Name: in situ mhc-tetramer staining and quantitative analysis to determine the location, abundance, and phenotype of antigen-specific cd8 t cells in tissues
Uploaded: 2017-09-22T15:00:00.000+00:00
Duration: 8 min 37 s
Description: Watch this Scientific Journal Video about In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues at JoVE

这项工作得到了国家卫生研究院 (T32 DA007097、R01AI096966、andUM1AI26617) 的公共卫生服务补助金的支持。

1. 1 天: 新鲜的组织切片和主要孵化 <ol> <li> 使用手术刀将新鲜的组织切成小 (大约0.5 厘米宽0.5 厘米高) 的碎片。分别将每个组织粘附到一个柱塞上, 并在 PBS 中嵌入 3-5 毫升的 4% low-melt 琼脂糖。用贴纸标记柱塞与组织信息。把它放在冰桶的冷冻柜里, 以固化. </li>
	<li> 打开切片并将剖面的粗细设置为200和 #181; m. 在切片上安装刀片式服务器, 并在切片浴缸中插入带有组织的柱塞. </li>
	<li> 用肝素 (pbs-H) 制备磷酸盐缓冲盐水, 加入100和 #181; 在 pbs 中添加 g/毫升或 18.7 U/毫升肝素以保存 RNA, 并允许下游潜在的应用。用 100-120 毫升的冷冻、无菌的 PBS 填充切片浴, 覆盖嵌入的组织。在浴缸中加入 PBS-H 冰块, 以保持 0-2 和 #176 的温度; c. 开始切片并将组织切成200和 #181; m 部分. 
	注意: 重要的是保持在冰上的组织冷冻, 以减少细胞活动的组织内, 因为新鲜的组织更容易切片时, 它是冷。如果没有针对下游的计划, 则可以单独使用 PBS. </li>
	<li> 或者, 对于不切片的组织 (例如, 肠道和肺部), 使用手术刀或刀片将组织切成薄条, 尽可能接近200和 #181; m. </li>
	<li> 用实验样本信息对24个组织培养皿的盖子进行标记, 并将组织室放置在相应的井中。使用画笔将剖面转移到一个24井组织培养板的井中, 其中含有1毫升的冷却 PBS-H. 
	注: 在开始染色前, 应先制作可重复使用的组织室。组织室可以使用14毫升聚丙烯圆底管和金属丝网。使用锋利的剃刀刀片削减底部的一个14毫升聚丙烯圆底的 snap 盖帽管。将金属丝网切割成一个圆, 以适应管子底部的孔。用本生燃烧器加热金属丝网圈, 直到 red-hot。将金属丝网圈迅速放下, 把管子推到网格上。检查金属丝网是否牢固地连接到管子的底部, 然后小心地用锋利的剃刀刀片在3毫升标记上切断管子的顶端。在每个组织室中放置多达3组织切片, 或每井组织多达 1 cm 2 。保持至少一个空井之间的不同的抗体组合, 以避免交叉污染. </li>
	<li> 在完成将所有切口部分转移到组织室后立即进行主聚丙烯和抗体染色。在任何时候保持1毫升的 PBS-H 的部分被淹没和冷冻. </li>
	<li> 在夜间用0.5 和 #181 孵育组织切片; FITC 共轭的、多肽负载的 MHC I 体在 PBS-H 中稀释, 2% 正常山羊血清 (NGS)。包括老鼠或其他 non-rabbit 抗体定向的细胞外表位的兴趣在这个孵化 ( 例如, 大鼠 anti-CD8 抗体稀释1:500 在 PBS H 与 2% NGS)。每井放置1毫升稀释的抗体. 
	注: 在选择 CD8 抗体时应注意, 一些可以增强, 有些可以抑制 MHC 体对 T 细胞受体的束缚 4 , 21 。在这里描述的大鼠人 CD8 抗体是不稳定的, 有时会导致一些微弱的染色。这是用于三重标记, 因为它是唯一的 non-rabbit 和 non-mouse CD8 抗体测试, 染色恒河猴 CD8 T 细胞. </li>
	<li> 使用1毫升的溶液每井的主要抗体和所有后续孵化, 并执行这一和所有随后的孵化在4和 #176; C, 在一个摇摆平台上的板块. 
	注: 组织应在会议厅自由浮动. </li>
</ol> 2。2天: 固定和二次孵化 <ol> <li> 在主孵化后, 用1毫升的冷冻 PBS 清洗两次, 每次洗涤20分钟。通过将组织腔转移到不同的24孔组织培养基中, 在相应的井中包含1毫升的冷冻 PBS H. 
	注意: 在组织室间移动时, 要注意不要将内容从一个实验样品滴入另一种。对于所有随后的孵化和洗涤, 类似的转移组织室到一个干净的板包含适当的解决方案。在手术过程中, 一定要对组织室的各部分进行监测, 以确保这些部分不会粘在组织室的两侧。如果他们这样做, 把他们推回解决方案. </li>
	<li> 用1毫升新鲜 PBS-缓冲4% 甲醛在室温下2小时修复部分 (不要 over-fix)。用冷的 PBS 冲洗两次, 5 分钟 
	警告: 甲醛有毒;穿戴适当的个人防护设备. 
	注: 如果需要抗原提取和性检测细胞表位, 抗原可以通过煮沸的切片0.01 摩尔尿素后, 甲醛固定. </li>
	<li> 将剖面转换为含有0.01 摩尔尿素的24井培养皿, 并将这些板放在微波炉中。煮沸的部分三次, 约十年代每个, 总共三十年代. 
	注意: 要非常小心, 煮沸的解决方案可以迫使部分出井。如果发生这种情况, 使用画笔将部分从组织室的两侧或从板的盖子推回到适当的组织室的底部. </li>
	<li> 在二次抗体孵化之前, permeabilize 并阻断组织切片, 将其孵化为含有 pbs-h、0.3% 洗涤剂 (pbs-T) 的阻断溶液和 2% NGS 在4和 #176 上的 1 H 的摇杆上; c. 执行随后的抗体孵化PBS-H-T/2%ngs. </li>
	<li> 对于次生孵育, 将组织室中的部分转移到含有家兔抗 FITC 抗体稀释1:10,000 的 PBS-H-T/2%ngs。夜间孵化. </li>
	<li> 在 PBS-H-T/2%ngs 中使用小鼠 anti-CD20 抗体稀释1:200 进行 counterstaining。对于这个选项, 如果需要的话, 检索表位, 渗透到细胞, 并在这种孵化之前阻止, 如上所述. </li>
</ol> 3。3天: 第三次孵化 <ol> <li> 在第二个孵育后, 在 PBS H 中以4和 #176 的三次清洗剖面; C 至少 20 min. </li>
	<li> 执行最后的孵化与适当的荧光标记抗体 ( 例如, 山羊兔共轭绿黄色, 山羊鼠共轭远红色染料, 和山羊 anti-mouse 共轭绿色染料抗体稀释 1:5, 000,1:5, 000, 和 1:2, 000, 分别在 PBS-H-T/2%ngs)。夜间孵化. 
	注意: 在这一点上, 孵化可以延长多达三天, 如果需要。在这个 incu 中, 通过在锡箔纸上包裹盘子来保持部分免受光线的保护。bation 步和尔后, 作为轻解渴荧光. </li>
</ol> 4。4天: 安装节 <ol> <li> 在 PBS H 中将部分三次洗涤, 至少 20 min. 
	注意: 如果计划在下游的 19 , 修复4% 甲醛中的部分, 1 小时, 以确保体和抗体到位, 然后清洗两次与 PBS h 的每节5分钟. 
	警告: 甲醛有毒;穿戴适当的个人防护设备. </li>
	<li> 使用画笔将剖面转移到显微镜幻灯片。注意不要把纸巾戳得太多。每节涂上甘油/明胶, 含有4毫克/毫升的无食子酸丙酯或另一种载有荧光防腐剂的安装介质。盖上片. </li>
	<li> 将幻灯片存储在-20 和 #176 的 light-protected 幻灯片容器中; c. 冲洗组织培养皿, 用酒精除去盖子上的标签. 
	注: 板可重复使用. </li>
</ol> 5。共焦显微镜图像的获取 <ol> <li> 使用共聚焦显微镜捕获高分辨率图像, 对每个荧光 ( 图 1A 和 B ) 采用适当的激光器和滤镜. 
	注: 在本例中, 使用了共焦显微镜 (参见 材料表 )。图像收集使用 561 nm 激光在20% 功率为绿色的黄色标记特异性 t 细胞, 488 nm 激光在10% 功率为绿标记的 CD20-expressing B 细胞, 和 640 nm 激光器在15% 功率为远红色标签的 CD8 t 细胞。20X 目标和数字孔径0.8 被使用了. </li>
	<li> 在多个 800 x 800 像素字段的三通道中依次收集 z 系列3和 #181; m (或其他) 间隔。构造收集字段的蒙太奇 ( 图 1C -e )。根据相应幻灯片的信息命名每个蒙太奇图像, 并保存以进行分析. </li>
	<li> 使用各自的共聚焦显微镜分析和量化软件或使用 ImageJ 进行量化图像分析. </li>
</ol> 6。定量图像分析 注意: 可以使用共聚焦显微镜分析和量化软件或使用 ImageJ 软件来完成定量图像分析。在这里, ImageJ 被用作一个例子. 
<ol> <li> 通过将其拖动到 ImageJ 窗口 ( 图 2A ) 来打开共焦蒙太奇. 
	注: ImageJ 可以直接打开蒙太奇收集的许多不同的共焦显微镜。如果 ImageJ 无法直接打开蒙太奇, 则将选定的 z 扫描作为 TIFF 文件导出以将其打开. </li>
	<li> 复制所选的 z 扫描以进行分析 (#34; 图像和 #34;-#62; #34;D 重复和 #34;) ( 图 2B ). </li>
	<li> 拆分不同的通道 (#34; 图像和 #34;-和 #62; #34; 颜色和 #34;-和 #62; #34; 分割通道和 #34;) ( 图 2C ). </li>
	<li> 在相应的通道中绘制量化分析的 roi 以实现目标, 并通过按下和 #34 将其添加到 roi 管理器中; #34; 在键盘上。测量该区域. 
	注: ImageJ 的 ROI 管理器显示了 #181 中的区域; m 2 ( 图 2D ). </li>
	<li> 调整要分析的通道的荧光亮度和对比度 (#34; 图像和 #34;-#62; #34; 调整和 #34;-#62; #34; 亮度/对比度和 #34;) ( 图 2E ). </li>
	<li> 拼合图像上的 roi (#34; roi 管理器和 #34; #62; #34; 拼合和 #34;) ( 图 2F ). </li>
	<li> 使用 and #34 量化图像中的正单元格; 多点和 #34; 工具 ( 图 2G ). </li>
</ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Vibratome+and+Compresstome+sectioning+of+fresh+primate+lymphoid+and+genital+tissues+for+in+situ+MHC-tetramer+and+immunofluorescence+staining.">Comparison of Vibratome and Compresstome sectioning of fresh primate lymphoid and genital tissues for in situ MHC-tetramer and immunofluorescence staining.</a> Biol Proced Online. 17, (2015).</li><li>Wang, F., 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=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> J Mol Diagn. 14 (1), 22-29 (2012).</li></ol>

作者没有什么可透露的。

IST 结合 IHC 提供了一个基本的工具, 以检测, 定性和量化抗原特异性 CD8 T 细胞在本机环境与其他细胞和组织结构的上下文。在这里, 我们描述了 IST 结合 IHC 的详细程序, 其次是定量图像分析, 以确定的位置, 丰度和表型的抗原特异性 CD8 T 细胞在淋巴结的恒河猴。类似的染色可以适用于人类, 老鼠, 或其他物种组织的 MHC I 体是可用的。此外, 肽 MHC 类 II 聚丙烯或 dextramer 染色可以使用相对相似的方法来标记抗原特异性 CD4 T 细胞在组织4,5,6,7,8,9,10,11,12,13. IST 还可以结合使用, 以确定, 例如,在体内effector-to-target 单元格级别18,19。将来, 通过将 ist/IHC 与高级原位RNA 和 DNA 检测方法相结合, 将进一步进行 ist/IHC 的兴趣。最近在原位杂交试验的进展包括 RNAscope 和 DNAscope24的开发。这些技术可以检测靶 RNA 和组织中的 DNA。将这些方法与 IST 和 IHC 结合, 同时检测 virus-specific-CD8 T 细胞、病毒 RNA、病毒 DNA 和抗体标记抗原, 将是令人兴奋的。
虽然我们最初描述的 IST 方法与新鲜组织, 组织是固定的短期和冷冻组织4, 在近几年, 我们完全使用新鲜的组织切片, 因为他们一贯生产的最高质量的污渍并允许在厚组织切片中检查细胞。作为这里介绍的程序的替代方法, 可以将体应用于组织, 在冻结介质(例如, OCT) 中进行夜间孵育、固定、嵌入和冷冻, 生成冻结的薄片, 并在以后执行 IHC22。同样, 我们通常只用体来染色组织切片的一个子集, 然后冻结 OCT 中的部分, 以便将来能有更多的 counterstaining 组合。此外, Qdot 655-共轭肽 MHC 体可用于直接可视化抗原特异性 T 细胞在低温保存组织切片13。
我们在这里描述了间接聚丙烯染色。使用 APC 或 PE 共轭体的直接染色也已显示为工作4,22。在这种情况下, 所需的 MHC 聚丙烯的浓度高于间接聚丙烯染色。在我们的手, 浓度20µg/毫升的 APC 标记聚丙烯是有效的检测抗原特异细胞。但染色强度远低于间接贴标, 包括抗 FITC 抗体的扩增。
我们发现, 使用 compression-based 切片 (参见材料表) 来进行新鲜的组织切割, 与使用振动切片23相比, 可以减轻切割新鲜组织切片的过程。然而, 在没有 compression-based 切片的情况下, 振动切片或手术刀可以用于新鲜的组织切片。
这项技术的一个主要限制是使用新鲜的组织。使用新鲜的组织比固定或冷冻组织要困难得多, 因为它们需要立即注意和处理。我们已成功地在组织培养培养基或 PBS-H 的冰上过夜的新鲜组织。然而, 有偶尔的问题与航运;例如, 暴风雪推迟了48小时或更高的组织的运输。在这些情况下, 我们发现, 新鲜的组织切片和那些染色 48 h 蒜一般显示特定的染色, 有一些组织退化的迹象;染色 72 h 蒜的组织太过退化。我们还发现, 由于冰块太冷或太靠近组织而运送的新鲜组织在运输过程中会冻结组织;这种冷冻通常会破坏组织的染色。因此, 这是极其重要的船舶新鲜组织冷冻, 但不冻结, 并启动 IST 染色24小时内. 新鲜的组织加工也需要大量的学生和工作人员的时间, 因为组织从多个动物或研究参与者不能收集和染色一起在一个较晚的日期。尽管有这些困难, 我们发现新鲜的组织切片是美丽的, 特定的 IST/IHC 染色的最佳选择。
本文所描述的 IHC 方法的另一个局限性是间接染色法。由于可用于产生二次抗体组合的不同种类动物的数量有限, 因此我们在一次只用三或四种荧光抗体染色方法来限制间接抗体染色法。这就限制了可以在一个组织板上收集的信息量。直接 IHC 染色克服了这一限制, 可以扩大能力, 同时检测八或更多抗体标记抗原, 尽管每个抗体产生一个比间接方法弱得多的荧光信号。因此, 间接 IHC 可作为 counterstaining ist 染色组织的间接 IHC 的替代品, 允许在结合抗原特异性 CD8 T 细胞的 IST 检测时检测出更多的细胞抗原。
在某些情况下, 大量的自发荧光和/或非特异性聚丙烯或抗体结合可能发生与 IST/。因此, 良好的积极和消极的控制是必要的, 以辨别特定污渍的背景和 autofluorescent 染色。良好的负性控制 mhc i 体包括阴性对照组织 (例如,感染组织; 与 mhc 染色的组织 i 体加载无关肽或不相干的 mhc 体; 或者, 在一个捏, 组织染色没有体, 但与放大抗体)。
总之, MHC I IST 结合 IHC 是一个有价值的工具, 以确定的位置, 丰度和表型抗原特异性 CD8 T 细胞的组织。这种方法允许在本机环境中检测抗原特异的 CD8 T 细胞, 并对其他细胞类型和组织结构保持相对定位。这种方法是广泛适用的, 因为它可以用于本地化, 表型, 并量化基本上任何抗原特异性 CD8 T 细胞的 MHC 体是可用的, 在任何组织。

T 细胞对许多免疫过程至关重要, 包括检测和消除病毒感染的细胞, 预防自身免疫, 协助 B 细胞和血浆细胞产生抗体, 以及检测和消除癌细胞。多肽/MHC I. 类抗原特异性 CD8 t 细胞聚丙烯染色的研究进展1和最近发展的 mhc 类 II 聚丙烯染色 CD4 t 细胞2流式细胞术彻底改变了我们的学习和理解能力T 细胞的免疫。流式细胞仪在确定抗原特异性 t 细胞的数量和表型时非常有用, 但不允许检测抗原特异性 t 细胞在组织中的其他细胞和结构的空间定位, 以及当前用于提取流式细胞术所需 T 细胞的分类技术在非组织中的有效性有限3。
我们和其他人已经开发的方法使用肽负载 MHC i 类和 II 类聚丙烯或 multimer 试剂染色抗原特异性 CD8 和 CD4 T 细胞在组织中4,5,6,7,8,9,10,11,12,13. 这些 IST 方法可以确定组织中抗原特异的 CD8 和 CD4 T 细胞的位置、丰度和表型, 并提供一种方法来检测这些细胞与组织中其他细胞和结构的关系。我们的小组广泛使用 MHC I 聚丙烯染色研究人体免疫缺陷病毒 (HIV) 和猴免疫缺陷病毒 (SIV) 特定的 CD8 T 细胞的淋巴, 生殖器和直肠组织获得了解艾滋病毒和 SIV 免疫并识别成功接种策略的相关性14,15,16,17。此外, 我们还开发了一种技术, 结合 IST 与原位杂交 (一), 以本地化和量化病毒特异性 CD8 T 细胞和病毒感染的细胞在组织和确定的在体内effector-to-target 水平18,19。
在这里, 我们描述了一个协议使用多肽负载 MHC I 体染色抗原特异性 CD8 T 细胞在新鲜的组织切片, counterstain 组织使用 IHC, 并量化细胞与特定的表型在特定的组织舱室。这些过程与我们最近出版的李et al.所使用的程序相同, 在该出版物中, 我们确定了在猕猴慢性 siv 感染过程中, 在淋巴组织中的 siv 特异性 T 细胞的位置、丰度和表型,20。
在这个过程中, 新鲜的组织被切片和培养过夜与肽负载 MHC I 体共轭的荧光素硫氰酸盐分子 (FITC)。然后固定在甲醛。在修复组织后, 从 MHC 体的信号被放大使用兔抗 FITC 抗体和孵化与荧光标记兔 IgG 抗体, 进一步放大信号从绑定体。IHC 与 IST 结合用于抗原特异 T 细胞和周围细胞的特征。抗体, 承认表位在细胞表面或在胞外空间是包括在初级孵化与体。识别细胞内抗原表位的抗体需要在染色前渗透细胞壁。使用共聚焦显微镜对染色组织切片进行成像, 并使用共焦软件进行分析。标记细胞使用共聚焦显微镜软件或 ImageJ 进行量化。所述的协议可用于在任何有 MHC I 体的组织中基本上染色任何抗原特异的 CD8 T 细胞。

<table><tbody><tr><td>MHC-I monomer</td><td>NIH tetramer core facility</td><td></td><td>Materials for MHC-tetramer preparation</td></tr><tr><td>ExtrAvidin-FITC</td><td>Sigma-Aldrich</td><td>E2716</td><td>Materials for MHC-tetramer preparation</td></tr><tr><td>Normal goat serum</td><td>Jackson Immunoresearch</td><td>005-000-121</td><td></td></tr><tr><td>Low melt agarose</td><td>Promega</td><td>V3121</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>SLBL6391V</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T-6878</td><td></td></tr><tr><td>Urea</td><td>J.T.Baker</td><td>4204-05</td><td></td></tr><tr><td>Glycerol gelatin</td><td>Sigma-Aldrich</td><td>SLBH2672V</td><td></td></tr><tr><td>n-propyl gallate</td><td>Sigma-Aldrich</td><td>P3130</td><td></td></tr><tr><td>rat-a-h-CD8 (1:500)</td><td>Acris</td><td>0714</td><td>Antibody unstable, use single use frozen aliquot</td></tr><tr><td>m-a-h-CD20 (1:500)</td><td>NOVOCASTRA</td><td>6026819</td><td></td></tr><tr><td>m-a-h-Ki67 (1:500)</td><td>Vector</td><td>6022201</td><td></td></tr><tr><td>goat-a-m-A488 (1:2,000)</td><td>Jackson Immunoresearch</td><td>124083</td><td></td></tr><tr><td>goat-a-rb-Cy3 (1:5,000)</td><td>Jackson Immunoresearch</td><td>106232</td><td></td></tr><tr><td>goat-a-rat-Cy5 (1:5,000)</td><td>Jackson Immunoresearch</td><td>118088</td><td></td></tr><tr><td>goat-a-h-IgM-Dylight649 (1:5,000)</td><td>Jackson Immunoresearch</td><td>86579</td><td></td></tr><tr><td>Compresstome: VF-300 Microtome</td><td>Precisionary Instruments, LLC</td><td>1079</td><td></td></tr><tr><td>Quick Set Instant Adhesive</td><td>Loctite</td><td>46551</td><td></td></tr><tr><td>24-well flat bottomed tissue culture plates</td><td>Falcon</td><td>353226</td><td></td></tr><tr><td>Microscope slide</td><td>Globe scienfitic Inc.</td><td>#1321</td><td></td></tr><tr><td>Razor&amp;nbsp; blade</td><td>Ted Pella, Inc</td><td>121-6</td><td></td></tr><tr><td>Feather Disposable Scalpel</td><td>FEATHER SAFETY RAZOR CO. LTD.</td><td>No. 21</td><td></td></tr><tr><td>Round paintbrush #2</td><td>PRINCETON ART &amp;amp; BRUSH CO.</td><td>4350R</td><td>Can trim as needed with razor</td></tr><tr><td>Confocal Microscope</td><td>Olympus</td><td>FV1000</td><td></td></tr><tr><td>FV10-ASW_Viewer4.0</td><td>Olympus</td><td></td><td></td></tr></tbody></table>

in situ mhc-tetramer staining and quantitative analysis to determine the location, abundance, and phenotype of antigen-specific cd8 t cells in tissues

T 细胞对许多免疫过程至关重要, 包括检测和消除病毒感染的细胞, 预防自身免疫, 协助 B 细胞和血浆细胞产生抗体, 以及检测和消除癌细胞。流式细胞术分析抗原特异性 t 细胞 MHC-聚丙烯染色的进展, 改变了我们研究和理解 t 细胞免疫的能力。流式细胞仪在确定抗原特异性 t 细胞的数量和表型时非常有用, 但不能确定抗原特异性 t 细胞在组织中的其他细胞和结构的空间定位, 以及当前的分类技术提取流式细胞术所需的 T 细胞在非组织中的有效性有限。原位MHC-聚丙烯染色 (IST) 是一种技术的可视化 T 细胞是特定的抗原感兴趣的组织。结合免疫组化 (IHC), IST 可以确定抗原特异性 CD8 和 CD4 T 细胞在组织中的丰度、位置和表型。在这里, 我们描述一个协议, 以染色和列举抗原特异的 CD8 T 细胞, 与特定的表型位于特定的组织隔间。这些程序与我们在最近出版的李et al.中使用的相同, 标题是 "在卵泡中的猿猴免疫缺陷病毒产生细胞在体内被 CD8 的+细胞部分抑制."所描述的方法是广泛适用的, 因为它们可以用于本地化, 表型, 并量化基本上任何抗原特异性 CD8 T 细胞的 MHC 体是可用的, 在任何组织。

图 1显示了如何使用共聚焦显微镜收集共聚焦图像。图 2演示使用 ImageJ 进行定量图像分析。图 3和4显示了来自 SIV 感染的恒河猴的淋巴结组织的代表性图像, 体、CD8 抗体和 CD20 抗体染色, 并有助于证明 mhc-聚丙烯染色的特异性。图 3比较 mhc 类 i 体载有一个多肽从 SIV 相比, 从同一组织染色的 mhc 类 i 体加载与一个不相关的肽的部分。图 4显示, 用 MHC/SIV-肽聚丙烯染色的细胞 co-stained 有 CD8 抗体, 而不是 CD20 抗体, 这会使 B 细胞染色。图 5显示了从多个共聚焦 z 系列字段创建的蒙太奇示例, 用于在淋巴结的不同解剖隔间中量化具有特定表型的聚丙烯染色细胞。扩大显示淋巴结的一个区域与 B 细胞卵泡划定的 CD20 染色, 周围 t 细胞区, 其中 MHC-聚丙烯染色细胞可以检测, 其中一些 co-express Ki67 这是一个标志的 t 细胞的活化和增殖。这种染色组合可以确定在 b 细胞毛囊内外的 siv 特异的 CD8 t 细胞的表型, 与 siv 特异的 CD8 t 细胞和其他 Ki67 表达细胞和 b 细胞的关系。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56130/56130fig1.jpg"> 
图 1:代表屏幕截图, 显示如何使用共聚焦显微镜收集共聚焦图像.(A) 用于收集共聚焦图像的捕获模式。(B) 用于图像收集的通道。(C) 20X 目标和 800 x 800 像素字段用于图像收集。(D) 按3µm 的间隔收集顺序 z 堆栈。(E) 采用平铺方式在多个字段中描绘和收集图像。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56130/56130fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56130/56130fig2.jpg"> 
图 2: 具有代表性的屏幕截图, 演示使用 ImageJ 的量化图像分析.(A) 通过将其拖动到 ImageJ 窗口来打开共焦蒙太奇。(B) 复制选定的 z 扫描进行分析。(C) 拆分不同的通道。(D) 在相应的通道中绘制量化分析的 roi 以实现目标, 并按 "T" 将其添加到 roi 管理器中。(E) 调整要分析的通道的荧光亮度和对比度。(F) 拼合图像的 ROI。(G) 计算图像中的正单元格。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56130/56130fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56130/56130fig3.jpg"> 
图 3: IST 与 IHC 结合在一个 SIV 感染的恒河猴腋窝淋巴结的部分.Mamu-A*02 体载有 SIV YY9 肽, 用于染色抗原特异性 CD8 T 细胞, 和类似的体加载与一个不相关的 FLP 肽从乙肝病毒被用作阴性控制 (红色)。小鼠 anti-CD20 抗体被用来染色 CD20+ B 细胞 (绿色), 和老鼠 anti-CD8 抗体被用来染色 CD8+ T 细胞 (蓝色)。(a) 来自腋窝淋巴结区的代表图像, YY9 体、CD20 和 CD8 抗体染色。(B) 与面板 A 中显示 YY9 聚丙烯污点的图像相同。(C) 来自同一腋窝淋巴结的部分的代表性图像, 染色 FLP 体和 CD20 和 CD8 抗体。(D) 与 FLP 聚丙烯染色的面板 C 相同的图像。缩放栏 = 100 µm.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56130/56130fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56130/56130fig4.jpg"> 
图 4: IST 结合 IHC, 显示 MHC-聚丙烯染色的特异性.有代表性的腋窝淋巴结染色 Mamu-A*02 体载有 YY9 肽, 以标签 SIV 特异 CD8 t 细胞 (红色), 小鼠 anti-CD20 抗体标记 B 细胞 (绿色), 和大鼠 anti-CD8 抗体标签 CD8 T 细胞 (蓝色) (A).特定于 SIV 的 CD8 T 单元格为聚丙烯 + (B)、CD20- (C) 和 CD8+ (D)。缩放栏 = 10 µm.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56130/56130fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56130/56130fig5.jpg"> 
图 5:IST 与 IHC 结合以显示抗原特异 CD8 T 细胞的位置、丰度和表型.(A) 有代表性的腋窝淋巴结染色 Mamu-A*02 体载有 YY9 肽, 用于标记 SIV 特异的 CD8 T 细胞 (红色), Ki67 抗体以标记增殖细胞 (绿色), IgM 抗体标记 B 细胞 (蓝色)。缩放栏 = 100 µm. (B) 在面板 a 中选定区域的放大. IgM 染色定义卵泡区, 这是标记为 "F;" extrafollicular 区域被标记为 "EF"。聚丙烯+单元格用箭头表示。刻度栏 = 100 µm. 代表聚丙烯+ Ki67-单元格 (C、 D、 E) 和聚丙烯+ Ki67+单元格 (F、 G、 H)。缩放栏 = 10 µm.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56130/56130fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues at JoVE

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

在这里, 我们描述一个方法, 结合原位MHC 聚丙烯染色与免疫组化, 以确定的定位, 表型和数量的抗原特异性 T 细胞在组织。本协议用于确定抗原特异性 CD8 T 细胞相对于其他细胞类型和组织结构的空间和表型特征。

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 ...

in-situ-mhc-tetramer-staining-quantitative-analysis-to-determine

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

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

12.2K 次观看.  University of Minnesota. 在这里, 我们描述一个方法, 结合原位MHC 聚丙烯染色与免疫组化, 以确定的定位, 表型和数量的抗原特异性 T 细胞在组织。本协议用于确定抗原特异性 CD8 T 细胞相对于其他细胞类型和组织结构的空间和表型特征。

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

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment.
The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8+ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Studying tumor microenvironment may identify prognostic or predictive biomarkers of clinical response to immunotherapy. Presented here, is an innovative method based on in situ fluorescence multispectral imaging to analyze and count automatically various subpopulations of CD8+ T cells. This reproducible and reliable technique is suitable for large cohort analyses.

肿瘤 PD-1 的多路免疫荧光分析及量化研究+ Tim-3+ CD8+ T 单元格

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it ...

科研

癌症研究

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 ...

免疫与感染

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

可视化抗原特异性CD4 + T细胞的MHC II类四聚四

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

生物学

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

肿瘤移植用于评估小鼠肿瘤浸润CD8 + T细胞的动力学

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

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

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

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 ...

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 ...

小鼠肺中抗原特异性 T 细胞的分离和表征

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

作者聚焦:使用四聚体检测小鼠肺中抗原特异性 T 细胞的优化方案

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...

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

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此视频中的章节

肿瘤 PD-1 的多路免疫荧光分析及量化研究⁺ Tim-3⁺ CD8⁺ T 单元格

肽：MHC四聚体为基础的抗原表位特异性的T细胞的富集

可视化抗原特异性CD4 + T细胞的MHC II类四聚四

肿瘤移植用于评估小鼠肿瘤浸润CD8 ⁺ T细胞的动力学

原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

猴免疫缺陷病毒特异性CD8分析

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

用肽:主要组织相容性复合物四聚体鉴定小鼠肺中的稀有抗原特异性 T 细胞