Name: evaluation of t follicular helper cells and germinal center response during influenza a virus infection in mice
Uploaded: 2020-06-27T13:00:00.000+00:00
Duration: 7 min 7 s
Description: Watch this Scientific Journal Video about Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice at JoVE.com

我们感谢上海巴斯德医院流动细胞学设施、ABSL2设施和SPF动物设施的工作人员提供的技术帮助和建议。这项工作得到了以下资助:中国科学院战略重点研究计划（XDB29030103）、中国国家重点研发计划（2016YFA0502202）、中国国家自然科学基金委员会（31570886）。

动物实验得到了中国上海巴斯德学院动物护理和使用委员会的批准。所有实验都是根据机构动物护理和使用委员会批准的动物议定书进行的。
注:小鼠病毒感染和器官分离应在ABSL2条件下进行。
1. 接种PR8流感病毒和记录小鼠体重
<ol><li>准备8周大的雄性C57BL/6小鼠感染ABSL2室。 注:此协议也适用于雌性小鼠的实验。</li>
	<li>
PR8病毒的稀释:从-80°C冰柜中取出病毒，在冰上孵化，直到它融化成液体。将股票病毒彻底循环，用无菌磷酸盐缓冲盐水（PBS、135 mM NaCl、2.7 mM KCl、10 mM Na2HPO 4、1.8 mMKH 2 PO 4）在预冷1.5mL管中稀释病毒至2 PFU/μL。</li>
	<li>
小鼠麻醉: 称量每只小鼠，并计算鼠标重量（4倍（μL）的五巴比妥钠（2毫克/mL）的体积。在腹内注射五巴比妥钠的计算体积。 注:这一步骤是让小鼠稳定、平静地呼吸，以便准确滴答作响的病毒可以在体内接种。心跳过快或速度过慢表示麻醉不当。此外，建议使用兽医软膏，以避免眼干。</li>
	<li>
鼻内接种:彻底使稀释的PR8病毒漩涡。管道 10 μL，并仔细执行一侧一滴一滴的鼻内接种。在完成一个笼子中所有小鼠（最多5只小鼠）的接种后，在另一侧重复接种（通过接种保持小鼠的呼吸平静和稳定）。每只小鼠总共感染了40个PFU的PR8病毒。</li>
	<li>将老鼠放在温暖的笼子里，以便更好地恢复。</li>
	<li>每天监测鼠标重量10天。（感染日记录为第 0 天）。</li>
</ol>2. 从脾脏和介质淋巴结中分离淋巴细胞 （mLN）
<ol><li>
老鼠安乐死:把老鼠放在一个小房间里，从腔底和平地 泵入二氧化碳，对老鼠实施安乐死。当老鼠不动的时候，把老鼠拿出来，进行颈椎脱位，以确保老鼠完全死亡。用75%的乙醇浸入小鼠，并转移到生物安全罩。</li>
	<li>用解剖针将小鼠固定在吸和纸覆盖的解剖泡沫板上。用解剖剪刀切开腹部中线和后腿的皮肤，用钳子伸展皮肤。用解剖针固定拉伸的皮肤。</li>
	<li>为每只老鼠准备两个6厘米的菜，并把它们放在冰上。在每道菜中放入 70μm 细胞过滤器，并添加 5 毫升的 DMEM 补充 1% 胎儿牛血清 （DMEM （1% FBS））</li>
	<li>脾脏隔离:切开腹膜，用解剖剪刀露出腹腔。拿脾脏放在准备的菜里。</li>
	<li>mln 隔离: 将隔膜和笼肋骨底部切到胸腺附近。把肋骨拉到一边，用解剖针固定，露出胸腔。将肺拉到右侧，用钳子取mLN，在心脏下面和气管的腹侧附近。</li>
	<li>将mLN放入准备好的菜中。</li>
	<li>获得单细胞悬架:通过 70μm 细胞过滤器用 3 mL 注射器轻轻网格脾脏或 mLN。用1毫升新鲜DMEM（1%FBS）冲洗细胞过滤器。重新悬浮细胞悬架并转移到 15 mL 离心管中。</li>
	<li>离心细胞悬架在350 x g 6分钟在4°C。 删除超强剂并添加 1 毫升 DMEM （1% FBS）。</li>
	<li>用 1 mL 移液器彻底重新悬念细胞颗粒。将 4 mL 的 DMEM （1% FBS） 添加到脾脏细胞悬架中，并将其保留在冰上以进行以下操作。 注:首先，需要用1mL的介质（而不是5mL）重新悬息细胞颗粒，以完全隔离单细胞与颗粒。 从此以后，所有操作都可以在常规实验室中执行。</li>
	<li>
脾细胞计数
	<ol>
<li>通过将管子向上和向下转动数次来重新悬念细胞。将 10 μL 放入 90μL 的红血球 （RBC） 裂解缓冲区 （10 m M 特里斯 - Hcl pH 7.5， 155m NH4Cl）。在室温 （RT） 下孵化 3 分钟，并添加 900μL 的冷 PBS 以终止反应。</li>
		<li>离心机在400 x g 6分钟在4°C和删除超纳坦。用 100μL 的冷 PBS 重新悬念。将 10 μL 的细胞放入 10 μL 的 0.4% w/v 试盘蓝色中，并从混合物中取出 10 μL，用于使用血细胞计进行细胞计数。</li>
		<li>计算:将细胞计算为常规方法。简言之，在血小球仪上的两个对角形方块中计算细胞数，每个角方块获得 N1、N2。5mL细胞悬浮液的细胞浓度应计算为（N1+N2）/2 x 10 4/mL。</li>
	</ol>
</li>
</ol>3. PD-1和CXCR5多克隆Tfh细胞的免疫
<ol><li>染色与生物素抗CXCR5抗体。<ol>
<li>通过上下转动管子来重新悬浮细胞悬架。将 2 x 106 个细胞放入 FACS 管中，并添加 2 mL 的染色缓冲区（PBS（1% FBS，1 mM EDTA）。在涡流振荡装置上通过漩涡清洗。</li>
		<li>离心机在350 x g 6分钟在4°C。 倒出液体，将管口浸入吸水纸上两次，从而丢弃超纳坦。</li>
		<li>点击管子底部，用残留液体松开细胞颗粒。把管子放在冰上的管架上。 注:残留液体的体积约为25微升。</li>
		<li>为每根管子添加 0.2 μL 的反鼠标 CD16/CD32（Fc 受体阻滞剂）。通过轻轻敲击管底，在冰上孵育10分钟，使漩涡。 注:用每根管5μL的染色缓冲液稀释，为多个样品准备抗体混合物。混合物配方应通过稀释（n/10+1） x 0.2 μL Fc 受体阻滞剂放入 （n/10+1） x 5 μL 染色缓冲区，并在每个管子中加入 5.2 μL 混合物。</li>
		<li>通过点击管底，将 0.3 μL 的生物素防鼠标 CXCR5 添加到每个管和漩涡的污渍缓冲区残留物 30 μL 中。 注:准备步骤 3.1.4 中描述的混合物。</li>
		<li>在冰上孵育1小时，在30分钟内轻敲管子轻轻重新悬生细胞。 注:30 分钟的漩涡是为了避免细胞聚集物更好地染色。</li>
		<li>在漩涡振荡装置上添加2mL的染色缓冲器和漩涡。离心机在350 x g 6分钟，在4°C，并丢弃在步骤3.1.2描述的超纳坦。通过敲击管子并在冰上孵化以进行后续染色，从而产生漩涡。</li>
	</ol>
</li>
	<li>与其他表面标记染色。<ol>
<li>准备抗体混合物（表1）如步骤3.1.4所述。</li>
		<li>将抗体混合物加入每个管子中。通过敲击管底并在冰上孵育30分钟的漩涡。</li>
		<li>用2mL的染色缓冲区清洗电池。离心机在350 x g 6分钟在4°C。</li>
		<li>丢弃超纳坦，加入 400μL 的染色缓冲区。在涡旋振荡装置上对管子进行涡流，并将管子保持在黑暗中，直到进行流细胞学分析。</li>
	</ol>
</li>
</ol>4. PR8流感病毒NP特异性Tfh细胞的免疫化
注:这个染色NP特异性Tfh细胞的协议是从以前的研究15，17。
<ol><li>执行第 3.1 步中描述的生物素-CXCR5 染色，但用于染色的细胞数为 3 x 106， 以便有足够的抗原特异性细胞记录在流细胞学中。</li>
	<li>从 3.1.7 向管中添加 0.3 μL 的 APC 结合 IAbNP311-325 MHC II 类 （NP311-325）四重奏。为多个样品准备混合物，如步骤 3.1.4 注:在添加抗CD4抗体之前，必须先对四聚物进行污渍，因为CD4和抗CD4抗体之间的结合会干扰最佳的四聚体染色。</li>
	<li>轻轻敲击管子，在RT的黑暗中孵育30分钟，从而重新悬生细胞混合物。 注:在管子口盖上湿纸以减少蒸发</li>
	<li>加入其他表面标记（表1）的混合物，在RT继续孵育30分钟。</li>
	<li>洗涤和重新悬念细胞，如步骤 3.2.3 和 3.2.4 中描述的。</li>
</ol>5. 多克隆Tfh细胞中Bcl6的免疫化 
<ol><li>执行第 3 节中描述的表面标记 （表 2） 染色，但最后用 2 mL PBS 清洗，而不是染色缓冲区。</li>
	<li>离心机在350 x g 6分钟在4°C。 轻轻敲击管底，丢弃超纳坦和重新悬浮的细胞颗粒。</li>
	<li>将 300 μL 的 3.7% 甲醛溶液（用 PBS 稀释为 37% 甲醛）放入管中进行细胞固定。涡旋振荡装置上的漩涡，在RT孵育20分钟。</li>
	<li>在 4 °C 下以 500 x g 在 6 分钟内添加 2 mL 的染色缓冲区，用于清洗和离心机。 轻轻敲击管子，丢弃超纳坦和重新悬浮的细胞。</li>
	<li>添加 300 μL 的 0.2% Triton-X 100，并通过涡旋振荡装置上的漩涡重新悬浮细胞。在RT孵化15分钟。</li>
	<li>添加2 mL的染色缓冲区进行清洗。离心机在500 x g 6分钟在4°C。 轻轻敲击管底，丢弃超纳坦和悬浮细胞。</li>
	<li>为每根管子添加 1.5 μL 的 PE 抗 Bcl6 抗体。轻轻敲击管底以重新悬念混合物，并在 RT 孵育 2 小时，每 30 分钟轻轻敲击一次管子。 注意:在管子口盖上湿纸，以减少混合物蒸发。</li>
	<li>添加 2 mL 的 PBS 补充 0.01% 特里顿 X 100 到管子中。漩涡和离心机在500 x g 6分钟在4°C。</li>
	<li>作为步骤 5.8 重复洗涤。带 400μL 染色缓冲器的重新悬念细胞。在冰上保持细胞黑暗，直到流细胞学分析。</li>
</ol>6. IL21的细胞内染色
<ol><li>用PMA（磷12-肌酸13-醋酸盐）和离子霉素刺激细胞。<ol>
<li>以2 x 106 细胞从充血细胞悬浮和离心机在350 x g 6分钟在4°C。 用500μL的完整T细胞介质丢弃超纳坦和重新悬浮的细胞颗粒。将细胞转移到24井板中。</li>
		<li>将 20 nmol PMA 和 2 μmol 离子霉素添加到 500 μL 的完整介质18 中，并通过上下管道彻底混合。</li>
		<li>将步骤 6.2 中准备的解决方案添加到 24 井板中的单元格悬架中，并通过摇动板混合。通过在细胞中添加 500μL 完整 T 细胞介质而不添加 PMA 和离子霉素来设置未刺激的控制。在 37°C 的 CO2 孵化器中孵育 4 小时。</li>
		<li>在每口井中加入10微米BFA（布雷费尔丁A，用甲醇溶解），以阻止高尔吉仪器介于蛋白质运输。将板放回细胞孵化器，孵育2小时。</li>
	</ol>
</li>
	<li>执行细胞表面标记染色。<ol>
<li>通过轻轻上下管道将细胞转移到 FACS 管中来重新悬浮细胞。在管子中加入 1 mL 的染色缓冲液，在 4 °C 下以 350 x g 将离心机加入 6 分钟。</li>
		<li>执行 Fc 受体阻滞剂染色为步骤 3.1.4。</li>
		<li>执行细胞表面标记染色（表3），如步骤3.2.1至3.2.3，但用2 mL PBS清洗细胞除外。</li>
		<li>离心机在350 x g 6分钟在4°C。 通过点击管底丢弃超纳坦和重新悬浮的细胞。</li>
	</ol>
</li>
	<li>从活/死可修复水死细胞染色套件中加入 0.2 μL 试剂，并在 RT 的黑暗中孵育管 10 分钟，以执行死细胞的染色。</li>
	<li>在涡旋振荡装置的管子和漩涡中加入 2 mL 的 PBS。离心机在350 x g 6分钟在4°C和丢弃超纳坦。</li>
	<li>执行步骤 5.3 和 5.4 中描述的单元格固定。</li>
	<li>将 300μL 的染色缓冲器添加到重新悬架的电池中，并将管子存放在 4 °C 冰箱过夜。离心机在 500 x g 6 分钟在 4 °C 去除超纳坦。 注:此步骤可以省略，并继续紧跟第 6.5 步之后的步骤 6.7。</li>
	<li>在涡旋振荡装置上的管子和漩涡中加入 1 mL 的皂素缓冲区（用 0.2% （w/v） 皂素补充的染色缓冲区。在冰上孵化20分钟，进行细胞渗透。</li>
	<li>离心机在 500 x g 6 分钟在 4 °C 和丢弃超纳坦。</li>
	<li>在每个管子中加入0.5μL的人类Fc-IL21受体。将抗体混合物准备多次作为步骤 3.1.4，但用皂素缓冲剂而不是染色缓冲区稀释抗体除外。</li>
	<li>在 RT 孵化 1 小时，在 30 分钟内轻轻敲击管底以重新悬生细胞。</li>
	<li>加入2 mL的皂素缓冲液，以500 x g的500 x g清洗电池和离心机6分钟。丢弃超纳坦，重复清洗一次。</li>
	<li>在每个管子中添加 0.1 μL 的 APC 抗人 Ig （H+L）。作为步骤 3.1.4 为多个样品准备混合物，除非用皂素缓冲区而不是染色缓冲区稀释抗体。</li>
	<li>在冰上孵化样品30分钟，作为步骤6.11清洗。</li>
	<li>带 400μL 染色缓冲器的重新悬念细胞。将样品在冰上保持黑暗，直到流细胞学分析。</li>
</ol>7. GC B 和血浆细胞染色
<ol><li>以细胞为例，执行抗Fc受体抗体染色，从3.1.1到3.1.4。</li>
	<li>执行表面标记染色 （表 4）作为步骤从 3.1.5 到 3.1.7，但潜伏时间是 30 分钟而不是 1 小时。</li>
	<li>带 400μL 染色缓冲器的重新悬念细胞。将样品在冰上保持黑暗，直到流细胞学分析。</li>
</ol>8. 血清与血液的隔离
<ol><li>在感染后的第14天（d.p.i 14），从面部静脉收集血液，并将血液样本保存在4°C冰箱过夜。 注:在ABSL2条件下进行采血，从此以后，所有程序都可以在常规实验室进行。</li>
	<li>在4°C下将血液在400 x g 的离心机中抽吸10分钟。 用 200 μL 移液器小心隔离血清，以避免红细胞污染。每个样品分为 3 个管子，并存储在 -80°C。</li>
</ol>9. 与埃利萨一起检测 HA 特异性抗体滴度
<ol><li>每口井配有 50μL 的 2μg/mL HA 蛋白质溶液的涂层 ELISA 板，并在 4 °C 冰箱中孵育过夜。</li>
	<li>用 200 μL 的 PBS 稀释 0.05% 补间 （PBST） 洗三次。将 100 μL 的 PBST 稀释的 5% 脱脂牛奶加入每个油井，并在 RT 孵育 2 小时，以阻止非特异性结合。</li>
	<li>血清稀释和孵化:在PBS中准备3%的BSA作为稀释缓冲。稀释缓冲液中的血清稀释为 1:50、1:150、1:450。。。。。。至1:36450（建议进行3倍串行稀释）。将 50 μL 稀释的血清添加到每个油井中，并在 4°C 冰箱中孵育过夜。</li>
	<li>丢弃血清，通过在每个油井中加入 200 μL 的 PBST 快速洗一次井（轻轻摇动，然后丢弃）。然后用 200μL 的 PBST 缓慢地在摇床上清洗盘子，每次 5 分钟。</li>
	<li>添加 100 μL 的 HRP 标记的次要抗体，具体用于总 IgG、IgM、IgG1、IgG2b、IgG2c（1:5000，用 PBST 稀释），并在 RT 孵育 1 小时。按 9.4 中描述的 PBST 清洗板。</li>
	<li>从 4 °C 存储中取出等量的缓冲区 A 和缓冲区 B （TMB），并在使用前在 RT 预热至少 30 分钟。混合 A 和 B，在每口井中加入 100 μL TMB，并通过轻柔摇动在 RT 孵育 10-30 分钟。 注:这是TMB基板试剂集（BD，555214）手册的简要说明。</li>
	<li>派珀特 100 μL 的 2M H2SO4 进入每个井以终止反应。通过仪器阅读OD450值。</li>
	<li>数据分析:通过减去背景信号（空井的OD450值）获得最终的OD450值。绘制与每个样品的抗体同型对应的曲线，X 轴上的稀释因子和 Y 轴上的 OD450 值。</li>
</ol>10. 形而论
<ol><li>在d.p.i 10时隔离麻将。在 RT 中将甲醛溶液在 3.7% 中修复 1 小时。丢弃固定缓冲器，在摇床上用 PBS 清洗 5 分钟三次。</li>
	<li>在 4°C 下将 PBS 中的黄素（10% 蔗糖）脱水 1 小时，然后在 4°C 的 PBS 中脱水（30% 蔗糖），轻轻摇晃，直到松果沉入 15 mL 管的底部。</li>
	<li>取出脱水的孢子，嵌入最佳切割温度化合物和低温。</li>
	<li>在 -20°C 前冷却丙酮。 用预冷丙酮孵育组织部分10分钟。用PBS清洗组织三次。</li>
	<li>用 PBS 将含有 0.2% 三顿 X-100 的组织部分渗透 20 分钟，然后用 PBS 洗三次。</li>
	<li>在 RT 中用含有 10% 正常山羊血清（阻塞缓冲区）的 PBS 阻止非特定结合 1 小时，然后用 PBS 洗一次组织部分。</li>
	<li>用斯特里普塔维丁/生物素阻塞套件阻止非特定绑定。 注意:不要让样品从这一步开始干燥。</li>
	<li>与原发性抗体染色:小心地在组织部位添加阻塞缓冲稀释生物素-PNA（25μg/mL）和大鼠抗鼠IgD（2.5μg/mL）。在4°C冰柜的湿室中孵育组织部分过夜。</li>
	<li>用PBST快速清洗组织部分一次。快速洗涤PBST中的组织部分，缓慢摇晃5分钟。重复洗涤三次。</li>
	<li>稀释亚历克萨氟488-链球菌（1:500）和亚历克萨氟555-山羊抗鼠IgG（1:500）抗体与阻塞缓冲区，并将其添加到组织部分仔细</li>
	<li>在RT孵育1小时。</li>
	<li>将组织部分作为步骤 10.8 清洗，并小心地用延长溶液安装。小心地用盖片盖住组织，并在4°C时将其保持在黑暗中，直到进行粘膜分析。</li>
	<li>通过计算每个区域大小的 GC 数字来分析 GC 反应的大小。</li>
</ol>

<ol>
	<li>Johnston, R. 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=Bcl6+and+Blimp-1+are+reciprocal+and+antagonistic+regulators+of+T+follicular+helper+cell+differentiation.">Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.</a> Science. 325 (5943), 1006-1010 (2009).</li><li>Nurieva, R. I., 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=Bcl6+mediates+the+development+of+T+follicular+helper+cells.">Bcl6 mediates the development of T follicular helper cells.</a> Science. 325 (5943), 1001-1005 (2009).</li><li>Yu, 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=The+transcriptional+repressor+Bcl-6+directs+T+follicular+helper+cell+lineage+commitment.">The transcriptional repressor Bcl-6 directs T follicular helper cell lineage commitment.</a> Immunity. 31 (3), 457-468 (2009).</li><li>Pedros, 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=A+TRAF-like+motif+of+the+inducible+costimulator+ICOS+controls+development+of+germinal+center+TFH+cells+via+the+kinase+TBK1.">A TRAF-like motif of the inducible costimulator ICOS controls development of germinal center TFH cells via the kinase TBK1.</a> Nature Immunology. 17 (7), 825-833 (2016).</li><li>Xu, H., 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=Follicular+T-helper+cell+recruitment+governed+by+bystander+B+cells+and+ICOS-driven+motility.">Follicular T-helper cell recruitment governed by bystander B cells and ICOS-driven motility.</a> Nature. 496 (7446), 523-527 (2013).</li><li>Lee, S. K., 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=B+cell+priming+for+extrafollicular+antibody+responses+requires+Bcl-6+expression+by+T+cells.">B cell priming for extrafollicular antibody responses requires Bcl-6 expression by T cells.</a> The Journal of Experimental Medicine. 208 (7), 1377-1388 (2011).</li><li>Zotos, 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=IL-21+regulates+germinal+center+B+cell+differentiation+and+proliferation+through+a+B+cell-intrinsic+mechanism.">IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism.</a> The Journal of Experimental Medicine. 207 (2), 365-378 (2010).</li><li>Vogelzang, 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=A+fundamental+role+for+interleukin-21+in+the+generation+of+T+follicular+helper+cells.">A fundamental role for interleukin-21 in the generation of T follicular helper cells.</a> Immunity. 29 (1), 127-137 (2008).</li><li>Ansel, K. M., 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=A+chemokine-driven+positive+feedback+loop+organizes+lymphoid+follicles.">A chemokine-driven positive feedback loop organizes lymphoid follicles.</a> Nature. 406 (6793), 309-314 (2000).</li><li>Shi, 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=PD-1+Controls+Follicular+T+Helper+Cell+Positioning+and+Function.">PD-1 Controls Follicular T Helper Cell Positioning and Function.</a> Immunity. 49 (2), 264-274 (2018).</li><li>Methot, S. P., Di Noia, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Somatic+Hypermutation+and+Class+Switch+Recombination.">Molecular Mechanisms of Somatic Hypermutation and Class Switch Recombination.</a> Advanced ImmunoChemical. 133, 37-87 (2017).</li><li>Crotty, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+helper+CD4+T+cells+(TFH).">Follicular helper CD4 T cells (TFH).</a> Annual Review of Immunology. 29, 621-663 (2011).</li><li>Calame, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+cells:+finding+new+light+at+the+end+of+B+cell+development.">Plasma cells: finding new light at the end of B cell development.</a> Nature Immunology. 2 (12), 1103-1108 (2001).</li><li>Yoo, J. K., Fish, E. N., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LAPCs+promote+follicular+helper+T+cell+differentiation+of+Ag-primed+CD4++T+cells+during+respiratory+virus+infection.">LAPCs promote follicular helper T cell differentiation of Ag-primed CD4+ T cells during respiratory virus infection.</a> The Journal of Experimental Medicine. 209 (10), 1853-1867 (2012).</li><li>Leon, B., Bradley, J. E., Lund, F. E., Randall, T. D., Ballesteros-Tato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxP3++regulatory+T+cells+promote+influenza-specific+Tfh+responses+by+controlling+IL-2+availability.">FoxP3+ regulatory T cells promote influenza-specific Tfh responses by controlling IL-2 availability.</a> Nature Communications. 5, 3495 (2014).</li><li>He, 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=Extracellular+matrix+protein+1+promotes+follicular+helper+T+cell+differentiation+and+antibody+production.">Extracellular matrix protein 1 promotes follicular helper T cell differentiation and antibody production.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (34), 8621-8626 (2018).</li><li>Le&#243;n, B., 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=Regulation+of+TH2+development+by+CXCR5++dendritic+cells+and+lymphotoxin-expressing+B+cells.">Regulation of TH2 development by CXCR5+ dendritic cells and lymphotoxin-expressing B cells.</a> Nature Immunology. 13 (7), 681-690 (2012).</li><li>Wang, H., 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=The+transcription+factor+Foxp1+is+a+critical+negative+regulator+of+the+differentiation+of+follicular+helper+T+cells.">The transcription factor Foxp1 is a critical negative regulator of the differentiation of follicular helper T cells.</a> Nature Immunology. 15 (7), 667-675 (2014).</li><li>Bouvier, N. M., Lowen, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+for+Influenza+Virus+Pathogenesis+and+Transmission.">Animal Models for Influenza Virus Pathogenesis and Transmission.</a> Viruses. 2 (8), 1530-1563 (2010).</li><li>Miyauchi, K., 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=Protective+neutralizing+influenza+antibody+response+in+the+absence+of+T+follicular+helper+cells.">Protective neutralizing influenza antibody response in the absence of T follicular helper cells.</a> Nature Immunology. 17 (12), 1447-1458 (2016).</li><li>Rodriguez, L., Nogales, A., Martinez-Sobrido, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+A+Virus+Studies+in+a+Mouse+Model+of+Infection.">Influenza A Virus Studies in a Mouse Model of Infection.</a> Journal of Visualized Experiments. (127), (2017).</li><li>Kitano, M., 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=Bcl6+protein+expression+shapes+pre-germinal+center+B+cell+dynamics+and+follicular+helper+T+cell+heterogeneity.">Bcl6 protein expression shapes pre-germinal center B cell dynamics and follicular helper T cell heterogeneity.</a> Immunity. 34 (6), 961-972 (2011).</li><li>Yusuf, I., 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=Germinal+center+T+follicular+helper+cell+IL-4+production+is+dependent+on+signaling+lymphocytic+activation+molecule+receptor+(CD150).">Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150).</a> The Journal of Immunology. 185 (1), 190-202 (2010).</li><li>Sun, J., Dodd, H., Moser, E. K., Sharma, R., Braciale, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+cell+help+and+innate-derived+IL-27+induce+Blimp-1-dependent+IL-10+production+by+antiviral+CTLs.">CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs.</a> Nature Immunology. 12 (4), 327-334 (2011).</li></ol>

作者没有什么可透露的。

由于在提供B细胞帮助产生高亲和力抗体方面具有特殊作用，Tbh细胞在分化和操纵机制方面进行了广泛的研究，为疫苗设计提供了新的策略。流感病毒感染诱发强大的Tfh和GC B细胞反应，因此是这一研究领域的一个适当模型。本文通过鼻内接种、流细胞学、免疫荧光和ELISA对Tfh相关反应的评价，描述了流感病毒感染的治疗方案。这些检测将有助于检测Tfh分化、GC B发育和流感病毒特异性抗体，并帮助?...

近十年来，许多研究都集中在新发现的CD4+T细胞子集Tfh细胞子集上，因为它在胚芽中心（GC）B发育中的重要作用。B细胞淋巴瘤6（Bcl6），主要被认为是一种基因抑制剂，是Tfh细胞的血统定义因子，以证明Bcl6的异位表达足以驱动Tfh分化，而Bcl6的缺乏导致Tfh分化1，2，3消失。与其他CD4+T助剂子集通过迁移到炎症部位来发挥其作用或功能不同，Tfh细胞主要在脾脏和淋巴结的B细胞卵泡区提供B细胞帮助。共刺激分子 ICOS 和 CD40L 在 Tfh 和 GC B 细胞之间的相互作用中起着重要作用。在Tfh分化过程中，ICOS从认知B细胞传输必要的信号，并充当接收来自旁观者B细胞的迁移信号的受体，用于B细胞区域定位4、5。CD40L是来自Tfh细胞的信号的调停器，用于B细胞增殖和生存6。与CD40L扮演类似角色的另一个因素是细胞因子IL21，它主要由Tfh细胞分泌。IL21直接调节GC B细胞的高亲和力抗体的开发和生产，但它在Tfh分化中的作用仍有争议。PD-1 和 CXCR5 现在最常用于识别流细胞学分析中的 Tfh 细胞，它们也在这个子集的分化和功能中发挥着重要作用。CXCR5是B细胞卵泡化学素的受体，并调解Tbh细胞在B细胞卵泡9的本地化。PD-1现已确定不仅具有卵泡导引功能，而且在GC B细胞亲和力成熟10的过程中传输临界信号。基于这些发现，评估这些分子的表达基本上可以反映Tfh细胞的成熟和功能。
GC 是继发淋巴器官中的诱导瞬态微解剖结构，高度依赖 Tfh 细胞，因此是评估 Tfh 反应的完美读出。在GC中，在接收细胞因子和共刺激分子调停的信号后，B细胞受到类开关和体细胞多造，以产生高亲和力抗体11。差异抗体类切换发生在差异细胞因子利基，其中IL4和IL21诱导IgG1类切换，而IFNγ诱导IgG2类开关12。等离子体细胞是分泌抗体的产生者，是最终分化的细胞。与Tfh细胞一样，GC中B细胞的发展与许多重要分子的动态表达有关。根据目前的研究，GC B细胞可以识别为B220+PNA+Fas+或B220+GL7+Fas+细胞和等离子体细胞，与其前体相比，B220的低调节表达和向上调节CD138表达13。此外，这两种特征都可以在流动细胞学和免疫荧光分析中检测出来，从而对GC反应进行适当的评估。
在流感病毒感染中诱导强大的细胞和幽默反应，Tfh和Th1细胞主导CD4+T细胞反应14，这使得它成为Tfh细胞分化研究的完美模型。甲型流感/波多黎各/8/34 H1N1（PR8），这是一种常用的适应鼠标的菌株，在这项研究中经常使用14，15，16。在这里，我们描述了流感病毒感染中Tfh研究相关检测的一些基本方案:1）PR8病毒的鼻内接种:2） 抗原特异性Tfh细胞、GC B和血浆细胞以及IL21检测与流细胞学:3） GC的组织学可视化:4） 检测抗原特异性抗体滴度在血清与 ELISA.这些协议为与Tfh相关的研究中的新研究人员提供了必要的技术。

<table><tbody><tr><td>&lt;strong&gt;Immunostaining of Tfh cells, NP-specific Tfh cells and Bcl-6&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>37% formaldehyde</td><td>Sigma</td><td>F1635</td><td></td></tr><tr><td>Anti-CD16/32 mouse</td><td>Thermo Fisher Scientific</td><td>14-0161-86</td><td></td></tr><tr><td>APC-conjugated-IAbNP311-325 MHC class II tetramer</td><td>NIH</td><td></td><td></td></tr><tr><td>Bcl-6 PE</td><td>Biolegend</td><td>358504</td><td>clone:7D1</td></tr><tr><td>Biotin-CXCR5</td><td>Thermo Fisher Scientific</td><td>13-7185-82</td><td>clone: SPRCL5</td></tr><tr><td>CD4 Percp-eFluor 710</td><td>Thermo Fisher Scientific</td><td>46-0041-82</td><td>clone:GK1.5</td></tr><tr><td>CD44 eVolve 605</td><td>Thermo Fisher Scientifi</td><td>83-0441-42</td><td>clone:IM7</td></tr><tr><td>CD44 FITC</td><td>Thermo Fisher Scientifi</td><td>11-0441-82</td><td>clone:IM7</td></tr><tr><td>CD62L FITC</td><td>BD Pharmingen</td><td>553150</td><td>clone:MEL-14</td></tr><tr><td>ICOS BV421</td><td>Biolegend</td><td>564070</td><td>clone:7E.17G9</td></tr><tr><td>PD1 PE/Cy7</td><td>Biolegend</td><td>135216</td><td>clone:29F.1A12</td></tr><tr><td>Streptavidin BV421</td><td>BD Pharmingen</td><td>563259</td><td></td></tr><tr><td>Streptavidin PE</td><td>BD Pharmingen</td><td>554081</td><td></td></tr><tr><td>&lt;strong&gt;Intracelluar staining of IL21&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>37% formaldehyde</td><td>Sigma</td><td>F1635</td><td></td></tr><tr><td>anti-human IgG</td><td>Jackson ImmunoResearch Laboratories</td><td>109-605-098</td><td></td></tr><tr><td>Brefeldin A</td><td>Sigma</td><td>B6542</td><td></td></tr><tr><td>human FCc IL-21 receptor</td><td>R&amp;amp;D System</td><td></td><td></td></tr><tr><td>ionomycin</td><td>Sigma</td><td>I0634</td><td></td></tr><tr><td>Live/Dead Fixable Aqua Dead Cell staining kit</td><td>Thermo Fisher Scientific</td><td>L34966</td><td></td></tr><tr><td>PMA</td><td>Sigma</td><td>P1585</td><td></td></tr><tr><td>Saponin</td><td>MP</td><td>102855</td><td></td></tr><tr><td>&lt;strong&gt;GC B and plasma cells staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>B220 APC</td><td>Thermo Fisher Scientific</td><td>17-0452-81</td><td>clone:RA3-6B2</td></tr><tr><td>CD138 PE</td><td>BD Pharmingen</td><td>561070</td><td>clone:281-2</td></tr><tr><td>CD95 (FAS) PE/Cy7</td><td>BD Pharmingen</td><td>557653</td><td>clone:Jo2</td></tr><tr><td>IgD eFluor 450</td><td>Thermo Fisher Scientific</td><td>48-5993-82</td><td>clone:11-26c</td></tr><tr><td>PNA FITC</td><td>Sigma</td><td>L7381</td><td></td></tr><tr><td>&lt;strong&gt;Assay of HA-specific antibody titer with ELISA&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PR8-HA</td><td>Sino Biological</td><td>11684-V08H</td><td></td></tr><tr><td>BSA</td><td>SSBC</td><td></td><td></td></tr><tr><td>Goat anti mouse Ig (SBA Clonotyping System-HRP)</td><td>SouthernBiotech</td><td>5300-05</td><td></td></tr><tr><td>Goat anti mouse IgM (SBA Clonotyping System-HRP)</td><td>SouthernBiotech</td><td>5300-05</td><td></td></tr><tr><td>Goat anti mouse IgG1 (SBA Clonotyping System-HRP)</td><td>SouthernBiotech</td><td>5300-05</td><td></td></tr><tr><td>Goat anti mouse IgG2b (SBA Clonotyping System-HRP)</td><td>SouthernBiotech</td><td>5300-05</td><td></td></tr><tr><td>Goat anti mouse IgG2c (SBA Clonotyping System-HRP)</td><td>SouthernBiotech</td><td>5300-05</td><td></td></tr><tr><td>TMB Substrate Reagent Set</td><td>BD Pharmingen</td><td>555214</td><td></td></tr><tr><td>&lt;strong&gt;Histology&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 555-Goat-anti rat IgG</td><td>Life Technology</td><td>A21434</td><td></td></tr><tr><td>anti-mouse IgD</td><td>Biolegend</td><td>405702</td><td></td></tr><tr><td>biotinylated PNA</td><td>Vector laboratories</td><td>B-1075</td><td></td></tr><tr><td>dilute Alexa Fluor 488-streptavidin</td><td>Life Technology</td><td>S11223</td><td></td></tr><tr><td>normal goat serum</td><td>SouthernBiotech</td><td>0060-01</td><td></td></tr><tr><td>Pro-long gold antifade reagent</td><td>Thermo Fisher Scientific</td><td>P3630</td><td></td></tr><tr><td>STREPTAVIDIN/BIOTIN blocking kit</td><td>Vector laboratories</td><td>SP-2002</td><td></td></tr></tbody></table>

evaluation of t follicular helper cells and germinal center response during influenza a virus infection in mice

T 卵泡帮助器 （Tfh） 细胞是一个独立的 CD4+ T 细胞子集，专门为细菌中心 （GC） 的发育和高亲和力抗体的生成提供帮助。在流感病毒感染中，诱导强大的Tfh和GC B细胞反应，以促进有效的病毒根除，这为Tfh相关研究提供了合格的鼠标模型。在本文中，我们描述了在小鼠感染流感病毒期间检测与Tfh相关的基本免疫反应的协议。这些协议包括:流感病毒的鼻内接种:流细胞染色和多克隆和抗原特异性Tfh细胞、GC B细胞和血浆细胞的分析:GC的免疫荧光检测;血清中流感病毒特异性抗体的酶相关免疫体检测（ELISA）。这些检测方法基本量化了Tfh细胞在流感病毒感染中的分化和功能，从而有助于研究区分机制和操作策略。

流感病毒感染中小鼠发病率的描述 流感病毒感染后，小鼠因疾病而不太活跃和厌食，这反映在严重的体重减轻上，这是监测小鼠发病率的常用症状19。如 图1a所示，PR8病毒感染的小鼠在第6天开始减肥，第8天达到最高损失水平，10日恢复到初始水平。不出所料，在PBS治疗的对照小鼠中，整个期间都没有观察到减肥。对于体内症状?...

Watch this Scientific Journal Video about Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice at JoVE.com

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

本文描述了在流感病毒感染小鼠模型中评估Tfh和GC B反应的协议。

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

evaluation-t-follicular-helper-cells-germinal-center-response-during

Research

JoVE Journal

Immunology and Infection

5.2K 次观看.  University of Chinese Academy of Sciences. 本文描述了在流感病毒感染小鼠模型中评估Tfh和GC B反应的协议。

视频: Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

科研

免疫与感染

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

医学

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

病毒合并感染后 肺炎链球菌 从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

摘要

探索更多视频

此视频中的章节

流感病毒的高通量检测方法

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

流感病毒在小鼠感染模型中的研究

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

宿主病原学反应及疫苗效果评价

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

病毒合并感染后肺炎链球菌从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

摘要

探索更多视频

此视频中的章节

流感病毒的高通量检测方法

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

流感病毒在小鼠感染模型中的研究

流感血凝素特异抗体诱导的 Fc 介导效应函数的一种评估方法

感染 Ifnar1 小鼠 Immunoprivileged 器官寨卡病毒特异 T 细胞反应的评价

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

宿主病原学反应及疫苗效果评价

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

病毒合并感染后 肺炎链球菌 从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

病毒合并感染后肺炎链球菌从定植者转变为病原体的小鼠模型概括了年龄加重的疾病