我们感谢戈登·弗里曼提供抗PD-L1的封闭抗体。我们感谢的临床和实验室工作人员在美国马萨诸塞州总医院和所有的研究参与者在这个项目中的不可估量的作用。 ，国家心肺和美国国立卫生研究院血液研究所（RO1 HL-092565; DEK），这项研究是由国家过敏研究所和美国国立卫生研究院（DEK PO1 AI-080192）的传染病支持。 DEK是由魁北克卫生研究基金的研究学者成就奖（FRQS）的支持。 FP是由马萨诸塞州总医院执行委员会的研究和哈佛全球健康研究所（HGHI）的奖学金资助。

1。通过聚蔗糖分离耗竭和外周血单个核细胞的分离<ol><li>通过将人类CD8 +耗竭鸡尾酒在全血50微升/毫升耗尽CD8 + T细胞。 </li><li>拌匀，静置20分钟，在室温（18-25℃）。 </li><li>培养结束后，混合全血用HBSS（Hank平衡盐溶液不含Ca 2 +或Mg 2 +）和层以上的Histopaque。转速为340 RCF 30分钟（无刹车，缓慢加速）。 </li><li>收集的PBMC并转移到新的50ml的锥形。洗涤2倍用45ml的RPMI 1640在340相对离心力纺纱10分钟添加50 IU青霉素，50微克/毫升链霉素，2mM的L-谷氨酰胺和1％HEPES。 </li></ol> 2。抗体阻断和细胞的抗原特异性刺激<ol><li>重悬的细胞以2×10 6每条件的RPMI 1640含10％人血清白蛋白补充有50 IU青霉素，50微克/毫升链霉素，2mM的L-谷氨酰胺和1％HEPES，占每股条件500微升。 </li><li>分装500μl的FACS管细胞悬液。 </li><li>根据调查的路径，添加PD-L1，IL-10Rα阻断抗体和/或同种型对照在10微克/毫升孵育15分钟，在37℃，5％CO 2。 </li><li>刺激细胞与1微克/毫升/肽终浓度的HIV-1 Gag肽池。还包括一个“无刺激”控制每个阻塞状态。在37℃，5％CO 2的48小时。 </li></ol> 3。收集和上清液分析<ol><li> 48小时后，降速FACS管中在340 RCF 7分钟。 </li><li>仔细收集在Eppendorf管中进行的Luminex的珠子阵列2×225微升上清，而不会干扰颗粒。 </li><li>灭活病毒中收集的上清液用25微升0.5％PBS-吐温，得到0.05％PBS-吐温的最终浓度。 </li><li>店收集上清液是不是立即数iately用在-80℃下冷冻。 </li><li>根据制造商的方案使用Millipore公司的Luminex试剂盒测量所需的细胞因子浓度。 </li></ol> 4。收集和细胞的表型分析通过流式细胞仪<ol><li>收集上清液后，3毫升PBS洗涤细胞。旋转细胞7分钟在340 RCF，倒出多余的洗。 </li><li>染色使用根据制造商的协议的LIVE / DEAD固定式死细胞染色试剂盒生存能力。 </li><li>在340相对离心力，并在4℃下进行7分钟的PBS洗涤细胞重悬在100μlPBS中的1％FBS中。 </li><li>如果加入1.4微升的Fc受体阻断剂和旋涡混合染色的单核细胞或其他抗原呈递细胞，阻断Fc受体。 </li><li>孵育10分钟，在4℃下</li><li>添加表面抗体，漩涡，并在黑暗中孵育20分钟，在4°C。 </li><li>用PBS 1％FBS洗涤细胞，倒出多余的冲洗，并加入200微升4％多聚甲醛。孵化在室温下20分钟在黑暗中。 </li><li>用PBS 1％FBS重悬在250μl的PBS中的1％FBS洗涤细胞，并获得在MULTILASER流式细胞仪（ 例如 BD LSRII或Fortessa）。 </li></ol> 5。通过定量RT-PCR检测细胞分析<ol><li>对于细胞通过流式细胞仪分析未：收集上清液后，裂解细胞在300微升含1％β-巯基乙醇Qiagen公司RLT缓冲。如果不与协议持续，细胞可在-80℃这一点后冷冻。 </li><li>使用RNA提取试剂盒，按照制造商的方案提取RNA。 </li><li>使用cDNA合成试剂盒，按照制造商的方案从RNA合成cDNA。 </li><li>使用SYBR Green技术进行定量PCR。 </li><li>创建一个引物混合物用于通过将引物与不含核酸酶的水，得到10的终浓度使用，包括管家基因（ 例如 IL-13，IL-10，IFN-γ，GAPDH）的各引物微米。置于冰上。 </li><li>加入10.5μL无核酸酶水，12.5微升SYBR绿，每盘1μl引拌匀创建一个主结构为每个引物。置于冰上。 </li><li>移液器24μl反应混合液到将要使用的板的各孔中。 </li><li>加入1μl的cDNA，以每孔根据模板。 </li><li>盖上井和离心机板3分钟在800 RCF。 </li><li>一个实时荧光定量PCR仪， 例如一个Stratagene公司MX3005P仪器上运行的板块。 </li></ol> 6。适应于内细胞因子染色在48小时添加并菌素莫能菌素6小时前细胞内细胞因子染色。布雷菲尔德菌素加入，以10微克/ ml的浓度而莫能根据制造商的说明被添加。为方便起见，布雷菲德菌素可加入染色在5微克/毫升的浓度之前12小时。 <ol><li>表面染色后，用PBS洗涤细胞1％FBS和污垢在胞内细胞因子如IL-12，IFN-γ，并使用一个固定/透化溶液试剂盒和协议的TNF-α。 </li><li>在250μl的PBS中的1％FBS洗涤细胞，重悬，并在MULTILASER运行流式细胞仪（ 例如 BD LSR II或Fortessa）。 </li></ol> 7。为适应单元格排序<ol><li>刺激后，污垢为存活16小时使用根据制造商的方案LIVE / DEAD固定式死细胞染色试剂盒。在整个过程中，细胞置于冰上。 </li><li>用PBS为7分钟，在340相对离心力，并在4℃下洗涤细胞重悬在100μlPBS中的1％FBS中。 </li><li>添加表面抗体，旋涡混合，孵育细胞在冰上避光20分钟。 </li><li>用PBS 1％FBS的340相对离心力，并在4℃下洗涤细胞，并重新悬浮于500μl含有10％胎牛血清冷RPMI 1640中补充有50 IU青霉素，50微克/毫升链霉素，2mM的L-谷氨酰胺和1％ HEPES。 </li><li>使用5毫升POLYSTY过滤单元刘若英圆底管与细胞滤网盖。 </li><li>通过加入含有10％胎牛血清补充了50 IU青霉素，50微克/毫升链霉素，2mM的L-谷氨酰胺和1％HEPES，每管200μl的RPMI 1640中准备收集管中为排序。保留所有试管置于冰上排序过程中。 </li><li>对仪器（ 如 BD FACS咏叹调 ​​二）位于搭载的生物危害性物质的设施感兴趣的细胞亚群现场进行排序。 </li><li>细胞已被排序之后，发生细胞放回培养箱的时间所需的金额，并按照与上清收集/ Luminex公司分析和细胞沉淀收集/ PCR分析。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

收集上清液这是实验设计的一个必要组成部分。当收获上清液用于与Luminex测定中，等分试样制作并保存于-80℃，以防止蛋白质降解是由于冻融循环。冻融可以苛刻的进程蛋白质，并通过最小化冷冻解冻，信号将被最大化的检测。因此，它更容易从已冻融的次数相同等份工作时，得到可重复的和更准确的数据。 之前我们已经进行细胞因子的分泌和mRNA水平的动力学分析刺激与HIV...

在持续的病毒感染，病毒特定的T细胞获得被称为T细胞耗竭过程的功能缺陷。早在慢性病毒感染的小鼠LCMV模型的体内研究表明，耗尽病毒特异性T细胞具有降低溶细胞功能针对病毒感染的细胞，失去它们的增殖能力和减少容量，以产生细胞因子如IL-2，TNF-α α和IFN-γ1,2。免疫途径，如PD-1和IL-10的一个复杂的网络，在慢性感染上调，促进T细胞功能障碍（在3,4审查）。 在封闭抗体对这些抑制途径在LCMV小鼠体内给药模型恢复耗尽病毒特异性T细胞的功能和增强的病毒清除率，表明T细胞耗竭是部分可逆的现象（在5评论）。 在日的调查结果ËLCMV模式被迅速推广到人类慢性病毒感染，如乙肝，丙肝和艾滋病病毒5。在慢性HIV-1感染，PD-1和IL-10途径的上调在感染的受试者和疾病进展的参数相关联，并直接与病毒载量成反比与CD4计数6-8。对PD-1或IL-10途径的 HIV-特异性CD4和CD8 T细胞的体外增殖恢复表明，类似于LCMV小鼠模型，在人体T细胞耗竭是部分可逆的现象的抗体阻断。然而，由于对人的样品实验微妙的性质以及在体外试验中，通过这些措施达到的功能恢复访问彻查的灵敏度局限性是显着存在。增殖试验已经，到目前为止，唯一可靠的体外测定在大多数研究中进行测试，但是有一个显着的证据不足对这些相互的影响关于干预措施：细胞毒性T细胞的1）的杀灭能力，2）T细胞对病毒复制的抗病毒效果，3）的细胞因子分泌的空间，以及4）对其他细胞亚群的CD4 T细胞的辅助作用。 细胞内细胞因子染色（ICS）是一种非常有用的和广泛使用的流式细胞仪为基础的检测，用于检测在小鼠和人类的各种细胞类型的细胞因子的产生。它也被用于研究抑制途径的抗体阻断的效果。在ICS的分析，细胞因子分泌受阻，加入菌素和/或莫能菌素的。细胞因子被困在细胞内，然后与用多色流式细胞术荧光抗体检测。该测定法的持续时间通常限制在抗原刺激后6或12小时，因为这两个布雷菲尔德菌素和莫能菌素，它​​们通常在2小时内抗原另外的添加，是对细胞有毒。 ICS的试验是一个强大的技术，一直是我非常有用n个封闭抗体干预在细胞一段时间抗体暴露后9提取后的小鼠体内给药的效果进行调查。但是，也有对本实验方法的应用来评估抑制性受体人样品中的封锁的影响一些局限性。当执行抑制途径的体外一个6或12小时的刺激（通常与人类样本的情况下）抗体干预，抑制性受体在大多数情况下的封锁如通过标准测定法ICS测量不改变反应的抗原特异性T细胞的频率6，7。每个细胞的细胞因子产生的作为由平均值或中位数荧光强度测量结果已经给出不一致的结果，6，7，10。另一方面，ICS时后在一晚的时间点进行刺激（ 即 6天），改变细胞因子分泌的c数英语学习者可以观察到。不同之处的改变增殖，存活，并积累在一段6在指定期间内效应器功能变化的综合结果。以部分地克服这些局限性的一种方法是孵育时间较长（ 例如 36小时，60小时等 ），与另外的细胞因子分泌阻断前的抗原，而无需等待扩散发生。我们已经成功地使用这种方法，通过HIV-特异性CD4和CD8 T细胞11,12，以确定细胞因子分泌的动力学，但是，这种方法是不能够检测到小的反应，也不会给予的总细胞因子的分泌产生的积分结果因为刺激。因此，ICS是不是足够敏感的方法检测抑制通路的阻断对细胞因子与细胞因子mRNA的定量或分泌细胞因子的测量在supernatan相比，由活化的T细胞产生量的影响吨在同一时间点。此外，大多数由活化的T细胞产生的细胞因子，通过促进或正或负反馈作用的自分泌方式循环通过与抗原呈递细胞相互作用。因此，除了菌素或莫能菌素的ICS测定期间防止细胞因子的分泌，因而T细胞的刺激不是最佳的。最后，检测多种细胞因子与ICS的是由可用的和抗体的灵敏度进行检测的细胞因子流式细胞术，以及通过荧光染料，可以同时使用在任何给定的面板的数目的限制的限制。 我们开发了一种高度灵敏的体外试验方法，评价在由HIV特异性CD4 + T细胞调节细胞因子的分泌，并随后CD4辅助抗原呈递细胞（APC）和自然杀伤细胞（NK细胞）免疫途径的影响。此方法也可用于评估IMPA克拉消耗者从外周血中CD4 + T细胞，或通过与最优肽只能由CD8 + T细胞识别上的刺激CD8 + T细胞功能的抑制分子的封锁。相反，细胞内细胞因子染色测定法，我们决定不与细胞因子分泌的HIV特异性CD4 + T细胞干预，以便能够：1）执行所产生的细胞因子水平的更精确的量化; 2）调查更广的面板细胞因子或效应分子，以及3）评价由HIV特异性CD4 + T细胞上的其他细胞亚群产生的细胞因子产生的影响。我们刺激CD8耗尽外周血单个核细胞，在封闭抗体对感兴趣的免疫调节途径的存在HIV-1 Gag肽池。孵育后，通常48小时所需要的时间，我们收集上清液测定细胞因子的分泌与磁珠阵列和收集细胞沉淀无论是对不同的细胞亚群的表型分析或转录分析。的注意，在t其48小时的时间点，不检测增殖T细胞12的显著人口。这是一种灵活的方法，该设计的几个适配可以应用于解决不同的假设。例如，单独的细胞亚群（如HIV特异性CD4 + T细胞或PD-1的高CD4 + T细胞， 等等 ）可以在进一步孵育36小时，然后收集上清液和细胞沉淀物的12小时温育后进行排序调查更具体的封锁措施对细胞因子分泌的外周血单个核细胞亚群定义的影响。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Hanks Balanced Salt Solution without Ca2+ or Mg2+</td>
			<td>Sigma</td>
			<td>H9394</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RPMI 1640</td>
			<td>Sigma</td>
			<td>R0883</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Histopaque-1077</td>
			<td>Sigma</td>
			<td>10771</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Human Serum AB</td>
			<td>Gemini BioProducts</td>
			<td>100-512</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Penicillin/Streptomycin</td>
			<td>Mediatech</td>
			<td>30 001 CI</td>
			<td></td>
		 </tr>
		 <tr>
			<td>L-glutamine</td>
			<td>Mediatech</td>
			<td>25 005 CI</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HEPES buffer</td>
			<td>Mediatech</td>
			<td>25060CI</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FcR blocking reagent, human</td>
			<td>Miltenyi</td>
			<td>130-059-901</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RosetteSep Human CD8+ Depletion Cocktail </td>
			<td>Stem Cell </td>
			<td>15663</td>
			<td></td>
		 </tr>
		 <tr>
			<td>LIVE/DEAD Fixable Dead Cell Stain Kit, for UV excitation</td>
			<td>Invitrogen</td>
			<td>L23105</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Rneasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Improm-II Reverse Transcription System</td>
			<td>Promega</td>
			<td>A3800</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Brilliant II SYBR qPCR Low Rox Master Mix</td>
			<td>Agilent</td>
			<td>600830</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td>
			<td>BD</td>
			<td>554714</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tween-20</td>
			<td>Fisher</td>
			<td>BP337100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>IFN-&gamma; primer</td>
			<td>Invitrogen</td>
			<td></td>
			<td>FOR: CGAGATGACTTCGAAAAGCTGA REV: TCTTCGACCTCGAAACAGCA</td>
		 </tr>
		 <tr>
			<td>GAPDH primer</td>
			<td>Invitrogen</td>
			<td></td>
			<td>FOR: TCATCATCTCTGCCCCCTCT REV: AGTGATGGCATGGACTGTGG</td>
		 </tr>
		</tbody>
 </table>

in vitro assay to evaluate the impact of immunoregulatory pathways on hiv-specific cd4 t cell effector function

T细胞耗竭是在慢性病毒感染的主要因素未能病原清除。免疫调节途径，如PD-1和IL-10，被上调时这种持续抗原暴露，并有助于损失扩散，降低溶细胞功能，并削弱细胞因子的产生由CD4和CD8 T细胞。在LCMV感染的小鼠模型中，封闭抗体对抗这两种途径增强T细胞应答的管理。然而，目前还没有在体外测定来测量这类封锁对细胞因子分泌的人类样本的影响在细胞。我们的协议与实验方法使我们能够准确，有效地量化因子的产生由艾滋病毒感染者HIV特异性CD4 + T细胞的恢复。 在这里，我们描述了体外实验的设计，使细胞因子分泌的测量由HIV特异性CD4 + T细胞及其对其他CEL的影响升子集。 CD8 + T细胞从全血耗尽，剩下的外周血单核细胞经聚蔗糖分离方法分离。 CD8贫化的PBMC再孵育阻断抗体对PD-L1和/或IL-10Rα，并刺激与HIV-1 Gag肽池后，将细胞培养在37℃，5％CO 2。 48小时后，弃上清，用有孔玻璃珠阵列和细胞沉淀收集的细胞因子分析，使用流式细胞术或转录分析利用定量RT-PCR方法收集或表型分析。如需更详细的分析，不同的细胞群从外周血单个核细胞选择性的子集枯竭或使用流式细胞仪正在评估在相同的实验前分选获得的。这些方法提供了一个高度敏感和特异的方法由抗原特异性T辅助细胞以确定细胞因子产生的调节，并确定免疫细胞的不同种群之间的功能性相互作用。

当使用这种在体外系统中进行的细胞因子的测量，就必须能够区分相比，无刺激对照的抗原特异性应答。在一般情况下，我们认为是抗原刺激的任何值给比无刺激对照和其中分别测定的线性范围内的至少3倍的细胞因子水平的阳性反应。我们使用高灵敏度微球阵列分析，可以检测出细胞因子低至0.16皮克/毫升的浓度，使我们能够观察到弱的抗原特异性CD4 T细胞应答。样品，以确保所获得的精?...

Watch this Scientific Journal Video about In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function at JoVE.com

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

我们开发了一种体外测定，调查免疫调节途径中的细胞因子分泌的HIV特异性CD4 + T细胞的调节中的作用。

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

in-vitro-assay-to-evaluate-impact-immunoregulatory-pathways-on-hiv

Research

JoVE Journal

Immunology and Infection

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

14.0K 次观看.  The Ragon Institute of MGH, MIT and Harvard. 我们开发了一种体外测定，调查免疫调节途径中的细胞因子分泌的HIV特异性CD4 + T细胞的调节中的作用。 

视频: In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

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

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 including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

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

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

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 ...

科研

免疫与感染

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

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

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

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

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

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

基于图像的流式细胞仪技术来评估变化粒细胞功能<em&gt;在体外</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

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&amp;#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 ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

长期培养的干扰素γ酶联免疫检测方法的评估效应和记忆T细胞应答的牛应用

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

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 ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

一个<em&gt;体外</em&gt;血 - 脑屏障使用阻抗谱型号：对T细胞，内皮细胞的相互作用为中心

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

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.

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

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.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

在体外试验，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响