这项工作得到了 R01AI118694 NIH 的资助, 包括566950分奖给尤里 Sykulev。我们感谢悉尼 Bioimaging 癌症中心共享资源, 他们的出色支持。

这项研究是按照《赫尔辛基宣言》进行的。从所有参与者获得书面知情同意, 在宾夕法尼亚大学的机构审查委员会 (IRB#809316, IRB 815056 号) 的批准下获得了血液和淋巴结样本。所有的人类臣民都是成年人。在托马斯杰斐逊大学产科 & 妇科的分娩和分娩中, 提供脐带血样本。所有样品均被取消鉴定。
1. 为图像分析分离 CD4+ T 细胞
<ol><li>
从采集的样品中解冻1毫升整除, 其中含有 107冷冻外周血单个核细胞 (PBMCs) 或淋巴结单个核细胞 (LNMCs)。在无菌罩中, 加入解冻的细胞到9毫升的 RPMI 补充青霉素/链霉素和谷氨酰胺。
	<ol>
<li>Centrifugate 细胞为10分钟在 300 x g在4°c, 吸入上清, 并且并用重悬细胞在5毫升补充的 RPMI 包含10% 血清 (完全培养基)。在37摄氏度的 CO2孵化器中, 在一夜之间孵化细胞。</li>
	</ol>
</li>
	<li>第二天, 根据制造商的指示, 使用一个商业上可用的工具包对 CD4+ T 细胞进行负磁分类纯化。</li>
	<li>要测量新鲜纯化的 CD4+ T 细胞的数量, 混合5µL 的细胞悬浮与相同体积的台盼蓝溶液。加载一个 hemocytometer 与细胞-台盼蓝混合物和计数的活细胞内的5个部分的 hemocytometer。</li>
	<li>取细胞数的平均值并确定原细胞悬浮液中的细胞数: cells/1 毫升的数量 = 平均计数 x 2 x 104。如果孤立单元格的总数太小, 请按不计算的那样使用单元格。</li>
	<li>离心细胞在 300 x g 10 分钟, 并并用重悬他们在一个化验缓冲区 (20 毫米 HEPES, pH 值 7.4, 137 毫米氯化钠, 2 毫米 Na2HPO4, 5 毫米 d-葡萄糖, 5 毫米氯化钾, 1 毫米氯化镁2, 2 毫米 CaCl2, 1% 人血清白蛋白) 105/c13> cells/50 µL 或更少, 并保持细胞在4°c (为 h), 直到准备使用的实验。</li>
	<li>根据有关的机构指引处理所有生物废物。</li>
	<li>如果想要作为控制细胞人口, 准备激活 CD8 T 细胞从脐血 PBMC, 安置 107个细胞在5毫升完全培养基在 T25 养殖瓶覆盖以 anti-CD3 和 anti-CD28 抗体混合物在10个µg/毫升和1个µg/毫升分别。</li>
	<li>第二天, 从烧瓶中取出活化脐带血细胞, 用新鲜完整培养基冲洗 1x, 并在重组 IL-2 (100 U/毫升) 的存在下, 将细胞扩大2周。</li>
	<li>根据制造商的指示, 使用商业上可用的工具包, 通过负磁分类纯化脐带血 CD8+ T 细胞。根据1.3–1.6 和 PB CD8+ T 细胞的步骤中所述, 计数单元格并将介质交换到检测缓冲区。</li>
</ol>2. 平面脂质双层的制备组件
<ol><li>准备3种脂质体如另有说明5: (a) 0.4 毫米 DOPC (12-dioleoyl-锡-glycero-3-phosphocholine) 脂质体, (b) 0.4 毫米 DOPC 脂质体, 含 33 (摩尔) 狗-NTA (12-dioleoyl-锡-磷脂酰-3-[(N-(5-氨基-1-carboxypentyl) iminodiacetic 酸) 酰] (铵盐)) 脂质, (c) 0.4 毫米 DOPC 脂质体含有 4 (摩尔) Biotinyl 帽 PE (12-dioleoyl-锡-磷脂酰-3-乙醇胺 N (Cap Biotinyl) (钠盐))。</li>
	<li>
准备5% 酪蛋白溶液, 如前所述5。
	<ol>
<li>将5克酪蛋白粉溶于100毫升的超纯水中, 加入350µL 10 米氢氧化钠。根据可用的刻度, 在室温下2小时, 然后在4摄氏度一夜之间, 以慢速的速度在普通磁力搅拌器上搅拌一切。将 pH 值调整为 7.3, 并在4摄氏度的 10万 x g ultracentrifuge 2 小时的溶液。用0.22 µm 无菌过滤器过滤上清液, 并将溶液储存在整除数-80 摄氏度。 注意: 氢氧化钠溶液可以引起化学烧伤, 并可能导致永久性失明后, 与眼睛接触。使用橡胶手套, 安全衣物和眼睛保护时, 处理此化学品或其解决方案。</li>
	</ol>
</li>
	<li>
标签 anti-CD3 抗体与生物素, 以产生单 bionylated 抗体分子与先前描述的方法8。
	<ol>
<li>在0.1 毫克/毫升的二甲基亚砜 (亚砜) 中制备 Biotin-PEO4-NHS 溶液。添加3.7 µL 的 Biotin-PEO4-NHS 溶液, 1 毫克的抗体0.5 毫升磷酸盐缓冲盐水 (PBS) 含有100毫米碳酸氢钠。</li>
		<li>在室温下孵育2小时的混合物。在亚砜中, 用10毫克/毫升的方法制备出 488 NHS 酯的溶液。添加 Alexa 488 NHS 酯溶液的抗体标记的 Biotin-PEO4-NHS 在10倍摩尔过剩。</li>
		<li>在室温下将混合料孵育1小时, 在常规磁力搅拌器上进行慢速搅拌。使用大小排除色谱法分离未绑定染料。</li>
		<li>通过测量 280 nm (280) 抗体溶液的光学密度来确定抗体浓度。测量 577 nm (577) 标记抗体的光学密度。</li>
		<li>使用以下公式确定染料与抗体的比值: <img alt="Equation" src="/files/ftp_upload/58143/58143eq1.jpg"> 注意: 在制造商的协议中查找其他详细信息。</li>
	</ol>
</li>
	<li>
在果蝇表达系统中表达重组可溶性 ICAM-1 蛋白, 如前所述3、4、9、10。
	<ol>
<li>克隆, 用 cDNA 编码, 胞外区 ICAM-1 成果蝇表达载体 pMT/V5-His 与诱导金属硫蛋白促进剂, 以产生重组蛋白追加了他的6标签在 C 端端。</li>
		<li>染 S2 细胞与所产生的 ICAM-1 含有质粒和 G418 表达载体。选择稳定的 transfectants 使用施耐德的果蝇媒体补充10% 胎小牛血清 (FCS) 和0.5 毫克/毫升的 G418 3 周。在无血清昆虫培养基中展开细胞, 并诱导0.5 毫米丘索4的蛋白表达 3 d。</li>
		<li>通过一个切向流选矿厂集中的文化上清10x 和透析对 PBS, 如前所述11。</li>
		<li>将浓缩培养上清液应用于含琼脂糖的一列, 其中含有共价键固定化 anti-ICAM-1 单克隆抗体, 洗脱 ICAM-1 与50毫米甘氨酸缓冲, pH 3.0。立即中和洗脱 ICAM-1 蛋白与2米的三个缓冲, pH 值8.0。</li>
		<li>透析洗脱材料对抗 PBS, pH 值为 8.0, 并将透析材料添加到含有镍 NTA 琼脂糖的柱上。洗脱可溶性 ICAM-1 与200毫米咪唑, pH 8.0。透析洗脱材料对抗 PBS 缓冲器, pH 值为8.0。</li>
		<li>根据制造商的指示, 用 Cy5 NHS 酯标记纯化的 ICAM-1。 注: 最佳最终染料蛋白比为1:1。</li>
	</ol>
</li>
	<li>用木瓜蛋白酶消化 anti-CD107a 抗体, 用离子交换色谱法纯化晶圆碎片, 如前3所述。根据制造商的指示, 用 Alexa 的 568 NHS 酯标记该工厂的碎片。</li>
</ol>3. 玻璃支持的平面脂质双层的形成
<ol><li>通过混合140毫升浓硫酸和60毫升30% 过氧化氢, 制备出一种新的酸性食人鱼溶液。通过浸泡在酸性食人鱼溶液中30分钟, 将玻璃盖玻片为流片冲洗. 用聚丙烯剪刀式镊子握住玻璃盖玻片。 注意: 食人鱼溶液是一种极强的氧化剂。在处理解决方案时, 切记在任何时候佩戴安全眼镜、护目镜或全脸防护罩和厚橡皮手套。只与食人鱼解决方案在通风罩下工作。避免在使用过程中的任何时间加热、运输或摇动, 因为它可能会爆炸。将食人鱼废料收集到带有含铅孔的玻璃瓶中。与机构安全委员会联系, 了解适当的废物利用情况。</li>
	<li>
用超纯水冲洗洗净的盖玻片 7x, 将它们依次转换成含有淡水的烧杯。把湿盖玻片放在一边, 让剩余的水滚出干净的玻璃, 留下干燥的玻璃。
	<ol>
<li>或者, 使用连接到真空泵的吸管尖端, 小心地从盖玻片中取出剩余的水滴。</li>
	</ol>
</li>
	<li>在无菌罩中, 进行各种脂质的稀释, 以产生脂质体混合, 使双层。首先, 结合37µL 的 DOPC 脂质体和3µL 的 Biotinyl 帽 PE 脂质体。第二, 混合14µL 的 DOPC 脂质体和15µL 的狗 NTA 脂质体。第三, 添加1µL 的第一个混合到29µL 的第二个组合, 以制造最终的脂质体混合物。</li>
	<li>
在消毒罩, 设置一个工作区与干燥盖玻片附近。整除2µL 的最终脂质体混合物 (见步骤 3.3) 精确地在一个自粘性滑动通道的中心。立即和非常精确地对齐干净和干燥的盖玻片与幻灯片, 并轻轻地降低盖玻片在粘性一侧的幻灯片。
	<ol>
<li>如果准备一张以上的幻灯片, 在一次幻灯片上工作, 因为脂质体混合物迅速蒸发。翻转幻灯片, 使用聚丙烯剪刀式钳外圈, 对盖玻片与滑块的外围接触施加温和压力, 确保滑块与滑轨紧密相连以防止泄漏。 注意: 请勿按幻灯片的通道, 以免破坏或开裂盖玻片。</li>
		<li>再次翻转幻灯片, 并标记形成的双层的位置, 这看起来像盖玻片和通道幻灯片之间的下降, 通过绘制4点, 并围绕在组件的外部幻灯片一侧的双层上的永久性标记。</li>
	</ol>
</li>
	<li>在第一次注入液体进入通道之前, 将通道的一个端口指定为入口端口, 另一个作为出口端口, 并在整个实验中保持这个指定。</li>
	<li>为避免形成气泡, 请将吸管尖端的末端直接插入通道的入口端口。用50µL (至少室温) 化验缓冲器慢慢填满幻灯片的通道 (请参阅步骤 1.5, 以了解缓冲成分)。</li>
	<li>准备0.5 米 (二) 氯化镍溶液。将酪蛋白溶液的2毫升整除在37摄氏度的水浴中解冻30分钟, 并在最终浓度为200µM 的情况下用氯化镍溶液补充。</li>
	<li>先将酪蛋白溶液的100µL 注入通道的入口端口, 然后立即将100µL 从幻灯片的出口端口上移除吹打, 以清洗双层。通过将酪蛋白溶液的100µL 注入每个通道的入口端, 并在室温下孵化出45分钟的滑块, 以相同的溶液阻断双层。</li>
	<li>解冻整除数的 Cy5-ICAM-1-His6和链亲和素蛋白。将检测缓冲器中的蛋白质组合成2µg/毫升的最终浓度。离心解决方案为30分钟在 2万 x g 和4°c, 以消除任何骨料。</li>
	<li>通过吹打从幻灯片通道的退出端口删除其余的阻止解决方案。将包含 ICAM-1 和链亲和素的解决方案注入100µL 进入入口端口。</li>
	<li>在室温下孵化45分钟的滑梯。从出口口取出任何多余的蛋白质溶液。先将检测缓冲区的100µL 注入通道的入口端口, 然后立即将100µL 移出出口端口, 清洗双层2x。</li>
	<li>将 Alexa-Fluor-488-labelled anti-CD3 抗体稀释为最终浓度为2µg/毫升的检测缓冲器。将100µL 的抗体溶液注入幻灯片的入口口, 室温下孵化45分钟。从出口口取出任何多余的蛋白质溶液。清洗双层2x 与100µL 的化验缓冲区, 如在步骤3.11。</li>
</ol>4. T 细胞与平面双层的相互作用的成像
<ol><li>
预热的阶段和目标的共焦或全内反射荧光 (TIRF) 显微镜, 直到温度平衡在37摄氏度。在加热的舞台上用双层 (s) 设置幻灯片。根据油墨的标记将舞台移动到合适的位置, 并将焦点放在使用 ICAM-1 分子的荧光的双层上。
	<ol>
<li>使用61X 目标为共焦显微镜, 或100X 目标 TIRF 显微镜, 与适当的过滤器设置。</li>
	</ol>
</li>
	<li>
对于 TIRF 显微镜下的颗粒释放成像, 在将细胞注入进入端口之前, 在最终浓度为4µg/毫升的细胞悬浮液中加入 Alexa-Fluor-568-labeled anti-CD107a 抗体。
	<ol>
<li>并用重悬从 LN 或铅或脐血中分离出的 CD4+ T 细胞, 并将50µL 的细胞悬浮液注入包含双层的滑动通道的入口口。</li>
	</ol>
</li>
	<li>选择所需的字段数, 并在注入后每2分钟为30分钟记录每个字段的图像。</li>
	<li>利用共焦显微镜的亮场、反射光和荧光通道 (Alexa 488 和 Cy5) 获取图像。使用 TIRF 模式 Alexa-Fluor-488 和 Alexa-Fluor-568 荧光和 widefield Cy5 荧光, 以及光明场成像, 在 TIRF 显微镜。</li>
</ol>5. 图像分析
<ol><li>使用适当的软件分析获取的图像。观察透射光图像中的细胞形态学, 从分析中排除聚集和明显受损或凋亡细胞。仅在分析中包括那些卓有成效地与双层表面相互作用的细胞 (即在界面上积聚 Alexa-Fluor-488 荧光 (anti-CD3 抗体) 的细胞)。</li>
	<li>在细胞双层相互作用启动后, 确定细胞黏附面积的大小为20分钟。 注: 粘附区域是在干涉反射显微镜 (IRM) 图像的细胞双层界面上开发的暗区。</li>
	<li>观察 Cy5-ICAM-1 荧光的任何积累和环状结的形成, 在细胞双层界面上分离 Cy-ICAM-1 分子。如果积累的 ICAM-1 分子在至少两个连续的图像上形成了粘接环结, 则指定这样的细胞作为细胞开发外周超分子活化簇 (pSMAC)12。</li>
	<li>通过在靠近细胞的接触区域外的背景荧光测量 T 细胞双层界面上的 Alexa-Fluor-568 荧光强度来评估颗粒释放。指定具有 Alexa-Fluor-568 信号与背景的比率至少为 degranulating 细胞1.3 的细胞。</li>
</ol>

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S., Keen, J. H., Sykulev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+Influence+the+Size+and+Dynamics+of+Signaling+Microclusters+in+a+Pyk2-dependent+Manner.">Integrins Influence the Size and Dynamics of Signaling Microclusters in a Pyk2-dependent Manner.</a> The Journal of Biological Chemistry. 290, 11833-11842 (2015).</li><li>Anikeeva, N., Lebedeva, T., Sumaroka, M., Kalams, S. A., Sykulev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+HIV-specific+T-cell+receptor:+expression,+purification+and+analysis+of+the+specificity.">Soluble HIV-specific T-cell receptor: expression, purification and analysis of the specificity.</a> Journal of Immunological Methods. 277, 75-86 (2003).</li><li>Monks, C., Freiberg, B., Kupfer, H., Sciaky, N., Kupfer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+segregation+of+supramolecular+activation+clusters+in+T+cells.">Three-dimensional segregation of supramolecular activation clusters in T cells.</a> Nature. 395, 82-86 (1998).</li><li>Riddell, S. R., Greenberg, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+anti-CD3+and+anti-CD28+monoclonal+antibodies+to+clone+and+expand+human+antigen-specific+T+cells.">The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells.</a> Journal of Immunological Methods. 128, 189-201 (1990).</li><li>Lin, S. J., Yu, J. C., Cheng, P. J., Hsiao, S. S., Kuo, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+interleukin-15+on+anti-CD3/anti-CD28+induced+apoptosis+of+umbilical+cord+blood+CD4++T+cells.">Effect of interleukin-15 on anti-CD3/anti-CD28 induced apoptosis of umbilical cord blood CD4+ T cells.</a> European Journal of Haematology. 71, 425-432 (2003).</li><li>Anikeeva, N., Sykulev, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+controlling+granule-mediated+cytolytic+activity+of+cytotoxic+T+lymphocytes.">Mechanisms controlling granule-mediated cytolytic activity of cytotoxic T lymphocytes.</a> Immunologic Research. 51, 183-194 (2011).</li><li>Huppa, J. B., 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=TCR-peptide-MHC+interactions+in+situ+show+accelerated+kinetics+and+increased+affinity.">TCR-peptide-MHC interactions in situ show accelerated kinetics and increased affinity.</a> Nature. 463, 963-967 (2010).</li></ol>

作者没有什么可透露的。

此处描述的新技术利用了在常规流单元5中构建平面双层所需的类似试剂, 并可成功地应用于 cell–bilayer、4 的初级人类 T 型接口的成像. ,15。该技术提供了显着减少荧光分子的使用, 并要求10–20x 较少的 T 细胞相比, 流细胞系统5, 创造机会, 以分析主要人类 T 细胞从血液和其他组织。
<p class=...

对 t 细胞免疫突触的结构特征及其与 t 细胞功能活动的联系的科学知识, 主要来源于从 PB 中提取的细胞系和克隆的研究。这些发现与从血液或人类淋巴组织获得的初级 t 细胞的程度仍不清楚, 因为在淋巴和其他组织中的 t 细胞的突触界面迄今尚未分析。重要的是, 新出现的数据表明, 与 PB6、7相比, 组织居民和淋巴器官衍生的 T 细胞在表型和功能活性上可能有显著差异。这进一步巩固了需要更好地了解 t 细胞突触界面的特点在原发性人类 T 细胞。
为此, 我们开发了一种新的小尺度方法, 利用多通道流幻灯片中的脂质双层, 使我们能够进行 t 细胞/双层界面的成像, 其分离出的人 PB 和 LN 不到 105个初级 t 细胞。这项新技术允许研究人类 T 细胞突触界面的生物物理特性, 以便更好地模型和了解体内细胞间的相互作用。

<table><tbody><tr><td>CD4 T cell isolation kit, human</td><td>Miltenyl Biotec</td><td>130-096-533</td><td></td></tr><tr><td>CD8 T cells Isolation Kit, human</td><td>Miltenyl Biotec</td><td>130-096-495</td><td></td></tr><tr><td>DOPC</td><td>Avanti Polar Lipids</td><td>850375C</td><td></td></tr><tr><td>DOGS NTA</td><td>Avanti Polar Lipids</td><td>790528C</td><td></td></tr><tr><td>Biotinyl Cap PE</td><td>Avanti Polar Lipids</td><td>870273C</td><td></td></tr><tr><td>Human Serum Albumin</td><td>Octapharma USA</td><td>NDC 68982-643-01</td><td></td></tr><tr><td>sticky-Slide VI 0.4</td><td>ibidi</td><td>80608</td><td></td></tr><tr><td>Coverslips for sticky-Slides</td><td>ibidi</td><td>10812</td><td></td></tr><tr><td>Bioptech FCS2 Chamber</td><td>Bioptech</td><td>060319-2-03</td><td></td></tr><tr><td>anti-CD3 antibody</td><td>Thermo Fisher Scientific</td><td>16-0037-81</td><td>OKT3 clone, hybridoma cells are available from ATCC</td></tr><tr><td>anti- CD28 antibody</td><td>Genetex</td><td>GTX14664</td><td>9.3 clone</td></tr><tr><td>Casein</td><td>Sigma</td><td>C5890</td><td></td></tr><tr><td>Biotin-PEO4-NHS</td><td>Thermo Fisher Scientific</td><td>21329</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650-5</td><td></td></tr><tr><td>Alexa Fluor 488 protein labeling kit with column for labeled protein purification</td><td>Thermo Fisher Scientific</td><td>A10235</td><td></td></tr><tr><td>Alexa Fluor 568 protein labeling kit with column for labeled protein purification</td><td>Thermo Fisher Scientific</td><td>A10238</td><td></td></tr><tr><td>Amersham Cy5 NHS Ester</td><td>GE Life Science</td><td>PA15101</td><td></td></tr><tr><td>pMT/V5-His A, B, C Drosophila Expression Vectors</td><td>Thermo Fisher Scientific</td><td>V412020</td><td></td></tr><tr><td>pcopneo, G418 Drosophila expression vector for positive selection</td><td>ATCC</td><td>37409</td><td></td></tr><tr><td>Serum free Drosophial media Insect-XPRESS</td><td>Lonza</td><td>12-730Q</td><td></td></tr><tr><td>Hybridoma YN1/1.7.4</td><td>ATCC</td><td>CRL1878</td><td>The hybridoma secrets antibody against ICAM-1.</td></tr><tr><td>Cyanogen bromide-activated-Sepharose 4B</td><td>Sigma-Aldrich</td><td>C9142</td><td>Utilized for preparation of Sepharose with covelently bound anti-ICAM antibody.</td></tr><tr><td>MasterFlex tangential flow concentrator</td><td>Cole-Parmer</td><td>77601-60 7592-40</td><td>Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant</td></tr><tr><td>Centramate Lab Tangential Flow Systems</td><td>Pall Laboratory</td><td>FS002K10 OS010T12 FS005K10</td><td>Used for ICAM-1 containing supernatant concentration and dialysis of ICAM-1 containing supernant</td></tr><tr><td>Ni-NTA Agarose</td><td>QIAGEN</td><td>30210</td><td></td></tr><tr><td>Dialysis tubing</td><td>Spectra/Por</td><td>131384</td><td></td></tr><tr><td>Papain from papaya latex</td><td>Sigma</td><td>P3125</td><td></td></tr><tr><td>mouse anti-human antibody against CD107a</td><td>BD Bioscences</td><td>555798</td><td>Clone H4A3</td></tr><tr><td>Ansell Natural Blue Gloves</td><td>Fisher Scientific</td><td>19-014-539</td><td></td></tr><tr><td>Nalgene Polypropylene Scissor-Type Forceps</td><td>Thermo Fisher Scientific</td><td>6320-0010</td><td></td></tr><tr><td>Streptavidin</td><td>ProZyme</td><td>SA10</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td></td><td>Nikon TiE inverted microscope equipped with PFS for long-term image stability control, 60X oil objectives, 4 lasers with excitation lines at 405, 458, 488, 514, 561, and 640 nm, 2 GaAsP detectors and 2 high sensitivity PMTs, DIC transmitted light, Programmable X,Y,Z stage for multiple positions and stitching of large areas, time lapse functions, Tokai-Hit temperature and CO&lt;sub&gt;2&lt;/sub&gt;-controlled chamber for live imaging, and anti-vibration isolation table</td></tr><tr><td>TIRF microscope</td><td>Andor</td><td></td><td>Andor Revolution XD system equipped with Nikon TIRF-E illuminator, Lasers with 405,488,561 and 640 lines, DIC transmitted light, Yokogawa CSU-X1 spinning disk head for confocal imaging, 100/1.49 NA objective, Andor iXon X3 EM-CCD camera, objective heater, and a piezoelectric motorized stage with Perfect Focus System (PFS)</td></tr><tr><td>MetaMorph Premier Image Analysis Software</td><td>Molecular devices</td><td></td><td></td></tr></tbody></table>

assessment of the synaptic interface of primary human t cells from peripheral blood and lymphoid tissue

目前对 t 细胞突触界面的动力学和结构特征的理解, 主要是通过使用玻璃支持的平面双层和体外衍生的 t 细胞克隆或1、2 线来确定的. ,3,4。这些发现如何适用于从血液或淋巴组织中分离出来的主要人类 T 细胞是不知道的, 部分原因是在获得足够数量的细胞进行分析5方面存在重大困难。在这里, 我们通过开发一种利用多通道流幻灯片来构建含有活化和粘附分子的平面脂双层来解决这一问题。低高度的流动幻灯片促进快速细胞沉淀, 以同步细胞: 双层附着, 从而允许研究人员的动态的突触界面形成和动力学的颗粒释放。应用这种方法分析了 104至 105只从淋巴结 (LN) 和外周血 (PB) 中分离出的低温保存 T 细胞的突触界面。结果表明, 新的平面脂质双层技术能够研究健康和疾病背景下从血液和组织中提取的人 T 细胞的生物物理特性。

首先, 我们比较了在传统的大型流动细胞系统中建立的由活化脐带-血源 CD8+ T 细胞形成的双层的突触界面结构 (见材料表详细资料)1 、2、3、4或多通道流幻灯片。双层含有荧光标记的 anti-CD3 和 ICAM-1, 分别为50分子/µm2和300分子/µm2。从人脐血...

Watch this Scientific Journal Video about Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue at JoVE.com

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

该协议描述了一种研究原发性多克隆人 T 细胞通过平面脂质双层形成突触界面的能力的技术。利用该技术显示了从淋巴结和外周血中提取的人原发性 T 细胞的差异突触形成能力。

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

assessment-synaptic-interface-primary-human-t-cells-from-peripheral

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.4K 次观看.  Thomas Jefferson University. 该协议描述了一种研究原发性多克隆人 T 细胞通过平面脂质双层形成突触界面的能力的技术。利用该技术显示了从淋巴结和外周血中提取的人原发性 T 细胞的差异突触形成能力。

视频: Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

科研

免疫与感染

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

荧光目标阵列的T细胞反应评价中的应用<em&gt;在体内</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

测量TCR-的pMHC结合<em&gt;原位</em&gt;使用基于FRET的显微镜分析

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

生物工程

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

使用成像流式细胞仪T细胞同种异体反应性测定

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

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

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

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

影像性流式细胞仪对人体免疫突触的定性和定量分析

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

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

成像人类免疫学突触

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

制备珠状支持脂质双分子层研究T细胞免疫突触的颗粒输出

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the lymph node microenvironment in B-cell non-Hodgkin&#39;s lymphoma is analyzed. Subsets of circulating T-follicular helper cells (cTfh), the circulating memory compartment of Tfh cells in the blood, are also perturbed in disease and therefore represent potential novel predictive biomarkers. Peripheral blood-based testing is advantageous because it is relatively non-invasive and allows simple serial monitoring.This article describes a method for isolating CD4+ T-cells from human blood, and further analysis by flow-cytometry to enumerate cTfh cells and the proportions of their various subsets (cTfhPD-1-/+/hi, cTfh1,2,17 and cTfh1/17). The level of these subsets was then compared between normal subjects and patients with lymphoma. We found that the method was robust enough to obtain reliable results from routinely collected patient material. The technique we describe for the analysis can be easily adapted to cell sorting and downstream applications such as RT-PCR.

Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper cTfh Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry

Here, a protocol for the isolation and characterization of CD4+ T-cell subsets from human peripheral blood is described. Purified CD4+ T-cells are analyzed by flow cytometry to determine proportions of T-follicular helper cell subsets.

cd4+ t 细胞的分离及6色流式细胞仪分析外周血中循环 t-滤泡 (ctfh) 细胞亚群

Aberrant T-follicular helper (Tfh) cell activity is detectable in autoimmune conditions and their presence is associated with clinical outcomes when the ...

生物学

Assessing Immunological Synapse Topology through Live-Cell Imaging

Source: Martinelli, R. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/53288">An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates live-cell imaging of immunological synapse topology, investigating the interactions between T lymphocytes and epithelial cells carrying fluorescent antigens. Fluorescence microscopy reveals red cytoplasm displacement and yellow membrane rings in endothelial cells, indicating the formation of immunological synapses.

通过活细胞成像评估免疫突触拓扑

1. Preparing Human CD4+&#160;Th1 Effector/Memory T Cells

	Apply a tourniquet to the arm of the donor, wipe the vein with alcohol, and insert a needle. ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

Analysis of Immune Synapses between Human T Cells and SEB-Loaded Raji Cells using Imaging Flow Cytometry

Source:&#160; Wabnitz, G. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55345">Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry.</a> J. Vis. Exp. (2019)
This video analyzes the immune synapse formation between T cells in unpurified pan-leukocyte preparations and Staphylococcus aureus enterotoxin B, SEB-loaded Raji cells. CD3 and F-actin enrichment is observed in the immune synapse using imaging flow cytometry.

使用成像流式细胞术分析人 T 细胞和载有 SEB 的 Raji 细胞之间的免疫突触

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Pan-leukocytes

	...

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

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此视频中的章节

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

荧光目标阵列的T细胞反应评价中的应用

测量TCR-的pMHC结合

使用成像流式细胞仪T细胞同种异体反应性测定

影像性流式细胞仪对人体免疫突触的定性和定量分析

成像人类免疫学突触

制备珠状支持脂质双分子层研究T细胞免疫突触的颗粒输出

cd4⁺ t 细胞的分离及6色流式细胞仪分析外周血中循环 t-滤泡 (ctfh) 细胞亚群

通过活细胞成像评估免疫突触拓扑

使用成像流式细胞术分析人 T 细胞和载有 SEB 的 Raji 细胞之间的免疫突触

人外周血和淋巴组织中原发性 T 细胞突触界面的评价

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一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

荧光目标阵列的T细胞反应评价中的应用

测量TCR-的pMHC结合

使用成像流式细胞仪T细胞同种异体反应性测定

影像性流式细胞仪对人体免疫突触的定性和定量分析

成像人类免疫学突触

制备珠状支持脂质双分子层研究T细胞免疫突触的颗粒输出

cd4+ t 细胞的分离及6色流式细胞仪分析外周血中循环 t-滤泡 (ctfh) 细胞亚群

通过活细胞成像评估免疫突触拓扑

使用成像流式细胞术分析人 T 细胞和载有 SEB 的 Raji 细胞之间的免疫突触

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

cd4⁺ t 细胞的分离及6色流式细胞仪分析外周血中循环 t-滤泡 (ctfh) 细胞亚群