我们要感谢大卫·沃特金斯支持，使该方法存在的优化实验。研究本出版物中报道了由迈阿密中心的美国国立卫生研究院艾滋病研究下奖号P30AI073961提供一个试点补助的部分资助。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

在这个手稿中使用的外周血单个核细胞（PBMC）样品从设在威斯康星州国家灵长类动物研究中心的印度猕猴获得。这些动物在威斯康星研究生院动物护理和使用委员会33的大学批准的方案下，根据Weatherall报告进行照顾。麻醉下进行所有动物的程序并作出一切努力，以减少潜在的痛苦。 1.治疗PKI 注意:这是可选的，但建议马牧-B * 017:01四聚体。查看结果和讨论部分 。 <ol><li>重悬的PBMC在1.6×10 7个细胞/ ml的浓度R10培养基。 </li><li>加入50μl该细胞悬浮液，以流式细胞术管相应的流量。加入50μl的PKI的100nM的解决方案，以每一管。 </li><li>涡每管。孵育在37℃下30分钟。由此结束步骤，每个管应具有100μl包含8.0×10 5个 PBMC和PKI的50nM的细胞悬浮液。 </li><li>继续执行的pMHC-I四聚体染色的步骤。 </li></ol> 2.染色用荧光染料标记的pMHC-I四聚体<ol><li>准备之前的pMHC-I四聚体预混，离心机在20000 XG至少15分钟的pMHC-I四管在4℃。这一步骤的目的是，以沉淀蛋白质的聚集体的的pMHC-I四聚体的解决方案，可能会增加背景染色。一旦离心步骤完成后，避免其中蛋白质聚集体将积累在管的底部移液。 </li><li>准备足够的pMHC-I四预混染色所有的实验管。制备的过量的此主混合物的总体积的15％至帐户移液误差。 </li><li>淡化色斑缓冲（ 如辉煌染色缓冲）的pMHC-I四聚体，使25微升的pMHC-I四聚体主结构是ADDEd可每次试验。 <ol><li>标签PBMC每个测试二的pMHC-I四聚体;一种共轭至别藻蓝蛋白（APC），而另一对亮紫（BV）421。 </li></ol></li><li>添加8.0×10 5个 PBMC至流式细胞管相应的流量在100μl的终体积。如果细胞进行上述的PKI的治疗，他们应已再悬浮于100微升在此步骤。 </li><li>加入25微升的pMHC-I四聚体预混到流式细胞仪管相应的流量。通过此步骤结束时，在每个管中的最终体积应为125微升。 </li><li>涡流每个管以匀化细胞悬浮液。 </li><li>孵育在黑暗中在室温下45分钟。 45分钟孵化期间，准备表面染色单克隆抗体的鸡尾酒。 </li></ol> 3.表面染色<ol><li>准备足够的单克隆抗体预混染色所有的实验管。制备的过量的此主混合物的总体积的15％，占移液误差。 </li><li>调整主结构与染色缓冲音量，使50微升的每个测试增加。 </li><li>对于非CD8 + T细胞的适当的排斥和存储器的子集的划分，使用滴定针对在表面染色主混合物以下分子的单克隆抗体的量。 注:缀合到各单克隆抗体的荧光染料提供作为参考:CD14 BV 510，CD16 BV 510，CD20 BV 510，CD8αBV 785，CD28 PE Cy7的，并且CCR7 FITC。需要注意的是对CD14，CD16，和CD20的单克隆抗体缀合到相同的荧光染料（ 即，BV 510），因为它们将被包括在"转储"门。 </li><li>包括在表面染色预混辨别死细胞可修复的染料[即胺活性染料（ARD）。确保ARD试剂缀合到荧光具有类似的发射光谱如在"转储"门使用的。在这种情况下，使用ARD水族。 </li><li>加入50微升染色预混在3.1-3.4描述的流式细胞仪管相应的流量。通过此步骤结束时，在每个管中的最终体积应为175微升。 </li><li>涡每管。孵育在黑暗中在室温下25分钟。 </li><li>洗涤用洗涤缓冲液细胞（含牛血清白蛋白的0.1％和0.45克/升NaN 3的PBS溶液）。 注意:叠氮化钠是一种有毒物质。暴露在即使微量可引起症状。根据在哪里正在执行这些实验机构的环境健康与安全（EHS）办公室指定的指导方针处理这种物质。 </li><li>离心管在510×g离心5分钟。上清液小心地倒入一个单独的废物容器中。一定不要打扰颗粒。 小心:不要滗洗涤缓冲液上清液成含漂白剂储，因为它可以与存在于洗涤缓冲液中的叠氮化钠反应，并导致在有毒气体34的形成。 Contact EHS的办公室那里正在为如何处置叠氮化钠的指导进行这些实验的机构。 </li><li>倾析后，旋涡振荡细胞保持在每个管中的剩余液体。 </li></ol> 4.细胞固定<ol><li>加入250微升2％的多聚甲醛（PFA）溶液的所有管，以固定细胞。 注意:PFA是一种有毒物质。暴露在即使微量可引起症状。根据在哪里正在执行这些实验的机构，由EHS办公室指定的指导方针处理这种物质。 <ol><li>由于2％PFA溶液必须是等渗的细胞，使用PBS制备该溶液。 2％的PFA溶液的百毫升就足够了多个实验。彻底振荡试管，以防止细胞聚集体的形成的另外2％PFA中后，立即。 </li></ol></li><li>孵育在黑暗中在4℃下20分钟。 </li><li> W¯¯灰细胞通过重复步骤3.7-3.9。一旦该步骤完成后，可以将细胞储存在黑暗中在4℃下24-48小时。在开始的阶段透，彻底涡管。 </li></ol> 5.通透细胞<ol><li>加入500μl通透缓冲。振荡试管。 </li><li>孵育在黑暗中在室温下10分钟。 </li><li>通过重复步骤3.7-3.9洗涤细胞。 </li></ol> 6.细胞内染色<ol><li>准备足够的单克隆抗体预混染色所有的实验管。 </li><li>调整用PBS体积或使细胞内的mAb主混合物的50微升被每个测试加入染色缓冲液中。 </li><li>在准备6.1和6.2中所述的单克隆抗体主结构采用滴定针对CD3和GZM B.单克隆抗体量 注:通过的pMHC-I四聚体的TCR接合可导致CD3内化，其可以与检测四聚体+ CD3 + CD8的干扰+T细胞，如果抗CD3单克隆抗体被加入到表面染色母液。为了避免这种情况，添加抗CD3单克隆抗体在此阶段，即，将细胞透化之后。缀合到每种mAb的荧光染料列出作为参考:CD3 PerCP经Cy5.5和GZM乙聚乙烯。 </li><li>加入50μl的mAb鸡尾酒到相应的管中。孵育在黑暗中在室温下30分钟。 </li><li>通过重复步骤3.7-3.9洗涤细胞。管子准备在流式细胞仪获得。 </li></ol>

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccine-induced+CD8++T+cells+control+AIDS+virus+replication.">Vaccine-induced CD8+ T cells control AIDS virus replication.</a> Nature. 491 (7422), 129-133 (2012).</li><li>Lissina, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+inhibitors+substantially+improve+the+physical+detection+of+T-cells+with+peptide-MHC+tetramers.">Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers.</a> J Immunol Methods. 340 (1), 11-24 (2009).</li><li>Weatherall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The use of non-human primates in research. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+induces+CD4+effector+memory+T+cell+production+and+tissue+emigration+in+nonhuman+primates.">IL-15 induces CD4 effector memory T cell production and tissue emigration in nonhuman primates.</a> J Clin Invest. 116 (6), 1514-1524 (2006).</li><li>Dolton, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+tricks+with+tetramers:+a+practical+guide+to+staining+T+cells+with+peptide-MHC+multimers.">More tricks with tetramers: a practical guide to staining T cells with peptide-MHC multimers.</a> Immunology. 146 (1), 11-22 (2015).</li><li>Wooldridge, L., Lissina, A., Cole, D. K., vanden Berg, H. A., Price, D. A., Sewell, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricks+with+tetramers:+how+to+get+the+most+from+multimeric+peptide-MHC.">Tricks with tetramers: how to get the most from multimeric peptide-MHC.</a> Immunology. 126 (2), 147-164 (2009).</li><li>Roederer, M., Herzenberg, L. A., Weir, D. M., Blackwell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS+Analysis+of+Leukocyes.">FACS Analysis of Leukocyes.</a> Weir's Handbook of Experimental Immunology, 5th ed. , 1-49 (1996).</li><li>Colantonio, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KIR+polymorphisms+modulate+peptide-dependent+binding+to+an+MHC+class+I+ligand+with+a+Bw6+motif.">KIR polymorphisms modulate peptide-dependent binding to an MHC class I ligand with a Bw6 motif.</a> PLoS Pathog. 7 (3), e1001316 (2011).</li><li>Schafer, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+a+Natural+Killer+Cell+Response+by+Simian+Immunodeficiency+Virus+Peptides.">Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides.</a> PLoS Pathog. 11 (9), 1005145 (2015).</li></ol>

The authors declare that they have no competing financial interests.

在此过程中值得讨论的有几个步骤，因为它们是产生最佳效果的关键。首先，由于生物样本的质量是任何流式细胞测定法38的成功的一个强有力的预测，都必须小心，以确保细胞是染色过程中存活，并在悬浮液中。与冷冻保存的样品时，因为它们更容易结块和通常含有死细胞的更高数量，这是特别相关的。在这些情况下，通过将细胞悬浮液通过的pMHC-I四聚体标记过程之前的70微米的细胞滤网可能大大改善的结果。第二，在测定中使用的荧光染料的适当的补偿是准确的数据分析的关键，尤其是当多个参数在相同的实验进行评估。在这方面，建议新鲜单色彩补偿控制来对每个的pMHC-I四聚体随时准备投入泰宁检测。我们主张，因为其广泛的抗Ig反应，事实上，它们产生正面和负面的人群明确的双峰分布的补偿款使用的珠子。由于这些珠粒不结合的MHC-I类分子，抗体缀合到相同的荧光团作为的pMHC-I四聚体（ 例如 ，APC和BV 421）应该被用作补偿控制。数的这种补偿珠粒可从多个供应商，并应根据制造商的说明使用。第三，由于在这里所描述的表型的策略需要基于三个分子（ 即，CD28，CCR7，以及GZM B）所示，选通控制，如荧光减一（FMO）试验的分级表达CD8 + T细胞亚群的划分或同种型匹配抗体，应该用于促进这些类型的分析。在我们的实验，例如，将含有的pMHC-I四聚体+ CD8 + T细胞群体感兴趣的分离的管重新总是与缀合到相同的荧光染料作为对CD28，CCR7，以及GZM B.重要的单克隆抗体同种型对照单克隆抗体染色，虽然上述准则可能有助于改善的整体质量的pMHC-I四聚体染色，它们不是唯一的变量可能会影响该测定的性能。考虑到每一个实验室环境的独特性，这里所描述的整个工作流程应在模拟样本进行至少一次，以允许实践和相应的调整。 奇怪的是，的pMHC-I四聚体阳性CD8 + T细胞在外周血单个核细胞偶尔观察到SIV幼稚（未感染的和未接种）恒河猴。这些情况下，不会出现是背景染色的基于数据的视觉检查的结果。相反，他们可能反映的pMHC-I分子和杀伤免疫球蛋白样受体（的KIRs）之间的相互作用猕猴NK细胞和CD8 + T细胞3的表面上表达9,40。事实上，马目-A1 * 002:01四聚体与对应于加格GY9和Env RY8表位的肽折叠已显示出结合到马目-KIRDL05 39。沿着这些线路，马牧-A1 * 002:01 /加格GY9和马牧-A1 * 002:01 /信封RY8更容易表现出上述银独立的pMHC-I四聚体染色。鉴于这种现象，重要的是控制用于在涉及以下感染或疫苗接种的SIV-特异性CD8 + T细胞应答纵向评估猴实验这个基线反应是很重要的。为了实现这一点，最好是在治疗的第一天获得的样品进行的pMHC-I四聚体染色。它也可能在相关的这些初始时间点的情况下，比较冻结PBMC在几个小规模分装与基线样品需要在未来的实验。 的pMHC-I四聚体染色的一个限制是已知的特异性和MHC-I限制性的c只有CD8 + T细胞一种通过该方法进行研究。这是因为它们的MHC基因位点的复杂组织猕猴尤为重要。事实上，一个单一的猕猴单倍型可以有几十个MHC-I基因，并非所有的编码与在细胞表面29上稳定表达的分子。其结果是，它可能难以确定该组由一个给定的动物表达功能的MHC-I类分子的。 01， 马牧-B * 008:01，和Mamu-不过，这警告可以通过设计实验，包括猴子是积极已知MHC-I等位基因，如马牧-A1 * 001， 马牧-A1 * 002来克服B * 017:01。 总之，本手稿提供了用于确定在恒河猴SIV-特异性CD8 + T细胞的频率和记忆表型的优化的pMHC-I四聚体染色协议。自的pMHC-I四聚体染色比IFN-γELISPOT和ICS测定更敏感的检测抗原特异性CD8的+ T-细胞，并且不需要在体外的Ag的刺激，在这里介绍的方法是研究的CD8 + T细胞应答在免疫缺陷病毒感染的结果的影响是特别有用的。从掌握该技术获得的实际知识也可能是丰富抗原特异性CD8 + T细胞作为功能研究的一部分，在各种疾病的设置6监测的 CD8 + T细胞应答是有用的。

CD8 + T细胞包含适应性免疫系统的重要组成部分，因为它们参与肿瘤免疫监视和向细胞内病原体1根除。简单来说，CD8 + T细胞表达特异性识别肽-主要组织相容性复合体I类（的pMHC-I）中的T细胞受体（TCR）的分子存在于宿主细胞的质膜。因为这些肽是从内源性合成的蛋白的水解产生的，细胞表面的pMHC-I复合物提供了一个窗口，进入细胞内环境。在病毒感染，例如，感染的细胞将显示含有病毒蛋白衍生肽，可以作为配体被巡逻的CD8 + T细胞中表达的TCR的MHC-I类分子。在一个特定的病毒特异性CD8 + T细胞遇到感染细胞呈递其的pMHC-1配体的情况下，T细胞受体接合将导致CD8 + T细胞的激活和大招三方共同导致靶细胞裂解。鉴于这些TCR /的pMHC-I相互作用的关键特性，确定了震级，特异性和响应CD8 + T细胞表型往往能揭示人类疾病的重要线索。 直到20世纪90年代初，抗原特异性CD8 + T细胞的量化依靠技术要求有限稀释法（LDA）2,3-。不仅在LDA需要数天才能完成，因此也未能检测缺乏增殖潜能的细胞。其结果是，在LDA大大低估抗原（抗原）特异性的 CD8 +参与免疫应答的T细胞的实际频率。虽然ELISPOT和细胞内细胞因子染色测定的发展极大地提高测量细胞免疫的能力，这些方法仍然需要在体外刺激用于定量抗原特异性T细胞4。但直到1996年的奥特曼，戴维斯和科尔eagues发表了具有里程碑意义的文章报告的pMHC-I技术的四聚体5的发展。临界该技术的成功是的pMHC-I类分子，其延伸的TCR /的pMHC-Ⅰ相互作用的半衰期，从而减少的pMHC-I四聚体中流式细胞测定法的洗涤步骤脱落的概率的多聚化。的pMHC-I四聚体在上述测定法的主要优点是能够精确地直接离体检测抗原特异性CD8 + T细胞，而无需在体外再刺激的能力。此外，具有多色彩的pMHC-I四聚体染色的组合流式细胞仪已经允许分化阶段，记忆表型，和Ag特异性CD8 + T-细胞2-4的激活状态的详细分析。在最近对通过表征的 CD8 + T细胞所有组成成分的技术进步的光的pMHC-Ⅰ多聚体染色6，应用FO广度R此方法很可能会继续扩大。 在生物医学研究领域很少有比艾滋病病毒免疫学7领域受益更多来自的pMHC-I四聚体染色。虽然CD8 + T细胞曾与由奥特曼，戴维斯和同事8,9出版时HIV病毒血症的初步控制了时间相关的，在随后的几年中使用的pMHC-I四聚体的显著扩大了我们的理解的HIV特异性CD8 + T细胞应答。例如，的pMHC-I四聚体染色帮助确认病毒特异性CD8 + T细胞反应在大多数HIV感染者10-12强劲的大小。这种方法还促进艾滋病毒和SIV特异性CD8 + T细胞应答的通过与在不存在的被称为"精英控制"抗逆转录病毒治疗，这种现象的病毒复制的自发控制相关的MHC-I类分子限制的表征<suP&gt; 13-15。此外，的pMHC-I四聚体是在建立程序性死亡-1（PD-1）/ PD配体1（PD-L1）的轴作为可逆通路中的HIV特异性CD8 + T细胞的不受控制的慢性感染的功能失调的表型器乐16,17。总的来说，这些研究强调的pMHC-I四聚体的效用用于监测针对爱滋病病毒的 CD8 + T细胞应答。 猕猴（ 猕猴 ）的实验SIV感染仍然是防治艾滋病毒/艾滋病18,19评估免疫干预的最佳动物模型。在过去的25年中，实质性的进展已在SIV-特异性CD8 + T细胞在此猴类的鉴定和表征制成，包括MHC-I等位基因的发现和肽结合基序13,20-27的定义。其结果是，的pMHC-I四聚体已经开发了用于SIV-特异性CD8 + T细胞的分析在这种动物模型28的反应。大多数这些试剂是由四猕猴MHC-I等位基因的基因产物: 马目-A1 * 001，马目-A1 * 002:01，马目-B * 008:01，和马目-B * 017:01。值得注意的是，猕猴不发表MHC-C座29。绝大多数在本实验中使用的的pMHC-I四聚体均在埃默里大学美国国立卫生研究院四聚体核心工厂生产。然而，一些这些试剂，包括结合到免疫的Gag CM9表位马目-A1 * 001四聚体，只能从由于许可协议商业来源获得。四个猕猴等位基因使用的pMHC-I四聚体以上列出的，我们已经成功地枚举CD8 + T细胞对共21 SIV抗原表位（ 表1），这是诱导疫苗接种或主SIV感染30,31（马丁等人 ，未发表意见）。 本稿件提供优化的pMHC-I四聚体染色方案，用于确定在恒河猴SIV-特异性CD8 + T细胞的频率和存储器的表型。为了降低TCR内化，从而改善的pMHC-I四聚体染色32;该测定用择孵育30分钟与蛋白激酶抑制剂（这里，达沙替尼使用PKI）的开始。 01四聚体:如下所述，使用马目-B * 017时这种处理是非常有用的。还提供了关于如何标记与荧光标记的pMHC-I四聚体和单克隆抗体（mAb）的细胞的指示。该协议还包括用于将细胞溶解相关分子颗粒酶B（GZM B）的细胞内检测一个细胞透步骤。抗CD3的mAb在此步骤中加入，以及改进这种TCR信号分子的检测。作为参考，在这种染色面板使用的所有荧光被列出。

<table border="0" cellpadding="0" cellspacing="0" width="1449">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dasatinib</td>
			<td style="width:236px;">Axon medchem</td>
			<td style="width:127px;">Axon&#160;1392</td>
			<td style="width:757px;">Must be resuspended in DMSO and immediately stored at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI w/ Glutamax</td>
			<td>gibco/ Life Technologies</td>
			<td>61870-036</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat Inactivated FBS</td>
			<td>gibco/ Life Technologies</td>
			<td>10082-147</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycinp-Amphotericin B</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO, Anhydrous</td>
			<td>Life Technologies</td>
			<td>D12345</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-mL Round-Bottom Polypropylene Tubes</td>
			<td>VWR</td>
			<td>60819-728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorochrome-conjugated pMHC-I tetramers</td>
			<td>NIH Tetramer Core or MBL, Inc.</td>
			<td></td>
			<td>Must be stored and maintained at&#160; 4 &#176;C. Centrifuge at 20,000 x g for 15 min before use. Do not freeze.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorochrome-conjugated mAbs</td>
			<td>Various companies</td>
			<td></td>
			<td>Must be stored and maintained at&#160; 4 &#176;C. Do not freeze.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD&#160;Fixable Aqua&#160;Dead&#160;Cell Stain Kit</td>
			<td>Life Technologies</td>
			<td>l34957</td>
			<td>Must be stored at -20 &#176;C. Resuspend each aliquot in 50 &#956;L of DMSO prior to use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Stain Buffer</td>
			<td>BD Biosciences</td>
			<td>563794</td>
			<td>Must be stored at&#160; 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td>VWR</td>
			<td>97064-158</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumine Bovine</td>
			<td>VWR</td>
			<td>700011-230</td>
			<td>Must be stored at&#160; 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Azide</td>
			<td>VWR</td>
			<td>97064-646</td>
			<td>Store at room temperature. Toxic substance. Do not mix with bleach.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bleach</td>
			<td>VWR</td>
			<td>89501-620</td>
			<td>Corrosive chemical, cannot be mixed with sodium azide. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td>Flammable, corrosive, and toxic reagent. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysorbate 20 (Tween-20)</td>
			<td>Alfa Aesar</td>
			<td>L15029</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permeabilization Solution 2&#160;</td>
			<td>BD Biosciences</td>
			<td>340973</td>
			<td>Toxic and corrosive reagent. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sarsdet Tubes 1.5mL screw top</td>
			<td>VWR</td>
			<td>72.692.005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.0ml DNA/RNA Low bind Tubes</td>
			<td>Eppendorf</td>
			<td>22431048</td>
			<td>The use of Sterile microtubes is preffered&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biosafety Cabinet&#160;</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milli Q Intergral Water Purification system</td>
			<td>EMD Millipore</td>
			<td>ZRXQ010WW</td>
			<td>Molecular Biology grade water from any provider may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 &#176;C&#160; refrigerator</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD LSR II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer must contain lasers and filters that are compatible with the staining panel used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum Foil</td>
			<td>VWR SCIENTIFIC INC.</td>
			<td>89068-738</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td></td>
			<td></td>
			<td>Must be able to maintain 37 &#176;C&#160; internal temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Diva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowjo software version 9.6</td>
			<td>Flowjo</td>
			<td></td>
			<td>Used to analyze FCS files generated by FACS Diva software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropippette tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of simian immunodeficiency virus-specific cd8+ t-cells in rhesus macaques by peptide-mhc-i tetramer staining

肽主要组织相容性复合体I类（的pMHC-I）四聚体一直一个宝贵的工具来研究CD8 + T细胞反应。因为这些试剂直接结合到T细胞受体CD8 + T淋巴细胞的表面上，荧光染料标记的pMHC-I四聚体使抗原的精确检测（银）特异性CD8 + T细胞，而无需在体外重新-stimulation。此外，当多色组合流式细胞仪，的pMHC-I四聚体染色可以揭示抗原特异性CD8 + T细胞的重要方面，包括分化阶段，记忆表型，和激活状态。这些类型的分析已经在HIV免疫学领域特别有用的 CD8 + T细胞可影响发展为艾滋病。猴免疫缺陷病毒的猕猴（SIV）的实验感染提供了一个宝贵的工具来研究针对艾滋病病毒的细胞免疫功能。其结果是，相当大的逐行SS已经在该动物模型定义和表征T细胞应答已取得。在这里，我们提出了一个优化的协议用于通过的pMHC-I四聚体染色猕猴列举SIV-特异性CD8 + T细胞。我们的测定法允许每测试二的pMHC-I四聚体+ CD8 + T细胞群，这可能是用于跟踪由疫苗接种或SIV感染产生SIV-特异性CD8 + T细胞应答有用的同时定量和存储器表型。考虑在生物医学研究非人灵长类动物的相关性，这种方法适用于多种疾病设定学习的 CD8 + T细胞应答。

这里所描述的协议已被用来确定在一个马目-A1 * 001 +猕猴的疫苗诱导的，的Gag CM9特异性CD8 + T细胞应答的大小和存储器的表型。对于这种分析，在一个8色流式细胞术染色面板用的APC-缀合马目-A1 * 001 /加格CM9四聚体。 图1A - F显示用于分析数据，这应适用于存在于每个测试两个四聚体门控策略。需要注意的是的pMHC-I四聚体+ CD8 + T细胞群以及以最小的背景（ 图1F和G）分离。记忆CD8 + T细胞在恒河猴可以被分类为基于CD28和CCR7的表面表达三个子集:中央存储器（T CM; CD28 + CCR7 +），过渡存储器（T EM1; CD28 + CCR7-）和效应内存（T EM2; CD28-CCR7-）35。该协议还包括用于GZM B和CD3的细胞内检测一个细胞透步骤。 图1G - 我展示了四聚体+门内记忆亚群和GZM B-表达CD8 + T细胞的划定。 背景染色可以产生在的pMHC-I四聚体标记检测结果不理想。为了避免这种情况，一些预防措施提出了建议。在协议部分所述，包含的pMHC-I四聚体试剂小瓶应始终在20000 XG离心前主混音的准备至少15分钟。这一步骤的目的是，以沉淀任何蛋白质聚集可能在的pMHC-I四聚体溶液。此外，还建议滴定在使用前每种试剂。理想情况下，MHC-I匹配的那个或不含有目的抗原特异性的 CD8 + T细胞群体应与有关的pMHC-I四聚体标记的细胞。此CAÑ由侧用不同量的一新获得的pMHC-I四聚体由SIV幼稚（阴性对照）和SIV感染（阳性对照）猕猴侧染色冷冻保存的PBMC来完成。在图2中 ，例如，1.0微升/ APC的共轭马目-B * 008的试验:01 / Nef的RL10四聚体，得到四聚体阳性和阴性CD8 + T细胞群之间的最佳的分离。最后荧光的选择也可显著影响从的pMHC-I四聚体染色所获得的结果。在这方面，的pMHC-I四聚体缀合变暗分子（ 例如 ，荧光素）应该避免。在我们的经验，缀合到明亮的荧光染料，如PE，APC和BV 421的pMHC-I四聚体，已经取得了最好的结果。 我们注意到，马牧-B * 017:当结合上面提到的明亮荧光01四聚体通常产生朦胧的染色均匀。这种情况的原因低荧光强度尚不完全清楚，但可能包括低亲和力TCR /马牧-B * 017:01相互作用并在四结合36,37快速TCR内化。沿着这些线路，PKI的已显示出抑制CD8 + T细胞32的表面上的TCR内化。其结果是，PKI的可以提高抗原特异性CD8 + T细胞的pMHC-I四聚体染色检测。我们已经证实在我们的实验中这种效果，其中，马目-B的质量* 017:01四聚体染色可通过预处理与PKI的PBMC（ 图3）可以大大改善。奇怪的是，与此PKI预处理没有显著改善这里测试（数据未示出）等的pMHC-I四聚体的染色，这表明马目-B * 017:01限制的CD8 + T细胞可能是在它们的灵敏度独特以PKI治疗。 <img alt="图1" src="/files/ftp_upload/54881/54881fig1.jpg" /><br/&gt; 图1:闸战略和成功的pMHC-I四聚体染色的代表性结果。我们从马目-A1 * 001 +猕猴评估疫苗诱导的Gag CM9特异性CD8 + T-细胞的大小和记忆表型PBMC中。这里，在一个8色流式细胞术染色面板用的APC-缀合马目-A1 * 001 /加格CM9四聚体。 （A - F）门控策略，流式细胞仪分析。首先，在对角聚集汗衫门被与FSC区（FSC-A），然后一边散射高度（SSC-H）与SSC区域绘制前向散射高度（FSC-H）创建（SSC-A; A和B） 。接下来，时间门被创造，其中包括只有那些记录了5 日和90 日在百分的事件。然后将所得的细胞门控的"转储通道"负，CD3 +细胞（C和D）。在这个阶段中，淋巴细胞群是基于它的FSC-A与SSC-A性能划定并随后分析是内CD8 +细胞（E和F）进行。概述的pMHC-I四聚体+细胞（G）之后，存储器表型分析，此门（H）内进行。猕猴记忆T细胞可以分为基于CD28和CCR7的表达三个子集:中央记忆（T CM; CD28 + CCR7 +），过渡存储器（T EM1; CD28 + CCR7-）和效应记忆（T EM2 ; CD28-CCR7-）。本协议还包括粒酶B（GZM B）的四聚体+ CD8 + T细胞（Ⅰ）中表达的细胞内评价用细胞透步骤。在直方图中的阴影区域对应于四聚体+ CD8 + T细胞与同种型匹配对照mAb染色。尽管BV 421结合的四聚体+人口，这是目前在这个染色在图中未示出，相同的选通策略应该被用来进行分析。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54881/54881fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54881/54881fig2.jpg" /> 图2:01 / Nef的RL10四:一个APC标记马牧-B * 008的滴定的代表性结果。 PBMC从两个马牧-B * 008:01+猕猴被用于这个实验:一只动物SIV天真并担任阴性对照，而另一个是SIV感染并作为阳性对照。以确定马目-B的量* 008:01 / Nef的RL10四聚能产生四聚体+和tetramer- CD8 + T细胞之间的最佳的分离，从每只动物的PBMC与所示量的的pMHC-I四聚体试剂的孵育。基于这些结果，1.0微升/测试わ选为最佳量s到在将来测定中使用。用于分析该数据的选通策略在图1中说明。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54881/54881fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54881/54881fig3.jpg" /> 图3:01四聚体:在马目-B * 017的荧光强度的PKI治疗的影响。 PKI的抑制剂抑制T细胞受体复合物的内化和由此通过荧光染料标记的pMHC-I四聚体32提高了抗原特异性CD8 + T细胞的检测。 （A - B）从两个马牧-B * PBMC 017:01+猕猴被用于这个实验:一只猴子是SIV天真并担任阴性对照组（A），而其他被感染SIV并担任阳性对照（B）。来自两个动物的PBMC在37℃下在PKI中的50nM的浓度存在下培养用于与APC缀合的马目-B * 017到染色前30分钟:01 / Nef的IW9四聚体，如在协议部分中描述。作为参考，另一套管进行同样的温育和染色条件，所不同的是二甲基亚砜（DMSO）溶液中加入，而不是公钥基础设施。用于分析该数据的选通策略在图1中说明。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54881/54881fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> SIV蛋白 </td><td> 氨基酸位置 </td><td> 氨基酸序列 </td><td> MHC I类分子（新命名） </td><td> MHC I类分子（老nomenclaturE） </td><td> 表位短名称 </td></tr><tr><td>插科打诨</td><td> 181-189 </td><td> CTPYDINQM </td><td>马牧-A1 * 001 </td><td>马牧-A * 01 </td><td>加格CM9 </td></tr><tr><td>插科打诨</td><td> 254-262 </td><td> QNPIPVGNI </td><td>马牧-A1 * 001 </td><td>马牧-A * 01 </td><td>加格QI9 </td></tr><tr><td>插科打诨</td><td> 72-79 </td><td> GSENLKSLY </td><td>马牧-A1 * 001 </td><td>马牧-A * 01 </td><td>加格GY9 </td></tr><tr><td>信封</td><td> 830-838 </td><td> FHEAVQAVW </td><td>马牧-B * 017:01 </td><td>马牧-B * 17 </td><td>信封FW9 </td></tr><tr><td>信封</td><td> 233-241 </td><td> CAPPGYALL </td><td>马牧-A1 * 001 </td><td>马牧-A * 01 </td><td>信封CL9 </td></tr><tr><td>信封</td><td> 620-628 </td><td> TVPWPNASL </td><td>马牧-A1 * 001 </td><td>马牧-A * 01 </td><td>信封TL9 </td></tr><tr><td>信封</td><td> 788-795 </td><td> RTLLSRVY </td><td>马目-A1 * 002:01 </td><td>马牧-A * 02 </td><td>信封RY8 </td></tr><tr><td>信封</td><td> 296-304 </td><td> RTIISLNKY </td><td>马牧-A1 * 002:01 </td><td>马牧-A * 02 </td><td>信封RY9 </td></tr><tr><td> Vif蛋白</td><td> 123-131 </td><td> RRAIRGEQL </td><td>马牧-B * 008:01 </td><td>马目-B * 08 </td><td> Vif蛋白RL9 </td></tr><tr><td> Vif蛋白</td><td> 173-181 </td><td> RRDNRRGL </td><td>马牧-B * 008:01 </td><td>马目-B * 08 </td><td> Vif蛋白RL8 </td></tr><tr><td> Vif蛋白</td><td> 66-73 </td><td> HLEVQGYW </td><td>马牧-B * 017:01 </td><td>马牧-B * 17 </td><td> Vif蛋白HW8 </td></tr><tr><td> Vif蛋白</td><td> 100-109 </td><td> VTPNYADILL </td><td>马牧-A1 * 001 </td><td>马牧-A * 01 </td><td> Vif的VL10 </td></tr><tr><td> Vif蛋白</td><td> 97-104 </td><td> WTDVTPNY </td><td>马牧-A1 * 002:01 </td><td>马牧-A * 02 </td><td> Vif蛋白WY8 </td></tr><tr><td>启</td><td> 13-22 </td><td> KRLRLIHLL </td><td>马牧-B * 008:01 </td><td>马目-B * 08 </td><td>冯KL9 </td></tr><tr><td>达</td><td> 28-35 </td><td> STPESAML </td><td>马牧-A1 * 001 </td><td>马牧-A * 01 </td><td>达SL8 </td></tr><tr><td>奈夫</td><td> 137-146 </td><td> RRHRILDIYL </td><td>马牧-B * 008:01 </td><td>马目-B * 08 </td><td>奈夫RL10 </td></tr><tr><td>奈夫</td><td> 246-254 </td><td> RRLTARGLL </td><td>马牧-B * 008:01 </td><td>马目-B * 08 </td><td>奈夫RL9b </td></tr><tr><td>奈夫</td><td> 8-16 </td><td> RRSRPSGDL </td><td>马牧-B * 008:01 </td><td>马目-B * 08 </td><td>奈夫RL9a </td></tr><tr><td>奈夫</td><td> 165-173 </td><td> IRYPKTFGW </td><td>马牧-B * 017:01 </td><td>马牧-B * 17 </td><td>奈夫IW9 </td></tr><tr><td>奈夫</td><td> 195-203 </td><td> MHPAQTSQW </td><td>马牧-B * 017:01 </td><td>马牧-B * 17 </td><td>奈夫MW9 </td></tr><tr><td>奈夫</td><td> 159-167 </td><td> YTSGPGIRY </td> &lt;TD&gt;马牧-A1 * 002:01 </td><td>马牧-A * 02 </td><td>奈夫YY9 </td></tr></tbody></table> 表1:可用于在猕猴监测SIV-特异性CD8 + T细胞应答的pMHC-I四聚体的列表。

Watch this Scientific Journal Video about Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining at JoVE.com

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

在这里，我们提出了针对艾滋病病毒枚举和表征猕猴CD8 + T细胞的优化协议。本文不仅对HIV免疫学领域，而且也适用于的 CD8 + T细胞应答已知影响疾病结果的生物医学研究的其它领域是有用的。

猴免疫缺陷病毒特异性CD8分析<sup&gt; +</sup&gt; T细胞猕猴由肽MHC-I四聚体染色

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

analysis-simian-immunodeficiency-virus-specific-cd8-t-cells-rhesus

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

9.1K 次观看.  University of Miami. 在这里，我们提出了针对艾滋病病毒枚举和表征猕猴CD8 + T细胞的优化协议。本文不仅对HIV免疫学领域，而且也适用于的 CD8 + T细胞应答已知影响疾病结果的生物医学研究的其它领域是有用的。 

视频: Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

多色流流式细胞仪分析细胞在恒河猴的免疫应答

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

科研

免疫与感染

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 ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

肽:MHC四聚体为基础的抗原表位特异性的T细胞的富集

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

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 ...

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 ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

原位MHC-聚丙烯染色和定量分析确定抗原特异性 CD8 T 细胞在组织中的位置、丰度和表型

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika 病毒特定诊断表位发现

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

猕猴免疫缺陷病毒感染 CD4+ T 细胞单细胞定量的 mRNA 和表面蛋白的表达

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs.
Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8+T cells and to discriminate between na&#239;ve and effector cells.

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Here, we present a protocol for the ex vivo qualitative detection of antigen-specific CD8+ T cells. Analysis is possible with single cell suspensions from organs or from small amounts of blood. A broad range of studies require the analysis of cytotoxic T cell responses (vaccination and cancer immunotherapy studies).

用病毒载体接种后, mhc i 四分体染色同时定量抗载体和抗转基因特异性 cd8+ t 细胞

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of ...