这是考虑到当选择的初始细胞数为3×10 5细胞。 感兴趣的基因的高表达水平评估为了评估量实现由腺病毒基因转移的过度表达，我们试图确定的microRNA-155（miR-155的）幼稚T细胞的表达水平，我们重新感染的miR-155表达或控制腺病毒不感染。我们选择了miR-155的，因为这个成熟的miRNA表达幼稚T细胞的中等水平，成为强烈诱导T细胞活化后。此外，它具有至关重要的作用，T细胞依赖的体液免疫反应25,26。 评估水平高表达的microRNA qPCR的。细胞感染后，休息40小时，40小时激活，或者激活40小时休息40小时。经过40小时的休息，miR-155的编码病毒感染的幼稚T细胞显示了约17倍的过表达miR-155的相比，与对照病毒感染的细胞或者非感染的细胞中（图7）。这几乎匹配的程度内源性miR-155的诱导T细胞活化后（图7和图 25,27）。不管这种强烈的诱导内源性miR-155的水平，细胞感染与MIR-155腺病毒仍然显示激活时表达的miR-155在约四倍的增加相比，控制病毒感染或非感染细胞。异位过度的观测水平，是其他小分子RNA（数据未显示）相媲美。请注意，大刀片，过度表达可能不太有效。 分析T细胞分化可以通过几种方式执行数据分析。分化分析中感兴趣的基因（ 即在受感染的细胞）分化在相同的的GFP 阳性栅极（使用感兴趣的基因的载体没有感染的细胞）的对照细胞相比，GFP 阳性的栅极是足以检测到显着的差异。感兴趣的基因来研究更微妙的影响，我们比较了与非感染的细胞中，从相同的（图8）以及在感染的细胞的分化。这些作为内部控制升，并可以被用来计算"相对分化以及内部。如果病毒具有分化没有影响，"相对分化"，因此理想的是1。相对分化为控制病毒在我们手中，有0.9至1.1的范围。相对分化应该只适用于基因息感染的样本，以控制病毒感染的样本进行比较。 <img alt="图1" fo:content-width="6.5in" fo:src="/files/ftp_upload/50455/50455fig1highres.jpg" src="/files/ftp_upload/50455/50455fig1.jpg" /> 图1。腺病毒生成的示意图。入学载体（载体pENTR）包含重组捐助的网站（AttL1 AttL2），侧翼的基因利益。的的目标向量pCAGAdDu包含重组的目标网站（attr1的AttR2）CCDB毒素基因在大肠杆菌中的阴性选择侧翼大肠杆菌 ，感兴趣的基因，它将被替换通过LR复合。的目标向量还包含一个人5型腺病毒基因组没有E1/E3基因，产生复制缺陷型腺病毒已被删除。PacI位线性化释放反转重复序列（ITR）而承受的腺病毒基因，以产生感染性的HEK293A细胞转染前腺病毒颗粒，导致细胞病变效应。腺病毒是收获生产细胞的冷冻与解冻周期。 <a href="https://www.jove.com/files/ftp_upload/50455/50455fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50455/50455fig2highres.jpg" src="/files/ftp_upload/50455/50455fig2.jpg" /> 图2。示意性表示T细胞的感染和分化的幼稚T细胞感染腺病毒裂解液1.5小时，用PB洗涤<html> S和T细胞的培养基，在5％CO 2培养箱中在37℃下40小时休息。然后将细胞激活72小时，在TGF-β和IL-2的存在下使用抗CD28和抗CD​​3抗体偶联有孔玻璃珠。固定和染色细胞，用流式细胞仪进行分析，并测定在感染与非感染细胞中Foxp3表达。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50455/50455fig3highres.jpg" src="/files/ftp_upload/50455/50455fig3.jpg" /> 图3。 HEK293A细胞腺病毒。CPE为10 5 3纳克的线性pCAGAdDu矢量HEK293A细胞转染后10天发生细胞病变效应 。左边的面板显示相衬图像，右侧面板显示的绿色荧光。 55/50455fig4highres.jpg"SRC ="/ files/ftp_upload/50455/50455fig4.jpg"/&gt; 图4。确定滴度腺病毒裂解物感染的A549细胞，感染10 5 A549细胞用所示的病毒稀释度，孵育48小时，感染标志物的表达GFP通过流式细胞术进行分析。 GFP 阳性细胞的数目是对使用的病毒量作图。计算从标准曲线上的线性范围内，用x = 1,000微升未稀释的病毒的滴度。 <img alt="图5" fo:content-width="6in" fo:src="/files/ftp_upload/50455/50455fig5highres.jpg" src="/files/ftp_upload/50455/50455fig5.jpg" /> 图5。腺病毒感染的MOI为1 - 50，具有对T细胞存活率没有影响或调节性T细胞的分化。朴素的T细胞从DO11.10甘油三酯纯化;CARΔ1tg小鼠在不同的组MOI感染腺病毒90分钟，用PBS洗涤，静置40小时，在T细胞培养基。细胞被活化后72小时中调节性T细胞的偏振条件。固定和染色细胞死细胞染料掺入（b）中的感染标记GFP（a）及（三）分化标记Foxp3的表达进行了分析， <a href="https://www.jove.com/files/ftp_upload/50455/50455fig5large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图6" fo:content-width="6.5in" fo:src="/files/ftp_upload/50455/50455fig6highres.jpg" src="/files/ftp_upload/50455/50455fig6.jpg" /> 图6。腺病毒感染不改变T细胞的激活状态。从DO11.10甘油三酯的幼稚T细胞，腺病毒的MOI 50感染了CARΔ1tg小鼠90分钟（线）或左未感染的（区域），用PBS洗涤休息40小时。细胞为EXPRE分析ssion前活化标志物（上面板）和之后（下面板）40小时的活化的调节性T细胞的偏振条件。 <img alt="图7" fo:content-width="2.5in" fo:src="/files/ftp_upload/50455/50455fig7highres.jpg" src="/files/ftp_upload/50455/50455fig7.jpg" /> 图7。相对的miR-155过表达前后T细胞激活幼稚T细胞DO11.10 TG; TGCARΔ1小鼠感染MOI为50的microRNA-155（miR-155的）表达或控制腺病毒，或不感染。细胞，然后休息40小时，激活40小时，休息40小时，40小时激活。相对确定的microRNA-155表达的qPCR SnoRNA202。 <img alt="图8" fo:content-width="5in" fo:src="/files/ftp_upload/50455/50455fig8highres.jpg" src="/files/ftp_upload/50455/50455fig8.jpg" /> <strong&gt;图8。数据分析使用相对分化的调节性T细胞的培养物。所占的百分比的Foxp3 +细胞中的GFP 阳性 （ 即感染的细胞）表达的相对百分比的Foxp3 +细胞中的GFP 阴性 （未感染的细胞）。相对分化等于1基因的过度表达，如果没有调节性T细胞分化的影响。大于1的值表明分化的促进作用，而值低于1显示了抑制作用。

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

15.8K 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

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  ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

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&#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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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&#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 ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

CD4的制备<sup&gt; +</sup&gt; T细胞通过显微镜分析GD3和GD2神经节苷脂膜表达的

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

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 ...