Name: transplantation of tail skin to study allogeneic cd4 t cell responses in mice
Uploaded: 2014-07-25T13:00:00.000+00:00
Duration: 6 min 48 s
Description: Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R. We thank E. Palmer and B.T.H. Hausmann for mice and technical expertise.

在这个视频发布和协议，所有动物的程序按照批准由州局巴塞尔城市州，瑞士动物的协议进行的。执行在无菌条件下尽可能的所有程序。 外科1。准备<ol><li>高压灭菌所有的手术器械，使用前纱布。 </li><li>热保暖垫，并组织手术器械上的表 （表材料/方法 ）。 </li><li>打开手指带绷带。应用凡士林的伤口垫用棉签（确保纱布完全覆盖）。 </li><li>称取小鼠和管理镇痛丁丙诺啡0.05毫克/公斤体重。注:这将立即移植后提供镇痛。 </li></ol> 2，准备的尾部皮肤移植<ol><li>每二氧化碳窒息和D安乐死供体小鼠（ 例如 IA BM12）isinfect尾部与前切除术消毒液。 </li><li>定位尾部的黑色条纹倒在​​一个干净的塑料板，切纵向贯穿中间路线，做一个肤浅的切口仅累及皮肤层。 </li><li>拉过使用镊子夹住尾巴的皮肤。 </li><li>把尾部皮肤在10cm的细胞培养皿填充用10ml HBSS中。 </li></ol> 3，外科手术<ol><li>通过吸入以透明感应箱3％的异氟烷溶液麻醉受体小鼠（ 例如，C57BL / 6）。注意:这需要3-5分钟的鼠标，达到手术麻醉平面。使用上的眼睛兽医药膏，以防止肌肤干燥，同时在麻醉下。 </li><li>将鼠标放在暖垫，并通过一个口罩和驴由脚趾捏适用1.5％异氟烷每个动物的条件反射。 </li><li>剃接受者小鼠的背部的网站（ 例如，C57BL / 6），用干纱布SW去除污染的毛AB。 </li><li>消毒剃部位无菌解决方案移植。 </li><li>捏在背部中间/右现场镊子皮肤和使用弯曲剪刀，使约1×0.6厘米的圆形切口。 </li><li>如果发生出血，然后清洗伤口，用无菌棉签。 </li><li>评估该切口的大小和切断从使用手术刀尾部皮肤的估计大小的移植。 </li><li>修剪边角圆的移植。 </li><li>加载移植上​​的手术刀，干多余的HBSS在无菌棉签，并将其放置在受体小鼠的移植床。 </li><li>避免供体和宿主皮肤的重叠和应用上的接触区域组织粘合剂完全。 </li><li>应用无石膏鼠标左右腰部皱褶。 </li><li>适用于粘接非弹性绷带来固定石膏鼠标（2毫米移位）。 </li><li>切开以约3毫米的绷带的上腹部站点的剪刀。注意:这是为了避免TR鼠标的牙齿在恢复阶段apping。 </li><li>取出麻醉口面罩，并保持移植的小鼠在加热垫，直到清醒。 </li><li>返回小鼠移植到自己的笼子里，只有当完全康复，并评估其流动性，以确保绷带是不是太紧张。 </li><li>继续进行下一个手术前消毒用70％乙醇所有的外科手术器械。 </li></ol> 4，术后护理<ol><li>添加含扑热息痛的糖浆的饮用水（4毫克/毫升），并管理它7天。 </li><li>返回鼠标的动物房一旦恢复。 </li><li>密切留意老鼠的临床症状包括抑郁症或其他行为改变（饮食或不动），外观异常（缺乏疏导）或姿势（PILO勃起或弯腰驼背的姿势），当时的绷带到位（1周）。 </li></ol> 5，去除绷带（6-7天移植后） <ol><li> ANESthetize小鼠通过吸入以透明感应框（见上文）3％的异氟烷​​溶液。 </li><li>切绷带和石膏与动脉剪刀，小心地将其删除。注意:避免拉伸移植部位。 </li><li>监测移植小鼠的临床症状（见4.3），得分为抑制皮肤移植（ 表1）。注:该接枝评分系统采用的尾部皮肤较背侧皮肤不同发质的优势。移植和排斥的外观分为3级。 C57BL / 6小鼠免疫功能的异体皮（MHC II类错配， 例如，爱荷华BM12）移植物通常在12-15天移植后（自己的数据和15-20）拒绝。 </li></ol>

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The authors declare that they have no competing financial interests.

皮肤移植是一种广泛使用的方法为研究抑制和容忍依赖于T细胞。自成立以来的皮肤移植模型中，几个修改和变化都得到了应用。在描述的过程中，使用麻醉气体（异氟烷）进行IA BM12尾部皮肤移植。使用气体麻醉的降低执行和小鼠恢复，从而减少了应力对移植小鼠的时间。该程序使用组织粘合剂来固定尾部皮肤，这容易卷曲超过耳或背皮肤组织。此外，它弄平切割和修整的边缘，因为这降低卷曲和提高了对背侧切除部位的尾部皮肤的定位之前尾部皮肤是非常重要的。 为了评估移植排斥或接受它需要准备尾部皮肤与黑色条纹（ 例如，C57BL / 6小鼠）在移植物的中间，方便TRA得分根据头发的质感差异nsplants。 在MHC II类IA BM12分子的突变完全激活免疫（相同遗传背景）和免疫缺陷小鼠CD4 + T细胞。一方面这有利于对结果的解释，但另一方面它限制了该方法对CD4 + T细胞的研究。为了延长T细胞活化其他T细胞亚群，尾部的皮肤从其他捐助者， 比如，相同的遗传背景，包括IA BM1小鼠和不同的遗传背景，包括免疫BALB / c或DBA小鼠可移植。 在急性排斥反应的研究中，我们受益于II类MHC槽和异源CD4 + T细胞，研究器官排斥小鼠的活化天然存在的突变。 C57BL / 6小鼠拒绝IA BM12皮肤移植中移植，这与大多数研究一致USI后14天吴IA BM12皮肤移植15-20。除了 ​​急性排斥模型被广泛用于测试的CD4 + T细胞从基因缺陷小鼠的同种异基因的潜力。事实上，IFN-γ - / -小鼠表现出延迟IA BM12皮肤移植排斥反应（未发表数据和16）。阻断IFN-γ通过阻断抗体同样地延迟排斥15,17,18的管理。这表明IFN-γ在发起在Ag呈递细胞和内皮细胞（版本12）II类MHC表达的关键作用。自穿孔缺陷的小鼠并没有耽误IA BM12皮肤移植排斥21，细胞因子的作用似乎是抑制更为重要。实际上，IL-6施用导致延长移植物存活，有可能通过下调IFN-γ生产15。此外，嗜中性粒细胞似乎在IA BM12皮肤移植物中的IL-4缺陷型小鼠19的排斥中起主要作用</suP&gt;表示微调固有免疫和适应性免疫细胞产生的细胞因子。这一假说是由观察到的ABT-100的块的细胞因子分泌和延迟IA BM12移植排斥小鼠20加强。管理的抗CD3抗体延误IA BM12皮肤移植排斥反应的免疫小鼠（未发表资料）。此外，我们发现，T细胞缺陷的CD3ε - / -和的Rag2 - / -小鼠接受异基因皮肤移植长达100天。公差在这些小鼠中可以很容易地恢复/由CD4 + T细胞的自免疫C57BL / 6小鼠过继转移破碎。转移2×10 4个CD4 + T细胞或抗原特异性细胞ABM是足以引起大约14天IA BM12移植物排斥。隔离Vα2Vβ8阳性细胞ABM但是表明在排斥前淋巴结广泛的增殖和IFN-γ的生产，拒绝是依赖于I-ABM12快递离子在皮肤移植。 总之，这种小鼠模型是研究抑制或耐受性在低活化T细胞数量的一个很好的方式。尾部皮肤移植是容易执行，并且可以被应用到小鼠不同的遗传背景，研究T细胞的同种异体反应而不会产生严重的生理效应。此外，这种模式是理想的干预，化学和生物物质，诱发或破皮肤移植耐受。

固体器官，如皮肤，心脏和肾脏移植是目前在全世界的医疗实践1的标准程序。成功移植的器官可以通过激活受体的免疫系统，其中确认捐助的主要组织相容性抗原被拒绝。因此移植的患者需要使用免疫抑制药物治疗2。异体皮移植在小鼠体内建立了梅达沃和他的同事于1955年，是识别后世誉为主要组织相容性复合体（MHC）I类和II目标分子的帮助。从那以后，皮肤移植模型得到了不断的修改和调整，研究T细胞亚群的作用和抑制移植排斥2-4化学和生物干预的相关性。从耳朵和躯干的皮肤都比较困难的准备，而且更容易受到缺氧和坏死较尾皮5;然而，移植过程是类似的。在另外的尾部皮肤移植物的监测是很容易由于皮肤的特征头发纹理。 本文提供的详细步骤MHC II类错配尾部皮肤移植，允许的CD4 + T细胞介导的移植排斥反应和耐受小鼠不同方面的研究。在MHC II类分子IA b将天然三点突变（称为IA BM12）6-9是足以引起排斥反应的皮肤移植物在C57BL / 6小鼠8。在IA BM12分子激活CD4 + T细胞与各种αβT细胞受体（TCR）从C57BL / 6小鼠链，其中Vα2Vβ8-TCR特异性T细胞，以产生一个TCR转基因小鼠10识别。 Vα2Vβ8-TCR特异性T细胞的过继转移已被用于建立一个排斥模型在免疫缺陷C57BL / 6的Rag2 - / -移植与IA BM12皮肤老鼠。 捐赠者和接受者之间的基因差异影响移植接受和拒绝的结果。有不同类型的移植:自体移植是从接收者个人自身移植; syngrafts和移植物分别从基因上相同和基因无关个体移植。不同的异体器官移植的接受已经证明了患者和小鼠模型11,3,4化学和生物干预。在一个基本的方法，抗-CD3抗体处理的C57BL / 6小鼠表现出IA BM12尾部皮肤（未发表数据），生存期延长。 CD4 +和CD8 + T细胞移植受体小鼠前的枯竭导致接受MHC I类和II不匹配的移植（修订版12）。有趣的是，抑制皮肤移植取决于CD4 +的存在</sup&gt; T细胞（版本12）。在此模型中，通过阻断共刺激分子的抗体或抑制与调节性T细胞靶向不同的免疫细胞之间的特异性相互作用可能诱导耐受（未发表数据）。事实上，无论阻断CD40和CD28分子导致了长期的皮肤移植耐受13,14。 相比于其它器官的移植尾部皮肤移植容易进行并易于监测。此外，尾部皮肤移植很容易准备，而且不太容易受到缺血较其他皮肤组织。而相比之下，注射麻醉剂，移植过程中使用麻醉气体（异氟烷）的缩短两者的程序和收件人的恢复时间。尾部皮肤移植，这可能会导致不完全的伤口愈合，移植物排斥的卷曲，可以防止通过应用组织粘合剂。此外，IA BM12尾部皮肤移植模型的专门激活的CD4 <sup&gt; + T细胞（相同遗传背景）两个免疫和免疫缺陷小鼠促进结果的解释。 本协议描述了一个可靠，重现性好，易于监控的小鼠模型，允许化学和生物干预。该模型的目的是调查排斥和耐受诱导的尾部皮肤移植手术。

<table><tbody><tr><td>Betadine standard solution</td><td>Mundipharma</td><td></td><td></td></tr><tr><td>Cotton swab</td><td>Carl Roth GmbH</td><td>31025060</td><td></td></tr><tr><td>Dafalgan , UPSA</td><td>Bristol Myers Squibb UPSA</td><td></td><td></td></tr><tr><td>Hansaplast finger strips</td><td>Beierdorf AG</td><td>REF.76861</td><td></td></tr><tr><td>Histoacryl tissue adhesive</td><td>Braun</td><td>REF.1050052</td><td></td></tr><tr><td>Leukotape classic , 2 cm x 10 m</td><td>BSN Medical SAS</td><td>REF.02204-00</td><td></td></tr><tr><td>PBS, Phosphate Buffered Saline, pH 7.4</td><td>Invitrogen</td><td>10010015</td><td>GIBCO</td></tr><tr><td>Sterile gauze, 5 x 5 cm, 8 ply</td><td>MaiMed GmbH</td><td>21010</td><td></td></tr><tr><td>Narrow pattern forceps</td><td>FST</td><td>11003-12</td><td></td></tr><tr><td>Fine iris scissor curved</td><td>FST</td><td>14095-11</td><td></td></tr><tr><td>Fine iris scissor</td><td>FST</td><td>14094-11</td><td></td></tr><tr><td>Mayo scissors</td><td>FST</td><td>14010-15</td><td></td></tr><tr><td>Artery scissors ball tip 11.5 cm</td><td>FST</td><td>14080-11</td><td></td></tr><tr><td>Tissue forceps</td><td>FST</td><td>11021-14</td><td></td></tr><tr><td>Surgical blade No. 20</td><td>Swann-Morton LTD</td><td>3006</td><td>Carbon steel</td></tr><tr><td>Surgical blade handles</td><td>Swann-Morton LTD</td><td></td><td></td></tr><tr><td>Syringe, 1 ml</td><td>ARTSANA</td><td></td><td>Disposable</td></tr><tr><td>Temgesic, buprenorphine</td><td>ESSEX Chemie AG</td><td></td><td>0.3 mg/ml</td></tr><tr><td>Tissue Culture dishes 10 cm, 60.1 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>TPP</td><td></td><td></td></tr><tr><td>Vaseline</td><td>Vifor SA</td><td></td><td></td></tr><tr><td>Warm pad</td><td>Solis</td><td></td><td>Type 223</td></tr></tbody></table>

transplantation of tail skin to study allogeneic cd4 t cell responses in mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

在第一种方法中的C57BL / 6小鼠移植IA BM12同种异体移植物和IA b syngrafts。拆除绷带后，移植物伤口愈合的迹象明显没有在C57BL / 6小鼠（ 图1A）的接触带的闭合。拆除绷带后，CD4 + T细胞介导的炎症导致的坏死区域的外观（红点）和内移植（ 图1B-C，实心）后13天C57BL / 6小鼠抑制IA BM12移植，而syngrafts被容忍长达100天（ 图1B-C，空心符号）。这些数据显示，IA BM12移植在免疫C57BL / 6小鼠的急性排斥反应的进展。 各种研究表明，抑制MHCⅡ类错配的尾部皮肤取决于同种异体CD4 + T细胞（修订版12）的存在。事实上，T细胞缺陷CD3ε - / - &lt;/ SUP&gt;小鼠和T-和B-细胞缺陷的Rag2 - / -小鼠表现出完整的伤口愈合和耐受性的IA BM12移植物达100天（ 图2），而C57BL / 6小鼠移植拒绝在13天（ 图2） 。 IA的BM12不匹配的尾部皮肤可以接受异基因CD4 + T细胞和它们的效应功能在没有严重的疾病的病症的小鼠模型的研究。 打破既定公差在CD3ε - / -和的Rag2 - / -小鼠中，CD4 + T细胞过继转移:2×10 4个多克隆的C57BL / 6的CD4 + T细胞足以诱导排斥IA BM12移植的14天内（数据未显示）。 50,000和100,000的C57BL / 6的CD4 + T细胞的转移不加速CD3ε的移植物排斥过程- / -和的Rag2 - / -小鼠。这些数据强调allogen的作用EIC CD4 + T细胞在急性皮肤移植排斥模型。 这个尾部皮肤移植模型的优点是，CD4 + T细胞的功能分析可以通过抗原-特异性CD4 + T细胞的过继转移容易进行。该转基因小鼠ABM 9被认为是一种理想的工具描绘的活化和人口的表型行为，而不是非特异性的CD4 + T细胞的影响。 2×10 4个抗原特异性ABM单元的传输是足以引起排斥反应的IA BM12移植物中的Rag2 -在13天（ 图3A-B）小鼠- /。此外，ABM细胞易于分离，并且可以使用Vα2Vβ8-TCR链（ 图3C）中的表达进行跟踪。细胞因子IA BM12的ABM细胞增殖和生产移植的Rag2 - / -小鼠进行了研究。反导细胞增殖后缀的vely由eFluor670稀释测定法（ 图3D），并确定在PMA刺激（ 图3E）反导细胞高达30％的产生的IFN-γ。 总而言之，这些数据表明，相对少的异源CD4 + T细胞是必需的，诱导抑制IA BM12皮肤移植，使该模型非常适合同种异体T细胞以最小的严重病理分析。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51724/51724fig1highres.jpg" width="500" /> 图1。移植小鼠异体同基因和尾部的皮肤。 （一）C57BL / 6小鼠移植与异体（IA BM12）和同基因（IA B）尾部皮肤（N = 5元组）。图片第7天拆除绷带后，说明移植（左）和syngrafts（右）（B）计分和（C）皮肤移植物存活7天，直到抑制或最终的监测（100天）的日子。 <a href="https://www.jove.com/files/ftp_upload/51724/51724fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="4in" src="/files/ftp_upload/51724/51724fig2highres.jpg" width="400" /> 图2是同种异体移植物的耐受性在没有T细胞的小鼠中的Rag2的生存- / - IA BM12 -小鼠皮肤移植物移植到C57BL / 6，CD3ε - / -和的Rag2 - / -小鼠（n = 4-6每组），并说明拒绝和接受移植。 <a href="https://www.jove.com/files/ftp_upload/51724/51724fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in"SRC ="/ files/ftp_upload/51724/51724fig3highres.jpg"宽度="500"/&gt; 图3抗IA BM12特异性CD4 + T细胞增殖，并在移植排斥反应产生IFN-γ（一）评分和IA BM12尾部皮肤移植中的Rag2（B）生存- / -小鼠（n = 5）继转移用2×10 4的抗原特异性细胞ABM（C）Vα2Vβ8染色ABM细胞从转基因小鼠的反弹道导弹的淋巴结中分离的（D）eFluor670稀释增殖ABM细胞和（E）的IFN-γ生产的PMA-刺激反导细胞IA BM12皮肤移植的Rag2的淋巴结中分离- / -小鼠在第9天之后反导继细胞转移（N = 5只小鼠） <a href="https://www.jove.com/files/ftp_upload/51724/51724fig3highres.jpg" target="_blank">请点击此处</a>查看该图的放大版本。 <table border="0" cellpadding="0" cellspacing="0" width="569"><tbody><tr height="21"><td height="21" style="height:21px;width:84px;">移植得分</td><td style="width:485px;">描述</td></tr><tr height="21"><td height="21" style="height:21px;"> 3 + </td><td>没有红区和一个闪亮的黑色条纹的存在</td></tr><tr height="21"><td height="21" style="height:21px;"> 2 + </td><td>红色的小区域，损失一个闪亮的黑色条纹和干燥的外观</td></tr><tr height="21"><td height="21" style="height:21px;"> 1 + </td><td>大红色区域的外观，没有黑条和收缩</td></tr><tr height="21"><td height="21" style="height:21px;"> 0 </td><td>移植排斥反应定义为80％坏死</td></tr></tbody></table> 表1。嫁接评分系统，移植和排斥反应的出现是美食gorized在3级，趁着不同发质的尾巴和背部皮肤。

Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

transplantation-tail-skin-to-study-allogeneic-cd4-t-cell-responses

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.4K 次观看.  University of Basel and University Hospital Basel. Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

视频: Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

小鼠皮肤移植

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

科研

生物学

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 ...

免疫与感染

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

肿瘤移植用于评估小鼠肿瘤浸润CD8 + T细胞的动力学

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

癌症研究

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 ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

成像的CD4 T细胞间质迁移发炎真皮

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

鼠全层皮肤移植

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

原位后肢移植中的鼠标

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

医学

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Described here is a method for producing exogenous cytokine in patient-derived xenograft (PDX) mice via weekly intraperitoneal injection of a cytokine-transduced stromal cell line. This method broadens the utility of PDX and provides the option for transient or sustained exogenous cytokine delivery in a multitude of PDX models.

通过细胞因子转导的基质细胞系注射外源性细胞因子在患者来源的异种移植物中的表达

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

骨髓移植平台研究树突状细胞在移植与宿主疾病中的作用

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Adoptive Cell Transfer: Introducing Donor Mouse Splenocytes to a Host Mouse and Assessing Success via FACS

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
Adoptive cell transfer is a method for introducing cells into a patient or study organism in order to treat a disease or study a biological process, such as haematopoiesis. Aims of adoptive transfer are various; it can be used in fundamental biology as well as in medical sciences (1, 2). In mouse models, migration and distribution of transferred cells can be studied and followed by a tracking system (cell surface marker, staining by CFSE, etc.). In cancer studies on mouse models, transfer of specific cell populations can be used as experimental treatment against tumors. Another example for this technique is creation of chimeric mice by transfer of bone marrow cells to irradiated mice or mice with a severe immunodeficiency phenotype. This mouse model can be used to assess the impact of gene deletion on a specific cell population for instance. Transfer of bone borrow cells is also used in human medical treatment. When patients are irradiated in case of cancer therapy, adoptive transfer of bone marrow allows immune system reconstitution.
The first step in this technique is to obtain the cell population of interest. The technique chosen to isolate this population depends on the level of specificity of the targeted population. The largest level of selection is the whole organ, in which all cell populations present in the organ are taken. A more precise method is selection of a target cell population, often selected by one cell surface marker. The ideal method to sort cells in this case is by magnetic sorting. Finally, the most stringent level is the selection of cells by several cell surface markers to sort very specific cell populations. Flow cytometry sorting is the most popular method for this level of selection. Once population of interest is obtained, it can be transferred to the host. Before adoptive transfer it is essential to ensure compatibility between host and donor. Indeed, regardless of transfer goal, compatibility is crucial to assure cells adoption by the host without cells rejection.
In this lab exercise, we demonstrate the adoptive cell transfer technique by transferring splenocytes from a CD45.2 mouse into a CD45.1 Rag&#947;c mouse (lacking lymphocytes) and four days later confirm the splenocytes transfer using flow cytometry (see Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10504/10504fig1.jpg" /> 
Figure 1: Schematic representation of adoptive transfer. (1) Splenocytes are isolated from CD45.2 mice and (2) transferred in CD45.1 Rag&#947;c mouse, control mouse is injected with PBS only. (3) 4 days after adoptive transfer, splenocytes are recovered from mice and (4) analyzed by flow cytometry. <a href="https://www.jove.com/files/ftp_upload/10504/10504fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

摘要

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此视频中的章节

小鼠皮肤移植

移植物抗宿主病的诱导和

肿瘤移植用于评估小鼠肿瘤浸润CD8 ⁺ T细胞的动力学

诱导小鼠肠道炎症由效应CD4的过继转移

成像的CD4 T细胞间质迁移发炎真皮

鼠全层皮肤移植

原位后肢移植中的鼠标

通过细胞因子转导的基质细胞系注射外源性细胞因子在患者来源的异种移植物中的表达

骨髓移植平台研究树突状细胞在移植与宿主疾病中的作用

收养细胞转移:将供体小鼠孢子细胞引入宿主小鼠，并通过FACS评估成功