这项工作是由美国心脏协会和儿童心肌病基金会（SDG2462390204001）和美国国立卫生研究院（HL114669）的支持。 CM是荣获由心肌炎基金会，新泽西州的博士后研究奖学金补助金的获得者。

伦理声明: 所有动物的程序均按照实验动物的护理和使用的指导原则进行，并经内布拉斯加林肯，内布拉斯加州林肯大学。 1，EAE的诱发<ol><li>以诱导EAE，准备在11所述进行了以下修改的肽/完全弗氏佐剂（CFA）乳化: 步骤1.1:制备PLP 139-151以1毫克/毫升在1×磷酸盐缓冲盐水（PBS）中的浓度步骤1.4:用200微升CFA乳液含有每只动物100微克的PLP 139-151的步骤1.4.1:免疫10的动物，通过混合1毫升PLP 139-151（1毫克/毫升在1x准备2毫升乳液。PBS）和CFA等体积的步骤1.5:两个腹股沟区（75〜每微升）和五到六周龄胸骨区域（约50μl）注入200μL乳液的分割剂量皮下注射FEMA乐SJL / J小鼠。步骤1.7和1.8不适用于诱导EAE的。 </li></ol> EAE小鼠和Cerebrums的收获2。临床评分<ol><li>监测动物外观的临床体征，并且每天从第9天免疫后的得分与疾病的严重程度如下:0 - 健康; 1 - 柔弱的尾部或后肢无力，但不能同时使用; 2 - 柔弱的尾部和后肢无力; 3 - 后肢局部麻痹; 4 - 后肢完全瘫痪; 5 -濒死或死亡12。 </li><li>安乐死的小鼠，当它们达到使用CO 2中的神经学评分为3或4。安乐死的小鼠，先用CO 2的预填充安乐死室转动的CO 2罐不超过6磅的调节，并允许动物留在室内约5分钟，以达到彻底窒息。浸泡在实施安乐死的动物中70％的酒精。立即，将动物上的吸收垫，并用对消毒为牛肝菌和剪刀通过胸腔切割暴露的心。穿刺右耳廓和注入10毫升冷的1X PBS进入心中的左心室灌注。 </li><li>挖掘出的头骨，利用一对弯曲的剪刀剪断的头盖骨以循环方式，并小心地，翻转骷髅骨头用钳子。受钝性分离，并用干净的手术刀，从单独的小脑大脑收获的大脑。将大脑中含有冷的1×PBS管和离开管置于冰上，直到处理。 </li></ol> 3， 在脑部分与MHC II类（IA S）/ PLP 139-151 Dextramers的原位染色<ol><li>制备4％的PBS缓冲低熔点琼脂糖，熔融，加热至50℃并保持在40℃的水浴中，直到使用。 </li><li>将大脑中的一次性深基模组织学卡带保持在冰上，浇上熔融（40℃）的琼脂糖。随即，按大脑轻轻用钳子淹没的组织。将磁带留在冰上15-20分钟，直到凝固完成。 </li><li>用冷冻的1X PBS填充vibratome浴。填充空间周围vibratome浴冰块保持PBS中0和2之间的温度 °C。固定清洁刀片进入vibratome和设置在叶片角度为15°。 </li><li>组织在vibratome位置。修剪多余的琼脂糖从包埋的组织的各个侧面及稍微露出大脑的腹面。 <ol><li>涂片用超级胶水vibratome组织块并通过将大脑的暴露腹面过胶横向切片转移琼脂糖包埋的组织在胶合表面上。别动组织，一旦组织被设置在该块。 </li><li>上放置装有冰的琼脂糖包埋组织的vibratome块，并让胶干燥15分钟。 </li></ol></li><li>在此期间，将组织湛的BER每孔一个24孔板。填装有组织腔用1ml冷的1×PBS的孔中，并离开该板在冰上。 <ol><li>通过将一个薄尼龙网用强力胶25毫升聚丙烯移液管的切断端准备组织商会;修剪多余的网格围绕吸管后，从网格切吸管为1厘米的长度。 </li></ol></li><li>降低vibratome叶片组织的顶表面的水平;设置vibratome速度0.22毫米/秒，并开始向前运动切片，使200微米厚的切片。每转用水彩软刷到组织室放置在24孔板的孔中以及在一侧的大脑部分。保持板置于冰上，直到切片完毕，以防止组织退化。注:请按照以下步骤从大脑背侧向腹侧表面的三个不同区域（ 图2）取得合共三次配对部分。从一个部分放置在三个单独的组织腔室的每对（总共三个部分）被指定为与特定dextramers染色（IA 秒/ PLP 139-151）和抗-CD4。同样地，另一组三个部分被放置在三个单独的组织腔室被指定用于与控制dextramers染色（IA 秒/ TMEV 70-86）和抗-CD4。 </li><li>传送含有指定用于特定dextramers在染色成三个孔在24孔板中与一个腔室，每孔的部分中的3组织室的，300微升含别藻蓝蛋白鸡尾酒（APC）缀合的PLP 139-151 dextramers（2.5微克/毫升）和抗-CD4-藻红蛋白（PE，5微克/毫升）在封闭液（1×PBS中，用2％正常山羊血清）先前的溶液。盖井有盖。 </li><li>同样，含有转指定用于控制dextramers染色分为三个井在24孔板与我们每一个室的部分三个组织商会LL，其中，300微升含APC标记TMEV 70-86 dextramers（2.5微克/毫升）和抗-CD4-PE（5微克/毫升）在封闭液鸡尾酒的以前添加。盖井有盖。注:请参阅Massilamany 等人 ，8准备dextramers，而葡聚糖分子准备dextramers被好心Immudex，哥本哈根，丹麦提供。 </li><li>标签的井，和包裹该板用铝箔，然后将板在摇摆平台设置为平缓旋转1.5小时在黑暗中在RT上。 </li><li>孵育期后，洗涤组织切片在摇动平台上，通过将所述组织腔到新鲜的孔含有1ml冷的1×PBS中，并避免运球1中的内容以及到另一个。重复洗涤三次，让每冲洗至少10分钟。确保组织切片没有得到干燥的，因为它们会粘在腔室的侧面。 </li><li>通过将组织湛修复部分的BER到一个新的井在24孔板中含有2小时，轻轻旋转摇摆平台上过滤4％的PBS缓冲的多聚甲醛（pH 7.4）中1毫升。 </li><li>如步骤3.10所述重复洗涤三次。 </li><li>用水彩软毛刷放在一个干净的显微镜载玻片染色和固定部分。擦去多余的水不接触的部分，并安装使用的安装介质中的一节。盖上盖玻片，显微镜，让幻灯片在室温O / N干在黑暗的房间。 </li><li>保存幻灯片在不透光的滑动储物盒，在4°C。 </li></ol> 4，感应EAM心和收集<ol><li>诱导EAM如参考图11所述。 </li><li>安乐死在使用清洁室装有二氧化碳气瓶21天接种后的小鼠，和浸泡的动物在70％的酒精。随即，将动物上的吸收垫，并用钳子和剪刀暴露心脏s由通过胸腔切割。穿刺右耳廓和注入10毫升冷的1X PBS进入心中的左心室灌注。收集的心和地方在含冷的1X PBS管。 </li></ol> 5， 在心脏部分与MHC II类（IA K）/ MYHC 334-352 Dextramers的原位染色<ol><li>制备4％的PBS缓冲低熔点琼脂糖，通过加热熔化，在50℃，并让它在40 &#160; °C的水中洗澡，直到使用。 </li><li>将心脏在一次性深基模组织学卡带放在冰上，倒入熔化的40℃的琼脂糖直至在步骤3.2中所述的组织被完全淹没。 </li><li>按照步骤3.3。 </li><li>修剪多余的琼脂糖从包埋组织的所有侧面和微微露出心脏的基础。用涂抹强力胶vibratome组织块和放置的暴露表面转移琼脂糖包埋组织在表面粘心脏的胶水。别动组织，一旦组织被设置在该块。 <ol><li>上放置装有冰的琼脂糖包埋组织的vibratome块，并让胶干燥15分钟。 </li></ol></li><li>按照步骤3.5和3.6。 </li><li>用水彩软刷到组织室置于24孔板的孔中转移的心脏部分。保持板置于冰上，直到切片完毕，以防止组织退化。注:请按照以下步骤获得合共八对部分从顶点到心脏基地（ 图2）。将组织商会的所有部分，分配高达每室3部分。从每对（共8部分）的一个部分被指定用于特定dextramers（IA K / MYHC 334-352）和抗-CD4染色。同样地，另一组八个部分被指定用于与控制dextramers（IA K / RNA酶43-56）及抗CD4染色。 </li><li>转让包含指定用于特定dextramers染色成三个孔在24孔板中，每孔1室中的部分3的组织腔的，300微升含APC标记MYHC 334-352 dextramers（2.5微克/毫升）的鸡尾酒和抗-CD4-PE（5微克/毫升）在封闭液中预先加入。盖井有盖。 <ol><li>同样，传输包含指定用于控制dextramers染色成三个孔在24孔板中以每孔1室中的部分中的3组织腔，其中，300微升含APC标记的RNase 43-56 dextramers（2.5微克鸡尾酒/ ml）和抗-CD4-PE（5微克/毫升）在封闭液中预先加入。盖井有盖。 </li></ol></li><li>标签的井，和包裹该板用铝箔，然后将板在摇摆平台设置为平缓旋转1.5小时在黑暗中在RT上。 </li><li>按照步骤，3.10至3.11 </li><li>重复洗涤THR如步骤3.10中描述的冰。 </li><li>将染色和固定部分（最多三段/幻灯片）上用水彩软毛刷清洁显微镜幻灯片。擦去多余的水不接触的部分，并使用安装介质安装部分。盖上盖玻片，显微镜，让幻灯片在室温O / N干在黑暗的房间。 </li><li>保存幻灯片在不透光的滑动储物盒在4 °C。 </li></ol> 6。Dextramer +分析（DEXT +）CD4 + T细胞通过激光扫描共聚焦显微镜（LSCM）（以双方共同的大脑和心脏部分） <ol><li>使用尼康A1-Eclipse的90I共聚焦显微镜系统进行例行扫描和采集图像。要启动单击打开，NIS元素的AR软件（4.13版）。 <ol><li>选择"TRITC"，并从面板"Cy5的"通道。分别为TRITC和Cy5通道的激发/发射波长，561.5纳米/ 553-618 nm和640.7纳米/ 663-738 nm的默认显示。 </li><li>为TRITC（CD4-PE）和Cy5（dextramer-APC）渠道分配pseudocolors"绿色"和"红色"，分别。 </li><li>将要检查在显微镜舞台上的幻灯片和下放大100倍手动对焦。设置"HV"110和偏移为"0"。点击"焦点访谈"和调整"电压"来优化激光器。 </li><li>点击"焦点访谈";扫描截面从顶部到底部，并设置"Z"电平，用于图像采集。点击"CH系列"和获取图像的顺序。识别DEXT + CD4 + T细胞中出现的基础上共同定位的同时从红色（dextramers）和绿色（CD4 +）通道产生的信号，为黄色斑点状细胞。 </li></ol></li><li>使用奥林巴斯FV500-BX60共聚焦显微镜DEXT + CD4 + T细胞的易定量枚举。开始单击打开，FLUOVIEW软件（版本4.3）。 <ol><li>从下拉菜单中选择染料"Cy3标记"和"Cy5标记"，然后单击"应用"来加载相应的激光器。注意:在激发/发射波长为Cy3标记（543纳米/伪彩色的"绿色"，CD4-PE）和Cy5（633纳米/伪彩色的"红色"; dextramer-APC）的信道。 </li><li>将要检查在显微镜幻灯片的幻灯片，并在低倍率（4X或10X）手动对焦了。点击"焦点访谈"，而Cy3的激光，并专注于炎症病灶。 </li><li>改变客观上放大100倍。从下拉菜单中设置文件大小为"五百一十二分之五百十二"。点击"焦点访谈"，调整为"PMT"，"增益"的价值观和"偏移"参数分别优化激光器Cy3和Cy5。 </li><li>点击"Z级"，然后单击"焦点访谈"而无论是激光器上。集在"Z载物台"的通过点击"向上"和"向下"箭头的厚度。将"Z"的时间间隔为2微米。 </li><li>点击"停止"，并选择"Seq123"。然后，单击"XYZ"，并开始采集"Z"系列图像内的炎症病灶选定的区域。拯救"Z序列图像"为AVI文件。要保存图像文件，从"Z系列图像"选择图像，然后另存为"TIFF"格式。 </li></ol></li><li>检查从其中一组三段染了IA 秒/ PLP 139-151 dextramers/anti-CD4，另一组三个部分与执行机构的S / TMEV 70-86 dextramers/anti-CD4执行大脑3成对的节定量分析。 <ol><li>同样，分析对心脏八对的部分，其中，一组八个部分沾上了IA K / MYHC 334-352 dextramers/anti-CD4和另一个SE吨八个部分用RNase 43-56 dextramers/anti-CD4。 </li></ol></li><li>每检查大脑中第10至15炎性病灶，并在每个重点，掌握了一套15〜25序列图像顺序。 <ol><li>检查共20炎性病灶从所有八个部分心脏，并从每个重点掌握10〜15序列图像顺序。通过扫描载玻片在一个"水平 - 垂直向上，水平 - 垂直向下"运动，以便避免在同一焦点的重复的"Z"系列走&#39;Z&#39;的序列图像。 </li><li>一旦"Z"连续拍摄多张照片，通过识别幻灯片编号，各部分的名称，以及所采取的"Z"系列的图像数命名文件。 <ol><li>使用&#39;ImageJ的"软件计算DEXT + CD4 + T细胞相对于CD4 + T细胞的总数量。选择"ImageJ的的&#39;类型的计数器"，检查＆＃8216;显示所有"，并开始通过点击每个小区的中心算起，继续在不同的计数"使用一侧的箭头，Z&#39;的序列图像。计算所有的CD4 + T细胞后，选择其他类型的计数器和计数的CD4 + DEXT + T细胞。单击结果选项卡以获取细胞与两个不同的计数器计数的总数。 </li><li>取得的总数，增加细胞中的所有"Z"序列表示所有三个脑的部分或所有的8心脏切片的图像的数目。 </li></ol></li></ol></li></ol>

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(88), e51654 (2014).</li><li>Massilamany, C., Steffen, D., Reddy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epitope+from+Acanthamoeba+castellanii+that+cross-react+with+proteolipid+protein+139-151-reactive+T+cells+induces+autoimmune+encephalomyelitis+in+SJL+mice.">An epitope from Acanthamoeba castellanii that cross-react with proteolipid protein 139-151-reactive T cells induces autoimmune encephalomyelitis in SJL mice.</a> J Neuroimmunol. 219 (1-2), 17-24 (2010).</li><li>Donermeyer, D. L., Beisel, K. W., Allen, P. M., Smith, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocarditis-inducing+epitope+of+myosin+binds+constitutively+and+stably+to+I-Ak+on+antigen-presenting+cells+in+the+heart.">Myocarditis-inducing epitope of myosin binds constitutively and stably to I-Ak on antigen-presenting cells in the heart.</a> J Exp Med. 182 (5), 1291-1300 (1995).</li><li>Liao, L., Sindhwani, R., Leinwand, L., Diamond, B., Factor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+alpha-myosin+heavy+chains+differ+in+their+induction+of+myocarditis.+Identification+of+pathogenic+epitopes.">Cardiac alpha-myosin heavy chains differ in their induction of myocarditis. Identification of pathogenic epitopes.</a> J Clin Invest. 92 (6), 2877-2882 (1993).</li><li>Cameron, T. O., Cochran, J. R., Yassine-Diab, B., Sekaly, R. P., Stern, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+detection+of+antigen-specific+CD4++T+cells+by+HLA-DR1+oligomers+is+dependent+on+the+T+cell+activation+state.">Cutting edge: detection of antigen-specific CD4+ T cells by HLA-DR1 oligomers is dependent on the T cell activation state.</a> J Immunol. 166 (2), 741-745 (2001).</li><li>Novak, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+human+epitope-specific+T+cells+identified+by+class+II+tetramers+reside+within+a+CD4high,+proliferating+subset.">Activated human epitope-specific T cells identified by class II tetramers reside within a CD4high, proliferating subset.</a> Int Immunol. 13 (6), 799-806 (2001).</li><li>Reddy, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+autoreactive+myelin+proteolipid+protein+139-151-specific+T+cells+by+using+MHC+II+(IAs)+tetramers.">Detection of autoreactive myelin proteolipid protein 139-151-specific T cells by using MHC II (IAs) tetramers.</a> J Immunol. 170 (2), 870-877 (2003).</li><li>Skinner, P. J., Daniels, M. A., Schmidt, C. S., Jameson, S. C., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+In+situ+tetramer+staining+of+antigen-specific+T+cells+in+tissues.">Cutting edge: In situ tetramer staining of antigen-specific T cells in tissues.</a> J Immunol. 165 (2), 613-617 (2000).</li><li>Skinner, P. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+tetramer+staining.">In situ tetramer staining.</a> J Immunol Methods. 268 (1), 29-34 (2002).</li><li>Skinner, P. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+staining+using+MHC+class+I+tetramers.">In situ staining using MHC class I tetramers.</a> Curr Protoc Immunol. Chapter 17, Unit 17.14.11 (2004).</li></ol>

没有利益冲突被宣布。

与MHC I类四聚体，采用MHCⅡ类四聚体的常规应用仍然是具有挑战性的，尤其是枚举低亲和力的自身反应性CD4 + T细胞，部分原因是由于其激活依赖6,15-17的前体频率。最近，这个问题是由创建下一代四聚体，称为规避"dextramers，"允许我们检测抗原特异性CD4 + T细胞为一些自身抗原，如PLP 139-151，MOG 35-55和MYHC 334 - 352 8。在此报告中，用于第一时间的推移，MHC II类dextramers的实用程序来检测和量化自反应性CD4 T细胞在原位被使用从EAE小鼠的脑切片，并从EAM小鼠心脏切片证实。上的反应的基础上，dextramers提供需要少量的MHC /肽复合物的优点。在这个协议中，每个反应只需要MHC /肽单体的0.75微克。此外，栈进不去与dextramers是一步反应，因为dextramers被共孵育用抗CD4，而且整个过程，从组织切片染色，可以在不到一天的时间完成。与此相反， 在原位染色与常规四聚物的发布的协议为通常涉及在其中的荧光信号使用的二抗的荧光扩增扩增过程。因此，染色程序最多可能需要三天完成18-20。 此外，在脑切片使用dextramers的允许检测抗原特异性T细胞深处的组织切片中，甚至高达50微米。据指出，被发现PLP 139-151 DEXT + CD4 +细胞位于血管周围两个，也是实质内。此外，当用PLP 139-151 dextramers在大脑​​秒相比，从MYHC 334-352 dextramers产生的信号的强度是低的心脏部分行动（ 图1），并且这样的变化可以反映独特的每个器官的特性。一个这样的变量是其存在于高含量的脑组织中，而不是包含主要横纹肌纤维，可以潜在地限制dextramers的容易扩散进入炎症病灶心脏组织的脂质含量。支持这一观点的，MYHC 334-352 DEXT +细胞中检测到只有一个深度达20微米的大多数心脏部分。此外，MYHC 334-352 DEXT +细胞存在散落通过心肌提示炎性浸润在心肌炎病变的扩散性质。 通常，两个主要因素可造成负面的抗原特异性T细胞的检测由LSCM影响原位 。首先，由荧光团发射的信号不足可导致需要使用荧光抗体来放大信号。第二，这种间接染色ing可以破坏的特异性，作为背景染色对照试剂也可以按比例增加18,19。这些限制是由直接染色与dextramers规避。虽然，检测CD4 + T细胞已经成​​功地用新鲜的部分所示，利用在冷冻组织dextramer试剂需要额外的研究，这可能是一个挑战，作为组织完整性的保留可能是因为对组织中得到的倾向，难以各种染色过程中解体步骤18,19。

使用的主要组织相容性复合体（MHC）Ⅱ类四聚体检测抗原特异性CD4 + T细胞一直是个挑战1-7。为了增强MHC II类四聚体的检测灵敏度，研究小组已经创造下一代四聚体，命名为"dextramers"8。 Dextramers含有葡聚糖主链，其中多个链亲和素-荧光基团缀合，使得一些肽-拴系的，生物素化的MHC单体可以连接到一个葡聚糖分子9。因此，包含数个MHC-肽复合物的MHC dextramers的大的聚集体可以与多个T细胞受体（TCR）进行交互。 这组最近产生dextramers各种自身抗原，并证明dextramers的检测灵敏度约为5倍以上的四聚体8。实验系统包括实验性自身免疫性脑脊髓炎（EAE）诱导蛋白脂质蛋白（PLP）在SJL小鼠139-151; EAE诱导的髓磷脂少突胶质细胞糖蛋白（MOG）在C57BL / 6小鼠35〜55;和实验性自身免疫性心肌炎（EAM）与诱导心肌肌球蛋白重链α（MYHC）中的A / J小鼠334-352段。这项研究表明，使用PLP 139-151的-和MYHC 334-352 dextramers来检测抗原特异性CD4 + T细胞在原位特异性通过开发一个直接染色方案，从而省去了使用荧光抗体放大信号，一接近常用配合使用四聚体。此外，评估组织的综合方法也被设计成可靠地列举抗原特异性CD4 + T细胞在原位 10的频率。检测抗原特异性T细胞在原位的允许由从所造成的旁观者激活特定抗原刺激事件的描述。同样地， 在原位技术也providES机会来跟踪在靶器官的抗原特异性T细胞浸润的动力学。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:619px;">CFA</td>
			<td style="width:364px;">Sigma Aldrich, St Louis, MO</td>
			<td style="width:219px;">5881</td>
			<td style="width:167px;">Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MTB&#160; H37Rv extract&#160;</td>
			<td>Difco Laboratories, Detroit, MI</td>
			<td>231141</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PT</td>
			<td>List Biologicals Laboratories, Campbell, CA</td>
			<td>181</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">1x PBS&#160;</td>
			<td>Corning, Manassas, VA</td>
			<td>21-040-CV</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Female A/J mice&#160;</td>
			<td>Jackson Laboratories, Bar Harbor, ME</td>
			<td>646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Female SJL/J mice</td>
			<td>Jackson Laboratories, Bar Harbor, ME</td>
			<td>686</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leur-lok sterile 1 ml syrringe</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leur-lok sterile 3 ml syrringe</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile needle, 18 G</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile needle, 27 1/2 G</td>
			<td>BD, Franklin Lakes, NJ</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-way stopcock&#160;</td>
			<td>Smiths Medical ASD, Inc. Dublin, OH</td>
			<td>MX5311L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kerlix gauze bandage rolls&#160;</td>
			<td>Covidien, Mansfield, MA</td>
			<td>6720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipes</td>
			<td>Kimberly-Clark Professional, Roswell, GA</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose, Low Melting Point, Analytical Grade</td>
			<td>Promega corporation, Madison, WI</td>
			<td style="width:219px;">V2111</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Immunopure normal goat serum</td>
			<td>Thermo Scientific, Waltham, MA</td>
			<td style="width:219px;">31873</td>
			<td>Store at -20 &#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Anti-mouse CD4 conjugated with PE dye</td>
			<td style="width:364px;">eBioscience, San Diego, CA&#160;</td>
			<td style="width:219px;">12004185</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences, Hatfield, PA</td>
			<td style="width:219px;">19202</td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Faramount Aqueous Mounting Medium</td>
			<td>Dako, Carpinteria, CA</td>
			<td style="width:219px;">10073021</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Conical tube (15ml)</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td style="width:219px;">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical tube (50 ml)</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td style="width:219px;">352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well flat bottomed tissue culture plates</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td>356775</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water color #2 brushes</td>
			<td>Charles Leonard, Inc. Hauppauge, NY</td>
			<td style="width:219px;">73502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plain Microscope slides, size: 3&#34; x 1&#34; x 1.2 mm</td>
			<td>Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:219px;">1255010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Cover glass, 22 x 22-1</td>
			<td>Fisher Scientific, Pittsburgh, PA</td>
			<td style="width:219px;">12548B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable deep base mold histology cassettes</td>
			<td>Laboratory prodcust, Rochester, NY</td>
			<td style="width:219px;">M-475-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Loctite Super glue</td>
			<td>Henkel Corporation, Westlake, OH</td>
			<td style="width:219px;">1471879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td>Gillette SuperSilver</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Myhc-a 334-352 (DSAFDVLSFTAEEKAGVYK)</td>
			<td style="width:364px;">Neopeptide, Cambridge, MA</td>
			<td style="width:219px;"></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">PLP 139-151 (HSLGKWLGHPDKF)</td>
			<td style="width:364px;">Neopeptide, Cambridge, MA</td>
			<td style="width:219px;"></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MHC class II/IAs PLP 139-151 or TMEV 70-86 dextramers conjugated with APC dye</td>
			<td></td>
			<td></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MHC class II/IAk Myhc 334-352 or&#160; or RNase 40-56 dextramers conjugated with APC dye</td>
			<td></td>
			<td></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Dextran conjugated SA-APC</td>
			<td>&#160;Immundex, Copenhagen, Demark</td>
			<td style="width:219px;"></td>
			<td>Store at 4&#959;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile surgical scissors and forceps</td>
			<td style="width:364px;">INOX tool Corporation</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro oven</td>
			<td style="width:364px;">GE Healthcare, Pittsuburgh, PA</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica VT 1200 Vibratome</td>
			<td>Leica Microsystems, Inc. Buffalo Grove, IL</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus BX 60&#160; Laser scanning confocal microscope</td>
			<td>Olympus America, Inc. Center Valley, PA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon A1-Eclipse 90i confocal microscope</td>
			<td>Nikon Inc. Melville, NY</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ detection of autoreactive cd4 t cells in brain and heart using major histocompatibility complex class ii dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

在这份报告中， 原位检测抗原特异性，自身反应性CD4 + T细胞通过直接染色与MHC II类dextramers的描述。刚割下的脑切片均来自EAE小鼠并用含PLP 139-151（具体的）或TMEV 70-86（控制）dextramers和抗-CD4的鸡尾酒。顶部面板在图1所示的costained用PLP 139-151 dextramers（红色）和CD4抗体（绿色），其中双阳性细胞呈黄色脑切片的LSCM分析。这样的功能是没有的沾满TMEV 70-86（控制）dextramers（底板）的部分。该costained（DEXT + CD4 +）细胞显示在周围点状的点。 PLP特异性T细胞在脑切片的定量分析包括以下步骤（ 图2）:1）三对切片制成，并且从每对1段单独进行染色用抗CD4和PLP 139-151 dextramers。同样地，另一组三个切片用抗CD4和控制（TMEV 70-86）dextramers。 2）从一个给定的部分中，炎症病灶（10到15）被确定基于抗CD4染色。 3）一组&#39;Z&#39;的序列图（15至25），是从各个焦点顺序从上到下为2μm的每个之间的间隔。从本质上说，10至15相似的套&#39;Z&#39;的序列图像代表相等数目的炎症病灶中的每个部分进行分析。 4）在单独的图像，CD4 +细胞（绿色），细胞阳性为dextramers（红色）和CD4（绿色），它表现为黄色点状细胞通过使用开源软件，ImageJ的标记它们被单独列举，最后，通过增加细胞中的所有"Z"序列图像计数的编号为每节获得了总计。因为在多个图像要讲述的细胞，该细胞被标记的可能性重叠的被淘汰出局。因此，DEXT +细胞相对于细胞表达CD4的总数的可靠枚举可以实现的。 本研究进一步扩大示范使用的dextramer试剂原位诱导EAM与MYHC在334-352的A / J小鼠，这也是一种T细胞介导的疾病13,14检测抗原致敏的T细胞。八对心脏切片得到，每200微米厚，由EAM小鼠（ 图2）。从每对，一个部分（共8节）染了MYHC 334-352 dextramers和抗CD4，而另一组部分（共8个）的染色用RNase 43-56 dextramers（控制）和抗-CD4。被收购的顺序如上（ 图2）20套"Z"对应于许多炎症病灶占全部八个部分序列图像。本质上，在给定的"Z"串行IM年龄由10到15个人代表一个单一的炎症病灶。这些图像通过激光共聚焦显微镜分析表明细胞阳性MYHC 334-352 dextramers（红色）的存在，和CD4（绿色）和一如预期，势必既dextramers和CD4细胞出现黄色点状细胞（ 图3，顶部面板） 。背景染色对照dextramers是微不足道的（ 图3，下图）。被确定为从所有的部分，每一个部分与计数DEXT + CD4 + T细胞的总数量和CD4 + T细胞的总数目加在一起以得到总数为每个子集（DEXT + CD4 + T细胞，CD4 + T细胞）。 <img alt="图1" src="/files/ftp_upload/51679/51679fig1highres.jpg" /> 图1 的原位检测PLP-特异性CD4 T细胞的 </st荣&gt; 染色PLP 139-151 dextramers。EAE是由免疫与PLP 139-151动物在终审法院诱发SJL小鼠。在终止时，从EAE小鼠收集cerebrums包埋在4％琼脂糖，和将切片用vibratome制成。含有两种PLP 139-151 dextramers/anti-CD4或TMEV 70-86 dextramers（控制）/抗-CD4和4％的PBS缓冲多聚甲醛固定的鸡尾酒染色后，切片洗净，装入审查通过激光共聚焦显微镜。顶部面板:沾上PLP 139-151 dextramers/anti-CD4两个独立的部分。底板:沾上TMEV 70-86 dextramers/anti-CD4两个独立的部分。左侧面板:CD4，绿色环保;中图:dextramers，红色;右侧面板:合并（箭头，DEXT + CD4 + T细胞）。原放大1,000倍（采用:。Massilamany等人 ，2014年直接染色与主要组织相容性复合II类Dextramers允许检测抗原特异性，自身反应性CD4 + T细胞s 在原位PLOS ONE 9（1）:e87519） <a href="https://www.jove.com/files/ftp_upload/51679/51679fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/51679/51679fig2highres.jpg" /> 图2。定量DEXT +细胞在大脑和心灵的一种方法。配对的部分是由大脑或外耳切除心脏制成如图所示（面板1）。从对应于所指示的器官每对一个区段被指定为与特定dextramers（大脑，PLP 139-151 dextramers;心脏，MYHC 334-352 dextramers）染色，另一组为对照dextramers部分（大脑，TMEV 70 - 86，心脏，RNA酶43-56）。固定和装配后，将切片检查，以找出基于与CD4通过激光共聚焦显微镜染色炎性病灶（原MAGnification，40X）（面板2）。作为可视化的三维（3D）视图（面板3）每个炎症病灶经受了一套&#39;Z&#39;的序列图像顺序（面板4）。中的所有图像，细胞阳性为特定或控制dextramers和CD4，CD4或单独使用&#39;ImageJ的&#39;软件（通过计数:。Massilamany等人 ，2014年直接染色与主要组织相容性复合II类Dextramers允许检测抗原的具体的，自身反应性CD4 + T细胞原位PLOS ONE 9（1）:e87519） <a href="https://www.jove.com/files/ftp_upload/51679/51679fig2highres.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/51679/51679fig3highres.jpg" /> 图3。通过原位检测心肌肌球蛋白特异性CD4 T细胞的 <st荣&gt;染色MYHC 334-352 dextramers EAM是诱发A / J小鼠免疫所用MYHC在334-352终审法院的动物;在终止在第21天，心收集灌注后，用4％的PBS缓冲琼脂糖制备的组织块。每200微米厚八个成对切片使用vibratome制成。从每对（共八节）一个切片用含有两种MYHC 334-352 dextramers/anti-CD4或RNA酶43-56（控制）dextramers/anti-CD4混合物。用4％的PBS缓冲的多聚甲醛固定后，将切片用LSCM洗涤并安装用于检查。显示阳性的细胞为dextramers和CD4或CD4单独的图像显示。顶部面板:沾上MYHC 334-352 dextramers/anti-CD4两个独立的部分。底板:染色用RNase 43-56 dextramers /抗-CD4两个独立的部分。左侧面板:CD4，绿色环保;中间面板:dextramers，红色;右侧面板:合并（箭头，DEXT + CD4 + </s高达&gt; T细胞）。原放大1,000倍（采用:。Massilamany等人 ，2014年直接染色与主要组织相容性复合II类Dextramers允许检测抗原特异性，自身反应性CD4 + T细胞原位PLOS ONE的9（1）:e87519）。 <a href="https://www.jove.com/files/ftp_upload/51679/51679fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers at JoVE.com

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

通过直接染色与主要组织相容性复合物Ⅱ类dextramers检测自身反应性CD4 + T细胞在大脑和心脏的协议已经本报告中进行了描述。为综合分析，一种可靠的方法来枚举抗原特异性CD4 + T细胞在原位的频率也被设计出来。

原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

in-situ-detection-autoreactive-cd4-t-cells-brain-heart-using-major

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

11.6K 次观看.  University of Nebraska, Lincoln. 通过直接染色与主要组织相容性复合物Ⅱ类dextramers检测自身反应性CD4 + T细胞在大脑和心脏的协议已经本报告中进行了描述。为综合分析，一种可靠的方法来枚举抗原特异性CD4 + T细胞在原位的频率也被设计出来。 

视频: In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

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

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

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

T细胞受体在小鼠T细胞的逆转录病毒转导

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

科研

免疫与感染

piggyBac Transposon System Modification of Primary Human T Cells

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

piggyBac Transposon System Modification of Primary Human T Cells

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

piggyBac转座子初级人类T细胞系统改造

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

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

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

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

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

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

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

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

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

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

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

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

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

使用madSTORM进行高分辨率，超分辨率的T细胞成像

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

增殖人类抗原特异性CD4+ T细胞使用卡博曲莱辛苏奇尼米酯的定量

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Assessing the Expression of Major Histocompatibility Complex Class I on Primary Murine Hippocampal Neurons by Flow Cytometry

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A well-studied function of major histocompatibility complex class I (MHCI) is the presentation of cell-derived peptides to the adaptive immune system, particularly in response to infection. More recently it has been shown that the expression of MHCI molecules on neurons can modulate activity-dependent changes in the synaptic connectivity during normal development and neurologic disorders. The importance of these functions to the brain health supports the need for a sensitive assay that readily detects MHCI expression on neurons. Here we describe a method for primary culture of murine hippocampal neurons and then assessment of MHCI expression by flow cytometric analysis. Murine hippocampus is microdissected from prenatal mouse pups at the embryonic day 18. Tissue is dissociated into a single cell suspension using enzymatic and mechanical techniques, then cultured in a serum-free media that limits growth of non-neuronal cells. After 7 days in vitro, MHCI expression is stimulated by treating cultured cells pharmacologically with beta interferon. MHCI molecules are labeled in situ with a fluorescently tagged antibody, then cells are non-enzymatically dissociated into a single cell suspension. To confirm the neuronal identity, cells are fixed with paraformaldehyde, permeabilized, and labeled with a fluorescently tagged antibody that recognizes neuronal nuclear antigen NeuN. MHCI expression is then quantified on neurons by flow cytometric analysis. Neuronal cultures can easily be manipulated by either genetic modifications or pharmacologic interventions to test specific hypotheses. With slight modifications, these methods can be used to culture other neuronal populations or to assess expression of other proteins of interest.

This protocol describes a method for culturing primary hippocampal neurons from embryonic mouse brain. The expression of major histocompatibility complex class I on the extracellular surface of cultured neurons is then assessed by flow cytometric analysis.

按流细胞测量评估主要穆林河马神经元的主要组织相容性综合类 I 的表达

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A ...

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &beta;2-Microglobulin

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and tumor development. The hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m) substitution from different species can be stabilized in vitro. This is a feasible means to study MHC I of mammals, when the homologous &#946;2m is not available. Meanwhile, it is indicated that mammalian &#946;2m substitution does not significantly affect peptide presentation. However, there is limited summarization regarding the methodology and the technology for the hybrid MHC I complexed with heterologous &#946;2-microglobulin (&#946;2m). Herein, methods to evaluate the feasibility of heterologous &#946;2m substitution in MHC I study are presented. These methods include preparation of expression constructs; purification of inclusion bodies and refolding of the MHC complex; determination of protein thermostability; crystal screening and structure determination. This study provides a recommendation for understanding function and structure of MHC I, and is also significant for T cell response evaluation during infectious disease and tumor immunotherapy.

Stability and Structure of Bat Major Histocompatibility Complex Class I with Heterologous &#946;2-Microglobulin

The protocol describes experimental methods to obtain stable major histocompatibility complex (MHC) class I through potential &#946;2-microglobulin (&#946;2m) substitutions from different species. The structural comparison of MHC I stabilized by homologous and heterologous &#946;2m were investigated.

蝙蝠主要组织兼容性复杂类 I 的稳定性和结构与异质β2- 微球蛋白

The major histocompatibility complex (MHC) plays a pivotal role in antigen peptide presentation and T cell immune responses against infectious disease and ...