合资是由来自美国S＆结肠炎克罗恩基金会（CCFA）的奖学金支持。 JRM是支持由中国连锁经营协会，癌症研究所（CRI）的，霍华德H.古德曼（MGH），马萨诸塞州的生命科学中心（MLSC）和美国国立卫生研究院主任最新创新奖助学金。

1。 前肠道归巢和控制T细胞的体内产生（见图1 ） <ol><li>隔离糖化从野生型小鼠脾脾。在PBS的细胞悬液，离心重悬为5 400 × G.取出上清液和裂解红细胞resuspending在2-3分钟的ACK裂解液4毫升细胞沉淀。之后，添加5毫升的PBS。 5 400 XG离心去除上清。 </li><li>总脾重悬在1 × 10 6 /毫升的IMDM + 10％FBS + 50毫米2 -巯基乙醇+青霉素/链霉素（完整的IMDM）。分开两组，其中之一是与RA（或合成的RAR -受体激动剂，如Am80或Am580）补充到终浓度为100-200纳米3细胞。活化的T细胞在RA或RAR -受体激动剂的存在将上调a4b7and CCR9，将成为肠道嗜T细胞，而没有天雨激活的T细胞将成为控制T细胞。 </li><li>添加1.5-2.0毫升细胞悬液，每孔以前涂有抗CD3（10毫克/毫升）和抗CD28（10毫克/毫升）24孔板和37 °彗星在5％CO2 2（涂层板加500毫升/抗CD3加抗CD28在PBS孵育2小时，在37˚C，然后用2 ml PBS洗两次）。 </li><li>为了避免T细胞的过度刺激，转移到一个新的无涂层的24孔板中的细胞悬浮液后2-3天。孵育2-3天。根据细胞密度和增殖的媒体可能成为酸（黄色），在这种情况下，它可能是需要更换新鲜完整的IMDM媒体的一半。收获后4-5天（从0天算起）的细胞。 </li><li>典型的最终产量是1-2 × 10 6个效应T细胞/孔。因此，应至少每肠道嗜T细胞和控制T细胞9-12井镀以10-20 × 10 6 T细胞每条件。 </li></ol> 2。肠道归巢受体激活T细胞的分析经过4-5天的文化，我们应该观察肠道归巢受体的T细胞激活在RA或RAR激动剂的存在，而控制T细胞表达非常低的水平， 这些受体4 a4b7and CCR9的高表达。 流式细胞术（FACS）分析。 <ol><li>收集0.2-0.5 × 10 6细胞和离心5分钟在300克之间，4 ° C 。 </li><li>抗CD4 - FITC，抗- a4b7 - PE（BD公司），抗CCR9 - APC（eBiosciences）和抗- CD8 - PercP孵育15分钟，4 °在C黑暗。 </li><li>孵化后，300克4 ° C离心5分钟染色缓冲液洗和重悬细胞，并用流式细胞仪分析。 </li></ol> 3。 T与CFSE和CMTMR细胞跟踪细胞标记<ol><li>所有的解决方案保持在37℃前开始。清洗肠道嗜T细胞（a4b7 高 CCR9 高 ）和控制T细胞（a4b7 低/负 CCR9 低/ NEG）的两倍，用PBS去除血清。调节T细胞浓度为10-15 × 10 6 /毫升的PBS。 </li><li> CFSE（羧基琥珀酰亚胺酯）或CMTMR（氯苯甲酰氨基-四甲）荧光团，无论是肠道嗜T和控制T细胞的终浓度为5毫米和10毫米，分别轻轻旋涡和20分钟的孵育37 ° C。建议交换在相同或在另一项实验中，为了排除潜在的内在影响，对T细胞迁移荧光团的标号。 </li><li>孵化后，淬火1 FBS和孵化量在室温下为1-5分钟。用温PBS稀释10倍，离心5分钟，300克。洗净，用PBS重悬在完成IMDM两次。 </li><li> 10-20 × 10 6细胞的每个T人口理想的情况下，应以1:1的比例混合，然后在5分钟的300 XG离心。最后，在200-250毫升的温暖PBS重悬细胞和经尾静脉注入。 </li><li>为了计算出准确的投入比例（理想接近1）保存5-10毫升注入细胞悬液和300毫升的PBS稀释，并用流式细胞仪分析。 </li></ol> 4。分析的T细胞归巢虽然可以在不同时间点（1小时，几天后T细胞注射）分析小鼠，肠嗜T细胞归巢是最好的记录之间的12-24小时后注射。更长的时间点增加影响的最后一个单元格的数字/比率，如T细胞凋亡和T细胞从组织退出，其他变量的潜在影响。后的小鼠安乐死，从几个组织的细胞悬浮液，应毫不迟延地隔离，以减少细胞死亡，这可能会影响最终的产量和结果。利益的组织是小肠，结肠，脾，淋巴结肿大，Peyer氏斑，肝，肺和血液。如果注射T细胞是真正的肠嗜T细胞，它们应该平均5-10倍（或更高）的小肠黏膜相比，控制T细胞的家庭。然而，其他组织，如血液和脾脏，不应该表现出了优惠的T细胞迁移。淋巴细胞从肠道固有层和上皮内舱分离先前已经描述5。 <ol><li>流式细胞仪染色：样品是根据收集的密度不超过3.0 × 10 6细胞/ ml，悬浮细胞的数量。如果congenic CD45.1 +或Thy1.1 +小鼠作为接受者，与相 ​​应congenic的标记（例如，CD45.2或Thy1.2结合一些宗族标记（例如，TCRbchain，CD4 +，CD8 +细胞染色） ，CD45.1 + / CD45.2 +）。 </li><li>在流式细胞仪分析，细胞是门上的congenic标记（例如CD45.1），然后对具体的血统标志物（如CD4/CD8比值）和CMTMR和CFSE阳性细胞之间的比率分析。 </li><li>通常表示为归位指数（HI），这是计算的比例CFSE / CMTMR除以相应的输入比例（见下文）每一个组织（或CMTMR / CFSE）的数据。如果输入的比例是非常接近1，然后组织比率将相当于所喜。 </li></ol>您好= CFSE 组织 / CMTMR 组织 ：CFSE 输入 / CMTMR 输入 血也应该加以分析，以确定两个T细胞群是否平等的代表在流通领域中，如果一个T细胞的人口减少了其他方面（例如，在肺/肝优惠诱捕或下降的可行性）。可能发生这种情况时比较天真或休息的记忆性T细胞与最近激活的效应T细胞，因为后者可能会在更大程度上被困在肺部。如果血液中的HI是明显大于1的不同，可以在血液您好您好正常化组织。血液可通​​过从小鼠心脏穿刺麻醉avertin或isofluorane之前安乐死，并应与前两次使用流式细胞仪染色的ACK缓冲裂解。 血液正常化=您好组织 /嗨血 5。代表性的成果受体小鼠（Thy1.1 +）euthanized18h后的细胞注射。从脾，外周淋巴结（PLN），肠系膜淋巴结（MLN）和小肠椎板propia（LP）产生的细胞悬浮液。之后染色Thy1.2和CD8细胞，然后用流式细胞仪分析可行Thy1.2 + CD8 +细胞，这是最后CMTMR +细胞（肠嗜T细胞）和CFSE之间的比例分析浇注 +细胞（控制T细胞）除以投入比。结果可以显示在图2所示，其中肠道热带和控制T细胞同样归属脾（您好关闭TO1，用一根红线表示），可以欣赏。相比之下，肠道嗜T细胞迁移的10倍左右，更有效的唱片相比，控制T细胞。 <img src="/files/ftp_upload/2619/2619fig1.jpg" alt="图1" /> 图1：被隔离。图显示肠道热带和非肠嗜T细胞的生成和标签的T细胞和野生型小鼠与anti-CD3/anti-CD28在100-200纳米的存在或缺席所有激活- 反式维甲酸（RA）的。经过4-5天的RA治疗T细胞获得肠道CCR9和a4b7归巢受体的表达。然后，肠道热带和控制T细胞的差异标记CFSE或CMTMR，洗净，以1:1的比例混合成收件人小鼠经尾静脉注射。一些标记的细胞被用来确定CFSE / CMTMR输入比率（应接近1）。 <img src="/files/ftp_upload/2619/2619fig2.jpg" alt="图2" /> 图2：归位分析的例子 。小鼠分析h后12-18 T细胞注射液和单细胞悬液，获得利益的机关。细胞染色，抗- Thy1.2（congenic标志），加上抗CD8，然后Thy1.2 + CD8 + T细胞CMTMR比例分析 +和CFSE +各组织细胞，用流式细胞仪。组织CMTMR / CFSE比率，然后输入CMTMR / CFSE比例归一，以获取指数（HI）的归位。在这个例子中CMTMR +和CFSE +细胞是肠道热带和控制效应T细胞，分别。

<ol>
	<li>Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-endothelial+cell+recognition:+Three+(or+more)+steps+to+specificity+and+diversity.">Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity.</a> Cell. 67, 1033-1036 (1991).</li><li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+signals+for+lymphocyte+recirculation+and+leukocyte+emigration:+the+multistep+paradigm.">Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.</a> Cell. 76, 301-314 (1994).</li><li>Iwata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+imprints+gut-homing+specificity+on+T+cells.">Retinoic acid imprints gut-homing specificity on T cells.</a> Immunity. 21, 527-538 (2004).</li><li>Mora, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+and+dynamic+control+of+CD8+T+cell+homing+by+dendritic+cells+from+skin-+and+gut-associated+lymphoid+tissues.">Reciprocal and dynamic control of CD8 T cell homing by dendritic cells from skin- and gut-associated lymphoid tissues.</a> J Exp Med. 201, 303-316 (2005).</li><li>Mora, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+imprinting+of+gut-homing+T+cells+by+Peyer's+patch+dendritic+cells.">Selective imprinting of gut-homing T cells by Peyer's patch dendritic cells.</a> Nature. 424, 88-93 (2003).</li><li>Mempel, T. R., Scimone, M. L., Mora, J. R., von Andrian, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+leukocyte+trafficking+in+blood+vessels+and+tissues.">In vivo imaging of leukocyte trafficking in blood vessels and tissues.</a> Curr Opin Immunol. 16, 406-417 (2004).</li></ol>

即使归巢的实验提供了非常宝贵的有关在任何特定的组织细胞总人口的迁移的信息，应牢记这些实验不直接分析内皮细胞粘附，因此不歧视的多级粘连步骤（S）级联（圈养/滚动，激活或坚持）一个给定的归巢受体行事。黄金标准中定义的粘附级​​联的归巢受体的具体的作用，是活体显微镜（IVM），其中个人荧光标记的T细胞（或其他细胞，甚至荧光标记的珠）直接观察与内皮细胞的实时交互。?...

动物：C57BL / 6小鼠的T细胞隔离。此外，杰克逊实验室（ME）的巴尔港，CD45.1和Thy1.1 congenic株都可以通过。 文化传媒：IMDM（Iscove的修改贝科的中等+ L -谷氨酰胺+ HEPES），加上10％ 的热量，灭活胎牛血清 （胎牛血清，低内毒素，GIBCO ®，Invitrogen公司，卡尔斯巴德，加利福尼亚）辅以100 ü / ml青霉素，100毫克/ ml链霉素（Hyclone公司抗生素，马萨诸塞州沃尔瑟姆），0.5毫克/毫升fungizone /两性霉素B（GIBCO），B -巯基乙醇和50毫米。 ACK红血细胞裂解液 （红细胞，10毫米KHCO 3 0.1毫米，150毫米NH 4 CL，EDTA，pH值8.0），调整pH值7.2-7.4，并储存在室温下）。 PBS（磷酸盐缓冲生理盐水，Hyclone公司，马萨诸塞州沃尔瑟姆，）。 流式细胞仪（FACS）媒体 （PBS或IMDM + 2％FBS + 5 mM的EDTA）。当染色选择- FC的嵌合体，媒体使用2毫米钙+ +应该是所有步骤中使用（包括流式细胞仪收购） 。在这种情况下，建议IMDM培养。 T细胞的标签和过继转移：CFSE（羧基荧光素二醋酸琥珀酰亚胺酯），CMTMR（（5 - （6 )-((( 4 -氯甲基）苯甲酰）氨基）四甲）自Molecular Probes ®，Invitrogen公司，卡尔斯巴德，CA ）。 1000 ×股票应是在DMSO（CFSE 5毫米，20毫米CMTMR），并储存在-20 ° C 多克隆T细胞的活化 ：24孔或96孔板（组织培养处理，聚苯乙烯，平底，有盖的，位于美国新泽西州富兰克林湖，屋宇署猎鹰）孵育2小时，分别在37℃， 50毫升PBS含有抗CD3加抗CD28抗体（10毫克/毫升）。然后，培养板，用PBS洗两次，并立即用于T细胞培养。另外，anti-CD3/anti-CD28（DYNAL，Invitrogen公司，卡尔斯巴德，加利福尼亚州）涂层磁珠可用于对T细胞的活化，而不是板结合的抗体。 流式细胞仪（FACS）染色：多克隆活化：CD3（1452C11），CD28（37.51）。天堂单克隆抗体：CD4（L3T4），CD8a（LY - 2），Thy1.2（CD90.2/53-1.2），CD45.2（104）。肠道归巢受体：纯化CCR9（CD199/eBioCW-1.2，eBioscience公司，圣迭戈，CA），a4b7（LPAM-1/DATK3），同型对照 （IgG2a，K）。皮肤归巢受体：P -选择素- FC（纯化的小鼠P -选择- IgG融合蛋白，BD Pharmingen公司，圣何塞，加利福尼亚州），E -选择素- FC（重组小鼠E-Selectin/Fc奇美拉，R＆D系统，明尼阿波利斯， MN），加上相应的辅助试剂山羊F（AB）2抗人IgG的R - PE（Invitrogen公司，卡尔斯巴德，加利福尼亚州）。 全反式维甲酸 （Sigma公司，圣路易斯，密苏里州）是悬浮于无水乙醇或使用一个黄色的灯泡或间接的光线来源，在筹建期间DMSO的。商店的玻璃小瓶分装于-80 ° C和避光在任何时候。合成的RAR -受体激动剂：Am80（和光化工，里士满，弗吉尼亚州）。

competitive homing assays to study gut-tropic t cell migration

为了发挥其功能的淋巴细胞，需要离开血液和迁移到身体的不同组织。淋巴细胞粘附于血管内皮细胞和组织外渗是一个多步骤的过程，不同的粘附分子控制（归巢受体）对淋巴细胞和各自的配体（addressins）1 2内皮细胞上显示。即使这些粘附受体的功能，可部分研究体外，其生理意义的最终测试，以评估他们的作用 ，在体内淋巴细胞的黏附和迁移。两个相辅相成的策略已经用于此目的：活体显微镜（IVM）和归巢的实验。虽然IVM必须确定精确的实时性和在不同组织中的粘附级联在特定的粘附受体的贡献，IVM的是耗费时间和劳动力密集，往往需要复杂的外科技术的发展，它需要同质化的前隔离细胞群，它允许在任何给定的时间只有一个组织/器官的分析。与此相反，有竞争力的归巢实验允许的直接同步比较，在相同的鼠标在两个移民（甚至更多）的细胞亚群，他们还允许许多组织和分析大量的细胞在相同的实验。 在这里，我们描述古典的竞争归巢的协议，用于特定组织，以确定一个给定的细胞类型的优势/缺点相比，控制细胞群。我们选择来说明肠道热带与非肠道嗜T细胞的迁徙，因为肠道黏膜是与外部环境最大的身体接触表面，它也是与最好的定义的迁徙要求额外的淋巴组织。此外，最近的工作已经确定，维生素代谢产物的全反式维甲酸（RA）诱导淋巴细胞的肠道特异性粘附受体（整合a4b7and趋化因子受体CCR9）负责的主要分子机制。因此，我们可以很容易地通过激活T细胞在RA的存在或缺乏肠道热带和非肠道嗜淋巴细胞体外大量产生，分别，终于可以在这里所描述的竞争归巢实验。

Watch this Scientific Journal Video about Competitive Homing Assays to Study Gut-tropic T Cell Migration at JoVE.com

Competitive Homing Assays to Study Gut-tropic T Cell Migration

竞争归巢实验允许直接评估一个鼠标的两个不同的细胞群的洄游特性。在这里，我们说明了此过程中，通过对比前体内产生的肠道热带与非肠嗜T细胞迁移。

竞争的原点实验研究肠嗜T细胞迁移

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

competitive-homing-assays-to-study-gut-tropic-t-cell-migration

Research

JoVE Journal

Immunology and Infection

12.4K 次观看.  Massachusetts General Hospital, Harvard Medical School. 竞争归巢实验允许直接评估一个鼠标的两个不同的细胞群的洄游特性。在这里，我们说明了此过程中，通过对比前体内产生的肠道热带与非肠嗜T细胞迁移。

视频: Competitive Homing Assays to Study Gut-tropic T Cell Migration

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

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.

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 ...

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 ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with &lt;em&gt;Mycobacterium bovis&lt;/em&gt; Bacille Calmette-Gu&amp;#233;rin

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

基于CFSE-试验鼠皮肤树突状细胞的迁移习进引流淋巴结在与感染<em&gt;牛分枝杆菌</em&gt;卡介苗

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

利用X射线晶体学，生物物理学，和功能分析，以确定管理机制T细胞癌抗原的受体识别

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

一种新的体外创面愈合试验评价细胞迁移

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

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

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

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

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

一种研究人体 T 细胞召回反应的吸水泡协议

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

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Methods Article

竞争的原点实验研究肠嗜T细胞迁移