Name: high-efficiency generation of antigen-specific primary mouse cytotoxic t cells for functional testing in an autoimmune diabetes model
Uploaded: 2019-08-16T14:00:00
Description: Watch this Scientific Journal Video about High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model at JoVE.com

这项研究得到了JDRF资助1-INO-2015-74-S-B、2-SRA-2016-238-S-B和SRA-2-S-2018-648-S-B，糖尿病教育和行动奖，以及贝勒医学院分子医学研究卡罗琳·威斯法律基金的支持。细胞分拣由贝勒医学院细胞测定和细胞分拣核心支持，由NIH（S10RR024574和P30CA125123）资助。所有肽-MHC四联体均从NIH四聚能核心设施获得。

小鼠在转基因小鼠设施的特定无病原体条件下进行，所有动物实验均按照贝勒医学院动物护理和使用委员会批准的规程进行。
注：实验需要同时制备病毒和T细胞。表 1总结了协议。关键试剂和缓冲液列在材料表中。我们专注于生成和扩展针对该协议中特定 APC 人群的 CAR-T 细胞。
1. 单链Fab抗体（scFab）-CARs的生成和验证。
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该协议描述了一种通过抗逆转录病毒转导产生抗原特异性CD8 CAR-T细胞的有效方法。我们协议的转导效率通常很高，通常观察到CAR的强健表达。膨胀的CAR T细胞保留了母细胞激活的T细胞的基本特征和抗体特异性，适用于体外和体内使用。我们已经在NOD小鼠7的1型糖尿病中应用了Ab-CAR CD8 T细胞。
我们的协议包含了对前面描述的方法的一些关键修改。首先，我们使...

鉴于不必要的脱靶效应可能降低的风险，抗原特异性免疫疗法（ASI）有望治疗T1D等自身免疫性疾病。累积的证据表明，在T1D1中，对（前亲）胰岛素的免疫反应可能特别重要。在过去十年中，来自多个组（包括我们自己的组）的研究表明，由特定的MHC II类分子（B：9-23/MHCII）呈现含有胰岛素B链氨基酸9至23的表位在T1D的发展中起着重要作用。老鼠和人类2，3，4，5。为了有选择地瞄准B：9-23/MHCII复合物，我们产生了一种名为mAb287的单克隆抗体，这种抗体对含有其他肽6的激素胰岛素或复合物没有交叉反应。MAb287阻断体外抗原的呈现，每周给糖尿病前期NOD小鼠的mAb287分，延迟了35%的受治疗小鼠T1D的发育。为了阻断体内抗原的呈现，通常需要频繁注射以保持高循环浓度。我们假设，我们可以克服这一困难，利用Ab287的高特异性重新编程T细胞，从而为T1D7提供更好的抗原特异性T细胞治疗。
据报道，细胞毒性T细胞能够杀死他们的目标，即使一个单一的副本，他们的共生配体表示8，9，10。因此，B：9-23/MHCII 特异性CD8 T细胞在消除不需要的抗原表达方面比母抗体具有更高的效率，后者可能需要与同一APC上的多个复合物结合以发挥其作用。CAR T细胞已用于治疗多种人类癌症11，12，13，也可能有效在自身免疫14。然而，对致病性肽-MHC复合物具有特异性的CAR-T细胞迄今尚未用于改变T1D的进展。通过使用下面描述的优化的CD8 T细胞转导技术，我们最近证明了原理证明，这确实代表了一种可行的方法7。
在此协议中，我们概述了一种高效、简化的转导和扩展方法。我们的协议适用于需要高效生成小鼠 CD8 CAR T 细胞的其他研究。

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol (50mM)</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td>50 uM</td>
		</tr>
		<tr>
			<td>5&#8217; RACE PCR</td>
			<td>Clontech</td>
			<td>634859</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD28 antibodies</td>
			<td>eBioscience</td>
			<td>14-0281-86</td>
			<td>final concentration at 1&#181;g/ml</td>
		</tr>
		<tr>
			<td>anti-mouse CD3e antibody</td>
			<td>eBioscience</td>
			<td>145-2C11</td>
			<td>final concentration at 1&#181;g/ml</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse IFN-&#947;</td>
			<td>BD Biosciences</td>
			<td>554410</td>
			<td>Working concentration at 0.5 &#181;g/ml</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Endo-free Maxi-Prep kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td>Final 50 &#181;g/ml.</td>
		</tr>
		<tr>
			<td>Heat inactivated FCS</td>
			<td>Hyclone</td>
			<td>SH30087.03</td>
			<td>Final 10% FCS</td>
		</tr>
		<tr>
			<td>HEPES (100X)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>IAg7-CLIP tetramer-BV421</td>
			<td>NIH tetramer Facility at Emory</td>
			<td>per approval</td>
			<td>Working concentration at 6 &#181;g/ml</td>
		</tr>
		<tr>
			<td>IAg7-insulin P8E tetramer-BV421</td>
			<td>NIH tetramer Facility at Emory</td>
			<td>per approval</td>
			<td>Working concentration at 6 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-Ethanolamine (ITS 100x)</td>
			<td>ThermoFisher</td>
			<td>51500056</td>
			<td>Final concentraion is 1x</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Separation Buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse CD8a+ T Cell Isolation Kit</td>
			<td>Miletenyi Biotec Inc</td>
			<td>130-104-075</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse CD8a+ T Cell Isolation Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-075</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM medium</td>
			<td>ThermoFisher</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (5000U/ml)</td>
			<td>ThermoFisher</td>
			<td>15070063</td>
			<td>50 U/ml</td>
		</tr>
		<tr>
			<td>Phoenix-ECO cells</td>
			<td>ATCC</td>
			<td>CRL-3214</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>pMIG II</td>
			<td>Addgene</td>
			<td>52107</td>
			<td></td>
		</tr>
		<tr>
			<td>pMSCV-IRES-GFP II</td>
			<td>Addgene</td>
			<td>52107</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse IFN-&#947;</td>
			<td>BD Biosciences</td>
			<td>551216</td>
			<td>Working concentration at 3 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Red cell lysis buffer</td>
			<td>Sigma</td>
			<td>R7767</td>
			<td></td>
		</tr>
		<tr>
			<td>RetroNectin</td>
			<td>Takara</td>
			<td>T100A</td>
			<td>Working concentration at 50 &#181;g/ml in PBS</td>
		</tr>
		<tr>
			<td>rhIL-2 (stock concentration 105 IU/ul)</td>
			<td>Peprotech</td>
			<td>200-02</td>
			<td>Final concentration at 200 IU/ml</td>
		</tr>
		<tr>
			<td>rmIL-7 ( stock concentration 50ng/ul)</td>
			<td>R&#38;D</td>
			<td>407-ML-005</td>
			<td>Final concentration at 0.5ng/ml</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell Strainers</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryple</td>
			<td>Gibco</td>
			<td>12605-028</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-efficiency generation of antigen-specific primary mouse cytotoxic t cells for functional testing in an autoimmune diabetes model

1型糖尿病（T1D）的特点是小岛特异性自身免疫，导致β细胞破坏和胰岛素生产绝对损失。在自发的非肥胖糖尿病（NOD）小鼠模型中，胰岛素是主要目标，而这些动物的基因操作，以去除单键胰岛素表位预防疾病。因此，选择性地消除带有这种致病性表位的专业抗原呈现细胞（APC）是抑制不需要的胰岛素特异性自身免疫反应的方法，并且可能具有更大的转化潜力。
嵌合抗原受体（CARs）可以重定向T细胞，选择性地靶向致病抗原。这项技术是最近尝试使用细胞工程治疗多种癌症的基础。在此协议中，我们描述了优化的T细胞逆转录病毒（RV）转导和体外扩张协议，该协议产生大量功能抗原特异性CD8 CAR-T细胞，从少量幼化细胞开始。以前已经描述了多个CAR-T细胞协议，但转导后转导效率和细胞生存能力通常相对较低。相比之下，我们的方案提供高达90%的转导效率，产生的细胞可以在体内存活两周以上，并在一次输注后显著延缓疾病的发病。我们提供了细胞维护和转导协议的详细说明，以便轻松遵循关键步骤。从主细胞分离到CAR表达的整个过程可以在14天内执行。通用方法可应用于已知目标的任何小鼠疾病模型。同样，具体应用（针对致病肽/MHC II 类复合物）适用于已识别关键复合物的任何其他自身免疫疾病模型。

通常，使用该协议的转导效率为+60-90%。在图3所示的实验中，在排序之前，70%的CD8 T细胞共同表达GFP。他们还共同表达了CD28和CD3（图3C）。重要的是，所有的"测试"GFP+细胞也与IA g7-B：R3四联体共同染色，但不与对照四联体（图4）。同样，排序的测试和控制CAR-T细胞每个分泌高水平的IFN-*只有...

Watch this Scientific Journal Video about High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model at JoVE.com

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

本文介绍了一种用于生成抗原特异性CD8 T细胞及其在体外扩张的协议，目的是产生大量功能性T细胞，用于体外和体内。

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 ...

该协议允许您生成大量功能性抗原特异性T细胞，这是治疗自身免疫性疾病所需的安全方法。该技术的主要优点是，它为您提供了高效的转导过程和大量的功能T细胞。该技术可以推广用于治疗1型糖尿病和其他已知抗原的自身免疫性疾病。此外，CAR T细胞可能是某些类型的癌症的有希望的治疗方法。此方法可用于生成表达其他 CAR 的免疫细胞。良好的接受技术，阅读整个协议，并提前规划整个实验，是十分重要的。这两周的协议包括小步骤，成功取决于每个步骤的正确性能。此方法的可视化演示对于学员正确遵循至关重要。准备凤凰细胞转染，如手稿中所述。将添加和移除介质的侧壁标记，以尽量减少从细胞上吹走，这是一种好的做法。零天从细胞中吸出上一液，用五毫升PBS清洗。小心地将七毫升减少的血清介质滴到板的侧壁上，并将细胞转移回培养箱。加入1.5毫升的减少血清介质到两个14毫米圆底聚丙烯管。将40微米的转染试剂加入其中一个管中，将15微克的抗体重定向到另一个管中，将5微克的包装质粒添加到另一个管中。在室温下孵育管五分钟，然后将转染试剂加入质粒管。通过轻轻将溶液上下移液三次混合，并在室温下孵育至少20分钟。将三毫升混合物加入凤凰细胞，并将其放入组织培养箱中。四到五个小时后，向细胞中加入一毫升FCS，并在37摄氏度下过夜培养它们。第二天，从细胞中去除上一代，加入四毫升的新鲜预谋培养培养。第二天，通过从凤凰细胞中收集含有介质的病毒并过滤它来清除残留的细胞碎片来收获病毒。将重组的人类IL-2库存添加到病毒中，最终浓度为每毫升200国际单位，并立即将其用于转导。在凤凰细胞中加入四毫升新鲜培养物，并把它们放在孵化器中过夜。第二天重复病毒收集，进行第二次转导并丢弃细胞。在播种木氨酸CD8 T细胞前一天，在24井板的每个井中加入一毫升抗小鼠CD3和CD28抗体的混合物，并在4摄氏度下孵育板过夜。在第零天，收获小鼠，并把它们放在一个细胞过滤器浸泡在10毫升PBS在冰上细胞培养皿。在细胞培养罩中，将每个脾脏切成三到五块，并使用注射器的无菌柱塞通过铁丝网压入组织。轻轻去除红血球，在一至四个稀释的红细胞解液缓冲液中重新悬浮血细胞，并在室温下孵育五分钟，然后用 Trypan Blue 稀释 10 微升细胞悬浮液，用于细胞计数。以350倍g离心，在7分钟内对其余细胞进行造粒。使用小鼠CD8 T细胞分离试剂盒来丰富CD8 T细胞，将细胞颗粒悬浮在400微升缓冲液中，每10倍将100微升生物素抗体鸡尾酒悬浮到第八细胞中。将细胞悬浮液混合好，在4摄氏度下孵育5分钟，使抗体结合。然后在第八细胞中每10次添加300微升标签缓冲液和200微升抗生物素微珠。混合好，在4摄氏度下再孵育10分钟。同时，在分离器上设置分离柱，用三毫升的标记缓冲液清洗。将 40 微米细胞过滤器放在柱的顶部，通过应变器将一毫升珠子和细胞混合物穿过该过滤器进入分离柱。将流量收集到预冷的 15 毫升管中，然后根据制造商的指示清洗柱，将污水收集到同一管中。数数细胞，并在350次g下离心收集细胞5分钟。用两毫升的T细胞介质清洗它们，然后再次离心。然后以每毫升25万至50万细胞的浓度重新在预谋的T细胞培养中再增。用一毫升无菌PBS清洗先前准备好的CD3和CD28抗体涂层板三次，并将两毫升的细胞悬浮液添加到每孔中。使用旋转运动均匀分配细胞。作为一种控制，将相同数量的CD8 T细胞板入板的单一非涂层井中，并在10%的二氧化碳气罐培养箱中孵育37摄氏度的细胞48小时。第一天，在24孔板的井中加入0.5毫升的纤维素片，在4摄氏度的夜间孵化，准备人类纤维素碎片涂层板。第二天，取出纤维素溶液，每井加入1毫升2%BSA和PBS。在室温下孵育板30分钟，用一毫升无菌PBS清洗处理过的孔。取出洗涤液后，板已准备就绪。使用 Trypan Blue 计算激活的 CD8 T 单元格。通过离心收集它们。并重新以每毫升500万个活细胞进行转导。将激活的 CD8 电池悬浮液的 100 微升添加到纤维素涂层板的每个井中。然后添加1.5到两毫升先前准备的含有介质的病毒，并使用旋转运动混合，以均匀分配细胞。将盘子密封在Ziploc袋中，在37摄氏度下以2000次g离心90分钟。从离心机上取下板，并放在生物安全柜。小心地取出塑料袋，确保板外没有被介质污染。将板转移到37摄氏度的二氧化碳培养箱。四小时后，从每井中取出一毫升的培养物，用一毫升预谋的完整T细胞介质代替，然后将盘子放回培养箱中。该协议的关键方面是高滴度病毒、健康激活的T细胞和适当的转导方法。细胞与PE大小七结合抗CD8、AF647结合抗CD3和BV 421结合抗CD28共同染色，用于流动细胞测量分析。大约，70%的CD8 T细胞共同表达GP，表示汽车表达。他们还共同表示CD28和CD3。重要的是，所有测试GP阳性细胞也与 I-Ag7 胰岛素 BR3 三角体共同染色，它表示 CAR 在细胞表面的表达，但不是与控制四分之一剂的表达。此外，分类测试和控制 CAR T 细胞每个分泌高水平的干扰素伽马，只有在与表达其同源配体的目标细胞共同培养后，确认转导细胞具有针对父抗体目标的 CD8 效应或 T 细胞表型。病毒产生细胞的转导、原发CD8T细胞的分离以及这些T细胞的激活是本实验最重要的步骤。拥有健康的活性T细胞和用适当的试剂转导这些T细胞可确保良好的结果。此方法可用于换导其他 T 细胞，但必须针对特定 T 单元类型进行自定义。该协议为预防1型糖尿病与抗原特异性嵌合抗原受体T细胞治疗铺平了道路。

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

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

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 ...

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

Generation of Induced Regulatory T Cells from Primary Human NaÃƒÆ’Ã‚Â¯ve and Memory T Cells

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

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

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac转座子初级人类T细胞系统改造

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

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

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

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

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 ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

从小鼠皮肤使用酶消化和梯度分离提取白细胞浸润的

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

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

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 ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

在体内应用免疫模型检测抗原特异性 T 细胞细胞功能的试验

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...