葡萄糖-6-磷酸异构酶（G6PI）诱导RA小鼠中iNKT细胞的采用免疫治疗
Summary
该协议使用G6PI混合肽构建类风湿关节炎模型，更接近CD4+T细胞和细胞因子中的人类类风湿性关节炎模型。高纯度不变天然杀伤性T细胞（主要是iNKT2），具有特定的表型和功能，通过体内诱导和体外纯化获得，用于采用免疫治疗。
Abstract
风湿性关节炎（RA）是一种复杂的慢性炎症性自身免疫性疾病。这种疾病的发病机制与不变的自然杀手T（iNKT）细胞有关。活性RA的患者存在较少的iNKT细胞，有缺陷的细胞功能，和Th1的极化过度。在这项研究中，使用hGPI325-339和hGPI469-483肽的混合物建立了RA动物模型。iNKT细胞通过体内诱导和体外纯化获得，然后输入RA小鼠进行采用免疫治疗。体内成像系统（IVIS）跟踪显示，iNKT细胞主要分布在脾脏和肝脏中。细胞治疗后的第12天，病情进展明显减缓，临床症状得到缓解，胸腺中iNKT细胞的丰度增加，胸腺中iNKT1的比例下降，TNF-α、IFN-α和IL-6水平下降。血清减少。iNKT细胞的接受免疫疗法恢复了免疫细胞的平衡，纠正了身体的过度炎症。
Introduction
风湿性关节炎（RA）是一种自身免疫性疾病，其特征是慢性、渐进性侵入性，发病率为0.5-1%，发病率为1，2。潜在的发病机制归因于自动反应CD4+和CD8+T细胞的异常增殖，表现为CD4+IFN++和CD4+IL-17A+T细胞比例增加，CD4+IL-4+CD4+CD4+CD25+FoxP3+T细胞比例增加。 因此，炎症细胞因子的分泌增加，过度的炎症反应破坏人体免疫系统的本生平衡和耐受功能。此外，穿透关节的助剂T淋巴细胞（Th）1细胞会加重炎症反应和关节损伤。因此，抑制过度的炎症反应和恢复免疫耐受性和免疫平衡是治疗RA3，4的关键。
iNKT细胞同时具有NK细胞和T细胞的功能和特性。iNKT细胞具有独特的、不变的T细胞受体（TCR）+链，具有有限的TCR+链表5，并识别抗原呈现细胞表面的主要组织相容性复合物（MHC）I类分子CD1d呈现的糖脂抗原。三三等人6日检测出大量iNKT细胞和功能缺陷，包括RA在内的许多自身免疫性疾病。Aurore等人7表明，iNKT细胞对维持自身免疫耐受性有积极作用，当iNKT细胞的数量和功能恢复时，疾病得到缓解。此外，Miellot-Gafsou等人8日发现，iNKT细胞不仅可使疾病发生，而且增加了疾病的进展。这些相互矛盾的结果表明，iNKT细胞是异质T细胞，不同子集的功能可能逆转。在RA的临床研究中，iNKT细胞的频率与疾病活性9的分数相关。结果还证实，在RA患者中，iNKT的频率降低，CD4+IFN-α+T细胞子集数量增加，炎症细胞因子IFN-α和TNF-α的分泌水平增加10，11。此外，Sharif等人12日调查了1型糖尿病（T1D），发现INKT细胞的选择性输注能调节炎症细胞因子IL-4的表达，保持免疫耐受性，并防止1型糖尿病的发展。因此，采用特定iNKT细胞的输注或针对iNKT细胞的活化可增加RA患者的iNKT细胞水平，这在RA治疗中是一个突破。
细胞免疫疗法目前备受关注，已广泛应用于癌症治疗。然而，iNKT细胞是罕见的，异质免疫调节细胞（只占PBMC总数的0.3%）13，这限制了潜在的临床应用。这些细胞主要分为三个亚群：1）iNKT1细胞，具有高表达的原发性白血病锌指蛋白（PLZF）和T-盒转录因子（T-bet）;2） 具有PLZF和GATA结合蛋白3（GATA3）中间表达的iNKT2细胞;3）iNKT17细胞，具有PLZF和视网膜相关孤立核受体（ROR）的低表达，即分泌IFN-*、IL-4和IL-17 14。激活的iNKT细胞分泌Th1、Th2和Th17样细胞因子，这些细胞因子决定了iNKT细胞15的不同免疫调节作用。iNKT细胞各亚群特异性活化的免疫调节和免疫治疗效果不同。因此，选择具有抗炎功能的iNKT细胞（主要是iNKT2）的特定表型，以调节人体的免疫反应，可以纠正RA的免疫不平衡和免疫障碍。
建立理想的动物模型对RA发病机制的治疗和研究具有重要意义。目前，最常用的和成熟的动物模型包括胶原蛋白引起的关节炎，辅助性关节炎，酶性关节炎，和多糖引起的关节炎16-17。然而，没有一个模型可以完全模拟人类RA的所有特征。II型胶原蛋白诱发关节炎（CIA）是一种经典的关节炎模型。CIA是通过对II型胶原蛋白特异性单克隆抗体的小鼠进行免疫接种，反映该疾病模型的抗体依赖性。Benurs等人描述了一个对葡萄糖-6-磷酸异构酶（G6PI）有全身免疫反应的模型，该模型在易感小鼠菌株18、19中诱导外周对称多关节炎。在这个模型中，关节炎的发展依赖于T细胞、B细胞和先天免疫18、19、20。Horikoshi21发现，在 CD4+ T 细胞和细胞因子（即 IL-6 和 TNF-α）方面，通过免疫 DBA/1 小鼠的 G6PI 多肽片段产生的 RA 模型比 CIA 模型更类似于人类 RA。为了增加对TCR识别部位的刺激作用，利用G6PI（hGPI325-339和hGPI469-483）的混合多肽片段对DBA/1小鼠进行免疫，以构建RA小鼠模型。这种方法的成功率可以很高，因为hGPI325-339和hGPI469-483是I-A q受限T细胞反应的免疫主导。因此，该模型可以模拟RA患者22的CD4+T细胞和iNKT细胞缺陷的增殖。RA免疫病理学的基础研究为我国进一步深入研究奠定了基础。
Protocol
Representative Results
Discussion
iNKT细胞是特殊的T细胞，桥接先天和适应性免疫，主要从CD4+/CD8+ 胸腺细胞开发。iNKT细胞具有不同的免疫调节功能，通过直接接触和分泌不同细胞因子23与其他免疫细胞相互作用，影响树突细胞（DC）、巨噬细胞、嗜中性粒细胞、B细胞、T细胞和NK细胞分化和发育2 4。β-GalCer是从海绵中提取的经典iNKT细胞特异性?…
Declarações
The authors have nothing to disclose.
Acknowledgements
本研究得到国家自然科学基金（国家自然科学基金）（81771755）、河北省高校科技重点研究项目（ZD2017009）和河北大学医学实验中心动物实验室的支持。我们感谢他们的支持。
Materials
| Alexa Fluor 647 Mouse Anti-PLZF | BD | 563490 | America |
| Anti-PE MicroBeads | Miltenyi | 130-048-801 | Germany |
| Columns | Miltenyi | MS | Germany |
| Cryogenic Centrifuge | Beckman | Allegra® X-15R | America |
| DiR | Thermo Fisher Scientific | D12731 | America |
| Embedding Center | Tianjin Aviation Electromechanical Co., Ltd. | BMJ-1 | China |
| FITC Hamster Anti-Mouse TCR β Chain | BD | 553170 | America |
| Flow cytometer | BD | Accuri C6 | America |
| Freund's complete adjuvant | Sigma | F5881 | America |
| hGPI325-339 (IWYINCFGCETHAML) | Karebay Biochem | 18062202 | China |
| hGPI469-483 (EGNRPTNSIVFTKLT) | Karebay Biochem | 18062203 | China |
| In Vivo Imaging System | PerkinElmer | caliper IVIS lumina II | America |
| Ionomycin Calcium | Cayman | 10004974 | America |
| KRN7000 | AdipoGen | AG-CN2-0013 | America |
| Mouse CD1d Tetramer-PE | MBL | TS-MCD-1 | Japan |
| Mouse percoll | Solarbio | P8620 | China |
| Optical Microscope | Olympus | Olympus-II | Japan |
| PerCP-CyTM5.5 Mouse anti-ROR-ϒt | BD | 562683 | America |
| PerCP-CyTM5.5 Mouse anti-T-bet | BD | 561316 | America |
| Pertussis toxin | Sigma | P7208 | America |
| phorbol esters | Cayman | 10008014 | America |
| Red Blood Cell Lysis Buffer | BD | 555899 | America |
| RPMI-1640 | Biological Industries | 01-100-1ACS | Israel |
| Th1/Th2/Th17 cytokines kit | BD | 560485 | America |
| Ultramicrotome | Leica | Leica EM UC6 | Germany |
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