作者要感谢普里Subrahmanyam的有益讨论。作者没有竞争的经济利益。支持这项工作是由美国癌症协会，NIH / NCI K01的CA131487，R21 CA162273 R21 CA162277，和P30肿瘤免疫学和免疫计划，以TJ韦伯的补助。的内容完全是作者的责任，并不一定代表官方意见，国家癌症研究所，美国国立卫生研究院。

1。生成AAPC <ol><li>在添加蛋白质珠，准备好所有试剂和缓冲液：0.1M 硼酸盐缓冲液 ，1X D-PBS（没有的Ca 2 +和Mg 2 +）; 珠洗涤缓冲液 （1X PBS +5％人AB血清+ 0.02％的叠氮化钠） ; 完全培养基 （RPMI培养基100 mM的丙酮酸钠，10mM的非必需的维生素溶液，100mM的MEM维生素溶液，1％2 -巯基乙醇，10μM环丙沙星，5％人AB血清）; MACS缓冲液 （1 L的PBS的免费的Ca 2 +和Mg 2 +，5克BSA，和2毫摩尔EDTA）。 </li><li>漂洗1毫升的Dynabeads M-450环氧树脂珠与3毫升无菌的0.1M硼酸盐缓冲液（硼酸和水，pH 7.0-7.4）中，在5毫升清楚硼硅酸盐玻璃螺纹样品瓶。 </li><li>在一个单独的1.5 ml离心管中，加100微克hCD1d-Ig的二聚体和20μg共刺激分子（例如：抗CD28mAb）至1 ml PBS W / O型的Ca 2 +和Mg 2 +。 </li> &lt;李&gt;将磁铁和吸出的硼酸盐缓冲液珠的珠含玻璃小瓶。从步骤1.2玻璃瓶和蛋白质混合物更换帽。立即颠倒瓶子混合，用封口膜覆盖帽，并将其放置在旋转器上，并于4℃孵育过夜。 </li><li>第二天，代替玻璃小瓶中的磁铁和除去蛋白质的混合​​物中，同时小心地避免珠。洗磁珠通过加入3ml珠洗涤缓冲液（PBS中，用5％AB血清的0.02％叠氮化钠），并在4℃下孵育5分钟，在旋转器上。重复两次。 </li><li>珠可以被存储在此珠洗涤混合物。为了使功能性人工APC，取出一小等分，并用血球计数珠。检查，蛋白质稳定地加载到珠粒通过染色的抗体（例如，PE标记的抗小鼠IgG1），进行流式细胞仪分析。 </li><li>要加载珠抗原，去除5×10 7珠，并添加一个小的1.5毫升的玻璃小瓶中，用1毫升无菌PBS冲洗珠。 ŕesuspend 1毫升无菌PBS洗珠，并添加抗原，例如：Loadα-GalCer/KRN7000（20,10,5）。 *注：虽然，我们并没有发现任何问题与脂溶性或胶束的形成，血脂，应按照制造商的建议。具体而言，KRN7000重新溶于DMSO（1mg/ml的）这些研究。 KRN7000和其他糖脂抗原也可以溶解在0.2毫克/毫升PBS中含有0.5％的Tween-20（在37℃下超声处理2小时。）。重要的是 - 人工APC至少48-72小时，使用前需要加载。 </li></ol> 2。 CD161 + CD3 +细胞的分离<ol><li>收集外周血单核细胞（PBMC）。 Ficoll密度梯度离心分离从血沉棕黄层或按患者组的淋巴细胞，首先稀释肝素化血液在室温下，用等体积的1X PBS。 </li><li>加入15毫升的Ficoll（温热至室温），50 ml锥形管。慢慢地覆盖25毫升的稀释血液的混合物的聚蔗糖的顶部。在2,000 rpm离心30分钟，在室温下与制动切断。 </li><li>用巴斯德吸管小心地取出淋巴细胞接口（白色环之间的媒体和Ficoll），并转移到一个新的50毫升锥形管。 </li><li>通过填充至50毫升的管在1,500 rpm离心5分钟，用PBS和离心洗涤细胞。丢弃上清液，并结合管从一个单一的个体单管，并再次用20ml PBS洗的外周血单核细胞（PBMC）。然后计算PBMC和重新悬浮在浓度为5×10 7细胞/ ml，在MACS缓冲液（1升PBS免费的Ca 2 +和Mg 2 +，5克BSA，和2毫摩尔EDTA）中。 </li><li>为了分离出的T细胞比例，开始用2毫升10 8个细胞（PBMC），加入100μl泛EasySep人类T细胞富集试剂盒的T细胞丰富的解决方案。在室温下孵育10分钟。 </lI&gt; <li>加入100μl的磁性粒子另一个10分钟的溶液中，并在室温下孵育。使终体积的溶剂至2.5毫升，并在紫色磁铁放置在管5分钟。快速倒出到一个15毫升锥形管的CD3 +馏分。 </li><li>洗涤细胞，加入5毫升冷MACS缓冲，数一数，活细胞，FACS染色移出一份。 </li><li>要选择CD161 +细胞，重悬丰富的T细胞在980μL冰冷的MACS缓冲，添加10微克抗CD161单克隆抗体，并在冰箱中孵育10分钟。 </li><li>细胞在4℃下以1,500 rpm离心5分钟。重组细胞沉淀在800μl的MACS缓冲。加入200μl的抗小鼠IgG1微珠孵育10分钟的溶液，于4℃下</li><li>在此温育步骤，平衡一个LS列通过加入3ml MACS缓冲器。 </li><li>接着，洗涤细胞，在4℃下以1,500 rpm离心5分钟。 RESU花的细胞MACS缓冲液3毫升。到LS MACS分离柱，然后移液管的细胞。请一定要避免产生气泡慢慢移液。通过加入3ml MACS缓冲液冲洗色谱柱。重复两次。 </li><li>加入3毫升新鲜的MACS缓冲和删除列磁铁。把柱子成15毫升锥形管。将柱塞推出的内容，以获得纯化的CD161 + CD3 +细胞。计数NKT细胞富集馏分。你应该有2-4亿个细胞。 </li></ol> 3。人工APC介导的NKT细胞扩增<ol><li>设置共培养16毫升完全培养基（完全培养基+，100 U / ml的IL-2）中加入10 6富集CD161 + CD3 + T细胞和10 6人工APC。混合物通过加入160μl/孔最终体积的96孔组织培养处理的聚苯乙烯，U型底的板的低蒸发盖盘。每个第 7天通过加入80μl的新鲜培养基进行培养基更换。 </li><li>收成细胞，计数，并进行FACS染色12日至14日。 </li><li>如上所述在步骤3.1中，可以扩大的NKT细胞再接种，特别是重悬在16毫升完全培养基的10 6扩展的T细胞和10 6人工APC。盘通过添加160μl/孔96孔U型底的板的混合物。继续刷新共存培养基中每一个第 7天通过加入80μl的新鲜培养基。 </li><li>最好是冻结额外的NKT细胞的膨胀后的第二轮（1×10 6/1毫升5％二甲基亚砜/ 95％FBS的离心管中。） </li></ol> 4。功能测试：的人工APC介导的刺激NKT细胞<ol><li>设置为5×10 4的NKT细胞/孔5×10 5在200μl的最终体积（完全培养基），在96孔U型底的板中的人工APC。 </li><li> 24-48小时后收获细胞培养上清ELISA。 </li></ol>

<ol>
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没有利益冲突的声明。

AAPC可以用来研究NKT细胞激活的基本要求和体外扩增的NK T细胞过继免疫治疗中具有潜在的临床价值。 mescher 等人描述的第一胎圈的系统，其中生物素化的小鼠MHC I类肽的单链结构，B7.1和B7.2通过链霉抗生物素蛋白与生物素化的共刺激分子结合的胶乳微球体14，15的表面。这种方法已被成功地使用从转基因小鼠的抗原特异性T细胞刺激。此外，由于此方法使用了一个单链，以确保均匀的负荷的MHC分子的MHC-肽复合物，每个目标肽抗原将需要一个新的表达所需的单链MHC-肽复合物的转染，从而限制的一般性的的方法。更重要的是，施内克博士的研究小组首创的珠基于人工APC，开发另一种非基于细胞珠的人工APC，由耦合到磁珠上的HLA-Ig的，信号1，和抗-CD28，信号2，。 HLA-Ig的，通过他的研究小组开发的一个独特的多聚体形式的免疫球蛋白分子的支架16，17的稠合的HLA。随后，他们开发的MHC-Ig的基于人工APC，这已被证明有效地扩大CMV和MART-1特异性CTL 18。在这里，我们已经证明，可用于扩展功能的NKT细胞CD1D-Ig的基于人工APC。一项研究使用了一个类似的系统来检查身体的NK细胞的相互作用与CD1D 19。 值得一提的是，我们设计了一个人工抗原呈递细胞，它是适用于任何的要求，我们找到所需的最佳NKT细胞的增殖。人工APC扩展方法提供了一种简单可靠的方法，扩大和丰富人类NKT细胞。我们的人工APC系统评估潜在的共刺激分子的面板的作用，并评估其对NKT细胞增殖的作用，可以修改TION和功能。因此，AAPC一个强大的通用技术，可用于诱导和扩大NKT细胞。 AAPCS需要的产生小于1周，适合用于生产大量珠。然而，生成人工APC的一个关键步骤是，以确认CD1D-Ig的被稳定地固定化的珠的表面上，并评估它们的功能，以确保从批次间的一致性。系统是有一定的局限性，没有一个机制，在关闭刺激，机械拆除的珠子以外的地方。具体而言，卡合的T细胞受体（TCR）与抗原：CD1d/MHC络合物通常生成的免疫突触在演唱会与配件/粘附分子，这可能会导致T细胞和诱导的抑制或抑制因素抗原呈递细胞。在的人工APC系统，这些因素都可能上调的T细胞，但珠不会表达的同源配体这些受体。 此外，CD4 + NKT细胞已显示出抑制在小鼠和人类的抗肿瘤反应，因此，它是可能的非选择性的活化NKT细胞的所有错误的子集（ 即全球刺激与α-神经酰胺）或激活可以导致不想要的免疫学的结果。因此，一个人必须的表型和功能特征的的人工APC发泡NKT细胞种群。正如在图4中所示，我们已发现，表达抗-CD28刺激的α-神经酰胺加载人工APC可以导致NKT细胞产生Th1细胞，Th2细胞，和Th17型细胞因子。小鼠的研究报告说，挑战与IL-33，最近发现的细胞因子，导致循环炎性细胞因子，如IL-5和IL-13水平的提高。治疗的NKT细胞与IL-33增强细胞因子的产生20。 IL-33是ST2中，一个特定的配位体，它已被证明，可溶性ST2约n块IL-33信号。因此，作为将来的应用程序的一个例子，可以人工APC表达ST2产生和使用，以确定是否有一个可以选择性地抑制Th2型细胞因子的生产的，而诱导Th1细胞因子分泌的NKT细胞。也已报道，静脉注射KB-表示人工APC于C57BL / 6小鼠，导致减少的肺转移的肿瘤21。更重要的是，这些数据表明，人工APC交通肺和能够激活效应T细胞亚群。因此，可以生成多种类型的人工APC和检查抗原特异性T细胞亚群之间的相互作用。总之，这些研究表明，CD1D-Ig的基于人工APC可以用来取代正常细胞的APC，和具有的潜力，以提高基于NKT细胞的过继性免疫治疗的目前的临床途径。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>用Ficoll-Paque PLUS </td><td> GE生命科学</td><td> 17-1440 </td><td></td></tr><tr><td> EasySep人类T细胞富集试剂盒</td><td>干细胞技术</td><td> 19051 </td><td></td></tr><tr><td>别藻蓝蛋白CD161人单克隆抗体</td><td> Pharmingen公司</td><td> 550968 </td><td></td></tr><tr><td> EasySep磁铁</td><td>干细胞技术</td><td> 18002 </td><td></td></tr><tr><td>抗小鼠IgG1微球所</td><td>美天旎生物技术公司</td><td> 130-047-101 </td><td></td></tr><tr><td>二聚体XI重组可溶性二聚体鼠标CD1D：Ig融合蛋白</td><td> BD Biosciences公司</td><td> 557599 </td><td></td></tr><tr><td抗CD28单克隆抗体</td><td> Biolegend </td><td> 302914 </td><td></td></tr><tr><td> M-450环氧珠</td><td> Life Technologies公司</td><td> 150-11 </td><td></td></tr><tr><td> α-半乳糖苷（KRN 7000） </td><td> AXXORA，LLC </td><td> BML-SL232-0100 </td><td></td></tr><tr><td>的RPMI 1640中等</td><td> Sigma Aldrich公司</td><td> ŕ0883 </td><td></td></tr><tr><td>丙酮酸钠</td><td> Gibco公司</td><td> 11360-070 </td><td></td></tr><tr><td>非必需氨基酸</td><td> Gibco公司</td><td> 11140-050 </td><td></td></tr><tr><td>维生素解决方案</td><td> Gibco公司</td><td> 11120-052 </td><td></td></tr><tr><td> 2 - 巯基乙醇</td><td> Gibco公司</td><td></td><td></td></tr><tr><td>环丙沙星</td><td>亚历克西斯生化试剂</td><td> 380-288-G025 </td><td></td></tr><tr><td> PE-Vα24Jα18的</td><td> Biolegend </td><td> 342904 </td><td></td></tr><tr><td> PE-Vα24（克隆C15） </td><td> Beckman Coulter公司</td><td> A66907 </td><td></td></tr><tr><td> FITC-Vβ11（克隆C21） </td><td> Beckman Coulter公司</td><td> A66905 </td><td></td></tr><tr><td> PE-CD1D </td><td> Biolegend </td><td> 123510 </td><td></td></tr><tr><td> PE抗小鼠IgG1 </td><td> BD Biosciences公司</td><td> 556650 </td><td></td></tr><tr><td> FITC标记的抗小鼠IgG2a </td><td> Biolegend </td><td> 407105 </td><td></td></tr><tr><td> DPBS，无钙无镁</td><td> Life Technologies公司</td><td> 14190250 </td><td></td></tr><tr><td>叠氮化钠</td><td> Sigma Aldrich公司</td><td> S8032 </td><td></td></tr><tr><td>牛血清白蛋白</td><td>美国生物分析</td><td> AB00440-00100 </td><td></td></tr><tr><td> EDTA </td><td> Sigma Aldrich公司</td><td> 431788 </td><td></td></tr><tr><td> IL-2（Proleukin） </td><td> BD Biosciences公司</td><td> 354043 </td><td></td></tr><tr><td>人AB血清</td><td>亚特兰大生物</td><td> S40110 </td><td></td></tr><tr><td> Labquake管肩</td><td> Fisher Scientific则</td><td> 13-687-10Q </td><td></td></tr><tr><td> BD猎鹰5毫升聚苯乙烯圆底管</td><td> Fisher Scientific则</td><td> 14-959-1A </td><td></td></tr><tr><td>章惠顿玻璃样品瓶</td><td> Fisher Scientific则</td><td>小（03-343-6A）大（03-343-6E） </td><td></td></tr></tbody></table>

artificial antigen presenting cell (aapc) mediated activation and expansion of natural killer t cells

自然杀伤T（NKT）细胞是一种独特的子集T细胞，自然杀伤（NK）细胞和T细胞的特征性标记物显示。不同于传统的T细胞，NKT细胞识别脂质抗原CD1分子2的上下文中。 NKT细胞的表达一个不变的TCRα链重新排列：Vα14Jα18在小鼠和Vα24Jα18的在人类中，这是与有限的多样性的Vβ链3-6，以及被称为作为规范的或不变的NKT细胞（ⅰNKT细胞）。类似传统的T细胞，NKT细胞从CD4-CD8-胸腺前体T细胞发展CD1D 7适当的信令。利用NKT细胞，用于治疗目的的潜力已显着增加的能力，以刺激和扩大人类NKT细胞的α-半乳糖（α-神经酰胺），以及各种细胞因子8。更重要的是，这些细胞保留了原有的phenotypE，分泌细胞因子，并显示抗肿瘤细胞系的细胞毒性功能。因此， 体外扩增 NKT细胞保持功能和可用于过继性免疫治疗。然而，基于NKT细胞的免疫已限制使用自体的抗原呈递细胞和刺激细胞的数量和质量，这些基本上可以变化。已报道从癌症患者的单核细胞衍生的DC的共刺激分子的表达水平降低，并产生较少的炎性细胞因子9,10。事实上，已被用于小鼠DC而不是自体的APC从CML患者11 NKT细胞的功能进行测试。然而，这种系统只能被用于在体外测试，因为NKT细胞不能进行扩展，由小鼠DC，然后用于过继性免疫治疗。因此，标准化的系统依赖于人工抗原提呈细胞（AAPC）可能产生的刺激效果的DC没有的缺陷，异体或异种CELLS 12，13。在这里，我们描述了一种的方法产生CD1D基于人工APC。由于CD1D - 抗原复合物的T细胞受体（TCR）的参与是一个基本的要求，NKT细胞活化抗原：CD1D免疫球蛋白复合物提供了一种可靠的方法来隔离，激活和扩大效应NKT细胞群。

我们在此描述了一种方法，用于产生CD1D-Ig的基于人工APC，由CD1D-Ig和抗-CD28单克隆抗体共价偶联到磁珠刺激NKT细胞，NKT细胞（ 图1）的传播作为一个标准化的方法。首先， 1必须展示出CD1D-Ig融合蛋白质被稳定地固定到表面的磁珠。如示于图2A中 ，CD1D-Ig和抗-CD28抗体的磁珠的表面上均表示。要检查的人工APC的刺激能力，我们NKT细胞共培养的杂交瘤与人工APC过夜，收获培养物上清液和测定IL-2的生产通过ELISA。我们发现基于CD1D-Ig的人工APC能够刺激NKT细胞的杂交瘤在水平等于或高于它们的细胞（ 图3中 ，数据未示出）。有趣的是，我们发现，我们的小鼠NKT细胞杂交瘤AAPC人CD1D（FI刺激gure 2B），它提供了简单的方法，用于测试每批人工APC。 接下来，我们试图证明的繁殖潜力的人工APC，从外周血中分离出这样的人的T细胞。首先，CD161 + CD3 + T细胞磁珠分离富集馏分。然后，将T细胞与α-神经酰胺加载人工APC刺激双周。更重要的是，我们发现，即使具有相对低的初始NKT细胞种群（0.03％），我们能够扩大细胞〜67％Vα24+Vβ11+（ 图3）。我们已经扩大了NKT细胞从许多健康志愿者和癌症患者的外周血单个核细胞，并发现，α-神经酰胺装AAPC能在这两个群体中的NKT细胞群扩大。值得一提的是，捐助的扩张速度是非常依赖。正如预期的那样，较高的初始种群中的Vα24+细胞的百分比越大的膨胀。另外，当使用200万细胞起始人口的CD161 + CD3 + T细胞，可以得到&gt; 10 7细胞的扩张两轮后（ 表1）。大约80-90％的膨胀的NKT细胞是CD4 +，〜5％的CD8 +，和剩余的大概是CD4-CD8-双负NKT细胞。这些膨胀的NKT细胞，可用于在图4A-C所示的功能研究。我们已经发现，我们的体外扩增 NKT细胞保持响应于α-GalCer的刺激，和IL-17，TNF-α，IFN-γ和是有效的生产者。应当指出，如果初始的T细胞富集人口是低的，并且一个是无法执行所述第二CD161富集步骤，的人工APC-介导的扩张可能不会产生预期的结果（参见图4D，供体1）。但是，如果循环NKT细胞的百分比是高于0.1％，一应仍是能够获得显着的膨胀的 i NKT细胞。总的来说，这些数据表明，CD1D基于人工APC可以用来有效地扩大和刺激主要的NKT细胞。 <img alt="图1" src="/files/ftp_upload/4333/4333fig1.jpg" /> 图1。示意图CD1D：基于Ig的的AAPCS胞外部分的CD1D分子的恒定区的融合到由一个短的氨基酸接头分离的免疫球蛋白重链蛋白质。这些分子可以很容易地加载与脂质抗原，如α-神经酰胺，简单地通过培养用过量的脂质的兴趣。人工APC作了耦合CD1D-Ig和抗-CD抗体磁珠。在这个系统中，CD1D-Ig的用于通过TCR和抗-CD28单克隆抗体提供共刺激信号，以提供的同源抗原特异性信号。 <img alt="图2" fo:content-width="4.25in" fO：SRC =“/ files/ftp_upload/4333/4333fig2highres.jpg”SRC =“/ files/ftp_upload/4333/4333fig2.jpg”/&gt; 图2。 FACS染色AAPCS表面蛋白A）AAPCS CD1D：IgG抗体二聚体存在（通过与PE标记的抗小鼠IgG1染色）以及抗-CD28抗体（使用FITC标记的抗小鼠IgG2a）进行了测试，打开直方图显示同型对照;填充柱状图代表指定的抗体。 CD1D-Ig的表达人工APC可以刺激NKT细胞的IL-2产生。 B）Vα14+鼠标NKT细胞杂交，DN32.D3，无论是中等，可溶性抗原（α-神经酰胺），卸载AAPC或α-神经酰胺加载AAPC共培养。收获培养物上清液，并使用标准夹心ELISA测定IL-2产生。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/4333/4333fig3highres.jpg" src="/files/ftp_upload/4333/4333fig3.jpg" /> 图3。 CD1D-Ig的涂层人工抗原递呈细胞，NKT细胞的扩展。 （A）主CD3 + CD161 +双阳性细胞的分离从外周血单个核细胞，利用磁分离。排序条件刺激细胞与α-GalCer的加载，14天CD1D-Ig的涂层人工APC。将细胞染色为Vα24和Vβ11以下的人工APC刺激。 （B）的B细胞淋巴瘤线主要的人类NKT细胞介导的​​裂解。 C1R-CD1D NKT细胞孵育的细胞在存在或不存在的抗原，神经酰胺（100毫微克/毫升），在20-24小时的96孔U型底的板在所指示的比率。 NKT细胞介导的​​细胞裂解液进行了评估标准的51Cr释放法。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/4333/4333fig4highres.jpg" src="/files/ftp_upload/4333/4333fig4.jpg" /> 图4。的人工APC扩展NKT细胞的细胞因子。 </strong&gt;的刺激与α神经酰胺装AAPC了两个星期，扩大NKT细胞（1×10 5 /孔），共培养与可溶性α-神经酰胺，PMA /离子霉素，anti-CD3/28微球，或α-神经酰胺装AAPC后（2×10 5 /孔）48小时。 （A）的IL-17A，（B）的TNF-α，和（C）的IFN-γ的产生是由标准的细胞因子ELISA测定。显示的数据是减去阴性对照组（媒体和空珠）后的净细胞因子的产生。 （D）原发性T细胞的分离外周血单核细胞磁珠分离。刺激了两个星期的指示，神经酰胺加载人工APC细胞。细胞进行染色，采用ABS具体的Vα24+Vβ11+，并用流式细胞仪分析。

Watch this Scientific Journal Video about Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells at JoVE.com

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

在这里，我们描述了一种方法激活和扩大人类NKT细胞的自体T细胞的人口使用人工抗原提呈细胞（AAPC）。 CD1D基于人工APC提供了使用功能的NKT细胞产生高的标准方法。

人工抗原提呈细胞（AAPC）的自然杀伤T细胞介导的​​激活和扩展

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

artificial-antigen-presenting-cell-aapc-mediated-activation-expansion

Research

JoVE Journal

Immunology and Infection

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

17.9K 次观看.  University of Maryland. 在这里，我们描述了一种方法激活和扩大人类NKT细胞的自体T细胞的人口使用人工抗原提呈细胞（AAPC）。 CD1D基于人工APC提供了使用功能的NKT细胞产生高的标准方法。

视频: Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

科研

免疫与感染

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. 
While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. 
Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy donor with aAPC for one week, CMV specific CTL can be increased dramatically in specificity up to 98% (Figure 2) and amplified more than 10,000 fold. If more CMV-specific CTL are required, further expansion can be easily achieved by repetitive stimulation with aAPC. Phenotypic and functional characterization shows these expanded cells have an effector-memory phenotype and make significant amounts of both TNF&alpha; and IFN&gamma; (Figure 3).

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

A new DC independent method for induction and expansion of antigen-specific T cells is described. HLA A2-Ig based artificial Antigen Presenting Cells (aAPC) are loaded with HLA-A2 restricted peptides to efficiently expand CTL of diverse antigen specificity. This technology holds great potential for CTL-based adoptive immunotherapy.

HLA - IG基于人工抗原提呈细胞可高效体外扩展人类的CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

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 ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

杀手人工抗原提呈细胞（KaAPC）为有效<em&gt;在体外</em&gt;人抗原特异性T细胞耗竭

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

人工抗原提呈细胞（AAPC）的自然杀伤T细胞介导的​​激活和扩展

人工抗原提呈细胞（AAPC）的自然杀伤T细胞介导的激活和扩展