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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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。 <p class="…
Representative Results
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…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 2-Mercaptoethanol (50mM) | Gibco | 21985-023 | 50 uM |
| 5’ RACE PCR | Clontech | 634859 | |
| anti-mouse CD28 antibodies | eBioscience | 14-0281-86 | final concentration at 1µg/ml |
| anti-mouse CD3e antibody | eBioscience | 145-2C11 | final concentration at 1µg/ml |
| Biotin Rat Anti-Mouse IFN-γ | BD Biosciences | 554410 | Working concentration at 0.5 µg/ml |
| BSA | Sigma | A7030 | |
| Endo-free Maxi-Prep kit | Qiagen | 12362 | |
| Gentamicin | Gibco | 15750-060 | Final 50 µg/ml. |
| Heat inactivated FCS | Hyclone | SH30087.03 | Final 10% FCS |
| HEPES (100X) | Gibco | 15630-080 | 1X |
| IAg7-CLIP tetramer-BV421 | NIH tetramer Facility at Emory | per approval | Working concentration at 6 µg/ml |
| IAg7-insulin P8E tetramer-BV421 | NIH tetramer Facility at Emory | per approval | Working concentration at 6 µg/ml |
| Insulin-Transferrin-Selenium-Ethanolamine (ITS 100x) | ThermoFisher | 51500056 | Final concentraion is 1x |
| Lipofectamine 2000 | Invitrogen | 11668019 | |
| LS Columns | Miltenyi Biotec | 130-042-401 | |
| MACS Separation Buffer | Miltenyi Biotec | 130-091-221 | |
| Mouse CD8a+ T Cell Isolation Kit | Miletenyi Biotec Inc | 130-104-075 | |
| Mouse CD8a+ T Cell Isolation Kit | Miltenyi Biotec | 130-104-075 | |
| Opti-MEM medium | ThermoFisher | 31985070 | |
| Penicillin-Streptomycin (5000U/ml) | ThermoFisher | 15070063 | 50 U/ml |
| Phoenix-ECO cells | ATCC | CRL-3214 | |
| Phosphate-buffered saline (PBS) | Gibco | 10010-023 | |
| pMIG II | Addgene | 52107 | |
| pMSCV-IRES-GFP II | Addgene | 52107 | |
| Purified Rat Anti-Mouse IFN-γ | BD Biosciences | 551216 | Working concentration at 3 µg/ml |
| Red cell lysis buffer | Sigma | R7767 | |
| RetroNectin | Takara | T100A | Working concentration at 50 µg/ml in PBS |
| rhIL-2 (stock concentration 105 IU/ul) | Peprotech | 200-02 | Final concentration at 200 IU/ml |
| rmIL-7 ( stock concentration 50ng/ul) | R&D | 407-ML-005 | Final concentration at 0.5ng/ml |
| RPMI-1640 | Gibco | 11875-093 | |
| Sterile Cell Strainers | Fisher Scientific | 22-363-548 | |
| Tryple | Gibco | 12605-028 |
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Citazione di questo articolo
Davidson, H. W., Cepeda, J. R., Sekhar, N. S., Han, J., Gao, L., Sosinowski, T., Zhang, L. High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model. J. Vis. Exp. (150), e59985, doi:10.3791/59985 (2019).