这项工作是由美国国立卫生研究院/ NCATS临床与转化科学奖，以佛罗里达州的TL1 TR000066和UL1 TR000064，从父格兰特R01AI056152多样性的补充来自美国国立卫生研究院的BD Biosciences公司试剂授予大学和大学的支持部份佛罗里达州。

所有动物使用是根据批准的机构动物护理和使用委员会的协议进行的。 1。混音及媒体的制备 <ol><li> 无菌PBS pH值:7.3（1升） 0.23克磷酸二氢钠 1.15克的Na 2 HPO 4 9.0克氯化钠 占用体积去离子水。消毒用高压锅。 </li><li> 细胞培养基（100毫升） 89毫升RPMI 10毫升10％FBS的 1毫升抗生素抗真菌素（ABAM）100微克50-巯基乙醇（3.5微克股票2 - 巯基乙醇为96.5微克PBS） </li><li>在无菌PBS 缓冲液FACS（荧光激活细胞分选）（将在流式细胞术中使用）2％FBS </li><li> iTh17混合 （根据样品数量，确定所需的所有混合和媒体音量） 1.5微克/毫升抗-CD28 20纳克/毫升的IL-6 5毫微克/毫升TGF-β的</li></ol> 2。细胞将一式三份在下列条件下进行电镀 <ol><li>板结合的抗CD3，抗-CD28（这是起动控制的混合）。 </li><li> iTH17:板结合的抗CD3，抗CD28，IL-6，TGF-β。 </li></ol> 3。板结合的抗CD3（10微克/毫升） 建议制备板结合的抗CD3板先于细胞将被添加到板的时间进行至少4小时。 <ol><li>加入30微升抗CD3到井（抗-CD3稀释在无菌PBS），一个新的96孔U型底微量测试组织培养板中，挖掘板块的两侧，以确保该井的均匀覆盖。在37℃下4小时，然后冷藏，直到它是时间的混合和细胞添加到板上。 </li></ol> 4。小鼠解剖 <ol><li>此协议的基础上，利用C57BL / 6麦克风E，3-8个月的年龄，并从杰克逊实验室（缅因州巴港）购买。 </li><li>使用CO 2窒息牺牲的鼠标，并确认死亡，随后颈椎脱位。 </li><li>消毒小鼠解剖工具和切口面积用70％乙醇，并开始清扫。 </li><li>从腹面观，抓住皮肤，先于尿道口，并开始用剪刀剪了腹中线，直到达到下巴部位。采取预防措施，避免撕裂或剪切到腹膜壁衬里。 </li><li>向后拉动皮肤和牵制，使搬迁过程中方便地访问到淋巴结。 </li><li>收集淋巴结和脾中都将用钳子取出，并放置在含有5ml autoMACS电泳缓冲液自Miltenyi Biotec公司购买的（参见图2和图3为淋巴结和脾切除的图）的无菌培养皿中。 <ol><li>拧下腋窝淋巴结靠近腋窝（腋下）每只小鼠的胸肌后面找到。 </li><li>除去存在于位于每个腋窝附近的结缔组织的肱淋巴结。 </li><li>除去在每只小鼠的颈中发现的颈浅淋巴结。 </li><li>除去位于髋部地区在3个血管一起腹股沟淋巴结。 </li><li>以访问肠系膜淋巴结，通过腹膜衬里向上腹侧中线切开。肠系膜淋巴结中发现保存肠内在一起的结缔组织。他们普遍认为在4-8节点的字符串，可能会显示为"珍珠串"。请务必拔出整个字符串。 </li><li>脾脏位于腹部，胃和肠的后面。从胰腺拉动和分离它删除它。 </li></ol></li><li>磨下使用2磨砂载玻片无菌罩器官。放置在淋巴结或脾脏1显微镜载玻片，并与第二滑动，直到解体的磨砂边擦的磨砂面。重复，直到所有的淋巴结和脾脏已经接地。 </li></ol>注:建议先从淋巴结和完成具有健脾，因为血液会让人很难看到autoMACs缓冲区中剩余的淋巴结。 <ol start="9"><li>要过滤地面器官成单细胞悬液，通过折叠一块40微米的尼龙材料数次，置15ml离心管的顶部开始。尼龙材料应大致3 x 3英寸</li><li>用5毫升注射器和21号针头抽吸中的5毫升autoMACs的电泳缓冲液和地下器官。慢慢地分装到折叠的尼龙材料。注意避免刺破材料用针。 </li></ol>注意:一旦单细胞悬液实现，解决方案应该显示为面色苍白，一致的解决方案，没有visib勒个坚实的组织。如果固体残留，再吸入和排出通过一个新的折叠一块尼龙放置在一个新的15ml离心管。 <ol start="11"><li>始终保持细胞在冰上在不使用时。 </li></ol> 5。细胞分离 <ol><li>为了达到最佳效果，获得至少80％的通过利用MACS CD4 + CD25 +调节性T细胞分离试剂盒，鼠标autoMACS临细胞分离纯的CD4 + CD25-细胞群。通过美天旎生物技术公司获得的协议进行的CD4 + CD25-细胞群的负保持。 </li></ol> 6。 Th17细胞分化 <ol><li>分离与autoMACS Pro的细胞分离后收集的CD4 + CD25-细胞群。 </li><li>计数细胞稀释1:1000的比例用锥虫蓝使用血细胞计数。 </li><li>一旦浓度已经确定从细胞计数，稀释细胞悬液，以1×10 6个细胞/细胞培养基毫升。 </li><li>取出96孔板板4小时后绑定的抗CD3。 </li><li>用200微升PBS洗抗-CD3-包被的孔。重复2次。 <ol><li>洗加200微升无菌PBS中的抗CD3-包被的孔，并除去PBS到废物容器中。 </li></ol></li><li>加入100μliTh17组合或激活控制组合（见点＃2）的井，一式三份。 </li><li>加入100微升细胞的每个孔中，无论是Th17细胞混合物或激活控制组合已被放置。 </li><li>孵育细胞至少72小时或5天（Th17细胞可以分化后72小时或5天后获得。） </li><li> Th17细胞分化可通过细胞内染色和流式细胞术分析，ELISA或定量PCR来评估</li></ol> 7。细胞激活 （仅在细胞内染色） <ol><li>从培养箱取出96孔板，无论是72小时或5天结束<ol><li>回想一下，Th17细胞differentiation可以后，无论是72小时或5天来实现。 </li></ol></li><li>每个条件（ 例如起动控制或iTh17），转移细胞，一式三份在24孔细胞培养板的一个孔中。细胞的一个条件现在已汇集成24孔培养板的一个孔，而不是作为一式三份于96孔U型底培养板。 </li><li>的每个孔中的24孔细胞培养板的总体积是现在600微升。提高每一个的体积以及到1ml的细胞培养基。 </li><li>加入PMA（佛波酯）（50毫微克/毫升），离子霉素（1微米），BFA（布雷菲尔德菌素-A）（10微克/毫升），每孔在24孔细胞培养板在上市的浓度。 </li><li>在37℃保温4小时。 </li></ol> 8。细胞内染色 <ol><li>染色的细胞具有所需胞外和胞内标记物的流式细胞术分析。为了检测存在øf IL-17，细胞内染色，是用抗-IL-17A抗体。推荐外表面标志物包括CD4，CD8和CD25。 <ol><li>使用内细胞因子染色入门套件 - 鼠标自BD Bioscience公司的IL-17的细胞内染色。 </li><li>对于每个样品，沉淀细胞，除去上清液，并在200μlFACS缓冲液（2％FBS的PBS）中重悬细胞沉淀。 </li><li>转移重悬细胞至96孔细胞流式细胞仪检测板。 </li><li>自旋向下的细胞为5分钟1200转，弃上清。 </li><li>加入200μl的PBS缓冲液流式细胞仪，离心5分钟1200转，弃上清。 </li><li>重悬细胞于100μlFACS缓冲液，并应用100微升细胞外抗体（抗体）的混合物（细胞外抗体的混合物是在FACS缓冲液）。孵育15分钟，在室温下，用铝箔纸覆盖。 </li><li>重复步骤8.1.4。 </li><li>重复步骤8.1.5 2倍。 </li><li>重悬在100μlBD Cytofix / Cytoperm缓冲的细胞。 Incub吃了20分钟在室温下，用铝箔纸覆盖。 </li><li>加入100μl的1×BD烫发/洗涤缓冲液，离心5分钟1200转，弃上清。重复。 </li><li>加入50微升的细胞内抗体的混合物。 （细胞内抗体的混合物是在1X BD彼尔姆/冲）孵育15分钟在室温下，用铝箔纸覆盖。 </li><li>重复步骤8.1.10。 </li><li>重悬在200μlBD染色缓冲液中的细胞。 </li><li>将重悬细胞成流式细胞含200微升BD染色缓冲液（终体积为400微升）管。 </li><li>储存于4°C，直到样品准备好被读取。 </li></ol></li></ol> 9。流式细胞仪分析 <ol><li>门活细胞群。 </li><li>从活细胞群，对CD4 + CD8人口大门。 </li><li>从CD4 + CD8-的人口，对IL-17A +人口的大门。 <ol><li>根据我们以前的实验结果，白细胞介素-17A +人口的100％将是CD25 +。 </li></ol></li></ol> *总绝对细胞计数池的样品一式三份后得到 * CD4 + CD25 + IL-17A +细胞的绝对数量通过由活门百分比和总细胞轴承谱系特异性标志物CD4，CD25和IL-17A的百分比，细胞的总数乘以所确定通过流式细胞仪检测。 10。定量PCR和ELISA法 <ol><li>不用于流式细胞术分析细胞的地方在1.5ml Eppendorf管中，离心分离机以13,000 rpm离心5-10分钟。在离心后的细胞沉淀物中发现的细胞，将用于定量PCR分析。使用PTC-200珀尔帖热循环仪（Biorad公司）进行定量PCR分析。 </li><li>从离心管的ELISA收集上清液。 IL-17A ELISA试剂盒采用双抗体TC11-18H10（捕获，目录号555068）进行，TC11-8H4生物素（检测，目录号555067）购自BD公司。 IL-17A ELISA站ndards购自eBioscience公司（目录号14-8171-80）获得。 </li><li>除去上清液后，重悬，以用于定量PCR在175微升的RNA裂解缓冲液（立即用于RNA提取，cDNA合成，并定量PCR，或储存于-80℃以备后用）的剩余细胞。 <ol><li>参照表II为对IL-17A和肌动蛋白（持家基因）的引物序列</li></ol></li></ol>

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没有披露声明。

在这里，我们所描述的协议，实现在体外 Th17细胞分化。 Th17细胞分化的研究是重要的，因为的T淋巴细胞分化成Th17细胞子集，是有效消除人类病原体13关键。相反，IL17生产已与自身免疫性疾病的进展13有关。因为它可以适用于免疫调节和自身免疫的许多不同的鼠模型中的Th17分化的方法适用于许多研究设置。此方法有助于回答有关的T淋巴细胞的能力，分化成Th17细胞的细胞，?...

CD4 + T淋巴细胞（T细胞）在与感染性微生物的免疫系统介导的防御中发挥至关重要的作用。相反，T细胞还密切与自身免疫性疾病如1型糖尿病，系统性红斑狼疮，类风湿关节炎的发病和进展相关。 CD4 + T淋巴细胞成为通过T细胞受体（TCR）与同源抗原/主要组织相容性复合体II（MHCII）的分子相互作用的结合活性，并与B7.1/B7.2的CD28受体相互作用的配体15。除了提供TCR刺激和CD28共刺激的，抗原呈递细胞还提供一种细胞因子环境，这就决定了T淋巴细胞的分化状态，从而指示该T淋巴细胞的反应，给定抗原。不同的病原体/抗原呈递细胞相互作用创造独特的细胞因子环境，这歪斜T淋巴细胞向下聚焦于ELI不同的途径税款发起病原体。不幸的是，T淋巴细胞效应的途径，原本旨在消除入侵的病原体，可错误地针对自身组织15。因此，更好地了解每个不同的CD4 + T细胞亚群的分化状态的是我们如何来调节消除病原体和宽容自我之间的平衡的理解是至关重要的。 除了TH1，TH2，以及诱导型调节性T细胞的分化途径，幼稚T淋巴细胞还可以通过细​​胞因子向下的Th17通路驱动。而Th1细胞战斗细胞内的病原体，Th2细胞消除细胞外的病原体，调节性T细胞（Treg细胞）减少炎症反应的1，16; Th17细胞在消除胞外细菌和真菌中发挥重要作用。 Th17细胞是通常用的谱系特异性转录因子RORγT和产生IL的表达-17A，促进巨噬细胞和嗜中性粒1,7的激活。 Th17细胞有牵连的一些自身免疫性疾病，以及它们相关的啮齿类动物模型。例如，已经证明了IL-23（它是必需的，以维持Th17细胞表型），而不是IL-12，是在中央的罪魁祸首在实验性自身免疫性脑炎（EAE），用于MS的啮齿动物的疾病模型。它随后被显示，减少的IL-17的产生是相关的，以预防EAE 2，6，17。此外，Th17细胞已与其他自身免疫性疾病，包括关节炎和系统性红斑狼疮（SLE）的10，16相关联。 IL-23缺陷的p19 - / -小鼠显示出具有非常低的数字Th17细胞，并具有耐显影不仅EAE，而且胶原诱导的关节炎，型号为风湿性关节炎10，18。此外，小鼠中和IL-17A抗体治疗船尾呃胶原诱导的关节炎的发病中也发现有关节损伤18号决议。应当指出的是Th17细胞在自身免疫性疾病的进展中的作用仍有待表征为最近的研究还表明Th17细胞在1型糖尿病9,11和肠道炎症14的保护作用。这些研究证实Th17细胞分化的自体免疫的重要性。 体外 Th17细胞分化为T细胞研究的一个必要方法，因为有至少两个需要进一步调查令人困惑的问题:1）究竟如何IL-6的调节Treg细胞和Th17细胞分化之间的平衡，以及2）什么是确切机制IL-17诱导的炎性疾病背后？我们的方法使用的CD4 + CD25-T细胞从C57BL / 6小鼠的脾和淋巴结。要注意的是很重要的，虽然有可能使用不纯的诱导Th17细胞分化人群中，获得至少80％纯的CD4 + CD25-T细胞群否定任何污染的忧虑，并确保更成功的Th17细胞分化的结果。为了实现正确的Th17分化，CD4 + CD25-T细胞在抗-CD3和抗-CD28，提供活化信号，1和2分别存在下，与IL-6和TGF-β。虽然已经报道了IL-23单独使用，可用于实现Th17细胞分化，但后来被证明IL-23是必要的Th17细胞的细胞群的稳定性，但IL-6和TGF-β对Th17细胞分化所必需3，18，19。鼠的研究表明，该IL-23受体表达于CD4 + T细胞，他们已经被刺激的IL-6和TGF-β13，18之后。此外，Th17细胞将成功地在IL-23阻断性抗体的存在下培养，只要IL-6和TGF-β的存在18,19。因此，本Th17的分化方案省IDES在适当的条件下，成功地诱导Th17细胞分化。开发更好的了解相关Th17细胞分化和IL-17的产生提供了机会为更好的治疗目的是自身免疫性疾病13发展的机制。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>Sterile Polyestrene Petri Dish</td>
 <td>Fisher Scientific</td>
 <td>0875713A</td>
 <td>60 mm x 15 mm</td>
 </tr>
 <tr>
 <td>autoMACS Running Buffer</td>
 <td>Miltenyi Biotec</td>
 <td>130-091-221</td>
 <td></td>
 </tr>
 <tr>
 <td>Premium Microscope Slides, Frosted</td>
 <td>Fisher Scientific</td>
 <td>12-544-3</td>
 <td>3" x 1"1 mm</td>
 </tr>
 <tr>
 <td>5 ml 21G1 Latex Free Syringe and Needle</td>
 <td>BD Biosciences</td>
 <td>309632</td>
 <td></td>
 </tr>
 <tr>
 <td>Corning 15 ml Centrifuge Tubes</td>
 <td>Sigma-Aldrich</td>
 <td>CLS430791</td>
 <td></td>
 </tr>
 <tr>
 <td>Nylon 40 microns</td>
 <td>Miami Aquaculture</td>
 <td>Nylon 40/26</td>
 <td></td>
 </tr>
 <tr>
 <td>Microtest tissue culture plate 96 well U bottom </td>
 <td>BD Biosciences</td>
 <td>35-3077</td>
 <td></td>
 </tr>
 <tr>
 <td>Corning Costar 24 well cell culture plates</td>
 <td>Sigma-Aldrich</td>
 <td>CL3524</td>
 <td></td>
 </tr>
 <tr>
 <td>Eppendorf Tubes 1.5 ml</td>
 <td>Fisher Scientific</td>
 <td>05-408-129</td>
 <td></td>
 </tr>
 <tr>
 <td>Purified NA/LE Hamster Anti-Mouse CD3e</td>
 <td>BD Biosciences</td>
 <td>553057</td>
 <td></td>
 </tr>
 <tr>
 <td>Purified NA/LE Hamster Anti-Mouse CD28</td>
 <td>BD Biosciences</td>
 <td>553294</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse IL-6 Recombinant Protein</td>
 <td>eBioscience</td>
 <td>14-8061-62</td>
 <td></td>
 </tr>
 <tr>
 <td>TGFbeta</td>
 <td>R&amp;D Systems</td>
 <td>240-B-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan blue solution 0.4 %</td>
 <td>Sigma-Aldrich</td>
 <td>66H2364</td>
 <td></td>
 </tr>
 <tr>
 <td>Pac Blue Rat Anti-Mouse CD4</td>
 <td>BD Biosciences</td>
 <td>558107</td>
 <td></td>
 </tr>
 <tr>
 <td>APC Rat Anti-Mouse CD8a</td>
 <td>BD Biosciences</td>
 <td>553035</td>
 <td></td>
 </tr>
 <tr>
 <td>PE Conjugated Anti-mouse CD25</td>
 <td>eBioscience</td>
 <td>E01155-516</td>
 <td></td>
 </tr>
 <tr>
 <td>Alexa Fluor 700 Rat Anti-mouse IL-17</td>
 <td>BD Biosciences</td>
 <td>560820</td>
 <td></td>
 </tr>
 <tr>
 <td>Intracellular Cytokine Staining Starter Kit-Mouse</td>
 <td>BD Biosciences</td>
 <td>51-2041AK (559311)</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS CD4+CD25+ regulatory T cell isolation kit, mouse</td>
 <td>Miltenyi Biotec</td>
 <td>130-091-221</td>
 <td></td>
 </tr>
 <tr>
 <td>ABAM </td>
 <td>Cellgro</td>
 <td>30-004-CI</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI</td>
 <td>Corning, Cellgro</td>
 <td>10-040-CM</td>
 <td></td>
 </tr>
 <tr>
 <td>B 2-MercaptoEthanol</td>
 <td>MP Biomedical</td>
 <td>194834</td>
 <td>Hazardous</td>
 </tr>
 <tr>
 <td>Phorbol 12-Myristate 13-Acetate (PMA)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ionomycin Calcium Salt</td>
 <td>Sigma-Aldrich</td>
 <td>13909-1ML</td>
 <td>Hazardous</td>
 </tr>
 <tr>
 <td>Brefeldin A (BFA)</td>
 <td>MP Biomedicals</td>
 <td>159027</td>
 <td></td>
 </tr>
 <tr>
 <td>ELISA IL-17A Capture mAb </td>
 <td>BD Biosciences</td>
 <td>555068</td>
 <td></td>
 </tr>
 <tr>
 <td>ELISA IL-17A Detection mAb </td>
 <td>BD Biosciences</td>
 <td>555067</td>
 <td></td>
 </tr>
 <tr>
 <td>ELISA IL-17A Standard</td>
 <td>eBioscience</td>
 <td>14-8171-80</td>
 <td></td>
 </tr>
 <tr>
 <td>IL-2 ELISA Kit</td>
 <td>BD Biosciences</td>
 <td>555148</td>
 <td></td>
 </tr>
 <tr>
 <td>TMB Substrate Reagent Set </td>
 <td>BD Biosciences</td>
 <td>555214</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>autoMACS Pro Cell Separator</td>
 <td>Miltenyi Biotec</td>
 <td>130-092-545</td>
 <td></td>
 </tr>
 <tr>
 <td>Sorvall Legend RT+ Centrifuge</td>
 <td>ThermoScientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Napco series 8000 WJ CO2 Incubator</td>
 <td>ThermoScientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>PTC-200 Peltier Thermal Cycler</td>
 <td>Biorad</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation and th17 differentiation of na&iuml;ve cd4 t lymphocytes

Th17细胞是T细胞已被发现产生白细胞介素17（IL-17）的一个独特的子集，并且不同的功能从其他T细胞亚群，包括Th1细胞，Th2细胞和调节性T细胞。 Th17细胞已与许多自身免疫性疾病相关的炎症过激的免疫反应成为中央罪魁祸首。在这个方法中，我们从净化C57BL / 6小鼠的脾脏和淋巴结T淋巴细胞，并刺激纯化的CD4 + T细胞在控制之下和Th17细胞诱导的环境。在Th17细胞诱导的环境包括在抗CD3和抗CD28抗体，IL-6和TGF-β的存在下刺激。培养至少72小时后长达五天在37℃，细胞随后通过流产生IL-17流式细胞仪，定量PCR和酶联免疫吸附能力进行分析。 Th17细胞分化的CD4 + CD25-T细胞可以被用来进一步阐明角色Th17细胞在自身免疫和宿主德的发生和发展过程中发挥FENSE。此外，CD4 + CD25-细胞从不同的小鼠基因敲除/疾病模型的Th17分化，可以促进我们的细胞命运可塑性的理解。

这Th17细胞分化的协议始于切除脾脏及腋窝，上臂，肠系膜，颈和腹股沟淋巴结肿大。每个位置的表示可以在图2和图3中找到。图1和5都提供在本协议中所述的方法的可视化表示。 此协议的Th17着重从CD4 + CD25-T淋巴细胞种群分化。要注意的是如何有效的autoMACS临细胞分离富集从总汇集淋巴结和脾（ 图4）我们所期望的...

Watch this Scientific Journal Video about Isolation and Th17 Differentiation of NaÃ¯ve CD4 T Lymphocytes at JoVE.com

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

与效应器功能从其它的T细胞亚群的不同的，Th17细胞已被集中牵连的炎性自身免疫性疾病。这种体外 Th17细胞分化协议提供了一种方法来确定初始CD4 + T淋巴细胞是否能够分化成Th17细胞，并进一步研究其在自身免疫和宿主反应的作用。

隔离和幼稚的CD4 T细胞的Th17细胞分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

isolation-and-th17-differentiation-of-nave-cd4-t-lymphocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

34.3K 次观看.  The University of Florida. 与效应器功能从其它的T细胞亚群的不同的，Th17细胞已被集中牵连的炎性自身免疫性疾病。这种体外 Th17细胞分化协议提供了一种方法来确定初始CD4 + T淋巴细胞是否能够分化成Th17细胞，并进一步研究其在自身免疫和宿主反应的作用。 

视频: Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

科研

免疫与感染

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

小鼠生殖道黏膜淋巴细胞分离

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

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 ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

从人类腭扁桃体B和T淋巴细胞有效隔离协议

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

分离和<em&gt;离体</emVδ1的&gt;企业文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT细胞，胸腺外的αβT细胞祖

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

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

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

成像的CD4 T细胞间质迁移发炎真皮

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

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

分离及小鼠淋巴细胞的激活

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

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.

隔离和幼稚的CD4 T细胞的Th17细胞分化

摘要

探索更多视频

此视频中的章节

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

定向分化诱导性多潜能干细胞向T淋巴细胞

小鼠生殖道黏膜淋巴细胞分离

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

鼠标朴素CD4

从人类腭扁桃体B和T淋巴细胞有效隔离协议

分离和

成像的CD4 T细胞间质迁移发炎真皮

分离及小鼠淋巴细胞的激活

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

隔离和幼稚的CD4 T细胞的Th17细胞分化

摘要

探索更多视频

此视频中的章节

β细胞死亡介导细胞毒性T淋巴细胞的方法来评估

定向分化诱导性多潜能干细胞向T淋巴细胞

小鼠生殖道黏膜淋巴细胞分离

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

鼠标朴素CD4

从人类腭扁桃体B和T淋巴细胞有效隔离协议

分离和

成像的CD4 T细胞间质迁移发炎真皮

分离及小鼠淋巴细胞的激活

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

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