Name: rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development
Uploaded: 2018-06-19T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

我们感谢我们的技术助理: U. Bröder 和 h. Shkarlet, 他在实验过程中帮助我们。这项工作部分是由欧盟赠款 (UniVax, 601738 号合同和 TRANSVAC2, 730964号合同) 和亥姆霍兹协会赠款 (海-IDR) 资助的。资金来源不影响研究设计, 生成手稿或决定提交出版。

所有在这项研究中使用的老鼠都是从 C57BL/6 的背景。所有的动物都被保存在无病原体条件下。所有实验都是根据德国动物保护法 (TierSchG BGBl) 的规范进行的。我是 1105;25.05.1998), 并获下萨克森动物实验伦理学委员会和国家办事处 (下萨克森国家消费者保护和食品安全办公室) 批准, 许可证号为 33.4-42502-04-13/1281 和162280。
1. CFSE 细胞染色及移植
注意: OT I 小鼠是 transgenically 生成的动物, 表达 T 细胞受体 (TCR) 与固定α和β链, 共同承认的免疫优势肽的卵子, SIINFEKL10,11。结果, 这些小鼠有相当大数量的 SIINFEKL 特异性 CD8+ T 细胞 (97%)12 , 当比较正常或卵免疫小鼠 (≤ 1%)13。
<ol><li>Thy1a (Thy1.1 ) 等位基因表达 T 淋巴细胞特异性的 CD8 t 细胞的分离<ol>
<li>弄死6-9 周大鼠, 由 CO2吸入, 其次为颈椎脱位14。解剖脾脏和主要淋巴结 (腹股沟, 腋窝和颈部对只) 通过切割皮肤与外科剪刀, 从身体分离的皮肤与外科钳和镊子的帮助下, 并将其放置在培养皿 (60 x 15 毫米) 每个包含一个100µm 孔网杯, 用于组织研磨和细胞释放。</li>
		<li>在3-5 毫升完整的 RPMI 培养基 (RPMI 1640, 10% 伏/v FCS, 100 U/毫升青霉素, 50 µg/毫升链霉素) 保持在冰上的培养皿 (每一个网杯)。</li>
		<li>在注射器柱塞或相似的不育仪器的帮助下捣碎器官 (不需要事先切开) 并且收集导致的单细胞悬浮在15毫升离心机管子。</li>
		<li>离心机细胞悬浮在 300 x g 10 分钟在4°c。放弃上清。将10毫升的冷 PBS 中的细胞通过离心和溶解, 在氯化铵缓冲液1毫升/脾中重新悬浮颗粒 (ACK 缓冲器, 商用), 将红细胞从脾脏中冲洗出来。在冰上孵化细胞1.5 分钟, 然后用离心法 (如前所做) 用10毫升的冷 PBS 冲洗细胞。</li>
		<li>在1毫升的 PBS (pH 7.2) 中, 用5% 胎牛血清进行磁隔离, 将颗粒重新悬浮在同一管中。</li>
	</ol>
</li>
	<li>要进行磁隔离, 请根据制造商指令或已发布的协议15, 使用磁性隔离套件进行 CD8+ T 细胞的阴性选择。</li>
	<li>要执行 CFSE 染色, 首先计算使用自动单元计数器 (粒子计数器) 从 OT I 小鼠获得的 CD8+ T 细胞数。着色 1-5 x 107细胞/毫升在1-5 毫升的容量与5µM CFSE 在 PBS 为7分钟在37°c, 保护免受光在15毫升猎鹰管。</li>
	<li>将相同体积 (1:1) 的胎牛血清添加到细胞中, CFSE 染色, 并在37摄氏度的保护下, 将其孵化7分钟。用10毫升 PBS 清洗细胞两次。</li>
	<li>使用自动单元格计数器计数单元格。将细胞数设置为 3-5 x 107细胞/毫升的 PBS, 以注射 3-5 x 106细胞/小鼠在100µL 静脉 (静脉注射) 通过尾静脉。</li>
	<li>进行尾静脉注射, 将小鼠固定在适当的抑制剂。加热后区和尾部的小鼠被注射使用红光灯, 放置在20至25厘米以外的小鼠1-3 分钟, 以允许尾部静脉舒张。 注意: 这有助于缓解静脉检测和细胞悬浮的管理。</li>
	<li>确保 (与鼠标和灯之间的手), 它是不太热的老鼠, 当尾巴静脉清楚可见, 进行注射。用25口径针16的1mL 注射器将细胞注入侧或背尾静脉。</li>
</ol>2. 免疫接种 (游离卵子 +/-佐剂)
<ol><li>将小鼠与 OT I 细胞 (步骤 1.7,图 1) 移植到麻醉室中, 用麻醉机14在氧气中管理异氟醚。</li>
	<li>当老鼠在麻醉下完全睡着的时候, 把它从房间里拿出来, 在臀部 superficialis (侧下背部) 的地方剃掉小鼠的皮毛, 用电动理发机, 以执行干净的注射。对应用领域有很好的看法。</li>
	<li>注射50µL 的疫苗, 在被剃光区域使用25口径针。</li>
</ol>3. 流式细胞术分析中淋巴细胞和染色的分离
<ol><li>淋巴细胞与脾的分离<ol>
<li>弄死联合2吸入免疫接种小鼠, 其次为颈椎脱位。</li>
		<li>提取引流和远端淋巴结和脾脏, 并将其放置在单独的培养皿中, 含有100µm 孔网杯用于组织研磨和细胞释放。按照步骤1.1.2 通过1.1.3。</li>
		<li>离心后醒酒上清液, 并在相同体积的残余 PBS 中重新悬浮细胞颗粒 (约100µL)。</li>
	</ol>
</li>
	<li>流式细胞术分析染色<ol>
<li>在一个15毫升离心管准备一个主混合含有染色抗体的浓度在表 1中, 从而产生2x 浓缩染色混合。准备足够数量的主混合, 每样有100µL。混合细胞和主混合1:1 和孵化它在 4°c 30 分钟。 注: 每个样品的最终染色量应为200µL (100 µL 细胞颗粒 +100 µL 抗体主混合)。</li>
		<li>1.1.3 中描述的离心两次清洗细胞, 增加10毫升的 PBS, 两次。</li>
		<li>在 0.5-1 毫升的 PBS 中重新悬浮染色细胞, 用于采集和转移悬浮液, 用于流式细胞仪管。总是把样品放在冰上, 以免被光线所保护。</li>
	</ol>
</li>
</ol>4. 流式细胞仪
<ol><li>始终使用70-100 µm 过滤器预先筛选单元格样本。</li>
	<li>为表 1 (珠子或细胞) 中详细的显影准备单一染色补偿控制。 注: 应在细胞仪或分析软件17、18、19中进行补偿, 并应用于所有样品。</li>
	<li>按照图 2中描述的浇口策略进行。简要地, 门人口在正向散射高度与正向散射区域 (图 2A), 并且再侧散布宽对侧散布区域 (SSA,图 2B) 为了排除双峰。</li>
	<li>通过绘制 BV 650 (自动荧光) 与 FITC (CFSE) 来区分来自高自动荧光细胞的真实 CFSE 染色细胞, 从图 2B中进行填充。包括用与 BV 650 共轭的抗体染色的珠子或细胞纳入补偿控制。这一剧情 (图 2C) 的 BV 650 vs FITC 是用来门 CFSE 染色细胞。</li>
	<li>从图 2C中的 Pe-Cy7 (Thy1.1-CD90.1) 与 APC (CD8) 进行比较, 以区分来自捐赠者和受体小鼠的细胞。 注: 从 OT I. 捐献小鼠获得的细胞为 Thy1.1+ (CD90.1), 而 C57BL/6、受体) 小鼠派生细胞为 Thy1.2+ (CD90.2) (图 2D)。一些实验室有 Thy1.2 背景下的小鼠和 C57BL/6 (Thy1.1)。在这种情况下, 你必须使用抗体对抗 Thy1.2 细胞门。</li>
	<li>通过在 450 (CD4) vs APC (CD8) (图 2E) 的栅极上绘制 Thy1.1+填充的 CD8+细胞。</li>
	<li>通过使用显示 CFSE (FITC, 530/15 通道-蓝色激光) 的 CD8+单元格的直方图显示, 在图 2E中显示填充门, 通过将强度从 102增至水平处, 将激增的人口包括在内不分割控制人口是 (强度 ~ 105, 取决于 CFSE 染色的功效和流量式细胞仪的电压设置) (图 2F)。</li>
	<li>制作一个额外的门 (不显示在图2中) 绘制 FITC 与 L/D 标记 (450/20 通道, 紫外线激光), 以评估死细胞平行的扩散评估。</li>
	<li>从流式细胞仪的样本中获取至少5000项补偿控制和10.000 事件 (门 E,图 2) 的事件。在低或中流运行细胞仪 (不超过20.000 事件/秒的流速)。</li>
</ol>

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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=Type+I+interferon+protects+antiviral+CD8++T+cells+from+NK+cell+cytotoxicity.">Type I interferon protects antiviral CD8+ T cells from NK cell cytotoxicity.</a> Immunity. 40, 949-960 (2014).</li><li>Volk, 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=Comparison+of+three+humanized+mouse+models+for+adoptive+T+cell+transfer.">Comparison of three humanized mouse models for adoptive T cell transfer.</a> The journal of gene medicine. 14, 540-548 (2012).</li><li>Safinia, N., 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=Humanized+Mice+as+Preclinical+Models+in+Transplantation.">Humanized Mice as Preclinical Models in Transplantation.</a> Methods Mol Biol. 1371, 177-196 (2016).</li><li>Grover, 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=Humanized+NOG+mice+as+a+model+for+tuberculosis+vaccine-induced+immunity:+a+comparative+analysis+with+the+mouse+and+guinea+pig+models+of+tuberculosis.">Humanized NOG mice as a model for tuberculosis vaccine-induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis.</a> Immunology. 152, 150-162 (2017).</li></ol>

作者没有什么可透露的。

现代疫苗是理想的 composedof 纯化抗原和佐剂, 有可能添加一个交付系统, 如脂质体, 病毒样粒子, 纳米粒子或活载体。设计疫苗的一个关键方面是根据临床需要选择合适的佐剂。部分的范围可能涉及倾向于体液与细胞免疫应答 (或两者), 选举局部与系统性免疫应答 (或两者), 以及这种疫苗必须在目标人群中产生的记忆。辅助评价的一个关键方面是迅速确定其产生 CTL 的能力。本文提出了一种基于已知?...

细胞毒性 T 淋巴细胞 (CTL) 诱导疫苗是为抗击某些类型的癌症而开发的关键治疗措施1。CTL 对预防性疫苗对细胞内病原体2也很重要。此外, ctl 是少数的免疫防御机制之一的功能活跃的风险人群, 如新生儿3,4谁也依赖 CTL, 以抗击早期生命感染5。在这方面, 与不引起 CTL 反应的佐剂一起研制的呼吸道合胞病毒疫苗 (明矾) 导致6婴儿感染严重并发症导致的疫苗完全失败。免疫接种的这些负面影响可以通过 CD8 细胞反应 7逆转。我们以前已经证明, 一些干扰素基因 (蜇) 激动剂诱发的主要细胞因子 (I. 干扰素) 对这些佐剂8产生的 CTL 反应至关重要, 部分是通过测量在疫苗接种后的 T 细胞, 并将这些结果作为在延长接种计划9中观察到的 CTL 诱导能力的量度。用羧基琥珀酰亚胺酯 (CFSE)染料稀释法测定野生型 (CD8) C57BL/6 受体小鼠中的 OT I. T 细胞的增殖是一种对疫苗佐剂能力的稳健估计, 可产生SIINFEKL 的交叉启动 (卵清蛋白的免疫主导肽, 卵)。这种技术的变异被广泛用于评估 CD8 和 CD4 T 细胞的增殖. 例如, 它已被用于没有选定的细胞因子 (高鼠) 或测量疫苗的功效后, 抗原召回的动物。我们设计了一个简短的协议 (4 天的实验), 在 CFSE 染色的 CD8+ T 细胞被动转移后, 皮下 (南卡罗来纳州) 免疫由一剂量50µg 的无内毒素卵子辅以测试佐剂被管理(图 1)。疫苗接种后48小时随访结果提供了可靠的证据证明佐剂产生 CTL 反应的能力。通过这一策略, 可以评估免疫后引流淋巴结局部免疫反应的效力, 以及测量脾脏 (或远端淋巴结) CTL 活动的反应程度。

<table><tbody><tr><td>BD LSR Fortessa Cell Analyzer</td><td>BD</td><td>Special Order</td><td>Flow Cytometer</td></tr><tr><td>CFSE</td><td>Molecular Probes</td><td>C34554</td><td>Proliferation Dye</td></tr><tr><td>MojoSort Mouse CD8 T Cell Isolation Kit</td><td>Biolegend</td><td>480007</td><td>Magnetic Isolation Beads and antibodies for negative selection of untouched CD8 T cells.</td></tr><tr><td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td><td>Molecular Probes</td><td>L23105</td><td>Dead Cell Marker</td></tr><tr><td>CD90.1 (Thy-1.1) Monoclonal Antibody (HIS51), PE-Cyanine7</td><td>eBioscience</td><td>25-0900-82</td><td>antibody</td></tr><tr><td>APC anti-mouse CD8a Antibody</td><td>BioLegend</td><td>100712</td><td>antibody</td></tr><tr><td>BV421 Rat Anti-Mouse CD4</td><td>BD</td><td>740007</td><td>antibody</td></tr><tr><td>Z2 coulter Particle count and Size Analyzer</td><td>Beckman Coulter</td><td>9914591DA</td><td>Cell counter. Z2 Automated particle/cell counter</td></tr><tr><td>EndoGrade Ovalbumin (10 mg)</td><td>Hyglos(Germany)</td><td>321000</td><td>Ovalbumin endotoxin free tested.</td></tr><tr><td>Cell Strainer 100&amp;micro;m nylon</td><td>Corning</td><td>352360</td><td>Cell strainer (100 &amp;micro;m pore mesh cups).</td></tr><tr><td>Sample Vials</td><td>Beckman Coulter</td><td>899366014</td><td>Sample vials for Z2 automated counter</td></tr><tr><td>C57BL/6 mice (CD90.2)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Company is now Envigo</td></tr><tr><td>OT-I (C57BL/6 background, CD90.1)</td><td>Harlan (Rossdorf, Germany)</td><td></td><td>Inbreed at our animal facility. Company from where adquired is now Envigo</td></tr><tr><td>FACS tubes</td><td>Fischer (Corning)</td><td>14-959-5</td><td>Corning Falcon Round-Bottom Polystyrene Tubes</td></tr><tr><td>Falcon 15 mL tubes</td><td>Fischer (Corning)</td><td>05-527-90</td><td>Falcon 15mL Conical Centrifuge Tubes</td></tr><tr><td>PBS (500 mL)</td><td>Fischer (Gibco)</td><td>20-012-027</td><td>Gibco PBS (Phosphate Buffered Saline), pH 7.2</td></tr><tr><td>Red lamp (heating lamp)</td><td>Dirk Rossmann GmbH (Germany)</td><td>405096</td><td>Heating infrred lamp (100 wats)</td></tr><tr><td>IsoFlo (Isoflurane)</td><td>Abbott Laboratories (USA)</td><td>5260.04-05.</td><td>Isoflurane anesthesic (250 mL flask).</td></tr><tr><td>&lt;strong&gt;Tabletop Anesthesia Machine/Mobile Anesthesia Machine with CO2 Absorber&lt;/strong&gt;</td><td>Parkland Scientific</td><td>V3000PK</td><td>Isoflurane anesthesia machine.</td></tr><tr><td>RPMI 1640 medium</td><td>Gibco (distributed by ThermoFischer)</td><td>11-875-093</td><td>Base medium with Glutamine (500 mL)</td></tr><tr><td>Pen-Strept antibiotic solution (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>15-140-148</td><td>Gibco Penicillin-Streptomycin (10,000 U/mL)</td></tr><tr><td>Fetal Bobine Serum (Gibco)</td><td>Gibco (distributed by ThermoFischer)</td><td>10082147</td><td>Fetal Bovine Serum, certified, heat inactivated, US origin</td></tr><tr><td>ACK Lysing Buffer (100 ml)</td><td>Gibco (distributed by ThermoFischer)</td><td>A1049201</td><td>Amonium Chloride Potasium (ACK) Whole Blood Lysis Buffer, suitable for erytrocyte lysis in spleen suspensions also</td></tr><tr><td>Plastic Petri Dishes</td><td>Nunc (distributed by ThermoFischer)</td><td>150340</td><td>60 x 15mm Plastic Petri Dish, Non-treated</td></tr><tr><td>Cell Clump Filter</td><td>CellTrics (Sysmex)</td><td>04-004-2317</td><td>CellTrics&amp;reg; 50 &amp;mu;m, sterile</td></tr></tbody></table>

rapid in vivo assessment of adjuvant's cytotoxic t lymphocytes generation capabilities for vaccine development

对现代分单元疫苗的评估表明, 中中和抗体的产生是重要的, 但不足以辅助选择。因此, 迫切需要具有体液和细胞免疫刺激能力的佐剂, 能够促进细胞毒 T 淋巴细胞 (CTL) 反应。因此, 对诱导交叉启动并随后增强 CTL 生成的佐剂候选者的忠实监测是疫苗开发的关键步骤。在这里, 我们提出一个方法的应用, 使用 SIINFEKL 特定 (OT) T 细胞监测模型抗原卵清蛋白 (卵)在体内的交叉呈现, 在不同的佐剂候选者在场。该方法是一种快速试验, 用于选择具有最佳交叉启动能力的佐剂。CD8+ T 细胞的增殖是交叉引发的最有价值的标志, 同时也被认为是佐剂诱导的交叉表达的相关性。这一特点可以评估在不同的免疫器官, 如淋巴结和脾脏。CTL 生成的程度也可以监测, 从而对局部 (引流淋巴结主要) 的性质或系统性反应 (远端淋巴结和/或脾脏) 有深入的了解。这项技术进一步允许对检测药物进行多次修改, 以抑制特定的交叉表达通路, 并提供了可能用于不同品系的常规和转基因小鼠。总之, 我们在这里提出的应用将是有用的在工业或学术界的疫苗实验室开发或修改疫苗研究和发展的化学佐剂。

为了测试使用不同佐剂 (ADJ1 和 ADJ2) 的治疗方法, 我们通过流式细胞仪测量过继转输转移的 CD8+ T 细胞的增殖来评估 CTL 的生成能力 (图 2)。为此, 我们以前从引流淋巴结和脾脏中分离出细胞 (表 1)。通过测量淋巴结和脾脏中 CD8+ T 细胞的增殖, 我们能够证实在引流淋巴结中 ADJ2 的 CTL 生成能力 (图 3) 与 A...

Watch this Scientific Journal Video about Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development at JoVE.com

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

我们在这里提出一个标准免疫技术 (CFSE 染色的应用), 旨在快速监测佐剂介导的细胞毒性 T 淋巴细胞 (CTL) 生成在体内.这种对 CTL 容量的快速估计有助于开发预防性疫苗, 防止细胞内病原体和治疗性癌症疫苗的发展。

疫苗开发中佐剂细胞毒性 T 淋巴细胞生成能力的快速体内评价

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

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Research

JoVE Journal

Medicine

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

8.6K 次观看.  Helmholtz Centre for Infection Research. 我们在这里提出一个标准免疫技术 (CFSE 染色的应用), 旨在快速监测佐剂介导的细胞毒性 T 淋巴细胞 (CTL) 生成在体内.这种对 CTL 容量的快速估计有助于开发预防性疫苗, 防止细胞内病原体和治疗性癌症疫苗的发展。

视频: Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

科研

免疫与感染

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

荧光目标阵列的T细胞反应评价中的应用<em&gt;在体内</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

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 ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

免疫刺激性剂评价: 淋巴组织提取与注射路径依赖性树突状细胞活化

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

鼻自组装纳米乳肿瘤疫苗体外和体内的制备、特性、毒性及疗效评价

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

体外 纳米乳剂疫苗佐剂蛇腓磷素D的细胞活性评价

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

使用牛单核细胞来源的树突状细胞测定疫苗免疫原性

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...

An Immunological Technique to Monitor Adjuvant-Mediated Cytotoxic T Lymphocyte Generation

Source: Lirussi, D. et al., <a href="https://app.jove.com/v/57401">Rapid In Vivo&#160;Assessment of Adjuvant&#39;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development.</a> J. Vis. Exp. (2018)
The video demonstrates an immunological method to monitor in vivo adjuvant-induced cytotoxic T lymphocyte or CTL production. A mouse is pre-injected with genetically engineered, proliferative dye-stained donor CD8+ T lymphocytes. Subcutaneous injection of ovalbumin with adjuvants generates cytotoxic CD8+ T lymphocytes that are identified post-harvesting through immunostaining and flow cytometry.

一种监测佐剂介导的细胞毒性 T 淋巴细胞生成的免疫学技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
All mice ...

疫苗开发中佐剂细胞毒性 T 淋巴细胞生成能力的快速体内评价

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