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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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><tbody><tr><td>PBS</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Human AB serum</td><td>Atlanta biologicals</td><td>S40110</td><td></td></tr><tr><td>EDTA</td><td>Sigma/Aldrich</td><td>E5134</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma/Aldrich</td><td>71289</td><td></td></tr><tr><td>M-450 epoxybeads</td><td>Invitrogen/Dynal</td><td>14011</td><td></td></tr><tr><td>MPC-1 magnet</td><td>Invitrogen/Dynal</td><td>12001D</td><td></td></tr><tr><td>Screw top glass vial</td><td>Fisher Scientific</td><td>03-338AA</td><td></td></tr><tr><td>MACSmix Tube Rotator</td><td>Miltenyi</td><td>130-090-753</td><td></td></tr><tr><td>anti-CD95 (clone CH11)</td><td>Milliore</td><td>05-201</td><td></td></tr><tr><td>HLA-A2-Ig dimer</td><td>BD/Pharmingen</td><td>551263</td><td></td></tr><tr><td>Cryo-tube 2 ml</td><td>Corning</td><td>430488</td><td></td></tr><tr><td>anti-mouse IgG1 PE</td><td>BD/Pharmingen</td><td>559320</td><td></td></tr><tr><td>anti-mouse IgM FITC</td><td>BD/Pharmingen</td><td>553408</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck</td><td>S271-3</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma/Aldrich</td><td>C5080</td><td></td></tr><tr><td>HEPES</td><td>cellgro/Mediatech</td><td>25-060-CI</td><td></td></tr><tr><td>RPMI</td><td>gibco/life technologies</td><td>11875-085</td><td></td></tr><tr><td>96-well Round bottom plate</td><td>Falcon</td><td>353077</td><td></td></tr><tr><td>AnnexinV FITC</td><td>PromoKine</td><td>PK-CA577-1001-1000</td><td></td></tr><tr><td>7-AAD</td><td>BD/Pharmingen</td><td>51-68981E</td><td></td></tr><tr><td>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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

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

9.7K 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

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

协同刺激信号封锁的同种抗原刺激诱导人外周血单个核细胞同种异体抗原特异性无能

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

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 ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular substrates offer key advantages over cell-based aAPC, including precise control of signal presentation parameters and physical properties of the aAPC surface to modulate its interactions with T cells. aAPC constructed from anisotropic particles, particularly ellipsoidal particles, have been shown to be more effective than their spherical counterparts at stimulating T cells due to increased binding and larger surface area available for T cell contact, as well as reduced nonspecific uptake and enhanced pharmacokinetic properties. Despite increased interest in anisotropic particles, even widely accepted methods of generating anisotropic particles such as thin-film stretching can be challenging to implement and use reproducibly.
To this end, we describe a protocol for the rapid, standardized fabrication of biodegradable anisotropic particle-based aAPC with tunable size, shape, and signal presentation for T cell expansion ex vivo or in vivo, along with methods to characterize their size, morphology, and surface protein content, and to assess their functionality. This approach to fabricating anisotropic aAPC is scalable and reproducible, making it ideal for generating aAPC for &#34;off-the-shelf&#34; immunotherapies.

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

CD8+ T 细胞活化的各向异性聚合物人工抗原呈现细胞的制备

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular ...

环境科学

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

癌症研究

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 ...

Enrich and Expand Rare Antigen-specific T Cells with Magnetic Nanoparticles

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of antigen-specific T cells, B) probe the dynamics of antigen-specific responses, C) understand how antigen-specific responses affect disease state such as autoimmunity, D) demystify heterogeneous responses for antigen-specific T cells, or E) utilize antigen-specific cells for therapy. The tool is based on a magnetic particle that we conjugate antigen-specific and T cell co-stimulatory signals, and that we term as artificial antigen presenting cells (aAPCs). Consequently, since the technology is simple to produce, it can easily be adopted by other laboratories; thus, our purpose here is to describe in detail the fabrication and subsequent use of the aAPCs. We explain how to attach antigen-specific and co-stimulatory signals to the aAPCs, how to utilize them to enrich for antigen-specific T cells, and how to expand antigen-specific T cells. Furthermore, we will highlight engineering design considerations based on experimental and biological information of our experience with characterizing antigen-specific T cells.

Antigen-specific T cells are difficult to characterize or utilize in therapies due to their extremely low frequency. Herein, we provide a protocol to develop a magnetic particle which can bind to antigen-specific T cells to enrich these cells and then to expand them several hundred-fold for both characterization and therapy.

利用磁性纳米粒子丰富和扩大罕见抗原特异性 t 细胞

We have developed a tool to both enrich and expand antigen-specific T cells. This can be helpful in cases such as to A) detect the existence of ...

工程学

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 ...

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 ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

杀手人工抗原提呈细胞（KaAPC）为有效

摘要

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此视频中的章节

协同刺激信号封锁的同种抗原刺激诱导人外周血单个核细胞同种异体抗原特异性无能

HLA - IG基于人工抗原提呈细胞可高效体外扩展人类的CTL

人类承认免疫失衡的早期国家禁止作为筛检工具

CD8+ T 细胞活化的各向异性聚合物人工抗原呈现细胞的制备

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

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

利用磁性纳米粒子丰富和扩大罕见抗原特异性 t 细胞

从外周血人类同种异体抗原特异性T细胞的生成

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

Generation of Human Regulatory T Cell Clones

杀手人工抗原提呈细胞（KaAPC）为有效

摘要

探索更多视频

此视频中的章节

协同刺激信号封锁的同种抗原刺激诱导人外周血单个核细胞同种异体抗原特异性无能

HLA - IG基于人工抗原提呈细胞可高效体外扩展人类的CTL

人类承认免疫失衡的早期国家禁止作为筛检工具

CD8+ T 细胞活化的各向异性聚合物人工抗原呈现细胞的制备

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

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

利用磁性纳米粒子丰富和扩大罕见抗原特异性 t 细胞

从外周血人类同种异体抗原特异性T细胞的生成

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

Generation of Human Regulatory T Cell Clones

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