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。

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		<col style="width:110pt" width="146" />
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			<td height="20" style="width:199pt;height:15.0pt" width="265">6- to 12-week old female C57BL/6 mice&#160;</td>
			<td class="xl69" style="width:110pt" width="146">Charles River</td>
			<td class="xl70" style="width:89pt" width="119">027</td>
			<td style="width:125pt" width="167">C57 Black 6 mice</td>
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			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">OT-1 6-12-week old female mice&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Jackson Labs</td>
			<td class="xl70" style="width:89pt" width="119">003831</td>
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			<td height="24" style="height:18.0pt">hgp10025&#8211;33&#160;</td>
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			<td class="xl70" style="width:89pt" width="119">834139</td>
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			<td height="24" style="height:18.0pt">OVA 257&#8211;264&#160;</td>
			<td class="xl69">CPCScientific</td>
			<td class="xl70" style="width:89pt" width="119">MISC-012</td>
			<td>SIINFEKL</td>
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		<tr height="20" style="height:15.0pt">
			<td height="20" style="height:15.0pt">Imiquimod cream 5%&#160;</td>
			<td class="xl69">Fougera</td>
			<td class="xl73">51672-4145-6</td>
			<td>Aldara Cream</td>
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		<tr height="20" style="height:15.0pt">
			<td height="20" style="height:15.0pt">CD40-specific mAb&#160;</td>
			<td class="xl69">BioXcell</td>
			<td class="xl70" style="width:89pt" width="119">BE0016-2</td>
			<td>clone FGK4.5</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td height="20" style="height:15.0pt">rhIL-2 protein&#160;</td>
			<td class="xl69">Hoffman LaRoche Inc</td>
			<td class="xl70" style="width:89pt" width="119">136</td>
			<td>Recombinant human IL-2 protein</td>
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		<tr height="20" style="height:15.0pt">
			<td height="20" style="height:15.0pt">70% isopropyl alcohol Prep&#160;</td>
			<td class="xl69">Kendall</td>
			<td class="xl70" style="width:89pt" width="119">S-17474</td>
			<td></td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td height="20" style="height:15.0pt">PBS</td>
			<td class="xl69">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">10010-023</td>
			<td>Phosphate Buffered Saline</td>
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		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">FBS</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">26140-079</td>
			<td>fetal bovine serum</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">RBC lysis buffer&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">A10492-01</td>
			<td>red blood cell lysis buffer</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">RPMI 1640 Media</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">11875119</td>
			<td></td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">L-Glutamine&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">25030081</td>
			<td></td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Pen/Strep</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">15140122</td>
			<td>penicillin/streptomycin</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">CFSE</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">C34554</td>
			<td>5-(and 6)-Carboxyfluorescein diacetate succinimidyl ester</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Bovine serum albumin (BSA)</td>
			<td class="xl70" style="width:110pt" width="146">Sigma&#160;</td>
			<td class="xl70" style="width:89pt" width="119">A4503</td>
			<td></td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">1.5 mL MCT graduated natural&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Fisher</td>
			<td class="xl70" style="width:89pt" width="119">05-408-129</td>
			<td>microcentrifuge tube</td>
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		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">70% ethanol</td>
			<td class="xl70" style="width:110pt" width="146">Fisher</td>
			<td class="xl73">BP8201500</td>
			<td>EtOH</td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Trypan blue solution, 0.4%&#160;&#160;</td>
			<td class="xl70" style="width:110pt" width="146">Life Technologies</td>
			<td class="xl70" style="width:89pt" width="119">15250-061</td>
			<td></td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">Hemocytometer</td>
			<td class="xl70" style="width:110pt" width="146">Fisher</td>
			<td class="xl70" style="width:89pt" width="119">267110</td>
			<td></td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">27 gauge needle</td>
			<td class="xl70" style="width:110pt" width="146">BD</td>
			<td class="xl70" style="width:89pt" width="119">305109</td>
			<td></td>
		</tr>
		<tr height="20" style="height:15.0pt">
			<td class="xl71" height="20" style="width:199pt;height:15.0pt" width="265">1 mL syringe</td>
			<td class="xl70" style="width:110pt" width="146">BD</td>
			<td class="xl70" style="width:89pt" width="119">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

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

跨体内迟发型变态反应检测方法的抗原特异性调节

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

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 ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

一个<em&gt;体外</em&gt;急性脑片的少突胶质细胞定向T细胞攻击模式

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

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 ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

发电<em&gt;德诺</em&gt;抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

检测梭菌感染后抗原特异性抗体应答的蛋白微阵列测定方法

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

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