使用荧光成像快速体外细胞毒性评估表达嵌合抗原受体的 Jurkat
Summary
一种评估靶向单个肿瘤抗原的表达嵌合抗原受体 （CAR） 的 Jurkat 细胞定量肿瘤细胞杀伤的方案。该方案可用作筛选平台，用于在外周血来源的T细胞中确认之前快速优化CAR铰链结构。
Abstract
嵌合抗原受体 （CAR） T 细胞处于肿瘤学的最前沿。CAR 由靶向结构域（通常是单链可变片段 scFv）构建，伴随链内连接子，然后是铰链、跨膜和共刺激结构域。链内连接子和铰链结构域的修饰可以对CAR介导的杀伤产生显着影响。考虑到 CAR 结构的每个部分都有许多不同的选项，因此有大量的排列。制造CAR-T细胞是一个耗时且昂贵的过程，制造和测试许多构建体是一项大量的时间和材料投资。该协议描述了一个平台，用于快速评估 Jurkat 细胞 （CAR-J） 中铰链优化的 CAR 构建体。Jurkat 细胞是一种永生化的 T 细胞系，具有高慢病毒摄取量，可实现高效的 CAR 转导。在这里，我们提出了一个使用荧光成像仪快速评估 CAR-J 的平台，然后确认 PBMC 衍生的 T 细胞的细胞溶解。
Introduction
据美国国家癌症研究所1 报道，CAR-T 细胞疗法在血液系统恶性肿瘤中显示出巨大的前景，自 2017 年以来，FDA 批准了 6 种 CAR-T 产品。有许多CAR-T细胞在临床试验中用于靶向实体瘤。设计新的CAR靶点和优化CAR结构对于CAR-T细胞的功效至关重要。为每种应用选择理想的 CAR 构建体对于准确靶向肿瘤相关抗原 （TAA） 至关重要，同时避免正常组织中 TAA 表达水平低2。
CAR 构建体主要由五个区室组成：（1） 靶向肿瘤抗原的细胞外单链可变片段 （scFv） 结构域;（2）铰链域;（3）跨膜结构域;（4）细胞内细胞质T细胞共刺激结构域;（5）信令域。修改这些结构域中的每一个都会影响 CAR-T 细胞与其靶细胞结合的精度 3。因此，评估这些CAR构建体的细胞毒性和交叉反应性对于选择正确的构建体进行体内实验至关重要。目前评估 T 细胞溶解的方法包括 51Cr 释放试验、乳酸脱氢酶释放试验、生物发光成像试验、基于实时阻抗的细胞分析和基于细胞的流式细胞术试验 4,5。这里描述的基于荧光成像的平台可识别活细胞与死细胞的数量，这是对 T 细胞溶解的直接定量，而不是评估 T 细胞溶解的间接方法。
这是一种简单、经济高效、快速且高通量的技术，只需最少的干预，即可评估表达表皮生长因子受体 （EGFR） CAR 的 Jurkat 细胞对 MDA-MB-231 三阴性乳腺癌 （TNBC） 细胞和 EGFR CRISPR 敲除 MDA-MB-231 细胞的细胞毒性。Jurkat 细胞是永生化的人 T 淋巴细胞6，已被广泛用于研究 T 细胞活化和信号转导机制7。此外，Jurkat 细胞已在多项研究8、9、10、11 中用于体外 CAR 测试。Jurkat 细胞很容易被慢病毒转导并具有持续的增殖，该系统被用于优化各种 EGFR CAR 构建体的铰链结构域。
该测定可用于筛选靶向各种肿瘤抗原的多种CAR构建体，并用于对抗多种贴壁肿瘤细胞系和各种效应子与肿瘤（E：T）比率。此外，可以评估多个时间点，并且可以修改重复次数，以确定各种CAR构建体中的最佳杀伤。需要使用外周血单核细胞 （PBMC） 衍生的 CD3 T 细胞来确认最佳构建体。开发这种方法的总体目标是以高通量方式快速优化CAR铰链几何形状，克服转导效率低等障碍，然后在PBMC衍生的T细胞中得到确认。
Protocol
Representative Results
Discussion
在这里，我们提出了一种快速方法来有效地评估 Jurkat 细胞中 CAR 表达诱导的靶标特异性细胞溶解活性。所有 CAR 构建体具有相同的 scFv，但不同的铰链和跨膜结构域，这些结构域已被证明会影响 CAR-T 细胞的效力13。通过用抗原敲除 （KO） 细胞培养这些 CAR-J 来进一步评估这些 CAR-J 的非特异性杀伤。这表明杀伤是肿瘤抗原特异性的，而不是由于 CAR-J 的基础激活。可以很容易地确定?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
MDA-MB-231 是 Shane Stecklein 博士的一份善意礼物。作者感谢堪萨斯大学癌症中心为进行这项研究提供资金。
Materials
| 15 mL Conical Tube (Sterile) | Midwest Scientific | #C15B | Any similar will work |
| 50 mL Conical Tube (Sterile) | Thermo Scientific | 339652 | Any similar will work |
| Black/Clear 96 well plate | Falcon | 353219 | Celligo has a list of compatible plates |
| Celigo 4 Channel Imaging Cytomenter | Nexcelcom Bioscience | 200-BFFL-5C | Any similar will work |
| Celigo Software | Nexcelcom Bioscience | Version 5.3.0.0 | Any similar will work |
| Cell Culture Incubator | Thermo Scientific | HeraCell 160i | Any similar will work |
| Cell Culture Treated Flasks (T75, various sizes, Sterile) | TPP | 90076 | Any similar will work |
| CFSE | Tonbo | 13-0850-U500 | Any similar will work |
| Cytek Muse Cell Analyzer | Cytek | 0500-3115 | Any similar will work |
| DMEM | Gibco | 11995-040 | Any similar will work |
| FBS | Gemini bio-products | 900-108 | Any similar will work |
| Flow Cytometer | Cytek, BD, etc | Aurora, LSR II, etc | Any similar will work |
| FlowJo Sortware | Becton Dickinson & Company | Version 10.7.1 | Any similar will work |
| Fluorobrite DMEM | Gibco | A18967-01 | Any similar will work |
| GraphPad Software | GraphPad | Version 9.3.1 (471) | Any similar will work |
| Multichanel Pipette | Thermo Scientific | Finnpipette F2 | Any similar will work |
| PBS | Gibco | 10010-031 | Any similar will work |
| PenStrep | Gibco | 15070-063 | Any similar will work |
| Pipette tips (Sterile, filtered, 1 mL, Various sizes) | Pr1ma | PR-1250RK-FL, etc | Any similar will work |
| Pipettors | Thermo Scientific | Finnpipette F2 | Any similar will work |
| Propidium Iodide | Invitrogen | P1304MP | Any similar will work |
| RPMI | Corning | 10-041-cv | Any similar will work |
| Serological Pipette Aid | Drummond Scientific | 4-000-105 | Any similar will work |
| Serological Pipettes (Sterile, various sizes) | Pr1ma | PR-SERO-25, etc | Any similar will work |
| Sodium Pyruvate | Corning | 25-000-CI | Any similar will work |
| Sterile Reservoirs | Midwest Scientific | RESE-2000 | Any similar will work |
| Table top centrifuge | Eppendorf | 5810R | Any similar will work |
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