这项工作已经得到了德国研究基金会（DFG）SCHU-2681 / 1-1（CS）和B＆K 146（周一至周五），国防拨款PC的国防部部040972（MO）和美国国立卫生研究院授予RO1 CA108835 ，RO1 Ai44129（JPS）

1代的HLA-A2-IG基于KaAPC的<ol><li>准备无菌的硼酸盐缓冲液。使硼酸在水中的0.1M的溶液，并调节pH至7.0。无菌滤器（0.22微米）缓冲液中，并在4℃下存储。 </li><li>准备不育珠洗涤缓冲液。用磷酸盐缓冲盐水（PBS），而镁和钙。添加人AB血清的3％终浓度。为2mM的最终浓度添加乙二胺四乙酸（EDTA）。添加叠氮化钠（NAN 3）为0.01％，终浓度。无菌滤器（0.22微米）缓冲液中，并在4℃下存储。 </li><li>转移250微升从库存环氧树脂珠（约100×10 6珠）进入无菌，螺杆顶端的玻璃小瓶中。 </li><li>加入250μl硼酸盐缓冲液。 </li><li>放在磁铁玻璃小瓶，让珠坚持2分钟;吸出缓冲液和从磁体取出玻璃小瓶中。 </li><li>用1ml硼酸盐缓冲液洗一次珠子。 </li><li>重悬珠到550微升0.018微克/ ml的HLA-A2-Ig和3.64微克/毫升抗人CD95单克隆抗体（克隆CH11）和通过轻轻振动混合。 （请参阅讨论部分以获取更多信息。） </li><li>孵育玻璃小瓶上的转子端部上结束时，在4℃下放置24小时。 </li><li>降速珠在300×g离心2分钟。 </li><li>将玻璃瓶上的磁铁2分钟，吸出“KaAPC加载解决方案”;从磁体取出玻璃小瓶中，加入1毫升珠洗涤缓冲液并通过摇动小心重悬。 </li><li>重复步骤1.10的两倍。 </li><li>孵育玻璃小瓶在旋转器上在4℃下放置24小时的1ml珠洗涤缓冲液。在这点上，所有的残余蛋白质结合位点会被包含在珠洗涤缓冲液中的人AB血清阻断。 </li><li>降速珠在300×g离心2分钟。 </li><li>广场的玻璃小瓶上的磁铁2分钟，吸珠洗涤缓冲液;从磁体取出玻璃小瓶中，并用1ml的PBS溶液更换。 </li></ol> 2，质量控制，肽加载，W灰化，存储和KaAPC稳定性<ol><li>转让1.5×10 5 KaAPC成流式细胞仪管，洗用1ml FACS洗涤缓冲液。 </li><li>重悬珠子于100μlFACS洗涤缓冲液中添加1μl的抗小鼠IgG1的PE（用于检测HLA-A2-Ig的Fc部分的）和1μl的抗小鼠IgM抗体FITC（克隆R6-60.2的，用于检测抗人CD95单克隆抗体）。 </li><li>孵育15分钟，在4℃下，用1ml FACS缓冲液洗涤，并重新悬浮在250μl的FACS洗涤洗涤缓冲液并通过流读染色流式细胞仪。 </li></ol><img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51859/51859fig1highres.jpg" width="500" /> ÂKaAPC批次制成的协议部分中所述的图1中的QC污点。染色，如在协议部分所述用抗小鼠的IgG1 PE单抗2 HLA-A2-Ig的检测，而抗人CD95 （抗Fas）用抗小鼠IgM抗体FITC单克隆抗体检测（见协议本身ction 2.2）（蓝线）。填充黑色曲线表示用抗小鼠的IgG1 PE单克隆抗体或抗 - 小鼠IgM抗体FITC单克隆抗体，分别空珠。在右上角的数字表示平均荧光强度（MFI）。 <a href="https://www.jove.com/files/ftp_upload/51859/51859fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <ol start="4"><li>对于肽荷载传递20×10 6 KaAPC到一个新的无菌玻璃小瓶中，将上磁铁2分钟，吸出PBS重悬珠到500微升新鲜的PBS，5微克肽（记住，只有HLA-A2限制性肽会结合到二聚体！）。 </li><li>店内装KaAPC在4℃下直到使用，为至少3天，以允许足够的肽装载到HLA-A2-Ig二聚体。 </li><li>在使用前立即，洗净装KaAPC如1.14所表示的肽;重复此步骤，至少6倍。 注意：这是关键的一步，以确保自由肽具有吨Ø被完全删除，以避免假阳性结果被杀;残余的游离肽已经显示出诱导细胞凋亡中的抗原特异性T细胞的培养物9。为了排除可溶性肽的效果，运行肽装上清对照样品和清洗KaAPC（见3.8）。 </li><li>计数使用血球珠和日期，名称和肽​​浓度的标签。 注：加载KaAPC保持功能，在4℃下至少一个月。一个月以后，重新加载具有相同的肽可以KaAPC将要用于长达一年。 </li></ol> 3 KaAPC 体外杀灭试验<ol><li>制备共培养介质。制备添加有3％的T细胞生长因子及3％供体的自体血浆（在实验室6制备）的完全RPMI培养基中。如果捐助者的自体血浆不可用，用热灭活人AB血清。 </li><li>准备AnnexinV结合缓冲液配制。溶解8.12克氯化钠（NaCl）和0.12克计算值鎓氯化物（ 氯化钙 ）成990毫升DDH 2 O.Add 10ml1M的HEPES缓冲液中。 </li><li>产生人的抗原特异性T细胞。 （关于详细的协议，请参阅使用人工抗原提呈细胞（人工APC）先前公布7）。确保肽特异性为至少&gt; 75％，这通过四聚体染色来确定。 </li><li>对于每个样品孵育2×10 5个抗原特异性T细胞/孔（在96孔圆底板）在200μl的共培养培养基中以1：在湿润的培养箱1的比例与KaAPC 48小时，在37℃ C和5％的CO 2。 </li><li>嘉实样品和转移到FACS管;洗用1ml AnnexinV结合缓冲液，离心后，在300×g离心5分钟，弃去上清，重悬在100μl的AnnexinV结合缓冲液</li><li>染色样品与1μl的AnnexinV FITC和5μl的7-AAD为10-15分钟，在室温洗1毫升AnnexinV结合缓冲液;降速为300×g离心5分钟，弃去上清液的在250μLAnnexinV结合缓冲液D重悬</li><li>立即读取样品的流式细胞仪。 </li><li>准备下面的示例通过KaAPC确定抗原特异性杀伤：只有T细胞; T细胞+可溶性抗-CD95; T细胞+卸载KaAPC; T细胞+非同源肽加载KaAPC; T细胞+同源肽加载KaAPC; T细胞+上清同源肽加载KaAPC。 </li></ol><img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51859/51859fig2highres.jpg" width="500" /> 图2 KaAPC杀测定肽加载KaAPC或控制珠（HLA-A2-Ig的只有珠）进行48小时培养，在以1:1的比例与CMVpp65特异性T细胞（〜80％），收获，并随后用AnnexinV和碘化丙啶（PI），以通过流式细胞术（见协议部分3.3-3.7）确定细胞凋亡。“同源”是指小珠装入CMVpp65和“非同源”是广告装载MART-1肽。数字表明可行的T细胞在各小区的左下象限的百分比。 <a href="https://www.jove.com/files/ftp_upload/51859/51859fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <ol start="9"><li>执行交错，实验中，产生两个不同特异性的抗原特异性T细胞。 </li><li>进一步评估KaAPC的抗原特异性耗尽能力与抗原特异性消除T细胞的异质性T细胞群的同时分析。 （关于详细的协议，请参阅茨等人10）。 </li></ol>

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CS和MF什么都没有透露。 根据NexImmune和约翰·霍普金斯大学，博士之间的授权协议。 Schneck和Oelke有权使用费的销售在这篇文章中所描述的aAPC产品收到大学的股份。他们还拥有NexImmune股票，这是受到大学的政策有一定的限制。 Schneck博士为公司董事的“董事局和科学顾问委员会的成员。这种安排的条款是由美国约翰·霍普金斯大学按照利益的政策的冲突管理。

在该协议中最关键的步骤是确保信号1（肽MHC）和信号2（抗Fas单克隆抗体）的适当的比例。我们已经进行了深入的滴定实验，以确定最佳比对信号1和2;很明显，最小的变化，由于在HLA-A2 Ig二聚体或抗Fas单克隆抗体的浓度差异可与KaAPC的功能干扰。因此，两种信号的浓度应该总是被验证和两种蛋白质的质量应经常测试，即使它是从商业来源订购。此外，该浓度应始终用相同的测定法测定的不同检测方法可能会导致在不同的浓度。在HLA-A2-Ig的功能也可以由于不完全的重折叠和/或低效的肽的加样损害。此外二聚体分子可以聚集到不同程度，这可能对蛋白质的功能活性的影响。因此，ACTU需要二聚体的人的数量为一个功能和特定KaAPC的产生可能会发生变化。此外，我们发现，理想的范围内为死亡KaAPC信号诱导抗Fas单克隆抗体是非常紧迫的。在我们手中用量大于3.64微克/毫升导致的Fas阳性T细胞的非特异性杀伤而低于3.64微克/毫升的量导致剂量依赖减少杀伤活性。虽然这些条件似乎相当紧张，注意这些细节会导致功能和具体KaAPC的产生。 而目前KaAPC表型是官能活化的抗原特异性T细胞，最初的实验靶向幼稚或休息的抗原特异性T细胞的耗竭证实没有显著诱导的抗原特异性细胞凋亡的，因为这些种群减少Fas表达。因此，人们可能设想添加一个共刺激信号到KaAPC诱导上调的Fas对静息T细胞，以使将它们到了KaAPC的目标。尽管这是可行的所有三个信号将必须小心滴定以确保功能KaAPC表型。 调整珠平台，生物相容性和生物可降解基质将加强KaAPC 体内的适用性。最近，Shen 等人报道的成功 KaAPC利用缀合的抗Fas（克隆JO2）和抗His / H 2-K B＆-TRP2 115μm的胶乳珠的体内使用。我们已经能够显示，人工抗原呈递细胞（人工APC）的一般概念可以成功地从微米尺寸到纳米尺寸的颗粒12传递，并且功能是由粒子的几何形状13的影响。因此，通过改变尺寸和/或未来的KaAPC的形状设计中的一个可能能够在体内的功能和生物分布，这可能是关键的成功治疗的器官特异性自身免疫性疾病的影响。 2，我二聚体分子以及正在进行确定新的自身免疫相关的抗原，将进一步增加，拓宽了适用的附加 ​​类HLA的发展KaAPC。此外有人也许会想到用KaAPC针对不同的抗原决定簇可以同时针对多个抗原。 总之，KaAPC代表一个灵活的平台技术为tigen特异性T细胞耗竭。它们的“乐高样”性质使得列入新的信号，例如在不同的矩阵额外的共刺激或抑制性信号，如PD1或TRAIL，其可以扩大并增强其随后在体外和体内的适用性。这里，我们提出的一步一步的协议来生成KaAPC，所述第一胎圈为基础的方法来消除由具有不同特异性的T细胞的混合物的抗原特异性T细胞。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51859/51859fig3highres.jpg" width="500" /> 图3 KaAPC消除CTL中的抗原特异性方式 PKH26标记的活化的Fas +效应记忆CTL和来自同一供体PKH67标记CMVpp65特异性CTL进行混合，以1：1的比例，并共培养以CMVpp65 KaAPC 48小时。对照培养物用卸载 KaAPC，或1微克/毫升处理过可溶性抗Fas蛋白单克隆抗体（克隆CH11）。活细胞的损失降到最低是在未经处理的混合培养的CTL（T 组合 ）来确定。显示原始的流式细胞仪的数据于T 搭配每个CTL人口。进行细胞凋亡分析，此前公布的由不同的门上都T细胞群10。在左上象限中描绘的数字表示％的总细胞。在示图中被来自于一个代表性实验了三次独立实验。这项研究最初发表于血。 。舒茨，C. 等杀手人工抗原提呈细胞：一种新的战略，以删除特定T细胞的血液。 2008; 111，3546-52。版权所有美国血液学会。 <a href="https://www.jove.com/files/ftp_upload/51859/51859fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

在过去的二十年中许多已经取得了进展理解自身免疫性疾病的发病机制。然而，最常见的治疗方法仍然依赖于皮质类固醇以及免疫抑制药物如嘌呤类似物，烷化剂和钙调神经磷酸酶抑制剂1。使用这类药物，大大提高了患者的预后，但治疗不提供潜在的免疫异常的一种治疗方法。此外，患者变得易受感染和癌症的发展。 因此，对于治疗T细胞介导的​​自身免疫性疾病和移植排斥反应的最终目标是专门针对引起T细胞的同时，在同一时间，余下的免疫系统不变，并有能力打免疫疾病不仅发病。利用抗原呈递细胞（APC），为治疗以抑制外周T细胞应答首次描述早20世纪70年代由几组。自那时以来许多基于细胞的方法已被开发（在茨等 2审查）。使用FasL的表达杀手的APC作为免疫细胞表现出令人鼓舞的结果表明治疗潜力用于治疗自身免疫性疾病，移植排斥反应和慢性感染的策略。然而，存在与使用FasL的杀手-APC已最终限制了其临床应用性策略显著限制; （I）产生的APC或隔离的时间，人力和成本密集型（二）病人患血细胞减少症，由于免疫抑制剂提供有限的数量和细胞的质量（三）涂层或APC的转导与细胞凋亡诱导信号如FasL的结果高度变量表型及（iv）杀手-APC是敏感的细胞毒性T细胞从而降低它们的治疗潜力的效应子功能。因此，难以保证限定可再现杀手-APC苯氧这可用于在体内的T细胞应答的抗原特异性的调制类型。 根据我们以前的工作与FasL的表达杀手-APC 3-5和人工抗原呈递细胞（人工APC）6,7我们开发了一种基于珠的方法（KaAPC）为在抗原特异性抑制自身反应性T细胞的消除或体外 。 KaAPC由一个HLA-A2-Ig二聚体（信号1）和抗Fas单克隆抗体（细胞凋亡诱导信号）共价固定于4.5微米的顺磁性的乳胶珠的表面。它们消耗在从多个特异性的T细胞的文化中的Fas / FasL系统介导的时尚抗原特异性T细胞在抗原特异性的方式与独立的活化诱导的细胞死亡（AICD）途径。有针对性的T细胞剂量依赖性凋亡后，已经30分8迅速诱导。 KaAPC可以以受控的方式很容易地产生，确保规定的现成phenotYPE，克服大多数的基于细胞耗竭策略的缺点。因此，KaAPC显示用于治疗T细胞介导的​​自身免疫性疾病和移植排斥反应的巨大潜力，促进T细胞的特异性治疗策略的领域。

<table border="0" cellpadding="0" cellspacing="0" width="654">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:185px;">Name</td>
			<td style="width:157px;">Company</td>
			<td style="width:148px;">Cat #</td>
			<td style="width:164px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human AB serum</td>
			<td>Atlanta biologicals</td>
			<td>S40110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Sigma/Aldrich</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium azide</td>
			<td>Sigma/Aldrich</td>
			<td>71289</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-450 epoxybeads</td>
			<td>Invitrogen/Dynal</td>
			<td>14011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPC-1 magnet</td>
			<td>Invitrogen/Dynal</td>
			<td>12001D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">screw top glass vial</td>
			<td>Fisher Scientific</td>
			<td>03-338AA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACSmix Tube Rotator</td>
			<td>Miltenyi</td>
			<td>130-090-753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD95 (clone CH11)</td>
			<td>Milliore</td>
			<td>05-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HLA-A2-Ig dimer</td>
			<td>BD/Pharmingen</td>
			<td>551263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryo-tube 2ml</td>
			<td>Corning</td>
			<td>430488</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse IgG1 PE</td>
			<td>BD/Pharmingen</td>
			<td>559320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse IgM FITC</td>
			<td>BD/Pharmingen</td>
			<td>553408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium chloride</td>
			<td>Merck</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride</td>
			<td>Sigma/Aldrich</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES&#160;</td>
			<td>cellgro/Mediatech</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>gibco/life technologies</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96well round bottom plate</td>
			<td>Falcon</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AnnexinV FITC</td>
			<td>PromoKine</td>
			<td>PK-CA577-1001-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7-AAD</td>
			<td>BD/Pharmingen</td>
			<td>51-68981E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS tubes</td>
			<td>Falcon</td>
			<td>352008</td>
			<td></td>
		</tr>
	</tbody>
</table>

killer artificial antigen presenting cells (kaapc) for efficient in vitro depletion of human antigen-specific t cells

目前治疗T细胞介导的​​自身免疫性疾病主要依赖于全球免疫抑制，其中，从长远来看，伴随着副作用的策略，如控制感染或恶性肿瘤的能力下降。因此，新的方法需要开发针对唯一疾病的细胞介导，并留下剩余的免疫系统完好无损。在过去十年中的各种基于细胞免疫治疗策略，以调节T细胞介导的​​免疫反应已经制定出来。大多数这些方法依赖于耐受性诱导抗原呈递细胞（APC）。然而，除了在技术上是困难和麻烦的，例如基于细胞的方法是给细胞毒性T细胞应答，这限制了它们的治疗能力高度敏感。这里我们提出了一个协议，用于非细胞杀伤人工抗原呈递细胞的产生（KaAPC），它允许对病理的T细胞的耗竭，同时使重剩余的免疫系统不变和功能。 KaAPC是一种替代的解决方案具有潜力用于通过在抗原特异性方式调节不希望的T细胞应答的治疗自身免疫性疾病和移植排斥反应的细胞免疫疗法。

此协议的功能包括定义一代KaAPC表型显示HLA-A2的Ig（信号1）和抗Fas单克隆抗体（细胞凋亡信号）的同时，其相比于基于细胞的方法不被涂层改变的或转导的功效，并且可以生产（ 图1）过程中容易被调制。此外，KaAPC，不给T细胞的细胞毒性效应器功能的敏感和它们的功能不依赖于自体抗原呈递细胞前，后在体外操作的质量。 KaAPC能够在满载着同源肽，非同源加载KaAPC不会耗尽T细胞（ 图2）消耗的抗原特异性T细胞在体外的。因此，KaAPC诱导T细胞凋亡中的抗原特异性的方式。接着，我们使用KaAPC为抗原特异性T细胞的选择性消耗从不同的T细胞SPE的混合物cificities，表明只有很少的细胞毒性，泄漏到相邻的T细胞（ 图3）。因此，KaAPC代表一个精美的工具用于体外抗原特异性人活化的抗原特异性T细胞的耗竭与潜在的临床应用性自身免疫性疾病和移植排斥的治疗。

Watch this Scientific Journal Video about Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells at JoVE.com

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

准则都用于杀伤人工抗原的产生呈递细胞，KaAPC，用于体外耗尽人抗原特异性T细胞的和在一个抗原的替代解决方案，以细胞免疫治疗对T细胞介导的自身免疫性疾病的治疗中的有效工具具体的方式不影响剩余的免疫系统。

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 ...

killer-artificial-antigen-presenting-cells-kaapc-for-efficient-vitro

Research

JoVE Journal

Immunology and Infection

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

9.6K 次观看.  Johns Hopkins University. 准则都用于杀伤人工抗原的产生呈递细胞，KaAPC，用于体外耗尽人抗原特异性T细胞的和在一个抗原的替代解决方案，以细胞免疫治疗对T细胞介导的自身免疫性疾病的治疗中的有效工具具体的方式不影响剩余的免疫系统。 

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

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

抗原特异性在体内使用CFSE标记的靶细胞的杀伤含量

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

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 ...

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

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 classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

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

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

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 ...

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 ...

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 ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

发电<em&gt;德诺</em&gt;抗原特异性人T细胞受体通过中心的Hemichain的逆转录病毒转导

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

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 ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

增殖人类抗原特异性CD4+ T细胞使用卡博曲莱辛苏奇尼米酯的定量

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

在自身免疫糖尿病模型中高效生成抗原特异性原小鼠细胞毒性T细胞细胞细胞，用于功能测试

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...