作者感谢Tin Min Qi女士和Veluchamy Amutha Barathi博士及其小组协助处理这些动物。这项工作得到NHIC-I2D-1409007，SingHealth Foundation SHF / FG586P / 2014和NMRC / CSA / 045/2012的支持。

根据机构指导和ARVO"眼科和视力研究中使用动物声明"处理动物。研究方案由SingHealth机构动物保护和使用委员会批准。 泪液提取物的制备注意:腹腔注射氯胺酮（75mg / kg）和赛拉嗪（10mg / kg）麻醉大鼠。通过趾捏和尾was确认正确的麻醉。将眼用凝胶施用于大鼠眼睛，以防止每次手术后的干燥。将麻醉的大鼠置于远红外线下，使动物保持温暖，直至完全恢复。在手术和恢复期间，动物被研究人员密切监测。所有的材料和手术工具在使用前是无菌的。在实验结束时，通过腹膜内注射戊巴比妥（80mg / kg）将大鼠安乐死。完全的安乐死是通过缺乏心脏脉搏和触摸眼球无眨眼反射来证实的。大鼠在标准条件下饲养:室温，21-23℃;相对湿度30-70％;亮暗循环，交替12小时（上午7点至晚上7点）。 <ol><li> 将安乐死的女性Sprague-Dawley大鼠（年龄范围从8周到16周）平坦放置，一只耳朵靠在桌子上，另一只耳朵朝上。在一个弹簧剪刀下，在暴露的耳朵下面做一个10mm切口。通过将其从周围的结缔组织和排水管中分离出来去除泪腺。 <ol><li>将腺体存放在-80°C，直到需要。在冰上解冻腺体。用剪刀在冰上尽可能的微妙。每个泪腺加入150μL含有1x蛋白酶抑制剂的PBS。 </li></ol></li><li>超声波样品在冰上5分钟，20kHz，超声仪设定为10秒，10秒关闭，30％振幅。离心子冰醋酸样品在13,000 xg和4℃下20分钟。 </li><li>移取上清液并将其转移到新管中。将上清液等分至1.5 mL管，并将其储存在-80°C。根据制造商的说明书，用二喹啉酸测定法22测定蛋白质浓度。 </li></ol> 2.乳液和百日咳毒素的制备<ol><li> 乳胶 <ol><li>将40mg热杀死的结核分枝杆菌H37Ra加入到10mL含有1mg / mL 结核分枝杆菌H37Ra的完全弗氏佐剂中并充分混合。 注意:最终的完全弗氏佐剂含有5mg / mL 结核分枝杆菌H37Ra。 </li><li>称量卵清蛋白，溶解在PBS中，制备含有2mg / mL卵清蛋白和10mg / mL泪腺提取物的抗原混合物。针对每只动物注射体积为200μL，制备主混合物b根据动物数字。 </li><li>将不完全弗氏佐剂或完全弗氏佐剂转移到50mL管中，确保抗原混合物的佐剂体积为1:1比例。将抗原混合物逐滴加入佐剂，同时以不会导致溢出的最高速度涡旋。在所有抗原加入后继续涡旋5分钟。 </li><li>将乳液转移到5 mL注射器，并通过注射器连接器与另一个5-mL注射器连接。将乳液从一个注射器推到另一个注射器进行混合。 注意:如果在放置在水中时单个液滴保留为球体，则乳液已准备就绪。应使用新鲜制备的在4℃下储存过夜的乳液或乳液。 </li></ol></li><li> 百日咳毒素 <ol><li>在500μL水中重建50μg百日咳毒素，使终浓度达到100 ng /μL。 Votex容器30秒以确保t牛油完全溶解。 注意:不要过滤灭菌，否则会导致材料损失。不要冻结该溶液在4℃下保持活性至少6个月。 </li><li>使用PBS稀释100 ng /μL百日咳毒素至3 ng /μL。将100μL稀释的百日咳毒素转移到带有27 G针的1 mL Luer-lock注射器中。 </li></ol></li></ol>刘易斯大鼠的免疫<ol><li>在第0天，将乳液混合几次。将含有1毫克泪腺提取物和200克卵清蛋白的完全弗氏佐剂的200μL乳液分配到具有27G针的1mL鲁尔锁定注射器中。在没有麻醉的情况下，在大鼠尾巴的底部皮下注射乳剂。 注意:注射前不需要预热。每只大鼠用完全弗氏佐剂中的1mg泪腺提取物和200μg卵白蛋白免疫h500μg结核分枝杆菌H37Ra。 </li><li>在第14天，以与第0天相同的方式，在不完全弗氏佐剂中注入200μL含有1mg泪腺提取物和200μg卵清蛋白的乳液。腹膜内注射100μLPBS中的300ng百日咳毒素每只老鼠在同一天。 </li></ol> 4.将抗原混合物注射入穹隆结膜下和泪腺以招募抗原特异性免疫细胞并引起局部炎症<ol><li>基于实验中的大鼠数量计算卵清蛋白和泪腺提取物的量。称量所需量的卵清蛋白，并将其与步骤1中制备的解冻的泪腺提取物组合，制备含有1mg / mL卵清蛋白和1mg / mL泪腺提取物的抗原溶液。 </li><li>用氯胺酮（75mg / kg）和甲苯噻嗪（10mg / kg）通过腹膜内注射麻醉。捏捏到以确保已经实现适当的麻醉（ 即，在捏合后没有观察到反应性运动）。将5μL的抗原注射到眼睛的穹隆结膜下。将20μL抗原注入泪腺。 </li></ol> 5.干眼功能评估注意:对于步骤5.1-5.3，麻醉的大鼠应该用手套轻轻握在平坦表面上的直立位置以避免移动。 <ol><li> 用酚红线测量泪液体积。 <ol><li>使用一对镊子夹住线，另一镊子拉扯大鼠的下眼睑。将螺纹放在下穹窿的近端拐角处1分钟，然后取下螺纹。 注意:眼泪使线的润湿部分的颜色变红。 </li><li>用毫米标记拍摄一个尺子的湿润部分的长度的图像。测量湿润长度到第十个ofa毫米使用图像软件（ 例如， ImageJ）。 </li></ol></li><li> 测量角膜/撕裂光滑度。 <ol><li>将大鼠放在装有环形照明器和相机的立体显微镜下。向大鼠角膜涂抹5μL生理盐水。通过用戴手套的手指移动上下眼睑〜5次，以便传播盐水被动地眨眼。 </li><li>将环形照明器聚焦在角膜的中间，放大1.6倍。 10秒后拍摄照片。 注意:环形照明器在动物的角膜上投射两列圆点图像。无失真点的规则间距表示平滑的角膜/泪液层。 </li></ol></li><li> 用荧光素染色测量角膜损伤。 <ol><li>向大鼠角膜中加入0.2μL的0.2％荧光素。用戴手套的手指将大鼠眼睑动物打开并闭合3次，将荧光素染料铺展在眼睛表面。 </li><l1分钟后，用3 mL注射器（无任何针头附着）取1 mL生理盐水，将注射器置于角膜前方2-3 mm处，轻轻将盐水推入眼睛。 </li><li>在背景灯关闭的眼睛成像显微镜的钴蓝色滤光片（ 即 〜400 nm）下获取图像。 注意:随后由以前的出版物23修改的分级系统分析图像。然后通过主观分级绿点的数量，面积和强度从0到2进行分析，其中0表示不存在，1表示点状染色小于50个点，2表示多于50个点的点状染色。 </li></ol></li><li>通过腹膜内注射戊巴比妥（80 mg / kg）安乐死大鼠。用剪刀去除上下眼睑。通过用镊子推下眼睑组织来修复和脱垂眼球。通过切断t来释放地球他的眼外肌，视神经和穹窿结膜。 </li><li>用镊子固定大鼠眼球的位置，并沿赤道圆周切口打开。取出镜片和玻璃体。将解剖的眼球放入冰中的1.5 mL管中。 </li><li>收集泪腺，如步骤1.1所述。立即将解剖的眼球组织和泪腺转移到实验室以准备流式细胞术分析。 </li><li>用胶原酶和Dispase II消化法分离免疫细胞24,25 。继续使用流式细胞仪分析眼球组织中的T细胞亚群26 。 注意:抗体组为:抗CD45APC-Cyanine7（OX-1），抗CD3 BV421（1F4），抗CD4 PE-Cyanine7（OX-35），抗CD45RC Alexa647（OX-22），抗-CD62 PE（HRL1），抗CD44 FITC（OX-50）和活细胞染料7-AAD。在CD45 + CD3中<sup&gt; + 7AAD-群体，初始（CD3 + CD45RC + ），效应记忆（T EM， CD3 + CD45RC - CD44 + CD62L - ）和中央记忆（T CM， CD3 + CD45RC - CD44 + CD62L + ）T细胞。 </li></ol>

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作者没有利益冲突。 LT收到了Alcon，Allergan，Santen，Bausch和Lomb，Eyelens和Eyedetec之前的资金和/或礼物。

该方案的关键步骤是确保乳液的均匀性。在精心制备的乳剂中，抗原完全涂覆油，确保注射的抗原的缓慢释放和持续的免疫刺激。该方案的另一个关键特征是使用Lewis大鼠。 Lewis大鼠比其他菌株更敏感于自身免疫性疾病27 。 该协议已经从以前发布的两种方案中进行了修改，该协议既可以使用泪腺提取物，也可以使用重组Klk1b22 20,21 。在目前的方案中，使用卵白蛋白加泪腺提取物作为抗原，抗原特异性免疫细胞被吸引到眼表和泪腺，诱发局部组织损伤。干眼发展缓慢，初次免疫后第48天达到〜85％。抗原性挑战眼睛和泪腺在第48天加剧干眼症，并确保其长达七十岁的长期性。 与Klk诱导的DED模型中的重组Klk1b22相比，当前模型中使用的泪腺提取物和卵清蛋白更便宜，更容易获得。泪腺提取物还含有除了Klk之外的其他可能诱导自身免疫的蛋白质，因此在诱导DED时，该提取物在理论上比Klk方法更有效。我们也尝试只用泪腺提取物免疫大鼠;尽管这些免疫的大鼠发展为DED，但与对照组相比，眼球组织中效应记忆T细胞没有显着增加。 这种技术的局限性在于实现模型需要70天的时间。效应记忆T细胞是正常大鼠眼中的主要T细胞亚群。在该模型中，自身免疫性DED导致眼球中CD3 +效应记忆T细胞的增加。优先的药物临床上抑制效应记忆T细胞，例如Kv1.3钾通道的选择性抑制剂，因此可以对自身免疫性DED 28具有治疗益处。

干眼症（DED）是眼泪和眼表的多因素疾病，导致不适，视觉障碍和泪膜不稳定症状，这可能导致眼表受损。它伴随着泪膜的渗透压增加和眼表1的炎症。与DED相关的症状是灼热，刺痛，烧伤，异物感，撕裂，眼睛疲劳和干燥2,3 。 DED的两个主要原因是减少泪液分泌腺体的泪液产生和撕裂膜4的过度蒸发。在患有自身免疫性疾病的患者中，例如干燥综合征，系统性红斑狼疮和类风湿关节炎对睑板腺的免疫损伤降低了对于泪液稳定性至关重要的脂质的表达。此外，对眼表的免疫损伤降低了产品对于表面润湿性很重要的粘蛋白。一起，这些过程累积地引起慢性干眼5,6,7 。 泪液替代和抗炎治疗是治疗的主要内容。然而，目前DED（ 即皮质类固醇和环孢菌素）的抗炎疗法具有广泛的免疫抑制作用，导致严重的不良反应8,9,10 。需要合适的动物模型来测试新型免疫调节剂以治疗自身免疫性干眼症。 具有特定遗传缺陷的小鼠11,12,13 ，缺乏特异性基因的小鼠14,15和过表达免疫原性的转基因小鼠ory基因已被用作自身免疫干眼症的模型16,17 。抗原诱导的自身免疫动物模型也在小鼠18 ，兔子19和大鼠20,21 中报道。在这里，我们描述了抗原诱导的慢性自身免疫干眼模型。这个模型是两个早期模型的修改;一个使用泪腺提取物，第二个使用自体抗原（ 即， klk1b22）来自泪腺20,21 。 通过用卵白蛋白，完全弗氏佐剂和含有Sprague-Dawley大鼠的泪腺提取物的乳剂（ 图1 ）皮下免疫6至8周龄的雌性Lewis大鼠诱导该疾病。在不完全弗氏佐剂中用相同抗原进行第二次免疫两周后给药。为了将抗原特异性免疫细胞募集到泪腺和眼表面，第6〜7周将泪腺提取物和卵白蛋白（1mg / mL）的混合物注射入穹隆结膜下和泪腺（ 图1 ）。超过85％的大鼠在第一次免疫后70天开始出现干眼症的特征。这些特征包括减少的泪液生成（ 图2 ），增加的角膜荧光素染色（ 图3 ）和降低的撕裂稳定性（ 图4 ）。通过流式细胞术对正常大鼠眼球中的T细胞的免疫分析显示CD3 +效应记忆T细胞的优势（ 图5和6 ）。具有自身免疫性DED的大鼠显示CD3 +效应记忆T细胞的增加以及初始和中央记忆T细胞相应的减少（ 图6 </stro纳克&gt;）。

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			<td height="21" style="height:21px;width:387px;">Reagents</td>
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			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Protease inhibitor cocktail</td>
			<td style="width:300px;">Sigma-Aldrich</td>
			<td style="width:143px;">P2714-1BTL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:300px;">Thermal Scientific</td>
			<td style="width:143px;">23227</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Mycobacterium tuberculosis H37Ra&#160;</td>
			<td style="width:300px;">Becton, Dickinson and company</td>
			<td style="width:143px;">231141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">complete Freund&#39;s adjuvant</td>
			<td style="width:300px;">Becton, Dickinson and company</td>
			<td style="width:143px;">231131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">ovalbumin</td>
			<td style="width:300px;">Sigma-Aldrich</td>
			<td style="width:143px;">A5503-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">incomplete Freund&#39;s adjuvant&#160;</td>
			<td style="width:300px;">Sigma-Aldrich</td>
			<td style="width:143px;">F5506-6X10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">pertussis toxin&#160;</td>
			<td style="width:300px;">Sigma-Aldrich</td>
			<td style="width:143px;">P7208-50UG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">fluorescein sodium solution</td>
			<td style="width:300px;">Bausch &#38; Lomb U.K Limited</td>
			<td style="width:143px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Name</td>
			<td style="width:300px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Equipment</td>
			<td style="width:300px;"></td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Sonicator</td>
			<td>Sonics&#160;</td>
			<td>Vibra-Cell</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">phenol red thread</td>
			<td>Tianjin Jingming New Technological development Co. LTD.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:387px;">Stereo microscope with ring light illuminator and camera</td>
			<td>Carl Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Micro IV microscope&#160;</td>
			<td style="width:300px;">Phoenix Research Labs</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

a chronic autoimmune dry eye rat model with increase in effector memory t cells in eyeball tissue

干眼病是一种非常常见的病症，导致发病率和医疗负担，降低生活质量。需要合适的干眼动物模型来测试新型治疗剂以治疗自身免疫性干眼症状。该方案描述了慢性自身免疫性干眼鼠模型。用含有泪腺提取物，卵清蛋白和完全弗氏佐剂的乳剂免疫Lewis大鼠。两周后给予不完全弗氏佐剂中具有相同抗原的第​​二次免疫。这些免疫接种在尾部的基部皮下施用。为了增强眼表面和泪腺的免疫反应，首次免疫6周后​​，将泪腺提取物和卵清蛋白注射入穹隆结膜下和泪腺。大鼠发展为干眼特征，包括泪液产生减少，撕裂稳定性降低，角膜损伤增加。免疫专业流式细胞分析显示，CD3 +效应记忆T细胞在眼球中占优势。

图1说明了实验设计。在第48天和第70天，在免疫的大鼠中评估干眼临床特征。撕裂体积由酚红线的湿部分的长度表示。 图2显示了来自对照和DED大鼠的酚红线的代表性图像。 DED组苯酚红线长度比对照组短，表明泪液体积较小。 荧光素与受损的角膜上皮结合。因此，通过角膜荧光素染色来测量角膜损伤。 DED大鼠角膜表面的荧光斑点从0级分为2级，与对照组比较。 DED的大鼠比对照大鼠具有更多的荧光素染色（ 图3 ），表明角膜损伤。 角膜光滑在DED和对照大鼠中通过环形照明器进行评估。如果角膜表面光滑，撕裂稳定性高，眼表上的照明环的图像是圆形和完美的。图像变形表示角膜平滑度降低和泪液不稳定。环的变形程度从0到2。在DED组中，注意到更高的环形变形水平（ 图4 ），表明较少的撕裂稳定性。 当干眼症的至少两个临床特征异常时，大鼠被定义为具有干眼症。在24只免疫的大鼠中，21只大鼠在第48天开发了DED。当在第70天评价时，结果是一致的。 流式细胞分析显示正常大鼠眼球组织中主要的T细胞亚群是效应记忆T细胞（ 图5 ）。在DED大鼠的眼球中，〜70％的CD3+ T细胞是效应记忆T细胞，而在对照大鼠中，这个数目是〜50％。 DED大鼠的眼球具有比对照大鼠显着更高的效应细胞记忆T细胞（ 图6 ）。 <img alt="图1" src="/files/ftp_upload/55592/55592fig1.jpg" /> 图1:实验设计示意图。 LG:泪腺DED:干眼病; CFA:完全弗氏佐剂; IFA:不完全弗氏佐剂。 <a href="http://ecsource.jove.com/files/ftp_upload/55592/55592fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55592/55592fig2.jpg" /> 图2:酚红线测量撕裂体积。将苯酚红线放置在两只老鼠眼睛的近端拐角处1分钟，然后取出。代表Source显示了来自对照组和DED组的酚红线与标尺的图像。使用ImageJ来测量酚红线的湿部分的长度。比例尺= 1 mm。 <a href="http://ecsource.jove.com/files/ftp_upload/55592/55592fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55592/55592fig3.jpg" /> 图3:荧光素染色测量的角膜上皮损伤的代表性图像。每个大鼠角膜用0.2％荧光素染色1分钟，并用至少1mL盐水冲洗。在具有钴蓝光的眼睛成像显微镜下拍摄图像。第一列显示控制角膜的代表性图像。第二列包含具有DED特征的大鼠的角膜染色的代表性图像。绿色荧光斑点表示角膜上皮<html> l伤害。所有图像都以相同的颜色尺度生产。荧光素染色的定量根据绿点的面积和密度进行。比例尺= 1 mm。 <a href="http://ecsource.jove.com/files/ftp_upload/55592/55592fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55592/55592fig4.jpg" /> 图4:显示环形照明器反射的代表性角膜图像。通过环形照明器测量大鼠角膜/撕裂平滑度。捕获图像中的环的变形程度是相对撕裂稳定性的量度。左栏显示对照动物的代表性图像，右列在诱导干眼后显示代表性的图像。比例尺= 1 mm。请点击此处查看此图的较大版本。 <img alt="图5" src="/files/ftp_upload/55592/55592fig5.jpg" /> 图5:流式细胞仪分析得到的点图。从眼球组织分离的T细胞用一组抗体染色。在CD45 + CD3 + 7AAD-群体中，选择CD3 + 7-AAD - T细胞。在CD3 + 7-AAD - T细胞中，确定了初始的中枢记忆和效应记忆T细胞群。 <a href="http://ecsource.jove.com/files/ftp_upload/55592/55592fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" src="/files/ftp_upload/55592/55592fig6.jpg" /> 图6:眼球中的T细胞亚群概况。 CD3 + CD45RC+初始T细胞，CD3 + CD45RC - CD44 + CD62L -效应记忆T（T EM ）细胞和CD3 + CD45RC - CD44 + CD62L +中央记忆T（T CM ）细胞以CD3 + T细胞的百分比表示。结果来自3只对照大鼠和6只DED大鼠。从分离的泪腺的T细胞的分析中获得了类似的结果（数据未显示）。未配对的Student&#39;s t检验用于统计学比较。误差条代表SD。 * p &lt;0.05，** p &lt;0.01。 <a href="http://ecsource.jove.com/files/ftp_upload/55592/55592fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue at JoVE.com

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

该报告描述了通过用大鼠泪腺提取物，卵清蛋白和完全弗氏佐剂的乳液免疫在Lewis大鼠中诱导慢性实验性自身免疫性干眼的方法，随后将泪腺提取物和卵清蛋白注射入穹隆结膜下和泪腺六周后。

慢性自身免疫性干眼鼠模型增加效应记忆T细胞在眼球组织

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

a-chronic-autoimmune-dry-eye-rat-model-with-increase-effector-memory

Research

JoVE Journal

Immunology and Infection

11.4K 次观看.  Singapore Eye Research Insitute, A Member of SingHealth. 该报告描述了通过用大鼠泪腺提取物，卵清蛋白和完全弗氏佐剂的乳液免疫在Lewis大鼠中诱导慢性实验性自身免疫性干眼的方法，随后将泪腺提取物和卵清蛋白注射入穹隆结膜下和泪腺六周后。 

视频: A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

免疫细胞在受伤的脊髓组织流式细胞仪定量评估:一个细胞的分离纯化方法的新颖使用

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

神经干/祖细胞在小鼠EAE的慢性全身性注射

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

慢性活性泼斯坦-巴尔病毒感染患者 NK 和 T 细胞系建立的高效简便方法

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

慢性自身免疫性干眼鼠模型增加效应记忆T细胞在眼球组织

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