提供了对 S.S.Z.国家卫生研究所 （RO1 AI073737 和 RO1 NS092835-01），多发性硬化症协会 （RG 4768、 RG 5179 和 RG 5180）、 Maisin 基金会和 Guthy 杰克逊慈善基金。

所有动物手术均符合由加利福尼亚大学、 旧金山体制动物护理和使用委员会核准的试验准则。C57BL/6 (H-2 b) 雌性小鼠，8 周大的时候，被购买和 C57BL/6 AQP4 -/- 小鼠由 A.Verkman.
 1.免疫的小鼠与 AQP4 肽 <ol><li>准备完全弗 &#39; s 佐剂 (CFA) 工作股票。
	<ol><li>细磨干燥的制备的 结核分枝杆菌 (H37Ra) 用研钵和杵。</li>
		<li>添加地面对不完全佐剂 H37Ra &#39; s 佐剂对终浓度为 4 毫克/毫升。终审法院可以存储在 4 ° C，长达 6 个月。</li>
	</ol></li> <li>溶解冻抗原 (Ag) AQP4 肽在磷酸盐缓冲液 (PBS) 至终浓度为 1 毫克/毫升。保持在 4 ° C 或在冰面上</li>
	<li>准备大会 2 针头锁注射器，与旋塞阀，加入含终审法院/多肽乳液。
	<ol><li>附加不到 2 个男性 luer-锁连接的 3 种方式尼龙旋塞阀柱塞的玻璃针头锁注射器。通过切换对注射器杠杆关闭旋塞阀。注射器，允许注射器作为测试管的开口端的位置。放在管架，增加稳定性。</li>
		<li>涡终审法院工作股票和 Ag 解决方案。加 1:1 体积的肽 / 打开注射器的终审法院。200 µ L 的终审法院/Ag (4 x 50 µ L) 每个都需要鼠标。 
		注意: 通常情况下，大约 1 毫升的制备是迷失在旋塞阀，这将减少可用免疫的金额。因此，它是重要的是融入到所需的总体积的计算. 
		示例: 为 5 小鼠免疫: （200 微升/动物 x 5 动物） + 1 毫升额外容量 = 总终审法院/Ag 所需的 2 毫升。</li>
		<li>插入玻璃柱塞进入注射器和同时举行的地方，以便旋塞阀是面向向上反转注射器。切换到未使用的螺纹连接旋塞阀杠杆。仔细按压玻璃柱塞以从注射器中删除多余的空气。液体填充第二个男性 luer-锁连接旋塞阀。</li>
		<li>旋塞阀的第二个男性 luer-锁连接到附加第二玻璃注射器 （柱塞完全插入）。这应该是一个封闭的系统 2 玻璃注射器与包含作为尽可能小过量空气旋塞阀连接。</li>
		<li>创建的乳液，混合推动柱塞传给液体交替之间大约 2 分钟寒意在大约 10 分钟冰上的注射器重复 2 注射器令人不寒而栗的和搅拌直至有明显是改变了粘度的制备导致非常僵硬，白色的乳液。冷藏的注射器包含在 4 ° C 的乳胶或维持在冰面上</li>
	</ol></li> <li>将所有的乳状液转移到一个玻璃注射器针头锁、 删除空的玻璃注射器和替换 1 毫升注射针头注射器。用乳液填充新的注射器、 删除从旋塞阀和附加一根针 (25 x 5/8)。</li>
	<li>&#34; 水测试 &#34; 乳液的一致性。一个小碟子里面的水驱逐一滴乳液性能的影响。乳液应该不能分散，指示它适合于注射。
	<ol><li>乳液分散，如果它不是准备注射; 转移液体回玻璃注射器并继续混合，然后再放松，直到乳液是僵硬。</li>
		<li>再次测试乳液，直到取得了适当的浓度。</li>
	</ol></li> <li>注入捐助 AQP4 -/- 小鼠皮下注射 （南卡罗来纳州） 与含 AQP4 肽乳液。每个鼠标接收 4 x 50 µ L s.c.注射 (200 µ L 总 （100 微克肽）)。4 网站包括双方的下腹部，漏入腹股沟淋巴结 (LN) 及两侧胸壁内侧到每个腋下漏入腋生巷过继转会需要 1-2 免疫的捐助小鼠每个收件人的老鼠。</li>
</ol> 2。T 细胞培养及炎性 T 细胞极化 <ol><li>10-12 天后免疫，从小鼠收集 LN。
	<ol><li>在冰盘，准备 60 毫米的培养皿装有一个细胞过滤器 (网格大小 70 µ m) 和含冰鲜含 10%热灭活的胎牛血清 (FBS) 的 RPMI 媒体和 100 U/mL 青霉素 100 微克/毫升链霉素 （钢笔-链球菌） (RFP)。</li>
		<li>Euthanize 小鼠暴露于 CO 2，其次是颈椎关节脱位。沉浸小鼠中 70%的乙醇，然后寄到夹层板每个于拦路贼。</li>
		<li>从腹股沟切正中皮肤切口，脖子和到每条腿。拉肌肤远离腹膜和别针把它拉紧地向董事会。解剖腹股沟和腋窝 LN 和放入培养皿中 RFP，在冰上。 19</li> <li>过继转移实验，结合 LN 从相同的免疫接种组内的所有动物。Vβ 使用流流式细胞仪分析，分开个别动物的 LN。</li>
	</ol></li> <li>放置包含到含 RFP 50 毫升离心管 LN 的细胞过滤器。使用无菌 5 毫升注射器柱塞平端按 LN 细胞通过过滤器，用 20-30 毫升的 RFP，以便恢复单元格的清洗。维持在加工过程中，文化的时间直到冰上 LN 细胞。</li>
	<li>洗两次，离心在 393 x g 5 分钟。后第二次洗，重悬 LN 细胞的 T 细胞媒体 (TCM)。中医包含用 RPMI 10%热灭活胎牛血清、 292 微克/毫升 L-谷氨酰胺、 110 微克/毫升丙酮酸钠，和 55 µ M 2-巯基乙醇和笔链球菌。通常情况下，使用的 5 毫升中医捐助鼠标每卷。</li>
	<li>LN 单元格进行计数。预期收益率是大约 3-6 x 10 7 LN 细胞免疫的小鼠捐精者。留出 3-4 x 10 6 增殖测定 （见下面第 3 节）。</li>
	<li>设置偏光文化为过继转移 (6 节)。
	<ol><li>准备 Th17 偏光文化 5 x 10 6 LN 细胞每毫升 10 微克/毫升 Ag，20 吴/mL 重组小鼠白细胞介素 (IL)-23 和 10 吴/mL 重组小鼠白细胞介素-6 在中医。</li>
		<li>另外，Th1 两极化，准备文化 5 x 10 6 LN 细胞每毫升 10 微克/毫升 Ag 和 10 吴/mL 重组小鼠白细胞介素-12 在中医。</li>
		<li>加入 12 孔板每两极化的文化混合井 2 毫升 （1 × 10 7 细胞）。LN 小鼠细胞的 2-4 捐助将填补一个 12 孔板。保持文化在湿的孵化器在 37 ° C，5%CO 2 为 72 h.</li>
		<li>目视检查 LN 细胞培养的激活在 48 小时和 72 h.活化细胞会形成广泛的集群和 T 细胞将增加规模，成为更多形性。代谢的增加将导致在媒体上，改从桃橙 ph 值降低。如果 pH 值低到足以变黄了媒体，添加 1 毫升新鲜中医防止对在其余 24 h.细胞毒性的</li>
		<li>节 6 继续为过继转移。极化效率可核实染色 (ICS) 的细胞内细胞因子作为描述第 5 节中的部分。</li>
	</ol></li> <li>设置文化流流式细胞术分析的表面 Vβ 表达 (节 4) 从个别老鼠使用 LN。
	<ol><li>准备 5 x 10 6 每毫升 10 微克/毫升 Ag 在中医与 LN 细胞。</li>
		<li>加入 12 孔板 2 毫升 （1 × 10 7 细胞） 的每口井的文化。文化应维持在一个湿的孵化器，在 37 ° C，5%CO 2 10 天。第 4 节继续。</li>
	</ol></li></ol> 3。增殖 注: 这种情况继续从 2.4 节。
<ol><li>准备的 LN 细胞在管中，在 2 × 10 3 4 毫升每毫升中医 6 细胞。</li>
	<li>轻轻的逆变管多次，混合细胞，然后换乘不育槽。</li>
	<li>使用到板 100 μ L 细胞每口井的多通道滴入 96 孔板圆底细胞培养板。通常情况下，板 15 口井，一式三份和试验的 4 种 Ag 浓度没有 Ag 参考。</li>
	<li>稀释 Ag 在中医在不同浓度，通常 80、 20、 5、 1 和 0 微克/毫升 （2 x 股票）。在一式三份的终浓度为 40，10，2.5 中添加每个浓度 100 μ 的 L。0.5 和 0 微克/毫升。孵化为大约 72 小时 37 ° C. 
	注: 步骤通过 3.7 3.5 涉及放射性材料。正确使用方法、 监测和处置放射性材料应进行，按照体制和政府规定。</li>
	<li>准备工作 3 H-胸苷股票溶液 (40 µCi/mL) 1 mCi 3 H-胸苷加入 25 毫升 RPMI，而存储这在 4 ° C.吸管 0.5 毫升的工作解决方案不育的槽里。</li>
	<li>添加 25 µ L 的 3 H-胸苷 (1 µCi) 每好使用多通道的吸管。孵育额外 18 h 在 37 ° C.</li>
	<li>收获细胞到使用 96 孔板细胞收割机玻璃过滤器垫子上，用 70%乙醇冲洗。干燥后，进用 5 毫升闪烁液的塑料样品袋密封过滤垫。在每个好使用闪烁计数器测量放射性。</li>
</ol> 4。细胞表面 TCR Vβ 表达的流式细胞分析 注: 这种情况继续从第 2.6 节。抗体的鼠标 TCR Vβ 2，3，4，5.1 和 5.2、 6、 7、 8.1 和 8.2、 8.3，9、 10b、 11、 12、 13、 14、 及 17a 条是商业上供测试 TCR Vβ 表达。
<ol><li>的 TCR Vβ 检测，取出 T 细胞培养板从吹打几次和细胞转移到一个管。</li>
	<li>用流式细胞仪缓冲区 (FB) 含 2%胎牛血清，0.1%叠氮化钠和 2 毫米在 PBS 中 EDTA 清洗。</li>
	<li>细胞转移到 V 形底 96年孔板，密度为 1 × 10 5 至 3 x 10 6 细胞每口井。这些细胞进入多个井 （Vβ 正在测试每一个井） 分布的整个 Vβ 小组正在进行测试，如果。</li>
	<li>通过染色用活死可行性染料，与自由胺暴露的坏死细胞的反应，从流式细胞术分析排除死细胞。按照制造商 &#39; s 说明离心和再悬浮在可行性染料。10-20 分钟，整个细胞染色程序冰上孵育，保护细胞免受光和保持在冰面上</li>
	<li>后孵化，洗板一次在 FB。若要检测 TCR Vβ，暂停在 100 μ L 的 FB 包含抗体 1: 100 稀释的 CD4 细胞 （克隆 RM4 5） 和 TCR Vβ。孵育 15-60 分钟在冰面上</li>
	<li>在 FB 一次洗板和固定细胞通过添加 200 微升 4%多聚甲醛在 PBS 稀释。孵育 20 分钟在冰上。洗板在 FB 和流式细胞分析。</li>
</ol> 5。流流式细胞仪分析细胞内细胞因子生产 <ol><li>为细胞内细胞因子染色 (ICS)，对待 T 细胞文化与蛋白质运输抑制剂试剂 (例如，GolgiPlug) 1: 1000 稀释中医，ICS，防止前 4 h分泌细胞因子。在那个时候，激活 T 细胞与 50 吴/mL 佛 12-肉豆蔻 13-乙酸酯 (PMA) 和 500ml 吴 ionomycin 为了诱导蛋白生产。</li>
	<li>后的文化在 37 ° C 的 4 h，取出细胞培养板吹打向上和向下，并将转移到离心管 （15 或 50 毫升）。</li>
	<li>用 FB 洗净。重悬细胞在 FB 和转移到 V 形底 96年孔板，密度为 5 × 10 5 3 x 10 6 细胞每井。</li>
	<li>染色细胞与可行性染料作为上述 （第 4.2 节）。孵育后洗板一次在 FB。</li>
	<li>添加抗体检测的表面标记，如下所示: <ol><li>的表面标志物的检测，暂停在 100 μ L 的 FB 包含抗体 1: 100 稀释的 CD4 细胞 （克隆 RM4-5）。</li>
		<li>（可选） 包括抗体为 B220 （克隆 30 F11） 和 CD11b （克隆 M1/70） 1: 100 稀释，采用提高浇注的 B 细胞和单核细胞/巨噬细胞，分别。一般来说，PE Cy7 抗 CD4、 FITC 抗 B220 和 PerCP Cy5.5 抗 CD11b 工作好。抗体/荧光复合物的选择应遵循的流式细胞仪分析，用于配置和最优的抗体浓度应可根据经验确定。</li>
		<li>孵育 15-60 分钟上冰。</li>
	</ol></li> <li>FB 用洗板和重悬 200 微升的在冰上 20 分钟的固定/通透解决方案中的单元格。这将修复细胞和通透细胞细胞膜。</li>
	<li>为了维持细胞膜在细胞内染色，通透而不是 FB 使用烫发/洗涤缓冲液 (PW) (稀释 1:10 从 10 X 股票)。洗井的 PW 200 微升。</li>
	<li>为 ICS，制备抗体的 1: 100 稀释液干扰素 (IFN)-γ 和 IL-17A 使用 1 x PW。一种选择是 APC 抗 IFN-γ （克隆 XMG1.2） 和 PE 抗-IL-17A (克隆 eBio17B7)，效果很好。悬在 100 μ L 的鸡尾酒的胞内抗体的细胞，然后孵育至少 15 分钟。它也是可能继续染色一夜之间在 4 ° C.</li>
	<li>洗井的 PW 200 微升和再悬浮在 FB 为流式。</li>
	<li>并行，准备污泥而不染的样品 （FB 没有抗体细胞） 对优化检测器电压和单染色样品为每个个别的荧光染料 （即细胞或补偿珠沾上只有一个抗体/荧光）确定补偿溢出值。</li>
	<li>使用流式细胞仪，获得 &#8805; 一万个细胞 （事件），浇注上可行 （活/死-阴性） CD4 + 细胞。为了确保准确浇注的 T 细胞，排除 B220 + 和 CD11b + 细胞 (B 细胞和单核细胞/巨噬细胞，分别)。在 CD4 + T 细胞亚群、 确定细胞表达 IFN-γ （Th1 血统） 或 IL-17A （Th17 沿袭） 的百分比。</li>
</ol> 6。过继转移致病 AQP4 特异性 T 细胞 注: 此步骤如下从第 2.5 节: T 细胞文化和炎性 T 细胞极化。
<ol><li>恢复极化 12 孔板细胞吹打向上和向下取出，并将它们传送到 50 毫升管。马要保持直到注射冰上的。</li>
	<li>单元格进行计数，两次在冷的 PBS，洗净，准备暂停 PBS 1 x 10 8 细胞/毫升。一般来说，在一个小时内注入细胞。</li>
	<li>轻轻的旋转混合细胞和转移到 1 毫升注射器注射次。静脉内管理的细胞 (2 x 10 7) 200 微升 （静脉注射） 向每个鼠标，通过尾静脉。</li>
	<li>注入每个收件人小鼠腹腔与 200 吴 B.百日咳 毒素稀释 200 µ L 的 PBS 在那一天和 2 天后。监测小鼠每日为临床疾病的迹象。</li>
</ol> 7。临床评估 ATCA <ol><li>检查收件人小鼠每日为中枢神经系统自身免疫的临床症状。一般情况下，小鼠将显示中枢神经系统自身免疫的迹象后 AQP4 特异性 T 细胞过继转移 5-8 天。老鼠会显示神经功能障碍的临床症状。它有利于对天真鼠标来定义鼠标的正常能力进行评价。</li>
	<li>评价小鼠尾巴损失的语气。轻轻握住小鼠尾基部，直立提起。枯萎不断是称为损失的尾巴尾巴音 （1 分）。尾巴的语气损失通常是临床疾病的第一迹象。</li>
	通过将鼠标放在一个平面上背上的 <li>测试为扶正反射。缓慢的纠正是躯干共济失调的标志 （2 分）。天真的鼠标会完全抵制任何企图将它放在它的后面，所以任何缓慢的权利能力得分为功能障碍。它是典型的测试鼠标这反射 3-4 倍。</li>
	<li>评估姿势和移动。老鼠开始显示变化导致降低或拖动的臀部在行走的姿势 （2.5 分）。进一步疾病结果在单瘫，一个后完全陷于瘫痪肢体 （3.0 分） 和截瘫，两后肢完全瘫痪肢体 （3.5 分）。适度四肢轻瘫为，所有的四肢，弱点会导致非常缓慢的向前移动 （4 分）。严重四肢轻瘫为允许几乎没有向前移动 （4.5 分）。最后，严重的疾病，他们可能成为垂死或死 （5 分）。</li>
	<li>管理对小鼠失去的能力或者，获得流体或固体食物 1 毫升的生理盐水腹腔 5%葡萄糖液使用补充凝胶杯，高热量的凝胶和培根软弱来抵消脱水和剥夺食物。安乐死垂死老鼠。</li>
</ol> 8。组织编制和组织学 <ol><li>麻醉小鼠 100 毫克/公斤氯胺酮和甲苯噻嗪 10 毫克/公斤，然后销到夹层板每个于拦路贼。</li>
	<li>开放了胸，暴露心脏。切开肝允许血液流出。附加到单独的储水槽用 PBS 和 10%的福尔马林 miniflow 变速泵油管的蝴蝶针。将针插入到心脏的左心室和灌注配合 5 毫升的 PBS，其次是 25 毫升 10%福尔马林。</li>
	<li>删除头部，然后眼睛。过程的眼睛和视网膜前面所述 20。抄近路穿过眼窝、 插入剪刀在鼻子和每个侧面切开前-后头骨。
	<ol><li>删除头骨、 揭露视神经、 切断视神经交叉置于之间 2 泡沫垫卡带。</li>
		<li>在 10%福尔马林 （24 小时），然后 50%乙醇 （24 小时），然后转账到 70%的乙醇浸没。</li>
	</ol></li> <li>移除大脑和脊柱，并将在 10%福尔马林。连续的部分大脑在冠状面;节脊髓索在矢状面 （大约一半） 和串行冠状面 （大约每次鼠标 20 截面）。</li>
	<li>过程 CNS 样品常规石蜡包埋。染色 8 µ m 厚截面与苏木精和曙红 （视神经） 或勒快速蓝苏木精和曙红 （脑和脊髓）。</li>
	<li>评价视神经炎症神经和周围脑膜 （视神经炎） 内或主要地在周围脑膜 （光纤 ③） 的存在。</li>
	<li>计数脑膜和实质的炎症病灶 (&#62; 10 聚集外周血单个核细胞) 在脑和脊髓样品，每个鼠标。</li>
</ol> 9。在体内视网膜成像的光学相干断层成像 (OCT) 注: 频域 OCT 视网膜成像的小鼠进行使用商用设备 （例如，Spectralis 与 TruTrack 眼动仪） 以实现一致的眼定位和减少运动伪影。
<ol><li>五分钟前成像，瞳孔与托吡卡胺 1%（每只眼睛看到一滴） 和放置鼠标成像在麻醉诱导室，提供源源不断的 2 升 / 分钟 （1.5%异氟醚）。打开热垫和准备麻醉后恢复笼。</li>
	<li>将鼠标放置在成像滚筒上，因此麻醉流重定向。保护眼睛与 0.3%羟丙基甲基纤维素，保持眼睛湿润，确保折射连续性。眼要检查上放置自定义的隐形眼镜。</li>
	<li>红外眼底图像，作指导直接激光对眼睛，确保梁中心视神经头上。垂直和水平 OCT 扫描应确认，视网膜激光奠定了垂直。</li>
	<li>执行 25 B 扫描在高分辨率模式和栅格化从 30 平均 A 扫描。</li>
	<li>后成像两只眼睛，取隐形眼镜，并涂眼用凝胶。在温暖的恢复笼子里离开鼠标。</li>
</ol> 10。处理和分析的 OCT <ol><li>使用模块化成像软件的自动分割。
	<ol><li>手动正确段对应内界膜 (ILM) 和内网状层 (IPL)，代表的限制内视网膜层 (IRL) 21.</li>
		<li>验证视网膜神经纤维层 (RNFL) 和神经节细胞层 (GCL) 驻留在 IRL 范围内并不重叠。</li>
	</ol></li> <li>计算 IRL 厚度与直径的 1、 2 和 3 毫米围绕视盘和导出到电子表格文件使用早期治疗糖尿病视网膜病变的研究 (ETDRS) 2 网格。软件计算每个视网膜层厚度由平均每个网格的部门。因此，中央部分，对应于视神经头，排除。</li>
	<li>分析组在每个时间点的统计差异。分析对于每个鼠标，用两只眼睛广义估计方程与交换相关矩阵和调整内部学科间眼相关 21.</li>
</ol>

<ol>
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S.A.萨根、 A.克鲁兹-Herranz，C.M.斯宾塞，皮皮岛何、 L.斯坦、 A.J.绿色和 R.A.索贝尔报告没有披露。S.S.Zamvil 是神经病学、 神经免疫学和神经炎症的副主编，是神经免疫学国际社会咨询委员会的成员。他曾在神经科学杂志和临床研究 》 杂志上，免疫学杂志编辑部和一直为国立的补助金审查委员会宪章成员卫生研究院 (NIH) 临床神经免疫学和脑肿瘤 (CNBT) 研究部分和国家多发性硬化症社会 (NMS)。他曾担任顾问，所得酬金从生物 Idec、 EMD 雪兰、 健赞公司、 诺华、 罗氏/基因泰克，Teva 制药股份有限公司，并已送达或为礼来公司、 BioMS、 梯瓦公司和 Opexa 疗法服务在数据安全监测委员会。目前，博士 Zamvil 从美国国立卫生研究院、 NMS、 Maisin 基金会、 生物和 Celgene 接收研究赠款支持。

AQP4 被认定为 NMO IgG 20053中的主要目标。然后，它承认必须建立 AQP4 针对性的中枢神经系统自身免疫动物模型。这种模式可能有用，探讨 AQP4 特异性 T 细胞和 B 细胞如何参与发展的中枢神经系统自身免疫测试候选人疗法为 NMO。虽然 AQP4 特异性 T 细胞抗原表位在野生型小鼠首次报道 2010年13，回应这些抗原的 T 细胞识别并没有造成临床或病理疾病14，17。无法生成基于免疫反应性对 AQP4 的中枢神经系统自身免疫模型仍然是个谜，直到 2015年当琼斯，等.25发现捐助 AQP4 p135 153 引物从 AQP4-/-小鼠 T 细胞能够 WT 小鼠引起的中枢神经系统自身免疫的临床和组织学迹象。感兴趣，AQP4 p135-153 预计将与高亲和力17绑定 MHC II (-A 和b)。只有一个其他 AQP4 的氨基酸序列，201-220，预计要绑定-Ab与类似的高亲和力。确实，我们观察到那 AQP4 p135 153 和 p201-220 两引出 AQP4-/-，但不是 WT，小鼠的鲁棒性扩散。在这里，我们展示了如何一个人可以隔离和扩大 encephalitogenic Th17 AQP4 p135-153-和 p201-220-反应性 T 细胞从 AQP4-/-小鼠。当转入 WT 受鼠，捐助 AQP4 无功 Th17 细胞诱导麻痹，陪同外周血单个核细胞浸润在脊髓和视神经。传入的视觉系统损伤患者 AQP4 阳性 NMOSD26而闻名。在这里，我们观察到视神经炎 AQP4 无功，MOG 无功 Th17 细胞诱导显著。而 AQP4 特定 Th17 细胞诱导光纤 ③，MOG 特定 Th17 细胞诱导严重视神经炎。我们还描述了用于串行 oct 监测视神经炎 AQP4 特定和 MOG 特异性 T 细胞诱导的技术。其他研究者现在应该能够应用在这里描述来推进自己的研究侧重于 ATCA 的发病机制的协议。
人很容易能避免我们的协议中的三个潜在缺陷。第一，收养转让 ATCA 要求 AQP4-/-捐助小鼠 AQP4 特异性 T 细胞的使用。具体来说，捐助 WT AQP4 p135-153-异性 T 细胞并没有造成 ATCA 收件人 WT 小鼠。其次，它是重要执行与前 AQP4-/-小鼠 (协议步骤 1.5) 免疫肽/终审法院乳液一滴"水测试"。当它是适合 s.c.注射，乳液不应在水中分散。如果乳液分散，一个人应该混合乳液再一次、 再冷静，重复水测试。最后，激活的捐助中枢神经系统抗原特异性 T 细胞比休息 T 细胞更有效地诱导中枢神经系统自身免疫。一应目测检查那些前收获过继转移捐助者 T 细胞的光镜下的文化。快速分裂的细胞可能形成集群，容易识别。此外，当文化包含许多活化的 T 细胞，媒体可能从过渡粉红色到橙色或甚至到黄色，由于 ph 值下降。一个还可以评估捐助 AQP4 引淋巴结 T 细胞增殖活化3H-胸苷纳入协议步骤 3 中所述。
我们发现，两种致病 AQP4 T 细胞抗原表位 （1） 预计将与高亲和力结合 MHC II 和 （2） 引出 AQP4-/-，但不是 WT 有力增殖反应，小鼠建议针对这些决定因素的 T 细胞通常是由胸腺阴性选择17控制。用于识别 AQP4 p135-153 和 p201-220 AQP4-/-小鼠 TCR 剧目是独特的 (图 2)，这也是符合介导胸腺髓质上皮细胞的克隆删除。其他耐受机制通常可能抑制 AQP4 的免疫应答。之后我们初次报告17，另一组也表明 AQP4 p201-220 包含 encephalitogenic T 细胞行列式27。当 α/β （TCRα-/-） T 细胞缺陷小鼠被重组与 AQP4-/- CD4+ T 细胞，它有可能引起 encephalitogenic AQP4 特异性 T 细胞的反应，但不是 AQP4 特异性体液免疫反应，言外之意就是在WT 小鼠 AQP4 特异性 B 细胞反应，类似于 AQP4 特异性 T 细胞反应，是受消极的选择。事实上，不观察 WT 小鼠与 ATCA AQP4 特异性 T 细胞单独诱导脊髓轴突和视网膜神经节细胞，损失可能需要参与致病 AQP4 特异性抗体。很显然，AQP4 针对中枢神经系统自身免疫的小鼠模型将继续随着我们了解更多关于通常控制 AQP4 特异性 T 细胞和 B 细胞免疫耐受机制。
其它型号的 AQP4 针对中枢神经系统自身免疫也正在开发的6，16，28，，2930。每一个可能会带来好处的研究与 NMO 发病机制相关的特定方面。AQP4 特异性 T 细胞已在 WT 大鼠6，，1629。这些 AQP4 特异性 T 细胞引起的中枢神经系统自身免疫的组织学变化，但是，类似于小鼠观察，WT 大鼠 AQP4 特异性 T 细胞不会造成显著的临床疾病的迹象。因此，限制在 WT 小鼠的 T 细胞和 B 细胞 AQP4 特异性免疫反应机制的也是耐受的业务在大鼠中的。不管怎么说，人们不应低估利用小鼠模型研究机制所涉及的疾病的发病机制中的权力。淘汰赛，转基因和记者小鼠的财富可以是有利的。此外应该认识到，在自体免疫的几个基本发现了使用鼠标 EAE 模型。举个例子，示范认为 T 细胞克隆特定的自身抗原可以调解自身免疫性疾病31，32，识别和发挥的作用的 T 细胞聚集在自身免疫性33的发现发展通路的 Th17 分化34首先介绍了使用鼠标 EAE 模型。使用我们已经开发的 ATCA 小鼠模型，现在就研究发展及其致病性 AQP4 特异性免疫反应在体内，调控的手段，应该提供与 NMO 发病机制相关的重要见解。

视神经脊髓炎 (NMO) 是中枢神经系统 (CNS) 自身免疫性炎症脱髓鞘疾病导致的瘫痪和视觉丧失导致永久性的神经功能障碍1反复发作。NMO 目前被认为主要是体液的自身免疫性疾病2 ，因为它是与抗体 (Igs) 针对水通道蛋白-4 (AQP4) 表达在星形胶质细胞3，4水通道相关联。然而，中枢神经系统炎症是中枢神经系统条目的 AQP4 Ig5，6的先决条件。因此，已不可能建立模型 NMO 由反 AQP4 Igs 单独转让。结果 （1） 致病 AQP4 特定 Igs NMO 患者为 IgG11，2，NMO 病变8，9 （3) NMO 中确定了子类7 (2) T 细胞 T 细胞依赖 Ig 是关联与某些 m h C ⅱ 基因 (如 HLA DR17 (DRB1 * 0301年))10和 (4) 炎性 AQP4 无功博士-限制的 Th17 细胞扩大 NMO 患者11，12所有表明 AQP4 特异性 T 细胞起着关键作用在 NMO 发病机制。因此，它是重要的是建立动物模型，来确定如何 AQP4 特异性 T 细胞可能有助于 NMO 发病机制。
几年前，在野生型 (WT) 小鼠13，14 ，大鼠15确定了多个 AQP4 T 细胞抗原表位。虽然有人认为 AQP4 反应性 T 细胞能诱导视神经炎症的天真收件人大鼠15，16，不观察中枢神经系统疾病的显著临床症状。同样，直接 WT 小鼠免疫与含 AQP4 T 细胞抗原表位14，17，肽或转移的促炎症反应 T 细胞靶向这些决定因素17，没有引起临床症状或组织学中枢神经系统自身免疫的证据。
最近，它与高亲和力18观察那免疫 C57BL/6 AQP4 缺 （AQP4-/-） 小鼠 AQP4 肽 (p) 135-153 或 p201-220，预测要绑定 MHC II (-A 和b) 的两个决定因素，引起了强烈的 CD4+ T 细胞反应17。与此相反的是，这些两个肽引起 WT 小鼠只谦虚的增殖反应。此外，用于识别这些行列式的 AQP4-/-小鼠 T 细胞的 T 细胞受体 (TCR) 曲目是独一无二的。总的来说，这些研究结果表明 T 细胞识别的 AQP4 受胸腺阴性选择。AQP4 p135-153-p201-220-特定或 AQP4-/-捐助小鼠 Th17 细胞诱导瘫痪近 100%的天真收件人的 WT 小鼠;这是与视神经浸润的 T 细胞、 B 细胞和单核细胞。串行视神经相干断层成像 (OCT) 表明动态视觉系统的参与。小鼠 T 细胞介导 AQP4 针对中枢神经系统自身免疫 (ATCA) 从瘫痪和视觉系统损伤恢复。与 EAE 诱导髓鞘少突胶质细胞糖蛋白 (MOG) p35-55-特定于 T 细胞，从而导致持续性的临床疾病，ATCA 诱导 T 细胞单独不是与轴突损失或减少的视网膜神经节细胞 （视网膜神经节细胞） 相关联的。我们的研究结果清楚地表明，有多个致病 AQP4 T 细胞决定因素。ATCA 这个新模型是用于控制致病 AQP4 特异性 T 细胞，学习如何这些细胞诱导中枢神经系统炎症和如何他们可能配合 AQP4 特异性 B 细胞和抗体促进 NMO 发病机制发育的机制研究.
在本报告中，我们描述了用于诱导和评价 T 细胞诱导 ATCA 的协议。我们开始与用于免疫、 T 细胞培养和 Th17 极化产生致病性 AQP4 特异性 T 细胞，流式细胞分析以确认极化和那些 T 细胞过继转移的技术。然后，我们描述用于评价临床和病理疾病和串行 OCT 监测视觉系统小鼠损伤的收件人使用的方法。

<table width="1066">
	<colgroup>
		<col style="width:246pt" width="328" />
		<col style="width:162pt" width="216" />
		<col style="width:104pt" width="139" />
		<col style="width:287pt" width="383" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">M. tuberculosis H37Ra</td>
			<td class="xl19" style="width:162pt" width="216">BD Difco</td>
			<td class="xl24" style="width:104pt" width="139">231141</td>
			<td class="xl22" style="width:287pt" width="383">Dessicated, killed M. tuberculosis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Incomplete Freund&#39;s Adjuvant</td>
			<td class="xl19" style="width:162pt" width="216">BD Difco</td>
			<td class="xl24" style="width:104pt" width="139">263910</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">AQP4 peptide p135-153</td>
			<td class="xl19" style="width:162pt" width="216">Genemed</td>
			<td class="xl24" style="width:104pt" width="139">Custom Synthesis</td>
			<td class="xl22">Peptide sequence: LVTPPSVVGGLGVTMVHGN</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">AQP4 peptide p201-220</td>
			<td class="xl19" style="width:162pt" width="216">Genemed</td>
			<td class="xl24" style="width:104pt" width="139">Custom Synthesis</td>
			<td class="xl22">Peptide sequence: HLFAINYTGASMNPARSFGP</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">MOG peptide p35-55</td>
			<td class="xl19" style="width:162pt" width="216">Genemed / Auspep</td>
			<td class="xl24" style="width:104pt" width="139">Custom Synthesis</td>
			<td class="xl22">Peptide sequence: MEVGWYRSPFSRVVHLYRNGK</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">3-way Stopcock</td>
			<td class="xl19" style="width:162pt" width="216">Kimble</td>
			<td class="xl24" style="width:104pt" width="139">420163-4503</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">HyClone Fetal Bovine Serum (Characterized)</td>
			<td class="xl19" style="width:162pt" width="216">GE Healthcare Life Sciences</td>
			<td class="xl24" style="width:104pt" width="139">SH30071</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Recombinant Mouse IL-23</td>
			<td class="xl19" style="width:162pt" width="216">R&#38;D Systems (BioTechne)</td>
			<td class="xl24" style="width:104pt" width="139">1887-ML</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Recombinant Mouse IL-6</td>
			<td class="xl19" style="width:162pt" width="216">R&#38;D Systems (BioTechne)</td>
			<td class="xl24" style="width:104pt" width="139">406-ML</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Recombinant Mouse IL-12</td>
			<td class="xl23" style="width:162pt" width="216">R&#38;D Systems (BioTechne)</td>
			<td class="xl24" style="width:104pt" width="139">419-ML</td>
			<td class="xl22"></td>
		</tr>
		<tr height="25" style="height:18.75pt">
			<td class="xl19" height="25" style="width:246pt;height:18.75pt" width="328">Thymidine [Methyl-3H]</td>
			<td class="xl19" style="width:162pt" width="216">PerkinElmer</td>
			<td class="xl24" style="width:104pt" width="139">NET027</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Glass Fiber Filtermats</td>
			<td class="xl19" style="width:162pt" width="216">PerkinElmer</td>
			<td class="xl24" style="width:104pt" width="139">1450-421</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Anti-mouse antibodies</td>
			<td class="xl19" style="width:162pt" width="216">eBioscience (Affymetrix)</td>
			<td class="xl24" style="width:104pt" width="139">[various]</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Anti-mouse TCR V&#946; Screening Panel</td>
			<td class="xl19" style="width:162pt" width="216">BD Biosciences</td>
			<td class="xl24" style="width:104pt" width="139">557004</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">LIVE/DEAD Fixable Dead Cell Stain</td>
			<td class="xl19" style="width:162pt" width="216">ThermoFisher Scientific</td>
			<td class="xl24" style="width:104pt" width="139">[various]</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Paraformaldehyde (16%)</td>
			<td class="xl19" style="width:162pt" width="216">Electron Microscopy Sciences</td>
			<td class="xl24" style="width:104pt" width="139">15710</td>
			<td class="xl22"></td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl19" height="42" style="width:246pt;height:31.5pt" width="328">Fixation/Permeabilization Solution Kit with GolgiPlug</td>
			<td class="xl19" style="width:162pt" width="216">BD Biosciences</td>
			<td class="xl24" style="width:104pt" width="139">555028</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Phorbol 12-myristate 13-acetate (PMA)</td>
			<td class="xl19" style="width:162pt" width="216">Sigma-Aldrich</td>
			<td class="xl24" style="width:104pt" width="139">P8139</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Iomomycin (calcium salt)</td>
			<td class="xl19" style="width:162pt" width="216">Sigma-Aldrich</td>
			<td class="xl24" style="width:104pt" width="139">I0634</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Pertussis Toxin from B. pertussis</td>
			<td class="xl19" style="width:162pt" width="216">List Biological Laboratories</td>
			<td class="xl24" style="width:104pt" width="139">181</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">10% Formalin</td>
			<td class="xl19" style="width:162pt" width="216">VWR</td>
			<td class="xl24" style="width:104pt" width="139">89370-094</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Variable-Flow Peristaltic Pump</td>
			<td class="xl19" style="width:162pt" width="216">Fisher Scientific</td>
			<td class="xl24" style="width:104pt" width="139">13-876-2</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Foam Biopsy Pads, Rectangular</td>
			<td class="xl19" style="width:162pt" width="216">Fisher Scientific</td>
			<td class="xl24" style="width:104pt" width="139">22-038-221</td>
			<td class="xl22"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Isothesia (isoflurane, USP)</td>
			<td class="xl19" style="width:162pt" width="216">Henry Schein Animal Health</td>
			<td class="xl24" style="width:104pt" width="139">050033</td>
			<td class="xl22">NDC : 11695-0500-2</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Tropicamide Ophthalmic Solution, USP (1%)</td>
			<td class="xl19" style="width:162pt" width="216">Akorn</td>
			<td class="xl24" style="width:104pt" width="139"></td>
			<td class="xl22">NDC: 17478-102-12</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:246pt;height:15.75pt" width="328">Spectralis Diagnostic Imaging Platform</td>
			<td class="xl19" style="width:162pt" width="216">Heidelberg Engineering</td>
			<td class="xl24" style="width:104pt" width="139"></td>
			<td class="xl22"></td>
		</tr>
	</tbody>
</table>

induction of paralysis and visual system injury in mice by t cells specific for neuromyelitis optica autoantigen aquaporin-4

虽然它承认那水通道蛋白 4 (AQP4)-特定的 T 细胞和抗体参与发病的视神经脊髓炎 (NMO)，人类中枢神经系统 (CNS) 自身免疫性脱髓鞘疾病，有针对性的 AQP4 与两个模型的建立中枢神经系统自身免疫的临床和病理表现已证明具有挑战性。野生型 (WT) 小鼠与 AQP4 肽免疫引起 T 细胞增殖，虽然那些 T 细胞不能转移疾病到天真收件人小鼠。最近，两个小说 AQP4 T 细胞抗原表位肽 (p) 135-153 鉴别、 p201-220，当学习 AQP4 AQP4 缺陷 （AQP4-/-） 小鼠，建议对这些抗原的 T 细胞活性的免疫应答通常由胸腺消极的选择。AQP4-/- Th17 极化引到 p135 153 或 p201-220 瘫痪收件人 WT 在小鼠中，这与主要软脑膜发炎的视神经和脊髓的 T 细胞。炎症周围视神经和视网膜内层 (IRL) 参与主要表现为串行的光学相干断层扫描 (OCT) 的变化。在这里，我们说明用于创建此新体内模型 AQP4 针对中枢神经系统自身免疫反应 (ATCA)，现在可以用来研究允许发展他们可能怎样配合 B 与致病性 AQP4 特异性 T 细胞的机制的途径NMO 发病机制中的单元格。

在此协议中，我们使用来自 C57BL/6 AQP4-/-小鼠 T 细胞。皮下免疫 AQP4 p135-153 或 p201-220，含有致病性 T 细胞表位，这些小鼠诱发强增生性 T 细胞反应在引流区淋巴结 (图 1)，而这些两个肽诱导多较弱的 T 细胞WT 小鼠增殖。相比较而言，AQP4 p91-110，接种含有非致病性 AQP4 T 细胞行列式13或激活 T 细胞，造成实验性自身免疫性脑脊髓炎 (EAE)22，23 髓鞘肽 MOG p35 55 ，24，诱发类似规模 AQP4-/-及 WT 小鼠 T 细胞增殖。TCR 利用流染色流式细胞术分析为单个 Vβ 或 Vβ 的家庭，证明 p135-153-和 p201-220-异性 T 细胞从 AQP4-/-小鼠利用独特的 TCR 演奏曲目。选择性超扩散 AQP4 p135 153 和 p201-220 AQP4-/-小鼠，以及独特的 TCR 利用率 (图 2)，表明这些决定因素致病性 T 细胞的反应通常受胸腺负选择，强调使用 AQP4-/- T 细胞在本议定书的重要性。
之前的 ATCA 引产的过继转移，AQP4 肽引小鼠淋巴结细胞进行体外 Th17 或 Th1 极化条件下的连续三天。程度的极化捐助 CD4+ T 细胞是由细胞内细胞因子染色 (ICS) 确认和计量的流式细胞术 (图 3)。2 x 107捐助 AQP4 肽特异性 T 细胞与天真收件人小鼠静脉注射注射。大约六天后，几乎 100%的收件人小鼠开发临床症状的中枢神经系统的自身免疫性疾病，包括无力的尾巴和后肢瘫痪 (图 4)。更严重的临床疾病比 Th1 极化 AQP4 特异性 T 细胞诱导 Th17 极化 AQP4 特异性 T 细胞。代表性的鼠标，收到特定于肽-AQP4 的 Th17 和开发完整的后肢麻痹 （截瘫） 如视频 1所示。与开发 EAE MOG 特定 Th17 细胞给药后的小鼠，收件人小鼠痊愈 AQP4 特定 Th17 细胞诱导的临床疾病。至于 MOG p35-55-特定 Th17 细胞诱导 EAE，临床疾病致 AQP4 p135-153-特定或 p201-220-异性 Th17 细胞与中枢神经系统实质和脑膜 (图 5) 外周血单个核细胞浸润。病变在中枢神经系统的自身免疫性 AQP4 特定 Th17 细胞诱导为薄壁细胞中比脑膜更丰富。
视神经参与证明通过组织学的评估和由串行 10 月 AQP4 特定和 MOG 特定 Th17 细胞外周血单个核细胞的存在，以两个诱导的视神经炎症。而 AQP4 特定 Th17 细胞引起视神经 ③，MOG 特定 Th17 细胞诱导严重视神经炎 (图 5)。使用串行 OCT，视神经炎症明显的肿胀和增加视网膜内层 (IRL) 厚度为临床疾病致 AQP4 特定或 MOG 特定 Th17 细胞 (图 6)。对于 AQP4 Th17 致中枢神经系统自身免疫有关，IRL 厚度恢复至基线作为小鼠恢复从临床疾病。相比之下，持久性的 EAE 诱导 MOG 特定 Th17 符合 IRL 细化，并正如我们先前表明17，伴视网膜神经节细胞的损失。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56185/56185fig1.jpg"> 
图 1: AQP4 p135-153 和 p201-220 引出强健的 T 细胞增殖中 AQP4-/-小鼠，但不是 WT 小鼠。免疫小鼠 s.c.与指示肽在终审法院。11 天后，淋巴结被删除，并且然后培养与要么没有抗原，或用于免疫肽。扩散法测定3H-胸苷纳入 （平均 ± SEM，代表 5 实验）。<a href="//ecsource.jove.com/files/ftp_upload/56185/56185fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56185/56185fig2.jpg"> 
图 2: 从 AQP4-/-小鼠 AQP4 特异性 T 细胞利用独特的 TCR 演奏曲目。AQP4-/-和 WT 小鼠接种与指示肽。11 天后，淋巴结被删除和与肽用于免疫接种培养。细胞收获。通过流式细胞仪分析了 TCR Vβ 利用 (意思是 ± SEM，n = 5)。<a href="//ecsource.jove.com/files/ftp_upload/56185/56185fig2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56185/56185fig3.jpg"> 
图 3: 捐助 AQP4 特异性 T 细胞的促炎症极化。AQP4 p135-153 或 p201-220，免疫后十一天收获、 培养与用于非极化条件、 Th1 或 Th17 偏振免疫肽淋巴结细胞。Th17 或 Th1 极化进行了检查 ICS 和流式细胞白细胞介素-17 或 IFN-γ，分别。<a href="//ecsource.jove.com/files/ftp_upload/56185/56185fig3large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56185/56185fig4.jpg"> 
图 4: Th17 极化 AQP4 特异性 T 细胞诱导 WT 收件人小鼠瘫痪。WT 受鼠收到 AQP4-/-小鼠 2 x 107捐助 Th17 极化 AQP4 p135-153 或 p201-220-异性 T 细胞。Th17 极化 MOG 特异性 T 细胞作为阳性对照。结果具有代表性的 8 组实验数据 (n = 5/组)。* p &#60; 0.05，* * p &#60; 0.01，* * * p &#60; 0.001。<a href="//ecsource.jove.com/files/ftp_upload/56185/56185fig4large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56185/56185fig5.jpg"> 
图 5: AQP4 特定 Th17 细胞诱导视神经小鼠炎症的 WT。收件人的小鼠收到 2 x 107捐助 Th17 AQP4 p135-153-或 p201-220-引 Th17 细胞从 AQP4-/-小鼠或 Th17 MOG p35-55-特定细胞和牺牲了 10 天后。脊髓和视神经组织编制并沾有 H &#38; E/LFB 评价为炎症和脱髓鞘的证据分别。结果是 5 小鼠/组的代表。条码秤 =<a href="//ecsource.jove.com/files/ftp_upload/56185/56185fig5large.jpg" target="_blank">请点击这里查看此图的大版本。</a> 50 µ m。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56185/56185fig6.jpg"> 
图 6: AQP4 特定 Th17 细胞诱导的视神经炎症可以监控的纵向视网膜-10-WT 受鼠 0 日收到 2 x 107捐助 Th17 极化 AQP4 p201-220-特定或 MOG p35-55-异性 T 细胞。他们检查 oct （前政府的 T 细胞） 的第 0 天开始然后在天 4，7，8，9 10 11 14 和 21。IRL 厚度是测量的 （平均 ± 扫描电镜）。统计数据表明与天真控制的比较。结果具有代表性的 3 个实验 （5 小鼠/组）。* p &#60; 0.05，* * p &#60; 0.01，* * * p &#60; 0.001。<a href="//ecsource.jove.com/files/ftp_upload/56185/56185fig6large.jpg" target="_blank">请点击这里查看此图的大版本。</a>

Watch this Scientific Journal Video about Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4 at JoVE.com

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

在这里，我们提出一项议定书，引起瘫痪和视神经炎症反应的水通道蛋白 4 (AQP4) 转让-具体从 AQP4-/-小鼠到 WT 小鼠的 T 细胞。此外，我们演示了如何使用串行光学相干层析成像技术来监视视觉系统功能障碍。

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 ...

induction-paralysis-visual-system-injury-mice-t-cells-specific-for

Research

JoVE Journal

Immunology and Infection

11.3K 次观看.  University of California. 在这里，我们提出一项议定书，引起瘫痪和视神经炎症反应的水通道蛋白 4 (AQP4) 转让-具体从 AQP4-/-小鼠到 WT 小鼠的 T 细胞。此外，我们演示了如何使用串行光学相干层析成像技术来监视视觉系统功能障碍。

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

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

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

确定最佳人力Her2neu特异性CD8 + T细胞的细胞毒活性的比较CR51发行含量xCELLigence系统

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

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 ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

肽:MHC四聚体为基础的抗原表位特异性的T细胞的富集

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

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 ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

巨细胞病毒特异性的自动化细胞富集的T细胞用于使用细胞因子捕获系统的临床应用

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

猴免疫缺陷病毒特异性CD8分析<sup&gt; +</sup&gt; T细胞猕猴由肽MHC-I四聚体染色

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

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

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

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

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

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

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

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

小鼠肺损伤的诱导,通过博霉素内切注射

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

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

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