SCJ是由一个国际社会对心脏和肺移植研究奖学金和医生的皇家学院和加拿大德特韦勒旅行Fellowship.SM外科支持部分由一个国际社会对心脏和肺移植事业发展奖（以SCJ）的支持。 SS由卫生研究牛津大学的生物医学研究Centre.JH国家研究院的支持是肾脏研究英国高级非临床奖学金的获得者。一威康信托基金会项目资助（082519Z07Z），英国心脏基金会的项目资助（PG / 10 / 62.28504）和欧盟框架计划7（; BioDRIM一项研究）:这项工作是由以下的赠款，以AB和KW资助。作者要感谢迈克尔·帕森斯和在Lunenfeld-的Tanenbaum研究所流式细胞仪核心设施，西奈山医疗系统，多伦多用于提供与马克的ImageStream仪器X获得支持。

1.准备试剂和材料要求<ol><li>制备含有2％胎牛血清（FBS）（"洗涤缓冲液"）的磷酸盐缓冲盐水（PBS）。用含有0.1％非离子型洗涤剂2％FCS的PBS制备（"烫发洗涤缓冲液";见材料的表）。用1.5％的甲醛制备PBS。 注意:小心！甲醛具有腐蚀性，可能致癌，并佩戴适当的个人防护装备必须被处理。 </li><li>制备含有2％FBS和0.5mM乙二胺四乙酸（EDTA），用于磁性细胞分离（"MCS缓冲液"）的PBS。 </li><li>制备50微克在二甲亚砜/ mL的鬼笔环肽 - 荧光素异硫氰酸酯（鬼笔环肽-FITC）（DMSO）。制备1mg / mL的核染色（ 例如，1毫克/在DMSO或双-苯甲亚胺染料毫升7氨基放线菌素D（7-AAD）;见材料的表）的DMSO。准备适合于感兴趣的细胞荧光染料标记的抗体和成像流式细胞仪。 </li><li>获得动物组织（ 例如，淋巴结和脾）和作为抗原呈递细胞或祖细胞的来源T细胞和异基因动物组织（ 例如，脾和骨髓）的源极。 </li><li>制备细胞培养基（ 例如，的Roswell Park Memorial Institute培养基（RPMI）1640或Dulbecco改进的Eagle培养基（DMEM），补充有10％FBS），50μM2-巯基乙醇，青霉素和链霉素和获得24-和/或96-孔细胞培养板。 </li></ol> 2.准备抗原呈递细胞 注:从理论上讲，任何APC人口可能用这种方法进行检查。未成熟的小鼠骨髓来源的树突状细胞（DC）作为APC是在这种情况下起诉。许多协议存在用于产生这些细胞（例如，参考文献34和35）。简言之，使用以下方案。 <ol><li>从股骨和胫骨成RPM冲洗骨髓我1640或DMEM。 </li><li>通过悬浮的细胞通过70-μm的细胞过滤器以去除小骨片和碎屑。 </li><li>沉淀通过离心细胞，然后用氯化铵缓冲液，在室温下5分钟裂解红细胞。 </li><li>沉淀通过离心（400×g下，5分钟）将细胞重悬细胞沉淀。 </li><li>洗涤细胞在5 - 10毫升洗涤缓冲液，沉淀物通过离心（400×g下，5分钟），并再悬浮细胞沉淀。 </li><li>通过标记与生物素化的抗CD3（5μg/ mL的），抗B220（5微克/毫升），抗MHC II类（1微克/毫升），和抗CD11b的细胞富集在细胞分离柱的造血前体（5微克/毫升）的抗体。 </li><li>通过离心（400×g下，5分钟）沉淀细胞并再悬浮细胞沉淀。 </li><li>孵育用抗生物素磁性微珠（参见材料的表）将细胞在4℃下进行10分钟。 </li><li>洗涤和沉淀细胞（400×g下，5分钟），然后重新suspenð他们在1个MCS毫升缓冲液使用大正选择去除所述标记的细胞用3mL缓冲MCS的引发和放置在其磁铁（LS）磁性细胞分离柱之前。用3mL缓冲MCS的洗涤柱3次;的流过将包含期望的细胞。 </li><li>培养通过该柱通过6天在RPMI 1640或DMEM将细胞用补充有2毫微克/毫升的重组小鼠粒细胞巨噬细胞集落刺激因子（GM-CSF）和2纳克/重组人转化生长因子β1的毫升（TGFβ1） 。每2天用含有2毫微克/毫升GM-CSF和TGFβ1新鲜的完全培养基替换一半培养基。 注:人TGF-β1具有活性的小鼠细胞。数据一直在使用这些未成熟DC产生。其他细胞（ 例如，B细胞和成熟DC）可以是适合作为APC，但尚未在该测定中进行测试。 </li><li>冷冻保存的DCs在90％血清/ 10％DMSO中，并存储在液氮;恢复上使用的当天。在使用之前，使用台盼蓝排除计数血球可行DC的数量。在适当的密度重悬在培养基中的细胞沉淀（参见步骤4.1）在第4在使用前。 </li></ol> 3.准备性T细胞<ol><li>使用阴性选择方法，以避免无意中发送激活或抑制性信号到细胞中。 注意:在这个例子中，CD4 + T细胞被准备用于分析。 <ol><li>从小鼠脾制备CD4 + T细胞，通过使用注射器的柱塞一个70微米的细胞过滤醪脾脏。洗用洗涤缓冲液的细胞滤网。 </li><li>沉淀通过离心悬浮细胞（400×g下，5分钟），然后通过重新悬浮于氯化铵缓冲液将沉淀在室温下5分钟裂解红细胞。 </li><li>通过离心（400×g下，5分钟）沉淀细胞并重新悬浮沉淀。 </li><li>洗净细胞S IN 5 - 10毫升洗涤缓冲液和沉淀物的通过离心（400×g下，5分钟）。重新悬浮沉淀。 </li><li>染色用生物素化抗体对CD8，主要组织相容性复合物II类（MHC II，1微克/毫升），和CD19（5微克/毫升）的细胞。孵育在4℃下10分钟。 </li><li>清洗细胞在10mL洗涤缓冲液和沉淀物的通过离心（400×g下，5分钟）。重新悬浮沉淀。 </li><li>孵育用抗生物素磁性微珠（参见材料的表），根据制造商的指示细胞。 </li><li>通过离心（400×g下，5分钟）清洗细胞在10mL洗涤缓冲液和沉淀物的;重新悬浮沉淀。 </li><li>重悬在1mL MCS缓冲器的细胞和丰富的CD4 + T细胞在具有上的磁体3 MCS毫升缓冲液引发的磁性细胞分离柱。用3mL缓冲MCS的洗涤柱3次。柱流过将包含富集的T细胞。 </li></ol></li><li>通过标准流程cytome尝试，使用阴性选择细胞的等分试样评估T细胞的纯度。使用荧光染料的链霉亲缀合物（以确定应已在塔中除去任何生物素标记的细胞）和一种或多种抗体来识别感兴趣的T细胞群体（CD4在这种情况下）将细胞染色;的≥85％的纯度是可以接受的23。 <ol><li>通过锥虫蓝排除（≥90％生存力是可以接受的）计数血球的T细胞。 注:MCS缓冲液含有EDTA，必须在分析前除去。为了实现这一点，沉淀通过离心（400×g下，5分钟）将细胞和在1毫升洗涤缓冲液洗涤。再次沉淀细胞（400×g下，5分钟），并在适当的密度培养基重悬浮（参见步骤4.1）。 </li></ol></li></ol> 4.共孵育性T细胞和DC <ol><li>在种子的T细胞和DC 2:1 T:DC比在24孔或96孔细胞培养板中。确保这件事<html>最终培养体积为≤500μL用于24孔板或≤50μL96孔板中。 注:小的体积促进细胞间的相互作用，并允许后的位置固定的缓冲空间。 <ol><li>精确调节细胞数量经验性地，但作为一般指导，使用1×10个6个 T细胞和每孔（96孔板）0.5×10 6的DC。这些应被视为小区的最小数目，因为使用较少的细胞，使免疫困难突触的枚举。 <ol><li>以增加细胞数量，设置重复孔和池后步骤5（固定）。 注意:当设置重复孔，最好是种子的DCs在所有的孔的第一和然后种子T细胞在所有孔;这最大限度地减少在井之间孵育时间的差异。 </li></ol></li></ol></li><li>在一个5％CO 2气氛中温育4个小时将板在37℃下。 </li></ol>e_title"&gt; 5.修正在板状电池<ol><li>在PBS中添加3倍1.5％甲醛的培养物体积到各孔中并在室温下温育30分钟; TT重要的是要固定的细胞从板中除去它们以最小化细胞 - 细胞相互作用的破坏之前。 </li><li>转移细胞在板成管用于随后的洗涤和染色。在该阶段，对于单染色对照预留附加细胞。除了这些控制，整个培养应与抗体混合物进行染色（参见步骤4.1.1）。 </li></ol> 6.细胞染色<ol><li>在100μL含在室温下30分钟的所需荧光染料缀合的抗体的混合物的洗涤缓冲液的染色细胞，避光。 注:鸡尾酒包括T细胞特异性和APC特异性抗体。荧光染料的选择应使得它们可以使用成像的配置流式细胞仪来区分。在T这里示出他的实验中，蓝色荧光团偶联的CD11b（5微克/毫升，见材料的表）和APC缀合的CD90.2（5微克/毫升）使用。 </li><li>清洗细胞在1mL洗涤缓冲液和离心分离机的5分钟，在400×g下。 倒出上清液。重悬在含有鬼笔环肽FITC烫发洗涤缓冲液将细胞在0.05-0.5微克/毫升孵育在室温下避光30分钟。 注:约0.1μg/ mL的鬼笔环肽的FITC浓度小鼠细胞工作良好，但是适当的浓度，预计由供应商，细胞类型而变化，并且成像流式细胞仪。 </li><li>洗在1mL渗透/洗涤缓冲液和离心分离机的细胞在400×g下5分钟。倒出上清液。重悬在渗透/洗涤缓冲液含有适当浓度（ 例如，约25微克/毫升7-AAD）核染料将细胞孵育在室温下避光30分钟。 </li><li>洗净细胞S IN 1毫升渗透/洗涤缓冲液和离心机在400×g下5分钟。倒出上清液。在洗涤缓冲液，沉淀物洗细胞一次，并在50个悬 - 洗涤缓冲液100微升，将细胞转移到小的，加盖的微量离心管。 </li><li>进行到数据采集立即或细胞储存在长达数天前采集在成像流式细胞仪避光4℃。 注:细胞已经成功地以这种方式存储长达7天。更长的存储是可能的，但还没有经过测试。 </li></ol> 7.采集数据<ol><li>初始化和配置根据制造商的说明流式细胞仪的成像流程。确保流动芯的稳定性收集任何数据之前。 </li><li>预留明场图像获取一个信道。获得单染色控制数据与明场通道关闭。 <ol><li>点击"Load"按钮和樱雪室温含有完全染色的样本到保持器（已染色的所有必需的荧光染料的样品）的管。 </li><li>在"工作区"窗口中，选择并创建具有纵横比在该区域以及栅极单峰新散点图，其中纵横比接近于1。创建（在水平轴强度的信道的）中使用的每个信道新的散点图。 </li><li>对于每一个荧光染料，检查了积极的人口，如果需要，在"照明"框调整激光的电压。 </li><li>卸载管并加载第一单染色管。 </li><li>在"获取设置"框中，输入样品名称和设定将要收集的事件的数量;如果它是一个单一的染色（用于补偿控制），1000 - 2000事件是足够的。 </li><li>在"频道"中，选择各样品已经染色与通道。对于单染色对照，所有信道应与brightf选择屈服和侧向散射掉。点击下的"收购"中的"录制"按钮;当事件的数量达到指定的阈值，采集将自动停止。 </li><li>点击"返回"按钮来卸载管。重复步骤7.2.4 - 7.2.6对于每个单染色控制。取决于仪和软件，它可能无法将所有采集过程中在图1所示的门的（更多的是分析过程中进行精确的选通;参见第8部分）。 </li></ol></li><li>获得的样品作为用于单染色的样品（步骤7.2），但在"通道"中，选择所需的所有信道，包括明场通道。 <ol><li>记录数据之前，检查，以确认信道强度是合适的用于识别所期望的细胞群。如果不是，使用新的设置调整激光器设置，并重新记录单染色对照，如在步骤7.2中描述。 </li><li>对于每一个样本采集活动的数万。 注:在大多数情况下，细胞-细胞接触事件是总细胞数（大部分是单电池）的一小部分。一般情况下，理想的是具有在最终的膜接触栅极至少100个事件。 </li></ol></li></ol> 8.分析数据<ol><li>分析在成像流式细胞仪使用可从制造商的网站免费分析软件获得的数据（的用户帐户必须创建;见材料的表）。 </li></ol><img alt="图1" src="/files/ftp_upload/55283/55283fig1.jpg" /> 图1.门控策略用于鉴定反应免疫突触。 A.在聚焦事件从所有事件通过检查基于根细胞图像门控使用明场瓒意味着像强度分布（梯度RMS）的变化率的平方NEL（通道4，CH04），如文中所述。 B.在对焦事件，双重从单个细胞通过绘制纵横比与用于明场通道区域区分。单一细胞聚集接近1的纵横比，并且具有更小的面积，而双峰是接近0.5，并具有更大的面积。 温度 。的APC的荧光强度（在这种情况下，树突细胞[DC]标记，CD11c的）随后作图的T细胞标志物的荧光强度（在这种情况下，CD90.2），和双阳性事件被选通。门的边框可以通过查看边界附近活动的图像进行细化。然后D. T-APC双峰被细化，使得它们仅包含一个APC通过绘制纵横比相对于APC标记（CD11c的，CH02）的面积。然后E.这些单APC双峰被细化，使得它们通过标绘的纵横比与所述区域仅包含一个T细胞T细胞标记物的（CD90.2，CH06）。 F.最后，通过对核染色通道（7-AAD，CH05）绘制斑点计数的直方图和选通仅包含2 7-AAD阳性斑点（ 即，核）事件选择仅含有两个核事件。在该栅极中的事件进行了分析为膜接触和突触形成，如在图2中描述。以盲方式对数据进行分析对于治疗的分配，并从先前公布的实验23。 <a href="https://www.jove.com/files/ftp_upload/55283/55283fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 注:门控策略在本节中描述并在图1中示出。应以盲方式相对于治疗分配来执行成像流式细胞术数据的分析。虽然我们相信免疫突触和非synaptiC触点通常容易区分（见下文和图2和3），致盲应尽量减少从固有的图像分析的主观性所产生的偏差。 <ol start="2"><li>通过单污点控制数据文件加载到补偿向导生成补偿矩阵。装载单染色文件后，选择在实验中使用的荧光通道。 注:该软件自动产生一个补偿矩阵，但应手动验证，以确保选择正确的积极的群体。 <ol><li>双击矩阵中的值和图形添加到分析区域。如果有必要排除任何死细胞/双峰/误报创建一个新的门;这个新的成品阳性群体所用的基质框在下拉每个通道菜单中选择。 </li><li>重复此对每个信道。 点击"完成"保存compensation矩阵（.ctm文件）。 </li></ol></li><li>通过装载.rif文件到分析软件和应用所述补偿矩阵获得从原始文件（.rif）中的数据分析文件（.daf）。 </li><li>一旦数据被加载，执行选通，以确定T细胞的APC接触事件。 注意:典型的门控策略涉及识别对焦的事件，基于大小的标准（该区域相对于所述事件的纵横比），和选择双峰选择T细胞的APC双峰作为是双阳性的T细胞的事件和APC标记物（ 图1A-C）。 注意:纵横比是一个事件到其高度，这允许单个细胞的判别（比接近1）从双峰（比接近0.5）的宽度的比值。 </li><li>通过绘制根指的是在明场通道（ 图1A）的成像强度分布（梯度RMS）的变化率的平方确定对焦事件。点击次È梯度RMS直方图，以显示个人箱细胞事件，然后将排除了焦事件的栅极。 </li><li>绘制图形的区域相对于明场通道的纵横比和绘制基于事件的形状和尺寸（ 图1B）标识双峰的栅极。只是里面和这个门的境外活动图像的审查可以帮助分析师细化门的大小和位置。然后，构建体，其中所述的T细胞标记物的强度出现在一个轴和APC标记物的强度出现在其它这些双峰事件的曲线图。绘制包含事件均为阳性标记物的T-APC双峰栅极。 </li><li>其中T细胞APC双重的事件，只包含一个T细胞和一个APC选择事件。 <ol><li>通过绘制纵横比与用于APC标记区域和通过门控上含有单一APC（ 图1D）双峰做到这一点。积的纵横比与区域的T细胞标记ER和栅极上含有单一的T细胞（ 图1E）的双峰。 注意:进一步改进可以通过与仅两个斑点（ 图1F）施加到核染色荧光和浇口上的事件标绘点计数功能的直方图来实现。 </li></ol></li><li>在一些情况下，双重峰中的两个小区将不会在接触;识别细胞相互接触，定义对象掩模APC和T细胞。 <ol><li>使用在分析菜单中的"模板"选项打开面罩管理器和定义一个新的面具。 输入名称，诸如"T细胞对象掩码。"点击"功能"按钮，在对话框中，选择"对象"。 </li><li>选择在其中检测到T细胞标记物的信道（ 例如，CH06）。点击"确定"。 注:"对象（M06，CH06，紧）"显示在功能框。这种默认的对象模板通常WORKS很好，但可能需要优化。 </li><li>重复此过程，以在适当的信道的APC对象掩码。 </li></ol></li><li>识别所述APC和T细胞的掩模后，确定由在对APC的荧光强度的APC对象模板在T细胞对象掩模绘制T细胞标志物的荧光强度的膜接触。在此图中，画出一个门，其仅包括在彼此接触的细胞。 注意:通常情况下，有相当明显的双阳性和双阴性群体（ 图2A）。 双阳性和双阴性群之间的边界可以通过查看在边界区域细胞的明视野图像，以确保细胞在接触是栅极内和细胞不接触被排除来设置。 </li><li>在此阶段，手动检查该栅极内的事件的鬼笔环肽FITC图像来区分从简单的细胞 - 细胞接触成熟免疫突触。 üSE中的"标签图像"功能，以纪念这些图像。 注意:这个门内部标记的事件（免疫突触）的百分比，可以使用如在T细胞群直接同种异体反应性的指标正在研究中。 </li></ol>

<ol><li>Cravedi, P., Heeger, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunologic+monitoring+in+transplantation+revisited%5BTitle%5D)+AND+%22Curr+Opin+Organ+Transplant%22%5BJournal%5D)">Immunologic monitoring in transplantation revisited.</a> Curr Opin Organ Transplant. 17 (1), 26-32 (2012).</li>
<li>Pearson, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+assessment+of+transplantation+tolerance+induced+by+anti-CD4+monoclonal+antibody+in+the+murine+model%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">The assessment of transplantation tolerance induced by anti-CD4 monoclonal antibody in the murine model.</a> Transplantation. 55 (2), 361-367 (1993).</li>
<li>Strober, S., Benike, C., Krishnaswamy, S., Engleman, E. G., Grumet, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+transplantation+tolerance+twelve+years+after+prospective+withdrawal+of+immunosuppressive+drugs%3A+studies+of+chimerism+and+anti-donor+reactivity%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Clinical transplantation tolerance twelve years after prospective withdrawal of immunosuppressive drugs: studies of chimerism and anti-donor reactivity.</a> Transplantation. 69 (8), 1549-1554 (2000).</li>
<li>Fussell, S. T., Donnellan, M., Cooley, M. A., Farrell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytotoxic+T+lymphocyte+precursor+frequency+does+not+correlate+with+either+the+incidence+or+severity+of+graft-versus-host+disease+after+matched+unrelated+donor+bone+marrow+transplantation%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Cytotoxic T lymphocyte precursor frequency does not correlate with either the incidence or severity of graft-versus-host disease after matched unrelated donor bone marrow transplantation.</a> Transplantation. 57 (5), 673-676 (1994).</li>
<li>Roelen, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relevance+of+cytotoxic+alloreactivity+under+different+immunosuppressive+regimens+in+clinical+islet+cell+transplantation%5BTitle%5D)+AND+%22Clin+Exp+Immunol%22%5BJournal%5D)">Relevance of cytotoxic alloreactivity under different immunosuppressive regimens in clinical islet cell transplantation.</a> Clin Exp Immunol. 156 (1), 141-148 (2009).</li>
<li>VanBuskirk, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+allograft+acceptance+is+associated+with+immune+regulation%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Human allograft acceptance is associated with immune regulation.</a> J Clin Invest. 106 (1), 145-155 (2000).</li>
<li>Poggio, E. D., Clemente, M., Hricik, D. E., Heeger, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Panel+of+reactive+T+cells+as+a+measurement+of+primed+cellular+alloimmunity+in+kidney+transplant+candidates%5BTitle%5D)+AND+%22J+Am+Soc+Nephrol%22%5BJournal%5D)">Panel of reactive T cells as a measurement of primed cellular alloimmunity in kidney transplant candidates.</a> J Am Soc Nephrol. 17 (2), 564-572 (2006).</li>
<li>Zitzner, J. R., Tambur, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+ELISPOT+Assays+in+Risk+Assessment+Pre-+and+Post-Kidney+Transplantation%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">Role of ELISPOT Assays in Risk Assessment Pre- and Post-Kidney Transplantation.</a> Cells. 1 (2), 100-110 (2012).</li>
<li>Newell, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+a+B+cell+signature+associated+with+renal+transplant+tolerance+in+humans%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Identification of a B cell signature associated with renal transplant tolerance in humans.</a> J Clin Invest. 120 (6), 1836-1847 (2010).</li>
<li>Brouard, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+a+peripheral+blood+transcriptional+biomarker+panel+associated+with+operational+renal+allograft+tolerance%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance.</a> Proc Natl Acad Sci U S A. 104 (39), 15448-15453 (2007).</li>
<li>Sagoo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+cross-platform+biomarker+signature+to+detect+renal+transplant+tolerance+in+humans%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans.</a> J Clin Invest. 120 (6), 1848-1861 (2010).</li>
<li>Halloran, P. F., Famulski, K. S., Reeve, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+assessment+of+disease+states+in+kidney+transplant+biopsy+samples%5BTitle%5D)+AND+%22Nat+Rev+Nephrol%22%5BJournal%5D)">Molecular assessment of disease states in kidney transplant biopsy samples.</a> Nat Rev Nephrol. 12 (9), 534-548 (2016).</li>
<li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+common+blood+gene+signatures+for+the+diagnosis+of+renal+and+cardiac+acute+allograft+rejection%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Identification of common blood gene signatures for the diagnosis of renal and cardiac acute allograft rejection.</a> PLoS One. 8 (12), e82153(2013).</li>
<li>Khatri, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+common+rejection+module+%28CRM%29+for+acute+rejection+across+multiple+organs+identifies+novel+therapeutics+for+organ+transplantation%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">A common rejection module (CRM) for acute rejection across multiple organs identifies novel therapeutics for organ transplantation.</a> J Exp Med. 210 (11), 2205-2221 (2013).</li>
<li>Halloran, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microarray+diagnosis+of+antibody-mediated+rejection+in+kidney+transplant+biopsies%3A+an+international+prospective+study+%28INTERCOM%29%5BTitle%5D)+AND+%22Am+J+Transplant%22%5BJournal%5D)">Microarray diagnosis of antibody-mediated rejection in kidney transplant biopsies: an international prospective study (INTERCOM).</a> Am J Transplant. 13 (11), 2865-2874 (2013).</li>
<li>Mastoridis, S., Issa, F., Wood, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+biomarkers+and+functional+assays+to+monitor+cell-therapy-induced+tolerance+in+organ+transplantation%5BTitle%5D)+AND+%22Curr+Opin+Organ+Transplant%22%5BJournal%5D)">Novel biomarkers and functional assays to monitor cell-therapy-induced tolerance in organ transplantation.</a> Curr Opin Organ Transplant. 20 (1), 64-71 (2015).</li>
<li>Miqueu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+the+peripheral+T-cell+repertoire+in+kidney+transplant+patients%5BTitle%5D)+AND+%22Eur+J+Immunol%22%5BJournal%5D)">Analysis of the peripheral T-cell repertoire in kidney transplant patients.</a> Eur J Immunol. 40 (11), 3280-3290 (2010).</li>
<li>Morris, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tracking+donor-reactive+T+cells%3A+Evidence+for+clonal+deletion+in+tolerant+kidney+transplant+patients%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Tracking donor-reactive T cells: Evidence for clonal deletion in tolerant kidney transplant patients.</a> Sci Transl Med. 7 (272), 272ra210(2015).</li>
<li>Dustin, M. L., Bromley, S. K., Kan, Z., Peterson, D. A., Unanue, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigen+receptor+engagement+delivers+a+stop+signal+to+migrating+T+lymphocytes%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Antigen receptor engagement delivers a stop signal to migrating T lymphocytes.</a> Proc Natl Acad Sci U S A. 94 (8), 3909-3913 (1997).</li>
<li>Goldsmith, M. A., Weiss, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+signal+transduction+by+the+antigen+receptor+without+commitment+to+T+cell+activation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Early signal transduction by the antigen receptor without commitment to T cell activation.</a> Science. 240 (4855), 1029-1031 (1988).</li>
<li>Grakoui, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+immunological+synapse%3A+a+molecular+machine+controlling+T+cell+activation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The immunological synapse: a molecular machine controlling T cell activation.</a> Science. 285 (5425), 221-227 (1999).</li>
<li>Stoll, S., Delon, J., Brotz, T. M., Germain, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+imaging+of+T+cell-dendritic+cell+interactions+in+lymph+nodes%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Dynamic imaging of T cell-dendritic cell interactions in lymph nodes.</a> Science. 296 (5574), 1873-1876 (2002).</li>
<li>Juvet, S. C., Sanderson, S., Hester, J., Wood, K. J., Bushell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+CD4%28%2B%29+T+Cell+Alloreactivity+and+Its+Control+by+Regulatory+T+Cells+Using+Time-Lapse+Microscopy+and+Immune+Synapse+Detection%5BTitle%5D)+AND+%22Am+J+Transplant%22%5BJournal%5D)">Quantification of CD4(+) T Cell Alloreactivity and Its Control by Regulatory T Cells Using Time-Lapse Microscopy and Immune Synapse Detection.</a> Am J Transplant. 16 (5), 1394-1407 (2016).</li>
<li>Ahmed, F., Friend, S., George, T. C., Barteneva, N., Lieberman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Numbers+matter%3A+quantitative+and+dynamic+analysis+of+the+formation+of+an+immunological+synapse+using+imaging+flow+cytometry%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Numbers matter: quantitative and dynamic analysis of the formation of an immunological synapse using imaging flow cytometry.</a> J Immunol Methods. 347 (1-2), 79-86 (2009).</li>
<li>Burbach, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+regulation+of+integrin-dependent+T+cell+conjugate+formation+and+NF-kappa+B+activation+by+the+adapter+protein+ADAP%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Distinct regulation of integrin-dependent T cell conjugate formation and NF-kappa B activation by the adapter protein ADAP.</a> J Immunol. 181 (7), 4840-4851 (2008).</li>
<li>Markey, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cross-dressing+by+donor+dendritic+cells+after+allogeneic+bone+marrow+transplantation+contributes+to+formation+of+the+immunological+synapse+and+maximizes+responses+to+indirectly+presented+antigen%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Cross-dressing by donor dendritic cells after allogeneic bone marrow transplantation contributes to formation of the immunological synapse and maximizes responses to indirectly presented antigen.</a> J Immunol. 192 (11), 5426-5433 (2014).</li>
<li>Moreau, H. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+in+situ+cytometry+uncovers+T+cell+receptor+signaling+during+immunological+synapses+and+kinapses+in+vivo%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Dynamic in situ cytometry uncovers T cell receptor signaling during immunological synapses and kinapses in vivo.</a> Immunity. 37 (2), 351-363 (2012).</li>
<li>Wabnitz, G. H., Nessmann, A., Kirchgessner, H., Samstag, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((InFlow+microscopy+of+human+leukocytes%3A+A+tool+for+quantitative+analysis+of+actin+rearrangements+in+the+immune+synapse%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">InFlow microscopy of human leukocytes: A tool for quantitative analysis of actin rearrangements in the immune synapse.</a> J Immunol Methods. , (2015).</li>
<li>Ford, W. L., Simmonds, S. J., Atkins, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+cellular+events+in+a+systemic+graft-vs.-host+reaction.+II.+Autoradiographic+estimates+of+the+frequency+of+donor+lymphocytes+which+respond+to+each+Ag-B-determined+antigenic+complex%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Early cellular events in a systemic graft-vs.-host reaction. II. Autoradiographic estimates of the frequency of donor lymphocytes which respond to each Ag-B-determined antigenic complex.</a> J Exp Med. 141 (3), 681-696 (1975).</li>
<li>Macedo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contribution+of+naive+and+memory+T-cell+populations+to+the+human+alloimmune+response%5BTitle%5D)+AND+%22Am+J+Transplant%22%5BJournal%5D)">Contribution of naive and memory T-cell populations to the human alloimmune response.</a> Am J Transplant. 9 (9), 2057-2066 (2009).</li>
<li>Noorchashm, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+direct+method+for+the+calculation+of+alloreactive+CD4%2B+T+cell+precursor+frequency%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">A direct method for the calculation of alloreactive CD4+ T cell precursor frequency.</a> Transplantation. 67 (9), 1281-1284 (1999).</li>
<li>Suchin, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+the+frequency+of+alloreactive+T+cells+in+vivo%3A+new+answers+to+an+old+question%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question.</a> J Immunol. 166 (2), 973-981 (2001).</li>
<li>Lee, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Indirect+recognition+of+allopeptides+promotes+the+development+of+cardiac+allograft+vasculopathy%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Indirect recognition of allopeptides promotes the development of cardiac allograft vasculopathy.</a> Proc Natl Acad Sci U S A. 98 (6), 3276-3281 (2001).</li>
<li>Pickl, W. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+and+functional+characteristics+of+dendritic+cells+generated+from+highly+purified+CD14%2B+peripheral+blood+monocytes%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Molecular and functional characteristics of dendritic cells generated from highly purified CD14+ peripheral blood monocytes.</a> J Immunol. 157 (9), 3850-3859 (1996).</li>
<li>Inaba, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+large+numbers+of+dendritic+cells+from+mouse+bone+marrow+cultures+supplemented+with+granulocyte%2Fmacrophage+colony-stimulating+factor%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.</a> J Exp Med. 176 (6), 1693-1702 (1992).</li>
<li>Francis, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+transplantation+tolerance+converts+potential+effector+T+cells+into+graft-protective+regulatory+T+cells%5BTitle%5D)+AND+%22Eur+J+Immunol%22%5BJournal%5D)">Induction of transplantation tolerance converts potential effector T cells into graft-protective regulatory T cells.</a> Eur J Immunol. 41 (3), 726-738 (2011).</li>
<li>Saitovitch, D., Bushell, A., Mabbs, D. W., Morris, P. J., Wood, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetics+of+induction+of+transplantation+tolerance+with+a+nondepleting+anti-Cd4+monoclonal+antibody+and+donor-specific+transfusion+before+transplantation.+A+critical+period+of+time+is+required+for+development+of+immunological+unresponsiveness%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Kinetics of induction of transplantation tolerance with a nondepleting anti-Cd4 monoclonal antibody and donor-specific transfusion before transplantation. A critical period of time is required for development of immunological unresponsiveness.</a> Transplantation. 61 (11), 1642-1647 (1996).</li>
<li>Faust, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+T+cell+TGFbeta+signaling+and+IL-17+in+allograft+acceptance+and+fibrosis+associated+with+chronic+rejection%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Role of T cell TGFbeta signaling and IL-17 in allograft acceptance and fibrosis associated with chronic rejection.</a> J Immunol. 183 (11), 7297-7306 (2009).</li>
<li>Yuan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+role+of+CD4+Th17+cells+in+mediating+cardiac+allograft+rejection+and+vasculopathy%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy.</a> J Exp Med. 205 (13), 3133-3144 (2008).</li>
<li>Emerson, R. O., Mathew, J. M., Konieczna, I. M., Robins, H. S., Leventhal, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+alloreactive+T+cell+repertoire+using+high-throughput+sequencing+of+mixed+lymphocyte+reaction+culture%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Defining the alloreactive T cell repertoire using high-throughput sequencing of mixed lymphocyte reaction culture.</a> PLoS One. 9 (11), e111943(2014).</li>
<li>Stanford, R. E., Ahmed, S., Hodson, M., Banner, N. R., Rose, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+indirect+allorecognition+in+lung+transplant+recipients+with+obliterative+bronchiolitis%5BTitle%5D)+AND+%22Am+J+Transplant%22%5BJournal%5D)">A role for indirect allorecognition in lung transplant recipients with obliterative bronchiolitis.</a> Am J Transplant. 3 (6), 736-742 (2003).</li>
<li>Benichou, G., Valujskikh, A., Heeger, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contributions+of+direct+and+indirect+T+cell+alloreactivity+during+allograft+rejection+in+mice%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Contributions of direct and indirect T cell alloreactivity during allograft rejection in mice.</a> J Immunol. 162 (1), 352-358 (1999).</li>
<li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contribution+of+direct+and+indirect+recognition+pathways+to+T+cell+alloreactivity%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">Contribution of direct and indirect recognition pathways to T cell alloreactivity.</a> J Exp Med. 177 (6), 1643-1650 (1993).</li>
</ol>

作者什么都没有透露。

成像流式细胞仪已经用于证明单克隆T细胞和之间的APC或超抗原24，25，26，27，28的存在的免疫突触形成。此方法采用的一个生产性T细胞的APC接触后，T细胞重新排列其肌动蛋白骨架，偏振它朝向触点21的部位的事实。这种重排不会发生没有TCR信号，因此它是T细胞活化19，20，21的早期相关。这里介绍的方法适应这种方法在多克隆T细胞群同种异体反应性的T细胞频率的测量。因此，它可能在未来作为试验的发展，为捐助reactivi基础<html> TY临床移植。 虽然直接比较尚未作出，同种异体免疫突触的检测似乎有优越的预测能力比传统的MLR。例如，先前的工作已经表明，在上述的耐受协议，MLR的结果不可靠地与移植物的结果2相关。 许多实验已经制定了在人类9，10，11操作性宽容的状态，虽然这些不响应同种异体抗原测定效应细胞功能。 与此相反，IFNγELISPOT测定法测量8的效应T细胞的功能，但不能捕获的细胞因子分泌的全谱，其可以是相关的急性和慢性同种异体移植物排斥，例如IL-17 38，&gt; 39。 有限稀释测定法4，这是劳动密集的，并且反式体内分析6，这需要的小鼠，有会妨碍在临床环境中它们的应用显著实际限制。使用T细胞在MLR响应的TCR序列分析增殖细胞的分析最近的改进可能是有价值的，但像这里介绍的试验中，将需要进一步的验证在临床研究18,40。 免疫突触检测法的进一步发展需要，一些重要的问题需要回答。首先，开发了测定仅测量直接同种异体反应性。直接途径涉及对供体 - 衍生的APCs的同种异体MHC /肽复合物的呈现。后者移植后很快被消除一般，进一步同种异体抗原表现是CARRI通过接受者的APCs呈递完整施主MHC（半直接途径）或自MHC（间接途径）处理供者抗原编出来。间接通路是慢性同种异体移植物排斥33,41的重要驱动力。 原则上，它应该是可以利用该法检测间接免疫突触，而是间接同种异体反应性T细胞具有比直接的人42，43低得多的频率，这意味着事件的较大数量的分析将是需要的。第二个考虑是，我们已经使用CD4 + T细胞，而CD8 + T细胞也是抗供者反应的一个重要组成部分只测试该测定。再次，应该是能够检测利用此方法的CD8 + T细胞的APC突触。另一个限制是，该方法需要在最终MEMB人工审查和细胞图像的分析RANE接触门，和我们目前正在对这一步骤的自动化。 最后，该方法需要在人类受试者的测试和开发，以及当前正在进行的人类样本初步研究。组合进一步的表型的T细胞亚群分析（ 即 ，执行器，存储器，规章等 ）在移植受者的免疫检测突触将代表一个有效的方法用于表征同种异体反应性T细胞所有组成成分，将成为一个重要的焦点未来的工作。

实体器官移植已经改变了患者的治疗与肾，肝，心脏和肺的终末期疾病。 由于在主要和次要组织相容性抗原的差异，然而，同种异体移植物及时通过受体T细胞拒绝如果不使用免疫抑制药物。这些药物有许多不利影响，包括癌症和器官功能障碍的风险。因此，一个重要的临床目标是免疫的剂量降低到防止移植排斥反应所需的最低水平。这个水平可能取决于先天免疫系统的活化的程度，以改变;供受体同种抗原不匹配的程度;和免疫功能，药代动力学和药效学的患者间的差异。 不幸的是，移植医生没有任何工具，个别患者1准确地评估捐助者的反应。 混合白细胞反应（MLR）可检测的反应性的供体，但它不能可靠地预测接枝结果2,3。有限稀释测定法，细胞因子ELISPOTS，和反式-体内试验测量任一的响应的有限范围或不实用有关操作公差9，10，11 4，5，6，7，8个 .Gene表达谱揭示签名， 12和排斥13，14，15，但这些并不总是跨越人口16一般化并可能最终在个别患者用处有限。 基于序列的ANALY在外周血中17或增殖在MLR 18的T细胞的T细胞受体（TCR）的SES也已经被开发，但是需要进一步的验证。 从概念上讲，这将是理想的是具有由供体抗原检测接受者T细胞活化的最早必要步骤的测定法。 由于培养细胞在数天（如在MLR）可以引入伪像，这样的测试将理想地不需要下游事件，诸如增殖或效应子功能的测量。然而，同样，它也将是理想的是，测试依赖T细胞功能的一些元件上，由于纯粹的描述的评估 （ 例如，TCR测序）可能无法无反应性和功能性的T细胞之间进行区分。 许多研究已经表明，延长的T-APC接触是所必需的形成中的免疫突触的，这是一个重要的第一步骤在T细胞应答19，20，21，22。我们最近报道的是，在体外时间推移成像，约5动态-小鼠的10％的CD4 + T细胞形成具有异体骨髓来源的树突细胞（BMDC的）23持久的接触。 长时间接触的频率在该拒绝移植物，而在小鼠中预先使其耐受同一抗原的动物增加，它仍然是在非移植小鼠23的水平。 延长相互作用收件人Treg的存在下还原，并在其不存在增加，并且我们使用人T细胞和单核细胞的同种异体衍生的DC（的MoDC）23观察到类似的现象。 然而，长时间的接触件的枚举多克隆T细胞populatio内作出n是耗时耗力。因此，我们利用成像流式细胞仪检查同种异体免疫突触的形成。成像流式细胞仪结合使用荧光显微镜的单细胞成像的能力的常规流的多参数数据采集和分析能力。此技术已经被用来通过其他研究者通过单克隆T细胞24，26，27或28的超抗原的存在来研究免疫突触的形成。在这样的设置，然而，反应性T细胞的频率从30-100％的范围内，而同种异体反应性T细胞通常估计来表示总T细胞所有组成成分29，30，31，32的5-15％。重要的是，我们发现，成像流式细胞仪能产生AVERY同种异体反应性T细胞的频率23和多克隆T细胞群体内的变化在突触频率是预测接枝结果23比较的量度。目前，这种方法已被优化，以测量CD4 + T细胞的直接同种异体反应性，但在原则上，它也可以被开发来检查CD8 + T细胞和间接的途径。间接同种异体反应性被认为是成为较长时间后移植33日益相关。我们目前正在开发这种方法使用人体细胞，这将允许在患者中测试。因此，在未来，总体方法可以是用于在移植前移植受者T细胞应答的功能评价有用;移植后立即而在长期，当药物的最小化成为一个重要的目标。

<table><tbody><tr><td>Phosphate-buffered saline</td><td>Various</td><td>Varies</td><td></td></tr><tr><td>Ethylenediamenetetraacetic acid, 0.5 M solution</td><td>Thermo Fisher Scientific</td><td>AM9260G</td><td></td></tr><tr><td>Triton X-100 nonionic detergent</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Beta-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F1635</td><td>Solution is 37% formaldehyde and so must be diluted 25 times for 1.5% solution</td></tr><tr><td>Cell strainers, 70 &amp;mu;m pore size</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>Phalloidin-fluorescein isothiocyanate</td><td>Sigma-Aldrich</td><td>P5282</td><td></td></tr><tr><td>7-aminoactinomycin D</td><td>Thermo Fisher Scientific</td><td>A1310</td><td>Reconstitute in DMSO</td></tr><tr><td>Allophycocyanin-conjugated anti-mouse CD90.2</td><td>eBioscience</td><td>17-0902</td><td></td></tr><tr><td>Pacific blue-conjugated anti-mouse CD11b</td><td>eBioscience</td><td>48-0112</td><td>Pacific blue has been replaced by eFluor 450</td></tr><tr><td>Biotinylated anti-mouse CD3</td><td>eBioscience</td><td>13-0032</td><td></td></tr><tr><td>Biotinylated anti-mouse MHC class II</td><td>eBioscience</td><td>13-5321</td><td></td></tr><tr><td>Biotinylated anti-mouse B220</td><td>eBioscience</td><td>13-0452</td><td></td></tr><tr><td>Biotinylated anti-mouse CD8</td><td>eBioscience</td><td>13-0081</td><td></td></tr><tr><td>Biotinylated anti-mouse CD19</td><td>eBioscience</td><td>13-0193</td><td></td></tr><tr><td>Anti-biotin microbeads</td><td>Miltenyi Biotec</td><td>130-090-485</td><td></td></tr><tr><td>LS columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>MidiMACS magnetic cell separator</td><td>MIltenyi Biotec</td><td>130-042-302</td><td></td></tr><tr><td>recombinant mouse GM-CSF</td><td>Peprotech</td><td>315-03</td><td></td></tr><tr><td>recombinant human TGF&amp;beta;1</td><td>Peprotech</td><td>100-21</td><td>Human TGF&amp;beta;1 has activity on mouse cells</td></tr><tr><td>Amnis ImageStream X Mark II</td><td>Amnis/EMD Millipore</td><td>N/A</td><td>Imaging flow cytometer; details available at http://www.emdmillipore.com/</td></tr><tr><td>IDEAS Software</td><td>Amnis/EMD Millipore</td><td>N/A</td><td>Free download (registration required): https://www.amnis.com/index.php/page/Display/login%20%20</td></tr><tr><td>Cell culture medium&amp;nbsp;</td><td>Various</td><td>Varies</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Various</td><td>Varies</td><td></td></tr><tr><td>Cell culture plates</td><td>Various</td><td>Varies</td><td></td></tr></tbody></table>

measurement of t cell alloreactivity using imaging flow cytometry

免疫反应的供者抗原在移植的测量很可能是为成功地减少或免疫抑制的撤出是至关重要的。在混合白细胞反应（MLR），有限稀释测定法，和反式体内迟发型超敏（DTH）测定已全部施加到这个问题，但这些方法具有有限的预测能力和/或减少其效用显著实际限制。 成像流式细胞仪是组合流的多参数定量权力流式细胞术用荧光显微镜的成像能力的技术。最近，我们利用的成像流式细胞仪的方法，以限定能够与供体的抗原呈递细胞（APC）的成熟免疫突触的受体T细胞的比例。使用良好表征的小鼠心脏移植模型，我们已经表明， 在体外免疫突触中T-APC membr频率ANE接触事件的强烈预测排斥反应，移植耐受性成果，并在移植的生存依赖于诱导调节性T细胞的情况。 的T-APC接触频率急性排斥期间与T细胞增加从小鼠和从呈现不响应同种异体抗原的小鼠的T细胞减少。在加入调节性T细胞的体外系统的减少延长的T-APC接触。重要的是，这种效果也可见与扩增的人多克隆的，天然存在的调节性T细胞，其是公知的，以控制排斥的人源化小鼠模型的人组织。这种方法的进一步发展，可以允许同种异体反应性T细胞室的移植受者更深的特征。在未来，进一步发展和使用人细胞该方法的评估可形成用于选择患者用于免疫抑制最小化测定法的基础上，并且它可以被用于测量耐受原的影响IC疗法在临床上。

使用该方法来研究异位心脏同种异体移植物移植之前使其耐受供者同种抗原的小鼠的CD4 + T细胞同种异体反应性。 CBA小鼠（H-2 k）的均（，H-2 B B6）接收B6心脏移植之前输血用非消耗CD4抗体1个月组合给定组成的供体特异性的耐受一个协议。这个方案导致长期同种异体移植物存活率是依赖于Foxp3 +调节性T细胞36，37。七天移植后，从耐受和B6心脏同种异体移植物的非耐受受者和共孵育根据该协议与B6骨髓衍生的DCs得到脾CD4 + T细胞。 图2示出从这个实验中的代表性数据。膜接触闸门设置在图2中所示A，用绿色十字线放置在突触事件（左面板中，1）和在非突触事件（右图）。 图2B示出此事件的明视野和荧光通道。为了减少偏见，数据由不了解治疗分配23观察者分析。如图几个例子在图3中，无论是从非耐受 （ 图3A-B）和耐受（ 图3C-D）B6心CBA收件人，突触由致密FITC阳性脊的在T-APC接口存在非突触联系容易辨别。这些结果表明，通过接受者的T细胞免疫制成突触的视觉检测与同种异体反应性的在受体的程度跟踪。 <img alt="图2" src="/files/ftp_upload/55283/55283fig2.jpg" /> 图2. T-APC双峰的识别与膜联系方式和免疫突触形成。在最终的双峰栅极（ 图1F）的事件进行了分析。在APC对象掩模A. T细胞标记物的荧光作图在DC对象掩模APC标记的荧光。 一些双峰事件有APC和T细胞无细胞 - 细胞接触，并显示在图的左下角（未示出的图像）。一种膜接触栅极因此可以得出，其包括仅在T细胞和的APC相接触双峰。在此栅极上的每个事件的图像被在鬼笔环肽-FITC通道肌动蛋白细胞骨架重排的证据综述，并且可以使用分析软件进行标记。左侧面板指示免疫突触事件（标记为1和由绿十字线表示），而右图表示膜接触事件，而不免疫突触形成（标记为2和由绿十字准线指示）。突触形成确定需要的这些人工审查图像，在B. B.所示的顶行示出了明场和荧光通道图像，用于与免疫突触一个双峰（对应于事件1中A）;底行示出了具有膜接触，但缺乏突触形成（对应于事件2中A）一种双峰。以盲方式对数据进行分析对于治疗的分配，并从先前公布的实验23。 <a href="https://www.jove.com/files/ftp_upload/55283/55283fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55283/55283fig3.jpg" /> 图3. T-APC突触形成的实例。 CBA小鼠接受来自供体B6心脏同种异体移植物之后或者没有前处理（AB）或耐受性诱导B6全血的非消耗性抗CD4抗体的掩护下后（ <st<html>荣&gt; CD）。 7天后，脾CD4 + T细胞对突触形成与B6的DC进行测试。 A.非突触双峰与来自非耐受动物膜接触的三个例子。 B.从非耐受动物免疫突触的三个例子。 C.非突触双峰与来自耐受动物膜接触的三个例子。 D.从耐受动物免疫突触的三个例子。突触形成由明亮的，FITC阳性脊的在T-APC接口（CH03）的存在来指示。以盲方式对数据进行分析对于治疗的分配，并从先前公布的实验23。 <a href="https://www.jove.com/files/ftp_upload/55283/55283fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> AYS"&gt; <tbody><tr><td> 抗体/染料 </td><td> 荧光 </td><td> 渠道 </td><td> 浓度 </td></tr><tr><td>表面CD11c </td><td> eF450 </td><td> CH02 </td><td> 5微克/毫升，滴定凭经验</td></tr><tr><td> CD90.2 </td><td> APC </td><td> CH06 </td><td> 5微克/毫升，滴定凭经验</td></tr><tr><td>鬼笔</td><td> FITC </td><td> CH03 </td><td> 0.05 - 0.5微克/毫升</td></tr><tr><td> 7-AAD </td><td> - </td><td> CH05 </td><td> 25微克/毫升</td></tr></tbody></table> 表1.抗体和染料这一研究进展。荧光标记抗体，染料，供应商和推荐的浓度在表中。成像流式细胞仪信道被用于检测每个荧光染料也被示出在表中。

Watch this Scientific Journal Video about Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry at JoVE.com

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

本文描述了用于在使用成像流式细胞仪的T细胞的混合群测量同种异体反应性的方法。

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

measurement-of-t-cell-alloreactivity-using-imaging-flow-cytometry

Research

JoVE Journal

Immunology and Infection

13.5K 次观看.  University of Toronto and University Health Network. 本文描述了用于在使用成像流式细胞仪的T细胞的混合群测量同种异体反应性的方法。 

视频: Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

协同刺激信号封锁的同种抗原刺激诱导人外周血单个核细胞同种异体抗原特异性无能

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

科研

免疫与感染

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

基于细胞的流式细胞仪检测，以测量细胞毒活性

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

从外周血人类同种异体抗原特异性T细胞的生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

流式细胞仪用来评估染色质的T细胞国家

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

在预处理肿瘤细胞的同种异体共培养环境中分析人 T 细胞活性

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

癌症研究

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

双氟铬流式细胞仪对七种免疫细胞子集的判别

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...

Analysis of T-cell Receptor-Induced Calcium Influx in Primary Murine T-cells by Full Spectrum Flow Cytometry

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to assess calcium signaling by band-pass flow cytometry. This protocol is designed to measure calcium responses in primary murine T-cells using full spectrum flow cytometry. Total splenocytes are labeled with the ratiometric calcium indicator dye Indo-1, along with a panel of fluorochrome-conjugated antibodies to cell surface molecules. Leveraging the capabilities of full spectrum flow cytometry provides a platform for utilizing a wide array of cell surface stains in combination with Indo-1. Cells are then analyzed in real-time at 37 &#176;C before and after the addition of an anti-CD3 antibody to stimulate the T-cell receptor. After unmixing the spectral signals, the ratio of calcium-bound to calcium-free Indo-1 is calculated and can be visualized over time for each gated population of splenocytes. This technique can allow for the simultaneous analysis of calcium responses in multiple cell populations.

Calcium influx, a measure of T-cell signaling, is an effective way to analyze responses to T-cell receptor stimulation. This protocol for multiplexing Indo-1 with panels of antibodies directed at cell surface molecules takes advantage of the highly flexible capabilities of full spectrum flow cytometry.

全谱流式细胞术分析原代小鼠T细胞受体诱导的钙内流

Calcium influx in response to T-cell receptor stimulation is a common measure of T-cell signaling. Several calcium indicator dyes have been developed to ...

Measuring Alloreactivity in a Mixed Population of T Cells

Source: Juvet, S. C. et al., <a href="https://app.jove.com/v/55283">Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry.</a>&#160;J. Vis. Exp.&#160;(2017)
This video demonstrates a method for assessing alloreactivity in mixed T cell populations by observing interactions with alloantigen-carrying dendritic cells, leading to the formation of immunological synapses. Differential staining and quantification of synapse-containing doublets via flow cytometry reveal T cell alloreactivity.

测量混合 T 细胞群中的同种异体反应性

1. Prepare Reagents and Materials Required

	Prepare phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) (&#34;wash buffer&#34;). ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

A Flow Cytometry Assay to Identify Immune Cell Subsets in Peripheral Blood Mononuclear Cells

Source: Torchia, M. L. G., et al. <a href="https://app.jove.com/v/58955">Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry.</a> J. Vis. Exp. (2019)
This video demonstrates a flow cytometry-based technique using two fluorochromes to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, natural killer (NK) cells, and monocytes in human peripheral blood mononuclear cells (PBMCs). Upon incubating the cells with fluorochrome-conjugated antibodies against the surface markers, the cell subsets are identified using an optimal flow cytometric gating strategy.

一种流式细胞术检测，用于鉴定外周血单核细胞中的免疫细胞亚群

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
NOTE: This protocol has been tested ...