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>

<ol>
	<li>Grakoui, A., 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=The+immunological+synapse:+a+molecular+machine+controlling+T+cell+activation.">The immunological synapse: a molecular machine controlling T cell activation.</a> Science. 285, 221-227 (1999).</li><li>Somersalo, K., 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=Cytotoxic+T+lymphocytes+form+an+antigen-independent+ring+junction.">Cytotoxic T lymphocytes form an antigen-independent ring junction.</a> Journal of Clinical Investigation. 113, 49-57 (2004).</li><li>Beal, A. M., 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=Protein+kinase+C+theta+regulates+stability+of+the+peripheral+adhesion+ring+junction+and+contributes+to+the+sensitivity+of+target+cell+lysis+by+CTL.">Protein kinase C theta regulates stability of the peripheral adhesion ring junction and contributes to the sensitivity of target cell lysis by CTL.</a> The Journal of Immunology. 181, 4815-4824 (2008).</li><li>Beal, A. M., 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=Kinetics+of+early+T+cell+receptor+signaling+regulate+the+pathway+of+lytic+granule+delivery+to+the+secretory+domain.">Kinetics of early T cell receptor signaling regulate the pathway of lytic granule delivery to the secretory domain.</a> Immunity. 31, 632-642 (2009).</li><li>Dustin, M. L., Starr, T., Varma, R., Thomas, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+planar+bilayers+for+study+of+the+immunological+synapse.">Supported planar bilayers for study of the immunological synapse.</a> Current Protocols in Immunology. , (2007).</li><li>Reuter, M. A., 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=HIV-Specific+CD8(+)+T+Cells+Exhibit+Reduced+and+Differentially+Regulated+Cytolytic+Activity+in+Lymphoid+Tissue.">HIV-Specific CD8(+) T Cells Exhibit Reduced and Differentially Regulated Cytolytic Activity in Lymphoid Tissue.</a> Cell Reports. 21, 3458-3470 (2017).</li><li>Buggert, M., 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=Limited+immune+surveillance+in+lymphoid+tissue+by+cytolytic+CD4++T+cells+during+health+and+HIV+disease.">Limited immune surveillance in lymphoid tissue by cytolytic CD4+ T cells during health and HIV disease.</a> PLoS Pathogens. 14, e1006973 (2018).</li><li>Carrasco, Y. R., Fleire, S. J., Cameron, T., Dustin, M. L., Batista, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LFA-1/ICAM-1+interaction+lowers+the+threshold+of+B+cell+activation+by+facilitating+B+cell+adhesion+and+synapse+formation.">LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation.</a> Immunity. 20, 589-599 (2004).</li><li>Anikeeva, N., 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=Distinct+role+of+lymphocyte+function-associated+antigen-1+in+mediating+effective+cytolytic+activity+by+cytotoxic+T+lymphocytes.">Distinct role of lymphocyte function-associated antigen-1 in mediating effective cytolytic activity by cytotoxic T lymphocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 6437-6442 (2005).</li><li>Steblyanko, M., Anikeeva, N., Campbell, K. 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 CO2-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

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

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

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

piggyBac Transposon System Modification of Primary Human T Cells

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

piggyBac转座子初级人类T细胞系统改造

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

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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

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

从外周血人类同种异体抗原特异性T细胞的生成

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

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

从人类母胎界面分离白细胞

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

从全血人单核细胞来源的树突状细胞的生成

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Analysis of Human Natural Killer Cell Metabolism

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to promote survival, cellular proliferation, production of cytokines such as interferon gamma (IFN&#947;) and/or cytotoxicity programs. NK cell activation by cytokine stimulation requires a substantial remodeling of metabolic pathways to support their bioenergetic and biosynthetic requirements. There is a large body of evidence that suggests that impaired NK cell metabolism is associated with a number of chronic diseases including obesity and cancer, which highlights the clinical importance of the availability of a method to determine NK cell metabolism. Here we describe the use of an extracellular flux analyzer, a platform that allows real-time measurements of glycolysis and mitochondrial oxygen consumption, as a tool to monitor changes in the energy metabolism of human NK cells. The method described here also allows for the monitoring of metabolic changes after stimulation of NK cells with cytokines such as IL-15, a system that is currently being investigated in a wide range of clinical trials.

In this paper, we describe a method to measure glycolysis and mitochondrial respiration in primary human Natural Killer (NK) cells isolated from peripheral blood, at rest or following IL15-induced activation. The protocol described could be easily extended to primary human NK cells activated by other cytokines or soluble stimuli.

人类自然杀手细胞代谢分析

Natural Killer (NK) cells mediate mainly innate anti-tumor and anti-viral immune responses and respond to a variety of cytokines and other stimuli to ...