Name: in vivo assay for detection of antigen-specific t-cell cytolytic function using a vaccination model
Uploaded: 2017-11-28T12:30:00.000+00:00
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Description: Watch this Scientific Journal Video about In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model at JoVE.com

这项工作得到 NIH 研究 (A1R03AI120027 (rn) 和 1R21AI20012 (rn)), 机构研究补助金 (rn), 启动补助金 (rn) 和 MD 安德森 CIC 种子补助金 (rn) 的支持。

这里描述的所有方法都得到了德克萨斯大学安德森癌症中心的机构动物保育和使用委员会 (IACUC) 的批准。
1. 疫苗用肽的制备
<ol><li>将冻干肽与磷酸盐缓冲盐水 (PBS) 溶于三十年代的适当浓度和涡旋。 注: 对于 hgp10025-33, 最终浓度为1毫克/毫升, 对于卵子257-264, 最终浓度为0.5 毫克/毫升。注射前重组肽。重建后不要存储。</li>
</ol>2. 转基因小鼠脾的分离
注意: 细胞与脾脏的分离必须以不育的方式进行。
<ol><li>
安乐 OT-1 转基因小鼠使用经批准的 CO2窒息法并切除脾脏。 注意: 在这一步中, 使用合适的转基因小鼠来选择特定的肽。<ol>
<li>将鼠标放在右侧。用70% 乙醇 (乙醇) 喷洒鼠标左侧。用钳子拉起皮肤, 用外科剪刀切开皮肤;脾脏在腹膜腔内可见。轻轻切开腹膜腔, 进入脾脏。用镊子取出脾。</li>
	</ol>
</li>
	<li>
将脾脏放在一个70µm 过滤器中, 用2毫升培养基 a (PBS 1% 胎牛血清 (FBS)), 用柱塞末端粉碎脾脏。
	<ol>
<li>从培养皿中收集脾, 并在50毫升锥形管中放置。用两次5毫升培养基冲洗培养皿, 收集所有细胞。加入培养基 A 至25毫升, 在室温下离心5分钟, 475 x g。</li>
	</ol>
</li>
	<li>在1毫升/每脾红血球 (RBC) 裂解缓冲液中吸出细胞和重。室温孵育5分钟. 在室温下加入10毫升的培养基 a 和离心机, 在 475 x g. 重在10毫升中 a 的颗粒, 并通过过滤细胞悬浮通过一个70µm 过滤到一个干净的50毫升圆锥。</li>
	<li>
使用台盼蓝和例计数细胞。PBS 中的重细胞最终浓度为 106 -1006单元/mL。对于卵子257-264特定的杀戮, 重在 106单元/mL 和为 hgp10025-33特定的杀戮, 重在 1006单元/ml。
	<ol>
<li>在室温下旋转剩余的细胞5分钟, 475 x g. 在15毫升冷 PBS 中吸取上清和重以洗涤细胞。再重复一次洗涤步骤。在室温下旋转细胞5分钟, 475 x g. 根据步骤2.4 中确定的最终体积, 在冷 PBS 中吸取上清和重。</li>
		<li>将单细胞悬浮液转移到1.5 毫升离心管上, 并将其保持在冰上直到注射入受体 C57/BL6 鼠。</li>
	</ol>
</li>
</ol>3. 转基因小鼠注射脾
<ol><li>将接受 C57/BL6 的老鼠放在抑制剂上, 背侧向上。喷淋尾基区与70% 异丙醇。管理100µL 单细胞悬架 (第2节) 静脉注射到尾部静脉使用27克针与斜面面朝上。</li>
</ol>4. Covax 行政
注: 如果在下午注射细胞, covax 应在第二天早上18小时内进行细胞注射。
<ol><li>将鼠标放在一个透明的麻醉箱中, 1-3% 异氟醚。小鼠完全麻醉后, 将小鼠转移到麻醉室中附着在歧管上的鼻锥。将小鼠的背侧保持克制。 注: 麻醉被确认为一个简短的脚趾捏, 以验证撤回反应没有被诱发。联邦法律限制异氟醚的使用顺序的特许兽医。</li>
	<li>喷淋尾基区与70% 异丙醇。注入100µL 的肽溶液 (从1节) 皮下注射使用27克针与注射器穿透 4-5 毫米进入尾部基地区域, 针锥面朝上。 注意: 小鼠在一个侧面接种了皮下注射在尾部的底部10。</li>
	<li>注射100µL anti-CD40 抗体 (0.5 毫克/毫升的股票) 侧向疫苗注射场。</li>
	<li>小心应用莫特霜50毫克/鼠标在疫苗接种点。在表面擦莫特奶油, 直到奶油不再可见并完全吸收。</li>
	<li>在下腹区注射100µL 10万毫升 rhIL-2 蛋白腹腔 (ip)。观察小鼠5分钟后, 他们完全恢复从麻醉。 注: 在这一点上, 实验暂停3天。</li>
</ol>5. CFSE 标靶脾的分离
注意: 细胞与脾脏的分离必须以不育的方式进行。
<ol><li>根据批准的 CO2窒息方法安乐 C57BL/6 小鼠。移除脾脏, 如步2.1.1。将脾脏和洗涤脾的步骤 2.2.1 2.2.3。溶解红血球, 如步骤 2.3-2.3.2。</li>
	<li>移除使用例计数的单元格, 并在 CFSE 标记解决方案 (步骤7中描述) 中计算 106单元/mL 的最后浓度的单元格。在室温下旋转剩余的细胞5分钟, 在 475 x g。</li>
</ol>6. 靶脾的多肽脉冲
注: 多肽脉冲必须以无菌的方式进行。
<ol><li>重细胞在 106细胞/毫升在完整的媒体 (RPMI 1640 媒体与 10% FBS, 1% l-谷氨酰胺 (l-酰), 1% 青霉素/链霉素 (笔/链球菌))。将细胞分成2管 (15 毫升 conicals)。将每个管的标签为脉冲或 unpulsed。</li>
	<li>
将肽添加到标有脉冲的管中。对于卵子257-264脉冲, 添加1µg/ml, 并为 hgp1002533脉冲, 添加2µg/ml。 注: unpulsed 细胞与脉冲细胞进行相同的孵育, 而不增加肽。<ol>
<li>孵育细胞 + 肽在37° c 为 1 h。</li>
	</ol>
</li>
	<li>添加10毫升的完整媒体 (RPMI 1640 媒体与 10% FBS, 1% l-谷氨酰胺 (l-酰), 1% 青霉素/链霉素 (笔/链球菌)), 以清洗两个脉冲和 unpulsed 靶细胞。在室温下旋转剩余的细胞5分钟, 在 475 x g。</li>
</ol>7. 用于标记目标脾的 CFSE 的制备
<ol><li>
在步骤6.3 中的细胞洗涤过程中准备 CFSE 标记溶液;脉冲单元格将被标记为 CFSEhi , unpulsed 为 CFSElo。为 106单元准备1毫升的 CFSE 标记解决方案。 注意: CFSE 是轻的敏感和应该保护免受光在任何时候。<ol>
<li>准备 CFSEhi -通过添加 1 uL/ml CFSE (5 毫米的库存解决方案) 为最终浓度为5µM/毫升在 RPMI 媒体1640与 2% FBS。</li>
		<li>准备 CFSElo -通过添加 1 uL/ml CFSE (0.5 毫米的库存解决方案) 为最终浓度为0.5 µM/毫升 RPMI 媒体1640与 2% FBS。</li>
	</ol>
</li>
</ol>8. 用 CFSE 标记目标脾
注: CFSE 标签必须以无菌的方式进行。
<ol><li>重脉冲和 unpulsed 目标单元格 (从步骤 6.3) 到 106单元格/mL CFSE 标记介质。重用准备好的 CFSEhi标记介质和带有准备好的 CFSElo标记介质的 unpulsed 单元进行脉冲电池的使用。</li>
	<li>混合细胞和 CFSE 标签媒体通过温和的反转或旋转。不要由涡流混合。孵育细胞和 CFSE 标记介质在37° c 15 min. 混合细胞每5分钟。</li>
	<li>在室温下将10毫升的完整介质添加到每个细胞悬浮和旋转细胞中, 5 分钟, 475 x g。</li>
	<li>在10毫升冷 PBS 中抽吸上清和重细胞。旋转细胞在4° c 为5分钟在 475 x g。</li>
	<li>重复步骤8.4。</li>
	<li>计数的细胞和混合肽脉冲, CFSEhi-标记为 unpulsed, CFSElo-标记的细胞在1:1 的比率注射到受体小鼠。将分 1x 106混合单元用于基线流式细胞仪评估 (第11节)。 注: 注射的最终体积为每只老鼠100µL。最后的细胞计数是10e6 细胞。需要考虑到注射器和针头的体积损失, 估计为500µL。</li>
</ol>9. 注射靶细胞
注意: 在注射之前和期间尽量保持 CFSE 标记的细胞免受光的保护。
<ol><li>将受体 C57BL/6 的老鼠放在抑制剂, 背侧向上。喷淋尾基区与70% 异丙醇。将100µL 的单细胞悬浮液静脉注射到尾静脉, 并朝上。</li>
</ol>10. 目标细胞的重新隔离
注意: 这一步的时间是关键的, 依赖于 CTL 的细胞毒性和刺激抗原的强度。为了评估杀死一个卵子257-264脉冲目标, 需要在注入后 4-6 小时内捕获细胞。由于 CFSE 标记细胞是轻的敏感, 过程脾脏在黑暗中。
<ol><li>根据批准的 CO2窒息方法, 安乐接受者 C57BL/6 小鼠。</li>
	<li>移除脾脏, 如步2.1.1。将脾脏和洗涤脾的步骤 2.2.1 2.2.3。溶解红血球, 如步骤 2.3-2.3.2。</li>
	<li>重细胞在1毫升荧光活化细胞分选 (外地) 缓冲 (1% BSA + PBS) 的评估流式细胞仪。</li>
</ol>11. 用流式细胞仪测定 CTL 活性的浇注逻辑
<ol><li>
使用标准流式细胞仪协议对 CTL 活动进行评估。使用 488 nm 激光器的流式细胞仪上的 FITC 通道获取单元, 用于激发11。
	<ol>
<li>使用前向散射 (FSC) vs 侧散射 (SSC) 参数的活淋巴细胞门。Subgate 在活淋巴细胞门之内为总 CFSE 阳性人口。 注: 注射 CFSE 标记的细胞将构成脾脏中总淋巴细胞的一个小子集。CSFE 阳性细胞应在对数刻度上显示为两个不同的种群。</li>
		<li>使用直方图格式确定 unpulsed (左峰值、CFSElo) 和脉冲 (右峰值、CFSEhi) 填充的百分比。CFSEhi单元格的丢失指示特定于抗原的 CTL 活动。</li>
	</ol>
</li>
</ol>

<ol>
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作者没有什么可透露的。

虽然该协议很简单, 但还有一些关键步骤必须仔细执行。covax 注射后的抗原特异性 T 细胞被测试是必要的, 看到任何杀害的脉冲目标。虽然水基 covax 疫苗可能造成急性炎症状态, 为慢性炎症阶段, 取代短命的水基配方与缓慢抗原释放油的方法可能会产生更好的结果7,12。
此外, 对于理想的流式细胞仪, 在抗原脉冲后的靶细胞的 CFSE 标记?...

存在多个协议来评估 CD8+或 CD4+ T 单元格的细胞 (CTL) 电位。这种评估可以很容易地做在体外在受控条件下1,2,3。此外, 传染病模型, 如 LCMV, 通过使用差异 CFSE (5-(和 6)-羧基双乙酸琥珀酰亚胺酯) 对 CTL 功能进行了经典的检查, 标记为 CFSEhi标记的单元格的目标单元格脉冲与多肽和 CFSElo标记的目标细胞是左 unpulsed。然后, 以1:1 的比率注入细胞, 并通过流式细胞仪4对 CFSEhi标记的脉冲目标的丢失进行评估。疫苗和排斥模型也使用类似的策略, 评估的在体内杀害的 CD8+和 CD4+ T 细胞以及 NK 细胞5,6。这是一个强有力的化验, 但需要使用的传染性药物, 在靶注射前免疫系统的首要。
这个协议, 另一方面, 不需要事先感染的主机, 而是利用疫苗接种策略, 以在目标注射前的免疫系统的首要。这种疫苗接种是由多肽疫苗的水基配方, 需要提供一种免疫鸡尾酒称为 covax7, 由 Toll 样受体 7 (TLR7) 激动剂 (莫特霜), 竞赛 anti-CD40抗体和 interleukin-2 (IL-2) 导致免疫剂的协同组合, 以诱导肽特异性启动和强健的免疫应答。因此, 这种化验提供了一个 CTL 功能的快速读数, 因为疫苗是在注射靶细胞前的三天内与细胞一起进行功能评估的。此外, covax 启动足够强, 注射后的抗原特异性 T 细胞的杀伤能力可以看到4和24小时。
该协议的主要限制是确定时间轴在体内检测目标杀死, 这是适当的抗原和假设被测试。必须进行仔细的评估, 因为抗原强度的变化以及在 T 细胞中测试的基因改变可能会导致不同的 CTL 功能, 这就需要对目标杀伤的定时检测。此外, 虽然适当浓度的肽接种已优化人类黑色素瘤抗原糖 100 (hgp10025-33) 和蛋白257-264 (卵257-264)8,9 ,使用另一个可能更适合于某项研究的抗原模型将需要进一步验证。由于预期的目标抗原的能力, 以刺激 CTL 效应功能与 covax 作为佐剂, 优化 IL-2 剂量浓度和剂量频率可能是必不可少的, 以达到预期的目标。总的来说, 该协议允许快速评估杀死功能在体内, 可以适应任何给定的抗原。

<table><tbody><tr><td>6 to 12 week old female C57BL/6 mice&amp;nbsp;</td><td>Charles River</td><td>027</td><td>C57 Black 6 mice</td></tr><tr><td>OT-1 6-12 week old female mice&amp;nbsp;</td><td>Jackson Labs</td><td>003831</td><td></td></tr><tr><td>hgp100&lt;sub&gt;25&amp;ndash;33&lt;/sub&gt;&amp;nbsp;</td><td>CPCScientific</td><td>834139</td><td>KVPRNQDWL</td></tr><tr><td>OVA &lt;sub&gt;257&amp;ndash;264&lt;/sub&gt;&amp;nbsp;</td><td>CPCScientific</td><td>MISC-012</td><td>SIINFEKL</td></tr><tr><td>Imiquimod cream 5%&amp;nbsp;</td><td>Fougera</td><td>51672-4145-6</td><td>Aldara Cream</td></tr><tr><td>CD40-specific mAb&amp;nbsp;</td><td>BioXcell</td><td>BE0016-2</td><td>clone FGK4.5</td></tr><tr><td>rhIL-2 protein&amp;nbsp;</td><td>Hoffman LaRoche Inc</td><td>136</td><td>Recombinant human IL-2 protein</td></tr><tr><td>70% isopropyl alcohol Prep&amp;nbsp;</td><td>Kendall</td><td>S-17474</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>10010-023</td><td>Phosphate Buffered Saline</td></tr><tr><td>FBS</td><td>Life Technologies</td><td>26140-079</td><td>fetal bovine serum</td></tr><tr><td>RBC lysis buffer&amp;nbsp;</td><td>Life Technologies</td><td>A10492-01</td><td>red blood cell lysis buffer</td></tr><tr><td>RPMI 1640 Media</td><td>Life Technologies</td><td>11875119</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Life Technologies</td><td>25030081</td><td></td></tr><tr><td>Pen/Strep</td><td>Life Technologies</td><td>15140122</td><td>penicillin/streptomycin</td></tr><tr><td>CFSE</td><td>Life Technologies</td><td>C34554</td><td>5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma&amp;nbsp;</td><td>A4503</td><td></td></tr><tr><td>1.5 mL MCT graduated natural&amp;nbsp;</td><td>Fisher</td><td>05-408-129</td><td>microcentrifuge tube</td></tr><tr><td>70% ethanol</td><td>Fisher</td><td>BP8201500</td><td>EtOH</td></tr><tr><td>Trypan blue solution, 0.4%&amp;nbsp;&amp;nbsp;</td><td>Life Technologies</td><td>15250-061</td><td></td></tr><tr><td>Hemocytometer</td><td>Fisher</td><td>267110</td><td></td></tr><tr><td>27 gauge needle</td><td>BD</td><td>305109</td><td></td></tr><tr><td>1 mL syringe</td><td>BD</td><td>309659</td><td></td></tr></tbody></table>

in vivo assay for detection of antigen-specific t-cell cytolytic function using a vaccination model

目前用于抗原特异性致死的方法仅限于在感染性疾病模型中使用或应用于体外。然而, 没有一项专门用于测量不感染抗原特异的杀伤的协议。本协议设计并描述了克服这些限制的方法, 允许通过 CD8+ T 细胞体内检测目标细胞的抗原特异性杀伤。这是通过将疫苗接种模型与传统的 CFSE 标靶杀伤试验结合起来完成的。这种结合使研究人员能够直接和快速地评估抗原特异的 CTL 电位, 因为这种检测不依赖于肿瘤的生长或感染。此外, 读数是基于流式细胞仪, 因此应该容易接近大多数研究员。该研究的主要限制是确定适合于所测试的假设的时间轴在体内。在 T 细胞的抗原强度和突变的变化, 可能导致差异细胞功能需要仔细评估, 以确定最佳时间的细胞收获和评估。对于 hgp10025-33和卵子257-264, 已优化了用于疫苗接种的适当浓度的肽, 但对于可能更适合于特定研究的其他肽, 则需要进一步验证。总的来说, 该协议允许快速评估杀死功能在体内, 可以适应任何给定的抗原。

在注射 CFSE 标记的靶细胞之前, 在流式细胞仪上运行1:1 单元混合物, 以确定 CFSEhi和 CFSElo目标单元格的基线频率。图 1A显示了用于检测 CFSE 种群变化的浇口策略, 使用 FSC 和 SSC 参数进行初始化。在评估频率变化之前, 总的 CFSE 阳性细胞 subgated, 因为相对于未标记的内生脾, 这个种群的数量比较小。CFSEhi和 CFSElo</su...

Watch this Scientific Journal Video about In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model at JoVE.com

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

该协议的目标是允许检测在小鼠模型中的靶细胞的体内抗原特异性杀伤。

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 ...

in-vivo-assay-for-detection-antigen-specific-t-cell-cytolytic

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

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

9.8K 次观看.  University of Texas MD Anderson Cancer Center. 该协议的目标是允许检测在小鼠模型中的靶细胞的体内抗原特异性杀伤。

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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

科研

免疫与感染

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

癌症研究

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

体内 通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

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 ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

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 ...

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 ...

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

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

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

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

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. ...

医学

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

Source: Lirussi, D. et al., <a href="https://appjove.unicartagenaproxy.elogim.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 ...

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

摘要

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此视频中的章节

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

体内通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

荧光目标阵列的T细胞反应评价中的应用

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

一种研究人体 T 细胞召回反应的吸水泡协议

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

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

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

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

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

摘要

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此视频中的章节

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

体内 通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

荧光目标阵列的T细胞反应评价中的应用

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

一种研究人体 T 细胞召回反应的吸水泡协议

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

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

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

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

体内通过脾T细胞流式细胞术筛选肿瘤来源的细胞外囊泡的免疫原性

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

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